Patent application title: Compositions and methods for the diagnosis and treatment of tumor
Inventors:
Gretchen Frantz (San Francisco, CA, US)
Kenneth J. Hillan (San Francisco, CA, US)
Heidi Phillips (San Carlos, CA, US)
Paul Polakis (Burlingame, CA, US)
Susan D. Spencer (Tiburon, CA, US)
P. Mickey Williams (Half Moon Bay, CA, US)
Thomas D. Wu (San Francisco, CA, US)
Zemin Zhang (Foster City, CA, US)
IPC8 Class: AG01N33574FI
USPC Class:
435 723
Class name: Involving a micro-organism or cell membrane bound antigen or cell membrane bound receptor or cell membrane bound antibody or microbial lysate animal cell tumor cell or cancer cell
Publication date: 2009-03-19
Patent application number: 20090075302
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Patent application title: Compositions and methods for the diagnosis and treatment of tumor
Inventors:
Paul Polakis
Thomas D. Wu
Zemin Zhang
Susan D. Spencer
Gretchen Frantz
Kenneth J. Hillan
Heidi Phillips
P. Mickey Williams
Agents:
Goodwin Procter LLP;Attn: Patent Administrator
Assignees:
Origin: MENLO PARK, CA US
IPC8 Class: AG01N33574FI
USPC Class:
435 723
Abstract:
The present invention is directed to compositions of matter useful for the
diagnosis and treatment of tumor in mammals and to methods of using those
compositions of matter for the same.Claims:
1. (canceled)
2. A method of diagnosing the presence of a tumor in a mammal, said method comprising contacting a test sample of tissue cells obtained from said mammal with an antibody that binds to a protein comprising the amino acid sequence of SEQ ID NO:93, and detecting the formation of a complex between said antibody and said protein in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.
3. The method of claim 2, wherein said antibody is detectably labeled.
4. The method of claim 2, wherein said test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.
5. The method of claim 2, wherein said tumor is a colon tumor.
6. A method of diagnosing the presence of a tumor in a mammal, said method comprising determining the level of expression of a gene encoding a protein comprising the amino acid sequence of SEQ ID NO:93, in a test sample of tissue cells obtained from said mammal and in a control sample of known normal cells of the same tissue origin, wherein a higher level of expression of said protein in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.
7. The method of claim 6, wherein the step of determining the level of expression of a gene encoding said protein comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.
8. The method of claim 6, wherein the step determining the level of expression of a gene encoding said protein comprises employing an antibody in an immunohistochemistry or Western blot analysis.
9. The method of claim 6, wherein said tumor is a colon tumor.
Description:
RELATED APPLICATIONS
[0001]This application is a continuation of U.S. patent application Ser. No. 10/872,972, filed Jun. 21, 2004, which claims priority to U.S. patent application Ser. No. 10/241,220, filed Sep. 11, 2002, now abandoned, which claims priority to U.S. Provisional Patent Application Nos. 60/323,268, filed Sep. 18, 2001, 60/339,227, filed Oct. 19, 2001, 60/336,827, filed Nov. 7, 2001, 60/331,906, filed Nov. 20, 2001, 60/345,444, filed Jan. 2, 2002, 60/369,724, filed Apr. 3, 2002 and 60/404,809, filed Aug. 19, 2002, the entirety of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002]The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same.
BACKGROUND OF THE INVENTION
[0003]Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)). Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
[0004]In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise membrane-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such membrane-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. In this regard, it is noted that antibody-based therapy has proved very effective in the treatment of certain cancers. For example, HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco, Calif.) are antibodies that have been used successfully to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN® is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30% of primary breast cancers. RITUXAN® is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both these antibodies are recombinantly produced in CHO cells.
[0005]In other attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify (1) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (2) polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (3) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both the cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue). Such polypeptides may remain intracellularly located or may be secreted by the cancer cell. Moreover, such polypeptides may be expressed not by the cancer cell itself, but rather by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Such secreted polypeptides are often proteins that provide cancer cells with a growth advantage over normal cells and include such things as, for example, angiogenic factors, cellular adhesion factors, growth factors, and the like. Identification of antagonists of such non-membrane associated polypeptides would be expected to serve as effective therapeutic agents for the treatment of such cancers. Furthermore, identification of the expression pattern of such polypeptides would be useful for the diagnosis of particular cancers in mammals.
[0006]Despite the above identified advances in mammalian cancer therapy, there is a great need for additional diagnostic and therapeutic agents capable of detecting the presence of tumor in a mammal and for effectively inhibiting neoplastic cell growth, respectively. Accordingly, it is an objective of the present invention to identify: (1) cell membrane-associated polypeptides that are more abundantly expressed on one or more type(s) of cancer cell(s) as compared to on normal cells or on other different cancer cells, (2) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) (or by other cells that produce polypeptides having a potentiating effect on the growth of cancer cells) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (3) non-membrane-associated polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (4) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both a cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue), and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals. It is also an objective of the present invention to identify cell membrane-associated, secreted or intracellular polypeptides whose expression is limited to a single or very limited number of tissues, and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals.
SUMMARY OF THE INVENTION
A. Embodiments
[0007]In the present specification, Applicants describe for the first time the identification of various cellular polypeptides (and their encoding nucleic acids or fragments thereof) which are expressed to a greater degree on the surface of or by one or more types of cancer cell(s) as compared to on the surface of or by one or more types of normal non-cancer cells. Alternatively, such polypeptides are expressed by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Again alternatively, such polypeptides may not be overexpressed by tumor cells as compared to normal cells of the same tissue type, but rather may be specifically expressed by both tumor cells and normal cells of only a single or very limited number of tissue types (preferably tissues which are not essential for life, e.g., prostate, etc.). All of the above polypeptides are herein referred to as Tumor-associated Antigenic Target polypeptides ("TAT" polypeptides) and are expected to serve as effective targets for cancer therapy and diagnosis in mammals.
[0008]Accordingly, in one embodiment of the present invention, the invention provides an isolated nucleic acid molecule having a nucleotide sequence that encodes a tumor-associated antigenic target polypeptide or fragment thereof (a "TAT" polypeptide).
[0009]In certain aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule encoding a full-length TAT polypeptide having an amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
[0010]In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule comprising the coding sequence of a full-length TAT polypeptide cDNA as disclosed herein, the coding sequence of a TAT polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).
[0011]In further aspects, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule that encodes the same mature polypeptide encoded by the full-length coding region of any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).
[0012]Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide(s) are disclosed herein. Therefore, soluble extracellular domains of the herein described TAT polypeptides are contemplated.
[0013]In other aspects, the present invention is directed to isolated nucleic acid molecules which hybridize to (a) a nucleotide sequence encoding a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the nucleotide sequence of (a). In this regard, an embodiment of the present invention is directed to fragments of a full-length TAT polypeptide coding sequence, or the complement thereof, as disclosed herein, that may find use as, for example, hybridization probes useful as, for example, diagnostic probes, antisense oligonucleotide probes, or for encoding fragments of a full-length TAT polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-TAT polypeptide antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide. Such nucleic acid fragments are usually at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a TAT polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the TAT polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which TAT polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such novel fragments of TAT polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably those TAT polypeptide fragments that comprise a binding site for an anti-TAT antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide.
[0014]In another embodiment, the invention provides isolated TAT polypeptides encoded by any of the isolated nucleic acid sequences hereinabove identified.
[0015]In a certain aspect, the invention concerns an isolated TAT polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide protein, with or without the signal peptide, as disclosed herein, an amino acid sequence encoded by any of the nucleic acid sequences disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein.
[0016]In a further aspect, the invention concerns an isolated TAT polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.
[0017]In a specific aspect, the invention provides an isolated TAT polypeptide without the N-terminal signal sequence and/or without the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture.
[0018]Another aspect of the invention provides an isolated TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture.
[0019]In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cells comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
[0020]In other embodiments, the invention provides isolated chimeric polypeptides comprising any of the herein described TAT polypeptides fused to a heterologous (non-TAT) polypeptide. Example of such chimeric molecules comprise any of the herein described TAT polypeptides fused to a heterologous polypeptide such as, for example, an epitope tag sequence or a Fc region of an immunoglobulin.
[0021]In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that competitively inhibits the binding of an anti-TAT polypeptide antibody to its respective antigenic epitope. Antibodies of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the antibodies of the present invention may be detectably labeled, attached to a solid support, or the like.
[0022]In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described antibodies. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described antibodies is further provided and comprises culturing host cells under conditions suitable for expression of the desired antibody and recovering the desired antibody from the cell culture.
[0023]In another embodiment, the invention provides oligopeptides ("TAT binding oligopeptides") which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding oligopeptides of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding oligopeptides of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding oligopeptides of the present invention may be detectably labeled, attached to a solid support, or the like.
[0024]In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described TAT binding oligopeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described TAT binding oligopeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired oligopeptide and recovering the desired oligopeptide from the cell culture.
[0025]In another embodiment, the invention provides small organic molecules ("TAT binding organic molecules") which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding organic molecules of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding organic molecules of the present invention preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding organic molecules of the present invention may be detectably labeled, attached to a solid support, or the like.
[0026]In a still further embodiment, the invention concerns a composition of matter comprising a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
[0027]In yet another embodiment, the invention concerns an article of manufacture comprising a container and a composition of matter contained within the container, wherein the composition of matter may comprise a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein. The article may further optionally comprise a label affixed to the container, or a package insert included with the container, that refers to the use of the composition of matter for the therapeutic treatment or diagnostic detection of a tumor.
[0028]Another embodiment of the present invention is directed to the use of a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT polypeptide antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, for the preparation of a medicament useful in the treatment of a condition which is responsive to the TAT polypeptide, chimeric TAT polypeptide, anti-TAT polypeptide antibody, TAT binding oligopeptide, or TAT binding organic molecule.
B. Additional Embodiments
[0029]Another embodiment of the present invention is directed to a method for inhibiting the growth of a cell that expresses a TAT polypeptide, wherein the method comprises contacting the cell with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, and wherein the binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes inhibition of the growth of the cell expressing the TAT polypeptide. In preferred embodiments, the cell is a cancer cell and binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes death of the cell expressing the TAT polypeptide. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.
[0030]Yet another embodiment of the present invention is directed to a method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.
[0031]Yet another embodiment of the present invention is directed to a method of determining the presence of a TAT polypeptide in a sample suspected of containing the TAT polypeptide, wherein the method comprises exposing the sample to an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide and determining binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide in the sample, wherein the presence of such binding is indicative of the presence of the TAT polypeptide in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the TAT polypeptide. The antibody, TAT binding oligopeptide or TAT binding organic molecule employed in the method may optionally be detectably labeled, attached to a solid support, or the like.
[0032]A further embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises detecting the level of expression of a gene encoding a TAT polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of the TAT polypeptide in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.
[0033]Another embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the mammal with an antibody, oligopeptide or small organic molecule that binds to a TAT polypeptide and (b) detecting the formation of a complex between the antibody, oligopeptide or small organic molecule and the TAT polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the antibody, TAT binding oligopeptide or TAT binding organic molecule employed is detectably labeled, attached to a solid support, or the like, and/or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.
[0034]Yet another embodiment of the present invention is directed to a method for treating or preventing a cell proliferative disorder associated with altered, preferably increased, expression or activity of a TAT polypeptide, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a TAT polypeptide. Preferably, the cell proliferative disorder is cancer and the antagonist of the TAT polypeptide is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide. Effective treatment or prevention of the cell proliferative disorder may be a result of direct killing or growth inhibition of cells that express a TAT polypeptide or by antagonizing the cell growth potentiating activity of a TAT polypeptide.
[0035]Yet another embodiment of the present invention is directed to a method of binding an antibody, oligopeptide or small organic molecule to a cell that expresses a TAT polypeptide, wherein the method comprises contacting a cell that expresses a TAT polypeptide with said antibody, oligopeptide or small organic molecule under conditions which are suitable for binding of the antibody, oligopeptide or small organic molecule to said TAT polypeptide and allowing binding therebetween.
[0036]Other embodiments of the present invention are directed to the use of (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT polypeptide or a vector or host cell comprising that nucleic acid, (c) an anti-TAT polypeptide antibody, (d) a TAT-binding oligopeptide, or (e) a TAT-binding small organic molecule in the preparation of a medicament useful for (i) the therapeutic treatment or diagnostic detection of a cancer or tumor, or (ii) the therapeutic treatment or prevention of a cell proliferative disorder.
[0037]Another embodiment of the present invention is directed to a method for inhibiting the growth of a cancer cell, wherein the growth of said cancer cell is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide (wherein the TAT polypeptide may be expressed either by the cancer cell itself or a cell that produces polypeptide(s) that have a growth potentiating effect on cancer cells), wherein the method comprises contacting the TAT polypeptide with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth-potentiating activity of the TAT polypeptide and, in turn, inhibiting the growth of the cancer cell. Preferably the growth of the cancer cell is completely inhibited. Even more preferably, binding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide induces the death of the cancer cell. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.
[0038]Yet another embodiment of the present invention is directed to a method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth potentiating activity of said TAT polypeptide and resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.
C. Further Additional Embodiments
[0039]In yet further embodiments, the invention is directed to the following set of potential claims for this application:
[0040]1. Isolated nucleic acid having a nucleotide sequence that has at least 80% nucleic acid sequence identity to:
[0041](a) a DNA molecule encoding the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0042](b) a DNA molecule encoding the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0043](c) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0044](d) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0045](e) the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
[0046](f) the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0047](g) the complement of (a), (b), (c), (d), (e) or (f).
[0048]2. Isolated nucleic acid having:
[0049](a) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0050](b) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0051](c) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0052](d) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0053](e) the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
[0054](f) the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0055](g) the complement of (a), (b), (c), (d), (e) or (f).
[0056]3. Isolated nucleic acid that hybridizes to:
[0057](a) a nucleic acid that encodes the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0058](b) a nucleic acid that encodes the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide; [0059](c) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0060](d) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0061](e) the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
[0062](f) the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0063](g) the complement of (a), (b), (c), (d), (e) or (f).
[0064]4. The nucleic acid of Claim 3, wherein the hybridization occurs under stringent conditions.
[0065]5. The nucleic acid of Claim 3 which is at least about 5 nucleotides in length.
[0066]6. An expression vector comprising the nucleic acid of Claim 1, 2 or 3.
[0067]7. The expression vector of Claim 6, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.
[0068]8. A host cell comprising the expression vector of Claim 7.
[0069]9. The host cell of Claim 8 which is a CHO cell, an E. coli cell or a yeast cell.
[0070]10. A process for producing a polypeptide comprising culturing the host cell of Claim 8 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
[0071]11. An isolated polypeptide having at least 80% amino acid sequence identity to:
[0072](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0073](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0074](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0075](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0076](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0077](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0078]12. An isolated polypeptide having:
[0079](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0080](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0081](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0082](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0083](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0084](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0085]13. A chimeric polypeptide comprising the polypeptide of Claim 11 or 12 fused to a heterologous polypeptide.
[0086]14. The chimeric polypeptide of claim 13, wherein said heterologous polypeptide is an epitope tag sequence or an Fc region of an immunoglobulin.
[0087]15. An isolated antibody that binds to a polypeptide having at least 80% amino acid sequence identity to:
[0088](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0089](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0090](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0091](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0092](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0093](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0094]16. An isolated antibody that binds to a polypeptide having:
[0095](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0096](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0097](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0098](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0099](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0100](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0101]17. The antibody of Claim 15 or 16 which is a monoclonal antibody.
[0102]18. The antibody of Claim 15 or 16 which is an antibody fragment.
[0103]19. The antibody of Claim 15 or 16 which is a chimeric or a humanized antibody.
[0104]20. The antibody of Claim 15 or 16 which is conjugated to a growth inhibitory agent.
[0105]21. The antibody of Claim 15 or 16 which is conjugated to a cytotoxic agent.
[0106]22. The antibody of Claim 21, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0107]23. The antibody of Claim 21, wherein the cytotoxic agent is a toxin.
[0108]24. The antibody of Claim 23, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0109]25. The antibody of Claim 23, wherein the toxin is a maytansinoid.
[0110]26. The antibody of Claim 15 or 16 which is produced bacteria.
[0111]27. The antibody of Claim 15 or 16 which is produced in CHO cells.
[0112]28. The antibody of Claim 15 or 16 which induces death of a cell to which it binds.
[0113]29. The antibody of Claim 15 or 16 which is detectably labeled.
[0114]30. An isolated nucleic acid having a nucleotide sequence that encodes the antibody of Claim 15 or 16.
[0115]31. An expression vector comprising the nucleic acid of Claim 30 operably linked to control sequences recognized by a host cell transformed with the vector.
[0116]32. A host cell comprising the expression vector of Claim 31.
[0117]33. The host cell of Claim 32 which is a CHO cell, an E. coli cell or a yeast cell.
[0118]34. A process for producing an antibody comprising culturing the host cell of Claim 32 under conditions suitable for expression of said antibody and recovering said antibody from the cell culture.
[0119]35. An isolated oligopeptide that binds to a polypeptide having at least 80% amino acid sequence identity to:
[0120](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0121](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0122](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0123](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0124](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0125](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0126]36. An isolated oligopeptide that binds to a polypeptide having:
[0127](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0128](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0129](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0130](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0131](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0132](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0133]37. The oligopeptide of Claim 35 or 36 which is conjugated to a growth inhibitory agent.
[0134]38. The oligopeptide of Claim 35 or 36 which is conjugated to a cytotoxic agent.
[0135]39. The oligopeptide of Claim 38, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0136]40. The oligopeptide of Claim 38, wherein the cytotoxic agent is a toxin.
[0137]41. The oligopeptide of Claim 40, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0138]42. The oligopeptide of Claim 40, wherein the toxin is a maytansinoid.
[0139]43. The oligopeptide of Claim 35 or 36 which induces death of a cell to which it binds.
[0140]44. The oligopeptide of Claim 35 or 36 which is detectably labeled.
[0141]45. A TAT binding organic molecule that binds to a polypeptide having at least 80% amino acid sequence identity to:
[0142](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0143](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0144](c) an extracellular domain of the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0145](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0146](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0147](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0148]46. The organic molecule of Claim 45 that binds to a polypeptide having:
[0149](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0150](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0151](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0152](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0153](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0154](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0155]47. The organic molecule of Claim 45 or 46 which is conjugated to a growth inhibitory agent.
[0156]48. The organic molecule of Claim 45 or 46 which is conjugated to a cytotoxic agent.
[0157]49. The organic molecule of Claim 48, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0158]50. The organic molecule of Claim 48, wherein the cytotoxic agent is a toxin.
[0159]51. The organic molecule of Claim 50, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0160]52. The organic molecule of Claim 50, wherein the toxin is a maytansinoid.
[0161]53. The organic molecule of Claim 45 or 46 which induces death of a cell to which it binds.
[0162]54. The organic molecule of Claim 45 or 46 which is detectably labeled.
[0163]55. A composition of matter comprising:
[0164](a) the polypeptide of Claim 11;
[0165](b) the polypeptide of Claim 12;
[0166](c) the chimeric polypeptide of Claim 13;
[0167](d) the antibody of Claim 15;
[0168](e) the antibody of Claim 16;
[0169](f) the oligopeptide of Claim 35;
[0170](g) the oligopeptide of Claim 36;
[0171](h) the TAT binding organic molecule of Claim 45; or
[0172](i) the TAT binding organic molecule of Claim 46; in combination with a carrier.
[0173]56. The composition of matter of Claim 55, wherein said carrier is a pharmaceutically acceptable carrier.
[0174]57. An article of manufacture comprising:
[0175](a) a container; and
[0176](b) the composition of matter of Claim 55 contained within said container.
[0177]58. The article of manufacture of Claim 57 further comprising a label affixed to said container, or a package insert included with said container, referring to the use of said composition of matter for the therapeutic treatment of or the diagnostic detection of a cancer.
[0178]59. A method of inhibiting the growth of a cell that expresses a protein having at least 80% amino acid sequence identity to:
[0179](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0180](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0181](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0182](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0183](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0184](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein, the binding of said antibody, oligopeptide or organic molecule to said protein thereby causing an inhibition of growth of said cell.
[0185]60. The method of Claim 59, wherein said antibody is a monoclonal antibody.
[0186]61. The method of Claim 59, wherein said antibody is an antibody fragment.
[0187]62. The method of Claim 59, wherein said antibody is a chimeric or a humanized antibody.
[0188]63. The method of Claim 59, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
[0189]64. The method of Claim 59, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
[0190]65. The method of Claim 64, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0191]66. The method of Claim 64, wherein the cytotoxic agent is a toxin.
[0192]67. The method of Claim 66, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0193]68. The method of Claim 66, wherein the toxin is a maytansinoid.
[0194]69. The method of Claim 59, wherein said antibody is produced in bacteria.
[0195]70. The method of Claim 59, wherein said antibody is produced in CHO cells.
[0196]71. The method of Claim 59, wherein said cell is a cancer cell.
[0197]72. The method of Claim 71, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.
[0198]73. The method of Claim 71, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.
[0199]74. The method of Claim 71, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.
[0200]75. The method of Claim 59 which causes the death of said cell.
[0201]76. The method of Claim 59, wherein said protein has:
[0202](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0203](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0204](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0205](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0206](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0207](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0208]77. A method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a protein having at least 80% amino acid sequence identity to:
[0209](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0210](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0211](c) an extracellular domain of the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0212](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0213](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0214](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising administering to said mammal a therapeutically effective amount of an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said mammal.
[0215]78. The method of Claim 77, wherein said antibody is a monoclonal antibody.
[0216]79. The method of Claim 77, wherein said antibody is an antibody fragment.
[0217]80. The method of Claim 77, wherein said antibody is a chimeric or a humanized antibody.
[0218]81. The method of Claim 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
[0219]82. The method of Claim 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
[0220]83. The method of Claim 82, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0221]84. The method of Claim 82, wherein the cytotoxic agent is a toxin.
[0222]85. The method of Claim 84, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0223]86. The method of Claim 84, wherein the toxin is a maytansinoid.
[0224]87. The method of Claim 77, wherein said antibody is produced in bacteria.
[0225]88. The method of Claim 77, wherein said antibody is produced in CHO cells.
[0226]89. The method of Claim 77, wherein said tumor is further exposed to radiation treatment or a chemotherapeutic agent.
[0227]90. The method of Claim 77, wherein said tumor is a breast tumor, a colorectal tumor, a lung tumor, an ovarian tumor, a central nervous system tumor, a liver tumor, a bladder tumor, a pancreatic tumor, or a cervical tumor.
[0228]91. The method of Claim 77, wherein said protein is more abundantly expressed by the cancerous cells of said tumor as compared to a normal cell of the same tissue origin.
[0229]92. The method of Claim 77, wherein said protein has:
[0230](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0231](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0232](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0233](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0234](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0235](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0236]93. A method of determining the presence of a protein in a sample suspected of containing said protein, wherein said protein has at least 80% amino acid sequence identity to:
[0237](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0238](b) the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0239](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0240](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0241](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS: 1-56, 113, 115, 117 or 119); or
[0242](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising exposing said sample to an antibody, oligopeptide or organic molecule that binds to said protein and determining binding of said antibody, oligopeptide or organic molecule to said protein in said sample, wherein binding of the antibody, oligopeptide or organic molecule to said protein is indicative of the presence of said protein in said sample.
[0243]94. The method of Claim 93, wherein said sample comprises a cell suspected of expressing said protein.
[0244]95. The method of Claim 94, wherein said cell is a cancer cell.
[0245]96. The method of Claim 93, wherein said antibody, oligopeptide or organic molecule is detectably labeled.
[0246]97. The method of Claim 93, wherein said protein has:
[0247](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0248](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0249](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0250](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0251](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0252](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0253]98. A method of diagnosing the presence of a tumor in a mammal, said method comprising determining the level of expression of a gene encoding a protein having at least 80% amino acid sequence identity to:
[0254](a) the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0255](b) the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0256](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0257](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0258](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0259](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), in a test sample of tissue cells obtained from said mammal and in a control sample of known normal cells of the same tissue origin, wherein a higher level of expression of said protein in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.
[0260]99. The method of Claim 98, wherein the step of determining the level of expression of a gene encoding said protein comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.
[0261]100. The method of Claim 98, wherein the step determining the level of expression of a gene encoding said protein comprises employing an antibody in an immunohistochemistry or Western blot analysis.
[0262]101. The method of Claim 98, wherein said protein has:
[0263](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0264](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0265](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0266](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0267](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0268](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0269]102. A method of diagnosing the presence of a tumor in a mammal, said method comprising contacting a test sample of tissue cells obtained from said mammal with an antibody, oligopeptide or organic molecule that binds to a protein having at least 80% amino acid sequence identity to:
[0270](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0271](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0272](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0273](d) an extracellular domain of the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0274](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0275](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), and detecting the formation of a complex between said antibody, oligopeptide or organic molecule and said protein in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.
[0276]103. The method of Claim 102, wherein said antibody, oligopeptide or organic molecule is detectably labeled.
[0277]104. The method of Claim 102, wherein said test sample sue cells is obtained from an individual suspected of having a cancerous tumor.
[0278]105. The method of Claim 102, wherein said protein has:
[0279](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0280](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0281](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0282](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0283](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0284](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0285]106. A method for treating or preventing a cell proliferative disorder associated with increased expression or activity of a protein having at least 80% amino acid sequence identity to:
[0286](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0287](b) the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0288](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0289](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0290](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0291](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising administering to a subject in need of such treatment an effective amount of an antagonist of said protein, thereby effectively treating or preventing said cell proliferative disorder.
[0292]107. The method of Claim 106, wherein said cell proliferative disorder is cancer.
[0293]108. The method of Claim 106, wherein said antagonist is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide.
[0294]109. A method of binding an antibody, oligopeptide or organic molecule to a cell that expresses a protein having at least 80% amino acid sequence identity to:
[0295](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0296](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide; (c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0297](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide; [0298](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0299](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein and allowing the binding of the antibody, oligopeptide or organic molecule to said protein to occur, thereby binding said antibody, oligopeptide or organic molecule to said cell.
[0300]110. The method of Claim 109, wherein said antibody is a monoclonal antibody.
[0301]111. The method of Claim 109, wherein said antibody is an antibody fragment.
[0302]112. The method of Claim 109, wherein said antibody is a chimeric or a humanized antibody.
[0303]113. The method of Claim 109, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
[0304]114. The method of Claim 109, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
[0305]115. The method of Claim 114, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0306]116. The method of Claim 114, wherein the cytotoxic agent is a toxin.
[0307]117. The method of Claim 116, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0308]118. The method of Claim 116, wherein the toxin is a maytansinoid.
[0309]119. The method of Claim 109, wherein said antibody is produced in bacteria.
[0310]120. The method of Claim 109, wherein said antibody is produced in CHO cells.
[0311]121. The method of Claim 109, wherein said cell is a cancer cell.
[0312]122. The method of Claim 121, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.
[0313]123. The method of Claim 121, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.
[0314]124. The method of Claim 123, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.
[0315]125. The method of Claim 109 which causes the death of said cell.
[0316]126. Use of a nucleic acid as claimed in any of Claims 1 to 5 or 30 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0317]127. Use of a nucleic acid as claimed in any of Claims 1 to 5 or 30 in the preparation of a medicament for treating a tumor.
[0318]128. Use of a nucleic acid as claimed in any of Claims 1 to 5 or 30 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0319]129. Use of an expression vector as claimed in any of Claims 6, 7 or 31 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0320]130. Use of an expression vector as claimed in any of Claims 6, 7 or 31 in the preparation of medicament for treating a tumor.
[0321]131. Use of an expression vector as claimed in any of Claims 6, 7 or 31 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0322]132. Use of a host cell as claimed in any of Claims 8, 9, 32, or 33 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0323]133. Use of a host cell as claimed in any of Claims 8, 9, 32 or 33 in the preparation of a medicament for treating a tumor.
[0324]134. Use of a host cell as claimed in any of Claims 8, 9, 32 or 33 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0325]135. Use of a polypeptide as claimed in any of Claims 11 to 14 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0326]136. Use of a polypeptide as claimed in any of Claims 11 to 14 in the preparation of a medicament for treating a tumor.
[0327]137. Use of a polypeptide as claimed in any of Claims 11 to 14 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0328]138. Use of an antibody as claimed in any of Claims 15 to 29 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0329]139. Use of an antibody as claimed in any of Claims 15 to 29 in the preparation of a medicament for treating a tumor.
[0330]140. Use of an antibody as claimed in any of Claims 15 to 29 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0331]141. Use of an oligopeptide as claimed in any of Claims 35 to 44 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0332]142. Use of an oligopeptide as claimed in any of Claims 35 to 44 in the preparation of a medicament for treating a tumor.
[0333]143. Use of an oligopeptide as claimed in any of Claims 35 to 44 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0334]144. Use of a TAT binding organic molecule as claimed in any of Claims 45 to 54 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0335]145. Use of a TAT binding organic molecule as claimed in any of Claims 45 to 54 in the preparation of a medicament for treating a tumor.
[0336]146. Use of a TAT binding organic molecule as claimed in any of Claims 45 to 54 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0337]147. Use of a composition of matter as claimed in any of Claims 55 or 56 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0338]148. Use of a composition of matter as claimed in any of Claims 55 or 56 in the preparation of a medicament for treating a tumor.
[0339]149. Use of a composition of matter as claimed in any of Claims 55 or 56 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0340]150. Use of an article of manufacture as claimed in any of Claims 57 or 58 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
[0341]151. Use of an article of manufacture as claimed in any of Claims 57 or 58 in the preparation of a medicament for treating a tumor.
[0342]152. Use of an article of manufacture as claimed in any of Claims 57 or 58 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
[0343]153. A method for inhibiting the growth of a cell, wherein the growth of said cell is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to:
[0344](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0345](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0346](c) an extracellular domain of the polypeptide shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0347](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0348](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0349](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, there by inhibiting the growth of said cell.
[0350]154. The method of Claim 153, wherein said cell is a cancer cell.
[0351]155. The method of Claim 153, wherein said protein is expressed by said cell.
[0352]156. The method of Claim 153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.
[0353]157. The method of Claim 153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein induces the death of said cell.
[0354]158. The method of Claim 153, wherein said antibody is a monoclonal antibody.
[0355]159. The method of Claim 153, wherein said antibody is an antibody fragment.
[0356]160. The method of Claim 153, wherein said antibody is a chimeric or a humanized antibody.
[0357]161. The method of Claim 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
[0358]162. The method of Claim 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
[0359]163. The method of Claim 162, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0360]164. The method of Claim 162, wherein the cytotoxic agent is a toxin.
[0361]165. The method of Claim 164, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0362]166. The method of Claim 164, wherein the toxin is a maytansinoid.
[0363]167. The method of Claim 153, wherein said antibody is produced in bacteria.
[0364]168. The method of Claim 153, wherein said antibody is produced in CHO cells.
[0365]169. The method of Claim 153, wherein said protein has:
[0366](a) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0367](b) the amino acid sequence shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0368](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0369](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0370](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0371](f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0372]170. A method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to:
[0373](a) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0374](b) the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0375](c) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
[0376](d) an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
[0377](e) a polypeptide encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
[0378](f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in anyone of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said tumor.
[0379]171. The method of Claim 170, wherein said protein is expressed by cells of said tumor.
[0380]172. The method of Claim 170, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.
[0381]173. The method of Claim 170, wherein said antibody is a monoclonal antibody.
[0382]174. The method of Claim 170, wherein said antibody is an antibody fragment.
[0383]175. The method of Claim 170, wherein said antibody is a chimeric or a humanized antibody.
[0384]176. The method of Claim 170, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
[0385]177. The method of Claim 170, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
[0386]178. The method of Claim 177, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
[0387]179. The method of Claim 177, wherein the cytotoxic agent is a toxin.
[0388]180. The method of Claim 179, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
[0389]181. The method of Claim 179, wherein the toxin is a maytansinoid.
[0390]182. The method of Claim 170, wherein said antibody is produced in bacteria.
[0391]183. The method of Claim 170, wherein said antibody is produced in CHO cells.
[0392]184. The method of Claim 170, wherein said protein has:
[0393](a) the amino acid sequence shown in anyone of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
[0394](b) the amino acid sequence shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0395](c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
[0396](d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIG. 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
[0397](e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIG. 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
[0398]Yet further embodiments of the present invention will be evident to the skilled artisan upon a reading of the present specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0399]FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAT207 cDNA, wherein SEQ ID NO:1 is a clone designated herein as "DNA67962".
[0400]FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT177 cDNA, wherein SEQ ID NO:2 is a clone designated herein as "DNA77507".
[0401]FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT235 cDNA, wherein SEQ ID NO:3 is a clone designated herein as "DNA87993".
[0402]FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT234 cDNA, wherein SEQ ID NO:4 is a clone designated herein as "DNA92980".
[0403]FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT239 cDNA, wherein SEQ ID NO:5 is a clone designated herein as "DNA96792".
[0404]FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT193 cDNA, wherein SEQ ID NO:6 is a clone designated herein as "DNA96964".
[0405]FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT233 cDNA, wherein SEQ ID NO:7 is a clone designated herein as "DNA105792".
[0406]FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT226 cDNA, wherein SEQ ID NO:8 is a clone designated herein as "DNA119474".
[0407]FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT199 cDNA, wherein SEQ ID NO:9 is a clone designated herein as "DNA142915".
[0408]FIGS. 10A-B show a nucleotide sequence (SEQ ID NO:10) of a TAT204 cDNA, wherein SEQ ID NO:10 is a clone designated herein as "DNA150491".
[0409]FIGS. 11A-B show a nucleotide sequence (SEQ ID NO:11) of a TAT248 cDNA, wherein SEQ ID NO:11 is a clone designated herein as "DNA280351".
[0410]FIG. 12 shows a nucleotide sequence (SEQ ID NO:12) of a TAT232 cDNA, wherein SEQ ID NO:12 is a clone designated herein as "DNA150648".
[0411]FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a TAT219 cDNA, wherein SEQ ID NO:13 is a clone designated herein as "DNA172500".
[0412]FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a TAT224 cDNA, wherein SEQ ID NO:14 is a clone designated herein as "DNA179651".
[0413]FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a TAT237 cDNA, wherein SEQ ID NO:15 is a clone designated herein as "DNA207698".
[0414]FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a TAT178 cDNA, wherein SEQ ID NO:16 is a clone designated herein as "DNA208551".
[0415]FIGS. 17A-B show a nucleotide sequence (SEQ ID NO:17) of a TAT198 cDNA, wherein SEQ ID NO:17 is a clone designated herein as "DNA210159".
[0416]FIGS. 18A-B show a nucleotide sequence (SEQ ID NO:18) of a TAT194 cDNA, wherein SEQ ID NO:18 is a clone designated herein as "DNA225706".
[0417]FIGS. 19A-B show a nucleotide sequence (SEQ ID NO:19) of a TAT223 cDNA, wherein SEQ ID NO:19 is a clone designated herein as "DNA225793".
[0418]FIG. 20 shows a nucleotide sequence (SEQ ID NO:20) of a TAT196 cDNA, wherein SEQ ID NO:20 is a clone designated herein as "DNA225796".
[0419]FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a TAT236 cDNA, wherein SEQ ID NO:21 is a clone designated herein as "DNA225886".
[0420]FIG. 22 shows a nucleotide sequence (SEQ ID NO:22) of a TAT195 cDNA, wherein SEQ ID NO:22 is a clone designated herein as "DNA225943".
[0421]FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a TAT203 cDNA, wherein SEQ ID NO:23 is a clone designated herein as "DNA226283".
[0422]FIGS. 24A-B show a nucleotide sequence (SEQ ID NO:24) of a TAT200 cDNA, wherein SEQ ID NO:24 is a clone designated herein as "DNA226589".
[0423]FIGS. 25A-B show a nucleotide sequence (SEQ ID NO:25) of a TAT205 cDNA, wherein SEQ ID NO:25 is a clone designated herein as "DNA226622".
[0424]FIGS. 26A-B show a nucleotide sequence (SEQ ID NO:26) of a TAT185 cDNA, wherein SEQ ID NO:26 is a clone designated herein as "DNA226717".
[0425]FIGS. 27A-B show a nucleotide sequence (SEQ ID NO:27) of a TAT225 cDNA, wherein SEQ ID NO:27 is a clone designated herein as "DNA227162".
[0426]FIG. 28 shows a nucleotide sequence (SEQ ID NO:28) of a TAT247 cDNA, wherein SEQ ID NO:28 is a clone designated herein as "DNA277804".
[0427]FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a TAT197 cDNA, wherein SEQ ID NO:29 is a clone designated herein as "DNA227545".
[0428]FIG. 30 shows a nucleotide sequence (SEQ ID NO:30) of a TAT175 cDNA, wherein SEQ ID NO:30 is a clone designated herein as "DNA227611".
[0429]FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a TAT208 cDNA, wherein SEQ ID NO:31 is a clone designated herein as "DNA261021".
[0430]FIG. 32 shows a nucleotide sequence (SEQ ID NO:32) of a TAT174 cDNA, wherein SEQ ID NO:32 is a clone designated herein as "DNA233034".
[0431]FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a TAT214 cDNA, wherein SEQ ID NO:33 is a clone designated herein as "DNA266920".
[0432]FIG. 34 shows a nucleotide sequence (SEQ ID NO:34) of a TAT220 cDNA, wherein SEQ ID NO:34 is a clone designated herein as "DNA266921".
[0433]FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a TAT221 cDNA, wherein SEQ ID NO:35 is a clone designated herein as "DNA266922".
[0434]FIG. 36 shows a nucleotide sequence (SEQ ID NO:36) of a TAT201 cDNA, wherein SEQ ID NO:36 is a clone designated herein as "DNA234441".
[0435]FIGS. 37A-B show a nucleotide sequence (SEQ ID NO:37) of a TAT179 cDNA, wherein SEQ ID NO:37 is a clone designated herein as "DNA234834".
[0436]FIG. 38 shows a nucleotide sequence (SEQ ID NO:38) of a TAT216 cDNA, wherein SEQ ID NO:38 is a clone designated herein as "DNA247587".
[0437]FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a TAT218 cDNA, wherein SEQ ID NO:39 is a clone designated herein as "DNA255987".
[0438]FIG. 40 shows a nucleotide sequence (SEQ ID NO:40) of a TAT206 cDNA, wherein SEQ ID NO:40 is a clone designated herein as "DNA56041".
[0439]FIGS. 41A-B show a nucleotide sequence (SEQ ID NO:41) of a TAT374 cDNA, wherein SEQ ID NO:41 is a clone designated herein as "DNA257845".
[0440]FIG. 42 shows a nucleotide sequence (SEQ ID NO:42) of a TAT209 cDNA, wherein SEQ ID NO:42 is a clone designated herein as "DNA260655".
[0441]FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a TAT192 cDNA, wherein SEQ ID NO:43 is a clone designated herein as "DNA260945".
[0442]FIG. 44 shows a nucleotide sequence (SEQ ID NO:44) of a TAT180 cDNA, wherein SEQ ID NO:44 is a clone designated herein as "DNA247476".
[0443]FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a TAT375 cDNA, wherein SEQ ID NO:45 is a clone designated herein as "DNA260990".
[0444]FIG. 46 shows a nucleotide sequence (SEQ ID NO:46) of a TAT181 cDNA, wherein SEQ ID NO:46 is a clone designated herein as "DNA261001".
[0445]FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a TAT176 cDNA, wherein SEQ ID NO:47 is a clone designated herein as "DNA261013".
[0446]FIG. 48 shows a nucleotide sequence (SEQ ID NO:48) of a TAT184 cDNA, wherein SEQ ID NO:48 is a clone designated herein as "DNA262144".
[0447]FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a TAT182 cDNA, wherein SEQ ID NO:49 is a clone designated herein as "DNA266928".
[0448]FIGS. 50A-B show a nucleotide sequence (SEQ ID NO:50) of a TAT213 cDNA, wherein SEQ ID NO:50 is a clone designated herein as "DNA267342".
[0449]FIGS. 51A-C show a nucleotide sequence (SEQ ID NO:51) of a TAT217 cDNA, wherein SEQ ID NO:51 is a clone designated herein as "DNA267626".
[0450]FIG. 52 shows a nucleotide sequence (SEQ ID NO:52) of a TAT222 cDNA, wherein SEQ ID NO:52 is a clone designated herein as "DNA268035".
[0451]FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a TAT202 cDNA, wherein SEQ ID NO:53 is a clone designated herein as "DNA268334".
[0452]FIG. 54 shows a nucleotide sequence (SEQ ID NO:54) of a TAT215 cDNA, wherein SEQ ID NO:54 is a clone designated herein as "DNA269238".
[0453]FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a TAT238 cDNA, wherein SEQ ID NO:55 is a clone designated herein as "DNA272578".
[0454]FIG. 56 shows a nucleotide sequence (SEQ ID NO:56) of a TAT212 cDNA, wherein SEQ ID NO:56 is a clone designated herein as "DNA277797".
[0455]FIG. 57 shows the amino acid sequence (SEQ ID NO:57) derived from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
[0456]FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from the coding sequence of SEQ ID NO:2 shown in FIG. 2.
[0457]FIG. 59 shows the amino acid sequence (SEQ ID NO:59) derived from the coding sequence of SEQ ID NO:3 shown in FIG. 3.
[0458]FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from the coding sequence of SEQ ID NO:4 shown in FIG. 4.
[0459]FIG. 61 shows the amino acid sequence (SEQ ID NO:61) derived from the coding sequence of SEQ ID NO:5 shown in FIG. 5.
[0460]FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived from the coding sequence of SEQ ID NO:6 shown in FIG. 6.
[0461]FIG. 63 shows the amino acid sequence (SEQ ID NO:63) derived from the coding sequence of SEQ ID NO:7 shown in FIG. 7.
[0462]FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived from the coding sequence of SEQ ID NO:8 shown in FIG. 8.
[0463]FIG. 65 shows the amino acid sequence (SEQ ID NO:65) derived from the coding sequence of SEQ ID NO:9 shown in FIG. 9.
[0464]FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived from the coding sequence of SEQ ID NO:10 shown in FIGS. 10A-B.
[0465]FIG. 67 shows the amino acid sequence (SEQ ID NO:67) derived from the coding sequence of SEQ ID NO:11 shown in FIGS. 11A-B.
[0466]FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived from the coding sequence of SEQ ID NO:12 shown in FIG. 12.
[0467]FIG. 69 shows the amino acid sequence (SEQ ID NO:69) derived from the coding sequence of SEQ ID NO:13 shown in FIG. 13.
[0468]FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived from the coding sequence of SEQ ID NO:14 shown in FIG. 14.
[0469]FIG. 71 shows the amino acid sequence (SEQ ID NO:71) derived from the coding sequence of SEQ ID NO:15 shown in FIG. 15.
[0470]FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived from the coding sequence of SEQ ID NO:16 shown in FIG. 16.
[0471]FIG. 73 shows the amino acid sequence (SEQ ID NO:73) derived from the coding sequence of SEQ ID NO:17 shown in FIGS. 17A-B.
[0472]FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived from the coding sequence of SEQ ID NO:18 shown in FIGS. 18A-B.
[0473]FIG. 75 shows the amino acid sequence (SEQ ID NO:75) derived from the coding sequence of SEQ ID NO:19 shown in FIGS. 19A-B.
[0474]FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived from the coding sequence of SEQ ID NO:20 shown in FIG. 20.
[0475]FIG. 77 shows the amino acid sequence (SEQ ID NO:77) derived from the coding sequence of SEQ ID NO:21 shown in FIG. 21.
[0476]FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived from the coding sequence of SEQ ID NO:22 shown in FIG. 22.
[0477]FIG. 79 shows the amino acid sequence (SEQ ID NO:79) derived from the coding sequence of SEQ ID NO:23 shown in FIG. 23.
[0478]FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived from the coding sequence of SEQ ID NO:24 shown in FIGS. 24A-B.
[0479]FIG. 81 shows the amino acid sequence (SEQ ID NO:81) derived from the coding sequence of SEQ ID NO:25 shown in FIGS. 25A-B.
[0480]FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived from the coding sequence of SEQ ID NO:26 shown in FIGS. 26A-B.
[0481]FIG. 83 shows the amino acid sequence (SEQ ID NO:83) derived from the coding sequence of SEQ ID NO:27 shown in FIGS. 27A-B.
[0482]FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived from the coding sequence of SEQ ID NO:28 shown in FIG. 28.
[0483]FIG. 85 shows the amino acid sequence (SEQ ID NO:85) derived from the coding sequence of SEQ ID NO:29 shown in FIG. 29.
[0484]FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived from the coding sequence of SEQ ID NO:30 shown in FIG. 30.
[0485]FIG. 87 shows the amino acid sequence (SEQ ID NO:87) derived from the coding sequence of SEQ ID NO:31 shown in FIG. 31.
[0486]FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived from the coding sequence of SEQ ID NO:32 shown in FIG. 32.
[0487]FIG. 89 shows the amino acid sequence (SEQ ID NO:89) derived from the coding sequence of SEQ ID NO:33 shown in FIG. 33.
[0488]FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived from the coding sequence of SEQ ID NO:34 shown in FIG. 34.
[0489]FIG. 91 shows the amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:35 shown in FIG. 35.
[0490]FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived from the coding sequence of SEQ ID NO:36 shown in FIG. 36.
[0491]FIG. 93 shows the amino acid sequence (SEQ ID NO:93) derived from the coding sequence of SEQ ID NO:37 shown in FIGS. 37A-B.
[0492]FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived from the coding sequence of SEQ ID NO:38 shown in FIG. 38.
[0493]FIG. 95 shows the amino acid sequence (SEQ ID NO:95) derived from the coding sequence of SEQ ID NO:39 shown in FIG. 39.
[0494]FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ ID NO:40 shown in FIG. 40.
[0495]FIG. 97 shows the amino acid sequence (SEQ ID NO:97) derived from the coding sequence of SEQ ID NO:41 shown in FIGS. 41A-B.
[0496]FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived from the coding sequence of SEQ ID NO:42 shown in FIG. 42.
[0497]FIG. 99 shows the amino acid sequence (SEQ ID NO:99) derived from the coding sequence of SEQ ID NO:43 shown in FIG. 43.
[0498]FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derived from the coding sequence of SEQ ID NO:44 shown in FIG. 44.
[0499]FIG. 101 shows the amino acid sequence (SEQ ID NO:101) derived from the coding sequence of SEQ ID NO:45 shown in FIG. 45.
[0500]FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derived from the coding sequence of SEQ ID NO:46 shown in FIG. 46.
[0501]FIG. 103 shows the amino acid sequence (SEQ ID NO:103) derived from the coding sequence of SEQ ID NO:47 shown in FIG. 47.
[0502]FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derived from the coding sequence of SEQ ID NO:48 shown in FIG. 48.
[0503]FIG. 105 shows the amino acid sequence (SEQ ID NO:105) derived from the coding sequence of SEQ ID NO:49 shown in FIG. 49.
[0504]FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derived from the coding sequence of SEQ ID NO:50 shown in FIGS. 50A-B.
[0505]FIGS. 107A-B show the amino acid sequence (SEQ ID NO:107) derived from the coding sequence of SEQ ID NO:51 shown in FIGS. 51A-C.
[0506]FIG. 108 shows the amino acid sequence (SEQ ID NO:108) derived from the coding sequence of SEQ ID NO:52 shown in FIG. 52.
[0507]FIG. 109 shows the amino acid sequence (SEQ ID NO:109) derived from the coding sequence of SEQ ID NO:53 shown in FIG. 53.
[0508]FIG. 110 shows the amino acid sequence (SEQ ID NO:110) derived from the coding sequence of SEQ ID NO:54 shown in FIG. 54.
[0509]FIG. 111 shows the amino acid sequence (SEQ ID NO:111) derived from the coding sequence of SEQ ID NO:55 shown in FIG. 55.
[0510]FIG. 112 shows the amino acid sequence (SEQ ID NO:112) derived from the coding sequence of SEQ ID NO:56 shown in FIG. 56.
[0511]FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a TAT376 cDNA, wherein SEQ ID NO:113 is a clone designated herein as "DNA304853".
[0512]FIG. 114 shows the amino acid sequence (SEQ ID NO:114) derived from the coding sequence of SEQ ID NO:113 shown in FIG. 113.
[0513]FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a TAT377 cDNA, wherein SEQ ID NO:115 is a clone designated herein as "DNA304854".
[0514]FIG. 116 shows the amino acid sequence (SEQ ID NO:116) derived from the coding sequence of SEQ ID NO:115 shown in FIG. 115.
[0515]FIG. 117 shows a nucleotide sequence (SEQ ID NO:117) of a TAT378 cDNA, wherein SEQ ID NO:117 is a clone designated herein as "DNA304855".
[0516]FIG. 118 shows the amino acid sequence (SEQ ID NO:118) derived from the coding sequence of SEQ ID NO:117 shown in FIG. 117.
[0517]FIGS. 119A-B show a nucleotide sequence (SEQ ID NO:119) of a TAT379 cDNA, wherein SEQ ID NO:119 is a clone designated herein as "DNA287971".
[0518]FIG. 120 shows the amino acid sequence (SEQ ID NO:120) derived from the coding sequence of SEQ ID NO:119 shown in FIGS. 119A-B.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0519]The terms "TAT polypeptide" and "TAT" as used herein and when immediately followed by a numerical designation, refer to various polypeptides, wherein the complete designation (i.e., TAT/number) refers to specific polypeptide sequences as described herein. The terms "TAT/number polypeptide" and "TAT/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides, polypeptide variants and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The TAT polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term "TAT polypeptide" refers to each individual TAT/number polypeptide disclosed herein. All disclosures in this specification which refer to the "TAT polypeptide" refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, formation of TAT binding oligopeptides to or against, formation of TAT binding organic molecules to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term "TAT polypeptide" also includes variants of the TAT/number polypeptides disclosed herein.
[0520]A "native sequence TAT polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding TAT polypeptide derived from nature. Such native sequence TAT polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence TAT polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific TAT polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In certain embodiments of the invention, the native sequence TAT polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons (if indicated) are shown in bold font and underlined in the figures. Nucleic acid residues indicated as "N" in the accompanying figures are any nucleic acid residue. However, while the TAT polypeptides disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the TAT polypeptides.
[0521]The TAT polypeptide "extracellular domain" or "ECD" refers to a form of the TAT polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a TAT polypeptide
[0522]ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the TAT polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a TAT polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention.
[0523]The approximate location of the "signal peptides" of the various TAT polypeptides disclosed herein may be shown in the present specification and/or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
[0524]"TAT polypeptide variant" means a TAT polypeptide, preferably an active TAT polypeptide, as defined herein having at least about 80% amino acid sequence identity with a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Such TAT polypeptide variants include, for instance, TAT polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide sequence as disclosed herein. Ordinarily, TAT variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids in length, or more. Optionally, TAT variant polypeptides will have no more than one conservative amino acid substitution as compared to the native TAT polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to the native TAT polypeptide sequence.
[0525]"Percent (%) amino acid sequence identity" with respect to the TAT polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific TAT polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
[0526]In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "TAT", wherein "TAT" represents the amino acid sequence of a hypothetical TAT polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "TAT" polypeptide of interest is being compared, and "X, "Y" and "Z" each represent different hypothetical amino acid residues. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
[0527]"TAT variant polynucleotide" or "TAT variant nucleic acid sequence" means a nucleic acid molecule which encodes a TAT polypeptide, preferably an active TAT polypeptide, as defined herein and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Ordinarily, a TAT variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.
[0528]Ordinarily, TAT variant polynucleotides are at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length.
[0529]"Percent (%) nucleic acid sequence identity" with respect to TAT-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the TAT nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
[0530]In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "TAT-DNA", wherein "TAT-DNA" represents a hypothetical TAT-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "TAT-DNA" nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent different hypothetical nucleotides. Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
[0531]In other embodiments, TAT variant polynucleotides are nucleic acid molecules that encode a TAT polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length TAT polypeptide as disclosed herein. TAT variant polypeptides may be those that are encoded by a TAT variant polynucleotide.
[0532]The term "full-length coding region" when used in reference to a nucleic acid encoding a TAT polypeptide refers to the sequence of nucleotides which encode the full-length TAT polypeptide of the invention (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures). The term "full-length coding region" when used in reference to an ATCC deposited nucleic acid refers to the TAT polypeptide-encoding portion of the cDNA that is inserted into the vector deposited with the ATCC (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures).
[0533]"Isolated," when used to describe the various TAT polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the TAT polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
[0534]An "isolated" TAT polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
[0535]The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
[0536]Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
[0537]"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
[0538]"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) overnight hybridization in a solution that employs 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10 minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) followed by a 10 minute high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.
[0539]"Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
[0540]The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a TAT polypeptide or anti-TAT antibody fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
[0541]"Active" or "activity" for the purposes herein refers to form(s) of a TAT polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring TAT, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring TAT other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT.
[0542]The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native TAT polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TAT polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a TAT polypeptide may comprise contacting a TAT polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TAT polypeptide.
[0543]"Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a TAT polypeptide-expressing cancer if, after receiving a therapeutic amount of an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the anti-TAT antibody or TAT binding oligopeptide may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.
[0544]The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR). Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0545]For bladder cancer, which is a more localized cancer, methods to determine progress of disease include urinary cytologic evaluation by cystoscopy, monitoring for presence of blood in the urine, visualization of the urothelial tract by sonography or an intravenous pyelogram, computed tomography (CT) and magnetic resonance imaging (MRI). The presence of distant metastases can be assessed by CT of the abdomen, chest x-rays, or radionuclide imaging of the skeleton.
[0546]"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
[0547]"Mammal" for purposes of the treatment of, alleviating the symptoms of or diagnosis of a cancer refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
[0548]Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
[0549]"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
[0550]By "solid phase" or "solid support" is meant a non-aqueous matrix to which an antibody, TAT binding oligopeptide or TAT binding organic molecule of the present invention can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
[0551]A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a TAT polypeptide, an antibody thereto or a TAT binding oligopeptide) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
[0552]A "small" molecule or "small" organic molecule is defined herein to have a molecular weight below about 500 Daltons.
[0553]An "effective amount" of a polypeptide, antibody, TAT binding oligopeptide, TAT binding organic molecule or an agonist or antagonist thereof as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" may be determined empirically and in a routine manner, in relation to the stated purpose.
[0554]The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, TAT binding oligopeptide, TAT binding organic molecule or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
[0555]A "growth inhibitory amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "growth inhibitory amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
[0556]A "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.
[0557]The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-TAT monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-TAT antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain anti-TAT antibodies, and fragments of anti-TAT antibodies (see below) as long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeable with antibody herein.
[0558]An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[0559]The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
[0560]The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
[0561]The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
[0562]The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
[0563]The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0564]The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.
[0565]An "intact" antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
[0566]"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[0567]Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
[0568]The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
[0569]"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[0570]"Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
[0571]The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0572]"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0573]A "species-dependent antibody," e.g., a mammalian anti-human IgE antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody "bind specifically" to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1×10-7 M, preferably no more than about 1×10-8 and most preferably no more than about 1×10-9 M) but has a binding affinity for a homologue of the antigen from a second non-human mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
[0574]A "TAT binding oligopeptide" is an oligopeptide that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).
[0575]A "TAT binding organic molecule" is an organic molecule other than an oligopeptide or antibody as defined herein that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).
[0576]An antibody, oligopeptide or other organic molecule "which binds" an antigen of interest, e.g. a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the antibody, oligopeptide or other organic molecule is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody, oligopeptide or other organic molecule to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody, oligopeptide or other organic molecule to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10-4 M, alternatively at least about 10-5 M, alternatively at least about 10-4 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
[0577]An antibody, oligopeptide or other organic molecule that "inhibits the growth of tumor cells expressing a TAT polypeptide" or a "growth inhibitory" antibody, oligopeptide or other organic molecule is one which results in measurable growth inhibition of cancer cells expressing or overexpressing the appropriate TAT polypeptide. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or organic molecules inhibit growth of TAT-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody, oligopeptide or other organic molecule being tested. In one embodiment, growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
[0578]An antibody, oligopeptide or other organic molecule which "induces apoptosis" is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is usually one which overexpresses a TAT polypeptide. Preferably the cell is a tumor cell, e.g., a prostate, breast, ovarian, stomach, endometrial, lung, kidney, colon, bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody, oligopeptide or other organic molecule which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.
[0579]Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
[0580]"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).
[0581]"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
[0582]"Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.
[0583]"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
[0584]The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases.
[0585]The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.
[0586]"Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
[0587]An antibody, oligopeptide or other organic molecule which "induces cell death" is one which causes a viable cell to become nonviable. The cell is one which expresses a TAT polypeptide, preferably a cell that overexpresses a TAT polypeptide as compared to a normal cell of the same tissue type. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody, oligopeptide or other organic molecule is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells. Preferred cell death-inducing antibodies, oligopeptides or other organic molecules are those which induce PI uptake in the PI uptake assay in BT474 cells.
[0588]A "TAT-expressing cell" is a cell which expresses an endogenous or transfected TAT polypeptide either on the cell surface or in a secreted form. A "TAT-expressing cancer" is a cancer comprising cells that have a TAT polypeptide present on the cell surface or that produce and secrete a TAT polypeptide. A "TAT-expressing cancer" optionally produces sufficient levels of TAT polypeptide on the surface of cells thereof, such that an anti-TAT antibody, oligopeptide or other organic molecule can bind thereto and have a therapeutic effect with respect to the cancer. In another embodiment, a "TAT-expressing cancer" optionally produces and secretes sufficient levels of TAT polypeptide, such that an anti-TAT antibody, oligopeptide or other organic molecule antagonist can bind thereto and have a therapeutic effect with respect to the cancer. With regard to the latter, the antagonist may be an antisense oligonucleotide which reduces, inhibits or prevents production and secretion of the secreted TAT polypeptide by tumor cells. A cancer which "overexpresses" a TAT polypeptide is one which has significantly higher levels of TAT polypeptide at the cell surface thereof, or produces and secretes, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. TAT polypeptide overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the TAT protein present on the surface of a cell, or secreted by the cell (e.g., via an immunohistochemistry assay using anti-TAT antibodies prepared against an isolated TAT polypeptide which may be prepared using recombinant DNA technology from an isolated nucleic acid encoding the TAT polypeptide; FACS analysis, etc.). Alternatively, or additionally, one may measure levels of TAT polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid based probe corresponding to a TAT-encoding nucleic acid or the complement thereof; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study TAT polypeptide overexpression by measuring shed antigen in a biological fluid such as serum, e.g, using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.
[0589]As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
[0590]The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody, oligopeptide or other organic molecule so as to generate a "labeled" antibody, oligopeptide or other organic molecule. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
[0591]The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.
[0592]A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of TAT-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
[0593]"Doxorubicin" is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,- 8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht- hacenedione.
[0594]The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
[0595]The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
TABLE-US-00001 TABLE 1 /* * * C-C increased from 12 to 15 * Z is average of EQ * B is average of ND * match with stop is _M; stop-stop = 0; J (joker) match = 0 */ #define _M -8 /* value of a match with a stop */ int _day[26][26] = { /* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z */ /* A */ { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0}, /* B */ { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3, 1}, /* C */ {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5}, /* D */ { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 2}, /* E */ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* F */ {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, /* G */ { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-1,-1,-3, 1, 0, 0,-1,-7, 0,-5, 0}, /* H */ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0, 2}, /* I */ {-1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-1, 0, 0, 4,-5, 0,-1,-2}, /* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4, 0}, /* L */ {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-1,-2}, /* M */ {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-1, 0,-2,-1, 0, 2,-4, 0,-2,-1}, /* N */ { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1}, /* O */ {_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,-1,-3,-1,-1,-5,-1, 0,-2, 0,-1,-3,-2,-1,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0}, /* Q */ { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1, 0,-2,-5, 0,-4, 3}, /* R */ {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, /* S */ { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1,_M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3, 0}, /* T */ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-3, 0}, /* U */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ { 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-1,-2,-2,-1, 0, 0, 4,-6, 0,-2,-2}, /* W */ {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4,_M,-6,-5, 2,-2,-5, 0,-6,17, 0, 0,-6}, /* X */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */ {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2,_M,-5,-4,-4,-3,-3, 0,-2, 0, 0,10,-4}, /* Z */ { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-4, 4} }; /* */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16 /* max jumps in a diag */ #define MAXGAP 24 /* don't continue to penalize gaps larger than this */ #define JMPS 1024 /* max jmps in an path */ #define MX 4 /* save if there's at least MX-1 bases since last jmp */ #define DMAT 3 /* value of matching bases */ #define DMIS 0 /* penalty for mismatched bases */ #define DINS0 8 /* penalty for a gap */ #define DINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap */ #define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16 -1 */ struct diag { int score; /* score at last jmp */ long offset; /* offset of prev block */ short ijmp; /* current jmp index */ struct jmp jp; /* list of jmps */ }; struct path { int spc; /* number of leading spaces */ short n[JMPS]; /* size of jmp (gap) */ int x[JMPS];/* loc of jmp (last elem before gap) */ }; char *ofile; /* output file name */ char *namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* best diag: nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main( ) */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /* bitmap for matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds path for seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char *getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment program * * usage: progs file1 file2 * where file1 and file2 are two dna or two protein sequences. * The sequences can be in upper- or lower-case an may contain ambiguity * Any lines beginning with `;`, `>` or `<` are ignored * Max file length is 65535 (limited by unsigned short x in the jmp struct) * A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA * Output is in the file "align.out" * * The program may create a tmp file in /tmp to hold info about traceback. * Original version developed under BSD 4.3 on a vax 8650 */ #include "nw.h" #include "day.h" static _dbval[26] = { 1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static _pbval[26] = { 1, 2|(1<<(`D`-`A`))|(1<<(`N`-`A`)), 4, 8, 16, 32, 64, 128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16, 1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24, 1<<25|(1<<(`E`-`A`))|(1<<(`Q`-`A`)) }; main(ac, av) main int ac; char *av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,"usage: %s file1 file2\n", prog); fprintf(stderr,"where file1 and file2 are two dna or two protein sequences.\n"); fprintf(stderr,"The sequences can be in upper- or lower-case\n"); fprintf(stderr,"Any lines beginning with `;` or `<` are ignored\n"); fprintf(stderr,"Output is in the file \"align.out\"\n"); exit(1); } namex[0] = av[1]; namex[1] = av[2]; seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1); xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile = "align.out"; /* output file */ nw( ); /* fill in the matrix, get the possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /* print stats, alignment */ cleanup(0); /* unlink any tmp files */} /* do the alignment, return best score: main( ) * dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983 * pro: PAM 250 values * When scores are equal, we prefer mismatches to any gap, prefer * a new gap to extending an ongoing gap, and prefer a gap in seqx * to a gap in seq y. */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx */ int *tmp; /* for swapping row0, row1 */ int mis; /* score for each type */ int ins0, ins1; /* insertion penalties */ register id; /* diagonal index */ register ij; /* jmp index */ register *col0, *col1; /* score for curr, last row */ register xx, yy; /* index into seqs */ dx = (struct diag *)g_calloc("to get diags", len0+len1+1, sizeof(struct diag)); ndely = (int *)g_calloc("to get ndely", len1+1, sizeof(int)); dely = (int *)g_calloc("to get dely", len1+1, sizeof(int)); col0 = (int *)g_calloc("to get col0", len1+1, sizeof(int)); col1 = (int *)g_calloc("to get col1", len1+1, sizeof(int)); ins0 = (dna)? DINS0 : PINS0; ins1 = (dna)? DINS1 : PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] = -ins0, yy = 1; yy <= len1; yy++) { col0[yy] = dely[yy] = col0[yy-1] - ins1; ndely[yy] = yy; } col0[0] = 0; /* Waterman Bull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] = -ins0; /* fill in match matrix */ for (px = seqx[0], xx = 1; xx <= len0; px++, xx++) { /* initialize first entry in col */ if (endgaps) { if (xx == 1) col1[0] = delx = -(ins0+ins1); else col1[0] = delx = col0[0] - ins1; ndelx = xx; } else { col1[0] = 0; delx = -ins0; ndelx = 0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis = col0[yy-1]; if (dna) mis += (xbm[*px-`A`]&xbm[*py-`A`])? DMAT : DMIS; else mis += _day[*px-`A`][*py-`A`]; /* update penalty for del in x seq; * favor new del over ongong del * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] - ins0 >= dely[yy]) { dely[yy] = col0[yy] - (ins0+ins1); ndely[yy] = 1; } else { dely[yy] -= ins1; ndely[yy]++; } } else { if (col0[yy] - (ins0+ins1) >= dely[yy]) { dely[yy] = col0[yy] - (ins0+ins1); ndely[yy] = 1; } else ndely[yy]++; } /* update penalty for del in y seq; * favor new del over ongong del */
if (endgaps || ndelx < MAXGAP) { if (col1[yy-1] - ins0 >= delx) { delx = col1[yy-1] - (ins0+ins1); ndelx = 1; } else { delx -= ins1; ndelx++; } } else { if (col1[yy-1] - (ins0+ins1) >= delx) { delx = col1[yy-1] - (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick the maximum score; we're favoring * mis over any del and delx over dely */ ...nw id = xx - yy + len1 - 1; if (mis >= delx && mis >= dely[yy]) col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij = dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset += sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; } else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset += sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = -ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy < len1) { /* last col */ if (endgaps) col1[yy] -= ins0+ins1*(len1-yy); if (col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps && xx < len0) col1[yy-1] -= ins0+ins1*(len0-xx); if (col1[yy-1] > smax) { smax = col1[yy-1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; } (void) free((char *)ndely); (void) free((char *)dely); (void) free((char *)col0); (void) free((char *)col1); } /* * * print( ) -- only routine visible outside this module * * static: * getmat( ) -- trace back best path, count matches: print( ) * pr_align( ) -- print alignment of described in array p[ ]: print( ) * dumpblock( ) -- dump a block of lines with numbers, stars: pr_align( ) * nums( ) -- put out a number line: dumpblock( ) * putline( ) -- put out a line (name, [num], seq, [num]): dumpblock( ) * stars( ) - -put a line of stars: dumpblock( ) * stripname( ) -- strip any path and prefix from a seqname */ #include "nw.h" #define SPC 3 #define P_LINE 256 /* maximum output line */ #define P_SPC 3 /* space between name or num and seq */ extern _day[26][26]; int olen; /* set output line length */ FILE *fx; /* output file */ print( ) print { int lx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) == 0) { fprintf(stderr,"%s: can't write %s\n", prog, ofile); cleanup(1); } fprintf(fx, "<first sequence: %s (length = %d)\n", namex[0], len0); fprintf(fx, "<second sequence: %s (length = %d)\n", namex[1], len1); olen = 60; lx = len0; ly = len1; firstgap = lastgap = 0; if (dmax < len1 - 1) { /* leading gap in x */ pp[0].spc = firstgap = len1 - dmax - 1; ly -= pp[0].spc; } else if (dmax > len1 - 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax - (len1 - 1); lx -= pp[1].spc; } if (dmax0 < len0 - 1) { /* trailing gap in x */ lastgap = len0 - dmax0 -1; lx -= lastgap; } else if (dmax0 > len0 - 1) { /* trailing gap in y */ lastgap = dmax0 - (len0 - 1); ly -= lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align( ); } /* * trace back the best path, count matches */ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly; /* "core" (minus endgaps) */ int firstgap, lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1; char outx[32]; double pct; register n0, n1; register char *p0, *p1; /* get total matches, score */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] + pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 = pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++; siz0--; } else if (siz1) { p0++; n0++; siz1--; } else { if (xbm[*p0-`A`]&xbm[*p1-`A`]) nm++; if (n0++ == pp[0].x[i0]) siz0 = pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++; p1++; } } /* pct homology: * if penalizing endgaps, base is the shorter seq * else, knock off overhangs and take shorter core */ if (endgaps) lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct = 100.*(double)nm/(double)lx; fprintf(fx, "\n"); fprintf(fx, "<%d match%s in an overlap of %d: %.2f percent similarity\n", nm, (nm == 1)? "" : "es", lx, pct); fprintf(fx, "<gaps in first sequence: %d", gapx); ...getmat if (gapx) { (void) sprintf(outx, " (%d %s%s)", ngapx, (dna)? "base":"residue", (ngapx == 1)? "":"s"); fprintf(fx,"%s", outx); fprintf(fx, ", gaps in second sequence: %d", gapy); if (gapy) { (void) sprintf(outx, " (%d %s%s)", ngapy, (dna)? "base":"residue", (ngapy == 1)? "":"s"); fprintf(fx,"%s", outx); } if (dna) fprintf(fx, "\n<score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT, DMIS, DINS0, DINS1); else fprintf(fx, "\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n", smax, PINS0, PINS1); if (endgaps) fprintf(fx, "<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n", firstgap, (dna)? "base" : "residue", (firstgap == 1)? "" : "s", lastgap, (dna)? "base" : "residue", (lastgap == 1)? "" : "s"); else fprintf(fx, "<endgaps not penalized\n"); } static nm; /* matches in core -- for checking */ static lmax; /* lengths of stripped file names */ static ij[2]; /* jmp index for a path */ static nc[2]; /* number at start of current line */ static ni[2]; /* current elem number -- for gapping */ static siz[2]; static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */ static char out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* set by stars( ) */ /* * print alignment of described in struct path pp[ ] */ static pr_align( ) pr_align { int nn; /* char count */ int more; register i; for (i = 0, lmax = 0; i < 2; i++) { nn = stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) { /* * do we have more of this sequence? */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /* leading space */
*po[i]++ = ` `; pp[i].spc--; } else if (siz[i]) { /* in a gap */ *po[i]++ = `-`; siz[i]--; } else { /* we're putting a seq element */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] = toupper(*ps[i]); po[i]++; ps[i]++; /* * are we at next gap for this seq? */ if (ni[i] == pp[i].x[ij[i]]) { /* * we need to merge all gaps * at this location */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] == pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn == olen || !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] = out[i]; nn = 0; } } } /* * dump a block of lines, including numbers, stars: pr_align( ) */ static dumpblock( ) dumpblock { register i; for (i = 0; i < 2; i++) *po[i]-- = `\0`; ...dumpblock (void) putc(`\n`, fx); for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ` ` || *(po[i]) != ` `)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars( ); putline(i); if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } } /* * put out a number line: dumpblock( ) */ static nums(ix) nums int ix; /* index in out[ ] holding seq line */ { char nline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn = ` `; for (i = nc[ix], py = out[ix]; *py; py++, pn++) { if (*py == ` ` || *py == `-`) *pn = ` `; else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? -i : i; for (px = pn; j; j /= 10, px--) *px = j%10 + `0`; if (i < 0) *px = `-`; } else *pn = ` `; i++; } } *pn = `\0`; nc[ix] = i; for (pn = nline; *pn; pn++) (void) putc(*pn, fx); (void) putc(`\n`, fx); } /* * put out a line (name, [num], seq, [num]): dumpblock( ) */ static putline(ix) putline int ix; { ...putline int i; register char *px; for (px = namex[ix], i = 0; *px && *px != `:`; px++, i++) (void) putc(*px, fx); for (; i < lmax+P_SPC; i++) (void) putc(` `, fx); /* these count from 1: * ni[ ] is current element (from 1) * nc[ ] is number at start of current line */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F, fx); (void) putc(`\n`, fx); } /* * put a line of stars (seqs always in out[0], out[1]): dumpblock( ) */ static stars( ) stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0] == ` ` && *(po[0]) == ` `) || !*out[1] || (*out[1] == ` ` && *(po[1]) == ` `)) return; px = star; for (i = lmax+P_SPC; i; i--) *px++ = ` `; for (p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) && isalpha(*p1)) { if (xbm[*p0-`A`]&xbm[*p1-`A`]) { cx = `*`; nm++; } else if (!dna && _day[*p0-`A`][*p1-`A`] > 0) cx = `.`; else cx = ` `; } else cx = ` `; *px++ = cx; } *px++ = `\n`; *px = `\0`; } /* * strip path or prefix from pn, return len: pr_align( ) */ static stripname(pn) stripname char *pn; /* file name (may be path) */ { register char *px, *py; py = 0; for (px = pn; *px; px++) if (*px == `/`) py = px + 1; if (py) (void) strcpy(pn, py); return(strlen(pn)); } /* * cleanup( ) -- cleanup any tmp file * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin * readjmps( ) -- get the good jmps, from tmp file if necessary * writejmps( ) -- write a filled array of jmps to a tmp file: nw( ) */ #include "nw.h" #include <sys/file.h> char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */ FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /* * remove any tmp file if we blow */ cleanup(i) cleanup int i; { if (fj) (void) unlink(jname); exit(i); } /* * read, return ptr to seq, set dna, len, maxlen * skip lines starting with `;`, `<`, or `>` * seq in upper or lower case */ char * getseq(file, len) getseq char *file; /* file name */ int *len; /* seq len */ { char line[1024], *pseq; register char *px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file,"r")) == 0) { fprintf(stderr,"%s: can't read %s\n", prog, file); exit(1); } tlen = natgc = 0; while (fgets(line, 1024, fp)) { if (*line == `;` || *line == `<` || *line == `>`) continue; for (px = line; *px != `\n`; px++) if (isupper(*px) || islower(*px)) tlen++; } if ((pseq = malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,"%s: malloc( ) failed to get %d bytes for %s\n", prog, tlen+6, file); exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] = `\0`; ...getseq py = pseq + 4; *len = tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == `;` || *line == `<` || *line == `>`) continue; for (px = line; *px != `\n`; px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ = toupper(*px); if (index("ATGCU",*(py-1))) natgc++; } } *py++ = `\0`; *py = `\0`; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); } char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, calling routine */ int nx, sz; /* number and size of elements */ { char *px, *calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if (*msg) { fprintf(stderr, "%s: g_calloc( ) failed %s (n=%d, sz=%d)\n", prog, msg,
nx, sz); exit(1); } } return(px); } /* * get final jmps from dx[ ] or tmp file, set pp[ ], reset dmax: main( ) */ readjmps( ) readjmps { int fd = -1; int siz, i0, i1; register i, j, xx; if (fj) { (void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr, "%s: can't open( ) %s\n", prog, jname); cleanup(1); } } for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for (j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j--) ; ...readjmps if (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset)); dx[dmax].ijmp = MAXJMP-1; } else break; } if (i >= JMPS) { fprintf(stderr, "%s: too many gaps in alignment\n", prog); cleanup(1); } if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax += siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = -siz; xx += siz; /* id = xx - yy + len1 - 1 */ pp[1].x[i1] = xx - dmax + len1 - 1; gapy++; ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP || endgaps)? -siz : MAXGAP; i1++; } else if (siz > 0) { /* gap in first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx += siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP || endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order of jmps */ for (j = 0, i0--; j < i0; j++, i0--) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1--; j < i1; j++, i1--) { i = pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j]; pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void) close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /* * write a filled jmp struct offset of the prev one (if any): nw( ) */ writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if (mktemp(jname) < 0) { fprintf(stderr, "%s: can't mktemp( ) %s\n", prog, jname); cleanup(1); } if ((fj = fopen(jname, "w")) == 0) { fprintf(stderr, "%s: can't write %s\n", prog, jname); exit(1); } } (void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }
TABLE-US-00002 TABLE 2 TAT XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the TAT polypeptide) = 5 divided by 15 = 33.3%
TABLE-US-00003 TABLE 3 TAT XXXXXXXXXX (Length = 10 amino acids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the TAT polypeptide) = 5 divided by 10 = 50%
TABLE-US-00004 TABLE 4 TAT-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequence identity = (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the TAT-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
TABLE-US-00005 TABLE 5 TAT-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison NNNNLLLVV (Length = 9 nucleotides) DNA % nucleic acid sequence identity = (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the TAT-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
II. Compositions and Methods of the Invention
[0596]A. Anti-TAT Antibodies
[0597]In one embodiment, the present invention provides anti-TAT antibodies which may find use herein as therapeutic and/or diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
[0598]1. Polyclonal Antibodies
[0599]Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups.
[0600]Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
[0601]2. Monoclonal Antibodies
[0602]Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0603]In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
[0604]The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[0605]Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0606]Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
[0607]The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).
[0608]Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.
[0609]The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
[0610]DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).
[0611]In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
[0612]The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
[0613]3. Human and Humanized Antibodies
[0614]The anti-TAT antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
[0615]Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
[0616]The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0617]It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
[0618]Various forms of a humanized anti-TAT antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.
[0619]As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.
[0620]Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.
[0621]As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
[0622]4. Antibody Fragments
[0623]In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.
[0624]Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
[0625]5. Bispecific Antibodies
[0626]Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a TAT protein as described herein. Other such antibodies may combine a TAT binding site with a binding site for another protein. Alternatively, an anti-TAT arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to the TAT-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TAT. These antibodies possess a TAT-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).
[0627]WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.
[0628]Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
[0629]According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.
[0630]In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).
[0631]According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
[0632]Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
[0633]Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
[0634]Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
[0635]Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
[0636]6. Heteroconjugate Antibodies
[0637]Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
[0638]7. Multivalent Antibodies
[0639]A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
[0640]8. Effector Function Engineering
[0641]It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
[0642]9. Immunoconjugates
[0643]The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
[0644]Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria
[0645]A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
[0646]Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.
Maytansine and Maytansinoids
[0647]In one preferred embodiment, an anti-TAT antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.
[0648]Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of which are hereby expressly incorporated by reference.
Maytansinoid-Antibody Conjugates
[0649]In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-21neu oncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansonid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
Anti-TAT Polypeptide Antibody-Maytansinoid Conjugates (Immunoconjugates)
[0650]Anti-TAT antibody-maytansinoid conjugates are prepared by chemically linking an anti-TAT antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
[0651]There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52:127-131 (1992). The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
[0652]Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
[0653]The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.
Calicheamicin
[0654]Another immunoconjugate of interest comprises an anti-TAT antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
Other Cytotoxic Agents
[0655]Other antitumor agents that can be conjugated to the anti-TAT antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).
[0656]Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
[0657]The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
[0658]For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-TAT antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0659]The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc99m or I123, Re186, Re188 and In111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
[0660]Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
[0661]Alternatively, a fusion protein comprising the anti-TAT antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
[0662]In yet another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).
[0663]10. Immunoliposomes
[0664]The anti-TAT antibodies disclosed herein may also be formulated as immunoliposomes. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
[0665]Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989).
[0666]B. TAT Binding Oligopeptides
[0667]TAT binding oligopeptides of the present invention are oligopeptides that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).
[0668]In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large oligopeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a polypeptide target. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides or oligopeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143.
[0669]Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren, Z-J. et al. (1998) Gene 215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can 128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci. 5:1833; Efimov, V. P. et al. (1995) Virus Genes 10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K. (1993) Methods in Enzymology, 217, 228--257; U.S. Pat. No. 5,766,905) are also known.
[0670]Many other improvements and variations of the basic phage display concept have now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol. Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833.
[0671]Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.
[0672]C. TAT Binding Organic Molecules
[0673]TAT binding organic molecules are organic molecules other than oligopeptides or antibodies as defined herein that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid chlorides, or the like.
[0674]D. Screening for Anti-TAT Antibodies, TAT Binding Oligopeptides and TAT Binding Organic Molecules with the Desired Properties
[0675]Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides have been described above. One may further select antibodies, oligopeptides or other organic molecules with certain biological characteristics, as desired.
[0676]The growth inhibitory effects of an anti-TAT antibody, oligopeptide or other organic molecule of the invention may be assessed by methods known in the art, e.g., using cells which express a TAT polypeptide either endogenously or following transfection with the TAT gene. For example, appropriate tumor cell lines and TAT-transfected cells may treated with an anti-TAT monoclonal antibody, oligopeptide or other organic molecule of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing 3H-thymidine uptake by the cells treated in the presence or absence an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention. After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways known in the art. Preferably, the tumor cell is one that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule will inhibit cell proliferation of a TAT-expressing tumor cell in vitro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, in one embodiment, at an antibody concentration of about 0.5 to 30 μg/ml. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or reduction of tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
[0677]To select for an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule which induces cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. TAT polypeptide-expressing tumor cells are incubated with medium alone or medium containing the appropriate anti-TAT antibody (e.g, at about 10 μg/ml), TAT binding oligopeptide or TAT binding organic molecule. The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 mm strainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Those anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules.
[0678]To screen for antibodies, oligopeptides or other organic molecules which bind to an epitope on a TAT polypeptide bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody, oligopeptide or other organic molecule binds the same site or epitope as a known anti-TAT antibody. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initially tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a TAT polypeptide can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.
[0679]E. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)
[0680]The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.
[0681]The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
[0682]Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
[0683]The enzymes of this invention can be covalently bound to the anti-TAT antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984).
[0684]F. Full-Length TAT Polypeptides
[0685]The present invention also provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as TAT polypeptides. In particular, cDNAs (partial and full-length) encoding various TAT polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.
[0686]As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC. The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the TAT polypeptides and encoding nucleic acids described herein, in some cases, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time.
[0687]G. Anti-TAT Antibody and TAT Polypeptide Variants
[0688]In addition to the anti-TAT antibodies and full-length native sequence TAT polypeptides described herein, it is contemplated that anti-TAT antibody and TAT polypeptide variants can be prepared. Anti-TAT antibody and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the anti-TAT antibody or TAT polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
[0689]Variations in the anti-TAT antibodies and TAT polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-TAT antibody or TAT polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the anti-TAT antibody or TAT polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
[0690]Anti-TAT antibody and TAT polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the anti-TAT antibody or TAT polypeptide.
[0691]Anti-TAT antibody and TAT polypeptide fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, anti-TAT antibody and TAT polypeptide fragments share at least one biological and/or immunological activity with the native anti-TAT antibody or TAT polypeptide disclosed herein.
[0692]In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
TABLE-US-00006 TABLE 6 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine
[0693]Substantial modifications in function or immunological identity of the anti-TAT antibody or TAT polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gin, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.
[0694]Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
[0695]The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the anti-TAT antibody or TAT polypeptide variant DNA.
[0696]Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244:1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
[0697]Any cysteine residue not involved in maintaining the proper conformation of the anti-TAT antibody or TAT polypeptide also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-TAT antibody or TAT polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
[0698]A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TAT polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
[0699]Nucleic acid molecules encoding amino acid sequence variants of the anti-TAT antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-TAT antibody.
[0700]H. Modifications of Anti-TAT Antibodies and TAT Polypeptides
[0701]Covalent modifications of anti-TAT antibodies and TAT polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an anti-TAT antibody or TAT polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the anti-TAT antibody or TAT polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking anti-TAT antibody or TAT polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-TAT antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0702]Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine, and histidine side chains [T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
[0703]Another type of covalent modification of the anti-TAT antibody or TAT polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence anti-TAT antibody or TAT polypeptide (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
[0704]Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
[0705]Addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for O-linked glycosylation sites). The anti-TAT antibody or TAT polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the anti-TAT antibody or TAT polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
[0706]Another means of increasing the number of carbohydrate moieties on the anti-TAT antibody or TAT polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
[0707]Removal of carbohydrate moieties present on the anti-TAT antibody or TAT polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
[0708]Another type of covalent modification of anti-TAT antibody or TAT polypeptide comprises linking the antibody or polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The antibody or polypeptide also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
[0709]The anti-TAT antibody or TAT polypeptide of the present invention may also be modified in a way to form chimeric molecules comprising an anti-TAT antibody or TAT polypeptide fused to another, heterologous polypeptide or amino acid sequence.
[0710]In one embodiment, such a chimeric molecule comprises a fusion of the anti-TAT antibody or TAT polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the anti-TAT antibody or TAT polypeptide. The presence of such epitope-tagged forms of the anti-TAT antibody or TAT polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the anti-TAT antibody or TAT polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
[0711]In an alternative embodiment, the chimeric molecule may comprise a fusion of the anti-TAT antibody or TAT polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an anti-TAT antibody or TAT polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0712]I. Preparation of Anti-TAT Antibodies and TAT Polypeptides
[0713]The description below relates primarily to production of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with a vector containing anti-TAT antibody- and TAT polypeptide-encoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-TAT antibodies and TAT polypeptides. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of the anti-TAT antibody or TAT polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-TAT antibody or TAT polypeptide.
[0714]1. Isolation of DNA Encoding Anti-TAT Antibody or TAT Polypeptide
[0715]DNA encoding anti-TAT antibody or TAT polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the anti-TAT antibody or TAT polypeptide mRNA and to express it at a detectable level. Accordingly, human anti-TAT antibody or TAT polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue. The anti-TAT antibody- or TAT polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
[0716]Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding anti-TAT antibody or TAT polypeptide is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
[0717]Techniques for screening a cDNA library are well known in the art. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.
[0718]Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.
[0719]Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
[0720]2. Selection and Transformation of Host Cells
[0721]Host cells are transfected or transformed with expression or cloning vectors described herein for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
[0722]Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaPO4, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
[0723]Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kanr; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
[0724]Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523 (Simmons et al.) which describes translation initiation regio (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.
[0725]In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TAT antibody- or TAT polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
[0726]Suitable host cells for the expression of glycosylated anti-TAT antibody or TAT polypeptide are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
[0727]However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0728]Host cells are transformed with the above-described expression or cloning vectors for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
[0729]3. Selection and Use of a Replicable Vector
[0730]The nucleic acid (e.g., cDNA or genomic DNA) encoding anti-TAT antibody or TAT polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
[0731]The TAT may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the anti-TAT antibody- or TAT polypeptide-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
[0732]Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
[0733]Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
[0734]An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0735]Expression and cloning vectors usually contain a promoter operably linked to the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding anti-TAT antibody or TAT polypeptide.
[0736]Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
[0737]Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
[0738]Anti-TAT antibody or TAT polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
[0739]Transcription of a DNA encoding the anti-TAT antibody or TAT polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the anti-TAT antibody or TAT polypeptide coding sequence, but is preferably located at a site 5' from the promoter.
[0740]Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-TAT antibody or TAT polypeptide.
[0741]Still other methods, vectors, and host cells suitable for adaptation to the synthesis of anti-TAT antibody or TAT polypeptide in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
[0742]4. Culturing the Host Cells
[0743]The host cells used to produce the anti-TAT antibody or TAT polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN® drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
[0744]5. Detecting Gene Amplification/Expression
[0745]Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
[0746]Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence TAT polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to TAT DNA and encoding a specific antibody epitope.
[0747]6. Purification of Anti-TAT Antibody and TAT Polypeptide
[0748]Forms of anti-TAT antibody and TAT polypeptide may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of anti-TAT antibody and TAT polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
[0749]It may be desired to purify anti-TAT antibody and TAT polypeptide from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the anti-TAT antibody and TAT polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular anti-TAT antibody or TAT polypeptide produced.
[0750]When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
[0751]The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on humanyl, γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX®resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE® chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
[0752]Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
[0753]J. Pharmaceutical Formulations
[0754]Therapeutic formulations of the anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic molecules and/or TAT polypeptides used in accordance with the present invention are prepared for storage by mixing the antibody, polypeptide, oligopeptide or organic molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml.
[0755]The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-TAT antibody, TAT binding oligopeptide, or TAT binding organic molecule, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti-TAT antibody which binds a different epitope on the TAT polypeptide, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
[0756]The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).
[0757]Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
[0758]The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
[0759]K. Diagnosis and Treatment with Anti-TAT Antibodies. TAT Binding Oligopeptides and TAT Binding Organic Molecules
[0760]To determine TAT expression in the cancer, various diagnostic assays are available. In one embodiment, TAT polypeptide overexpression may be analyzed by immunohistochemistry (IHC). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a TAT protein staining intensity criteria as follows:
[0761]Score 0--no staining is observed or membrane staining is observed in less than 10% of tumor cells.
[0762]Score 1+--a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane.
[0763]Score 2+--a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.
[0764]Score 3+--a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.
[0765]Those tumors with 0 or 1+ scores for TAT polypeptide expression may be characterized as not overexpressing TAT, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing TAT.
[0766]Alternatively, or additionally, FISH assays such as the INFORM® (sold by Ventana, Ariz.) or PATHVISION® (Vysis, Ill.) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of TAT overexpression in the tumor.
[0767]TAT overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g., by administering a molecule (such as an antibody, oligopeptide or organic molecule) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.
[0768]As described above, the anti-TAT antibodies, oligopeptides and organic molecules of the invention have various non-therapeutic applications. The anti-TAT antibodies, oligopeptides and organic molecules of the present invention can be useful for diagnosis and staging of TAT polypeptide-expressing cancers (e.g., in radioimaging). The antibodies, oligopeptides and organic molecules are also useful for purification or immunoprecipitation of TAT polypeptide from cells, for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill and eliminate TAT-expressing cells from a population of mixed cells as a step in the purification of other cells.
[0769]Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Anti-TAT antibody, oligopeptide or organic molecule therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. The tumor targeting anti-TAT antibodies, oligopeptides and organic molecules of the invention are useful to alleviate TAT-expressing cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, the anti-TAT antibody, oligopeptide or organic molecule can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy. Anti-TAT antibody, oligopeptide or organic molecule treatment can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. Chemotherapeutic drugs such as TAXOTERE® (docetaxel), TAXOL® (palictaxel), estramustine and mitoxantrone are used in treating cancer, in particular, in good risk patients. In the present method of the invention for treating or alleviating cancer, the cancer patient can be administered anti-TAT antibody, oligopeptide or organic molecule in conjuction with treatment with the one or more of the preceding chemotherapeutic agents. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EP0600517) is contemplated. The anti-TAT antibody, oligopeptide or organic molecule will be administered with a therapeutically effective dose of the chemotherapeutic agent. In another embodiment, the anti-TAT antibody, oligopeptide or organic molecule is administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages of these agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
[0770]In one particular embodiment, a conjugate comprising an anti-TAT antibody, oligopeptide or organic molecule conjugated with a cytotoxic agent is administered to the patient. Preferably, the immunoconjugate bound to the TAT protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
[0771]The anti-TAT antibodies, oligopeptides, organic molecules or toxin conjugates thereof are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody, oligopeptide or organic molecule is preferred.
[0772]Other therapeutic regimens may be combined with the administration of the anti-TAT antibody, oligopeptide or organic molecule. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.
[0773]It may also be desirable to combine administration of the anti-TAT antibody or antibodies, oligopeptides or organic molecules, with administration of an antibody directed against another tumor antigen associated with the particular cancer.
[0774]In another embodiment, the therapeutic treatment methods of the present invention involves the combined administration of an anti-TAT antibody (or antibodies), oligopeptides or organic molecules and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).
[0775]The antibody, oligopeptide or organic molecule may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen independent, the anti-TAT antibody, oligopeptide or organic molecule (and optionally other agents as described herein) may be administered to the patient.
[0776]Sometimes, it may be beneficial to also co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy, before, simultaneously with, or post antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-TAT antibody, oligopeptide or organic molecule.
[0777]For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of antibody, oligopeptide or organic molecule will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody, oligopeptide or organic molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, oligopeptide or organic molecule, and the discretion of the attending physician. The antibody, oligopeptide or organic molecule is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous infusion or by subcutaneous injections. Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-TAT antibody. However, other dosage regimens may be useful. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
[0778]Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody". See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.
[0779]There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector.
[0780]The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example). For review of the currently known gene marking and gene therapy protocols see Anderson et al., Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein.
[0781]The anti-TAT antibodies of the invention can be in the different forms encompassed by the definition of "antibody" herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In fusion antibodies an antibody sequence is fused to a heterologous polypeptide sequence. The antibodies can be modified in the Fc region to provide desired effector functions. As discussed in more detail in the sections herein, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used.
[0782]In one embodiment, the antibody competes for binding or bind substantially to, the same epitope as the antibodies of the invention. Antibodies having the biological characteristics of the present anti-TAT antibodies of the invention are also contemplated, specifically including the in vivo tumor targeting and any cell proliferation inhibition or cytotoxic characteristics.
[0783]Methods of producing the above antibodies are described in detail herein.
[0784]The present anti-TAT antibodies, oligopeptides and organic molecules are useful for treating a TAT-expressing cancer or alleviating one or more symptoms of the cancer in a mammal. Such a cancer includes prostate cancer, cancer of the urinary tract, lung cancer, breast cancer, colon cancer and ovarian cancer, more specifically, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma. The cancers encompass metastatic cancers of any of the preceding. The antibody, oligopeptide or organic molecule is able to bind to at least a portion of the cancer cells that express TAT polypeptide in the mammal. In a preferred embodiment, the antibody, oligopeptide or organic molecule is effective to destroy or kill TAT-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to TAT polypeptide on the cell. Such an antibody includes a naked anti-TAT antibody (not conjugated to any agent). Naked antibodies that have cytotoxic or cell growth inhibition properties can be further harnessed with a cytotoxic agent to render them even more potent in tumor cell destruction. Cytotoxic properties can be conferred to an anti-TAT antibody by, e.g., conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described herein. The cytotoxic agent or a growth inhibitory agent is preferably a small molecule. Toxins such as calicheamicin or a maytansinoid and analogs or derivatives thereof, are preferable.
[0785]The invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. For the purposes of treating cancer, compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-TAT antibodies present as an immunoconjugate or as the naked antibody. In a further embodiment, the compositions can comprise these antibodies, oligopeptides or organic molecules in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides formulations comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.
[0786]Another aspect of the invention is isolated nucleic acids encoding the anti-TAT antibodies. Nucleic acids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are encompassed.
[0787]The invention also provides methods useful for treating a TAT polypeptide-expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule to the mammal. The antibody, oligopeptide or organic molecule therapeutic compositions can be administered short term (acute) or chronic, or intermittent as directed by physician. Also provided are methods of inhibiting the growth of, and killing a TAT polypeptide-expressing cell.
[0788]The invention also provides kits and articles of manufacture comprising at least one anti-TAT antibody, oligopeptide or organic molecule. Kits containing anti-TAT antibodies, oligopeptides or organic molecules find use, e.g., for TAT cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For example, for isolation and purification of TAT, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT in vitro, e.g., in an ELISA or a Western blot. Such antibody, oligopeptide or organic molecule useful for detection may be provided with a label such as a fluorescent or radiolabel.
[0789]L. Articles of Manufacture and Kits
[0790]Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of anti-TAT expressing cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-TAT antibody, oligopeptide or organic molecule of the invention. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the antibody, oligopeptide or organic molecule composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
[0791]Kits are also provided that are useful for various purposes, e.g., for TAT-expressing cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For isolation and purification of TAT polypeptide, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.
[0792]M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding Nucleic Acids
[0793]Nucleotide sequences (or their complement) encoding TAT polypeptides have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA probes. TAT-encoding nucleic acid will also be useful for the preparation of TAT polypeptides by the recombinant techniques described herein, wherein those TAT polypeptides may find use, for example, in the preparation of anti-TAT antibodies as described herein.
[0794]The full-length native sequence TAT gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length TAT cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of TAT or TAT from other species) which have a desired sequence identity to the native TAT sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence TAT. By way of example, a screening method will comprise isolating the coding region of the TAT gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as P or S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the TAT gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below. Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
[0795]Other useful fragments of the TAT-encoding nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TAT mRNA (sense) or TAT DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of TAT DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).
[0796]Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. Such methods are encompassed by the present invention. The antisense oligonucleotides thus may be used to block expression of TAT proteins, wherein those TAT proteins may play a role in the induction of cancer in mammals. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
[0797]Preferred intragenic sites for antisense binding include the region incorporating the translation initiation/start codon (5'-AUG/5'-ATG) or termination/stop codon (5'-UAA, 5'-UAG and 5-UGA/5'-TM, 5'-TAG and 5'-TGA) of the open reading frame (ORF) of the gene. These regions refer to a portion of the mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation or termination codon. Other preferred regions for antisense binding include: introns; exons; intron-exon junctions; the open reading frame (ORF) or "coding region," which is the region between the translation initiation codon and the translation termination codon; the 5' cap of an mRNA which comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage and includes 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap; the 5' untranslated region (5'UTR), the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene; and the 3' untranslated region (3'UTR), the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.
[0798]Specific examples of preferred antisense compounds useful for inhibiting expression of TAT proteins include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is herein incorporated by reference.
[0799]Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative United States patents that teach the preparation of such oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference.
[0800]In other preferred antisense oligonucleotides, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
[0801]Preferred antisense oligonucleotides incorporate phosphorothioate backbones and/or heteroatom backbones, and in particular --CH2--NH--O--CH2--, --CH2--N(CH3)--O--CH2-- [known as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --O--N(CH3)--CH2--CH2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH2--] described in the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are antisense oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
[0802]Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)rCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred antisense oligonucleotides comprise one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2).
[0803]A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (--CH2--)n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
[0804]Other preferred modifications include 2'-methoxy (2'-O--CH3), 2'-aminopropoxy (2'-OCH2CH2CH2 NH2), 2'-allyl (2'-CH2--CH═CH2), 2'-O-allyl (2'-O--CH2--CH═CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.
[0805]Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C≡C--CH3 or --CH2--C≡CH) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of modified nucleobases include, but are not limited to: U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is herein incorporated by reference.
[0806]Another modification of antisense oligonucleotides chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, cation lipids, phospholipids, cationic phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) and U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
[0807]It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Preferred chimeric antisense oligonucleotides incorporate at least one 2' modified sugar (preferably 2'-O--(CH2)2--O--CH3) at the 3' terminal to confer nuclease resistance and a region with at least 4 contiguous 2'-H sugars to confer RNase H activity. Such compounds have also been referred to in the art as hybrids or gapmers. Preferred gapmers have a region of 2' modified sugars (preferably 2'-O--(CH2)2--O--CH3) at the 3'-terminal and at the 5' terminal separated by at least one region having at least 4 contiguous 2'-H sugars and preferably incorporate phosphorothioate backbone linkages. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
[0808]The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.
[0809]Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
[0810]Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0811]Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
[0812]Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
[0813]Antisense or sense RNA or DNA molecules are generally at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length.
[0814]The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related TAT coding sequences.
[0815]Nucleotide sequences encoding a TAT can also be used to construct hybridization probes for mapping the gene which encodes that TAT and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
[0816]When the coding sequences for TAT encode a protein which binds to another protein (example, where the TAT is a receptor), the TAT can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor TAT can be used to isolate correlative ligand(s). Screening assays can be designed to find lead compounds that mimic the biological activity of a native TAT or a receptor for TAT. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
[0817]Nucleic acids which encode TAT or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding TAT. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for TAT transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding TAT introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding TAT. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
[0818]Alternatively, non-human homologues of TAT can be used to construct a TAT "knock out" animal which has a defective or altered gene encoding TAT as a result of homologous recombination between the endogenous gene encoding TAT and altered genomic DNA encoding TAT introduced into an embryonic stem cell of the animal. For example, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques. A portion of the genomic DNA encoding TAT can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the TAT polypeptide.
[0819]Nucleic acid encoding the TAT polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
[0820]There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science 256, 808-813 (1992).
[0821]The nucleic acid molecules encoding the TAT polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each TAT nucleic acid molecule of the present invention can be used as a chromosome marker.
[0822]The TAT polypeptides and nucleic acid molecules of the present invention may also be used diagnostically for tissue typing, wherein the TAT polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type. TAT nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.
[0823]This invention encompasses methods of screening compounds to identify those that mimic the TAT polypeptide (agonists) or prevent the effect of the TAT polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the TAT polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins, including e.g., inhibiting the expression of TAT polypeptide from cells. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
[0824]The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
[0825]All assays for antagonists are common in that they call for contacting the drug candidate with a TAT polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.
[0826]In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the TAT polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the TAT polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the TAT polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
[0827]If the candidate compound interacts with but does not bind to a particular TAT polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER®) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
[0828]Compounds that interfere with the interaction of a gene encoding a TAT polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.
[0829]To assay for antagonists, the TAT polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the TAT polypeptide indicates that the compound is an antagonist to the TAT polypeptide. Alternatively, antagonists may be detected by combining the TAT polypeptide and a potential antagonist with membrane-bound TAT polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The TAT polypeptide can be labeled, such as by radioactivity, such that the number of TAT polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the TAT polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the TAT polypeptide. Transfected cells that are grown on glass slides are exposed to labeled TAT polypeptide. The TAT polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
[0830]As an alternative approach for receptor identification, labeled TAT polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
[0831]In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled TAT polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.
[0832]More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with TAT polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the TAT polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the TAT polypeptide.
[0833]Another potential TAT polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature TAT polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix--see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of the TAT polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the TAT polypeptide (antisense--Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the TAT polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
[0834]Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the TAT polypeptide, thereby blocking the normal biological activity of the TAT polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
[0835]Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
[0836]Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra.
[0837]These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.
[0838]Isolated TAT polypeptide-encoding nucleic acid can be used herein for recombinantly producing TAT polypeptide using techniques well known in the art and as described herein. In turn, the produced TAT polypeptides can be employed for generating anti-TAT antibodies using techniques well known in the art and as described herein.
[0839]Antibodies specifically binding a TAT polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders, including cancer, in the form of pharmaceutical compositions.
[0840]If the TAT polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
[0841]The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
[0842]The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
[0843]All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
EXAMPLES
[0844]Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Tissue Expression Profiling Using GeneExpress®
[0845]A proprietary database containing gene expression information (GeneExpress®, Gene Logic Inc., Gaithersburg, Md.) was analyzed in an attempt to identify polypeptides (and their encoding nucleic acids) whose expression is significantly upregulated in a particular tumor tissue(s) of interest as compared to other tumor(s) and/or normal tissues. Specifically, analysis of the GeneExpress® database was conducted using either software available through Gene Logic Inc., Gaithersburg, Md., for use with the GeneExpress® database or with proprietary software written and developed at Genentech, Inc. for use with the GeneExpress® database. The rating of positive hits in the analysis is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined from an analysis of the GeneExpress® database evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
TABLE-US-00007 upregulation of Molecule expression in: as compared to: DNA96792 (TAT239) colon tumor normal colon tissue DNA96792 (TAT239) rectum tumor normal rectum tissue DNA96792 (TAT239) pancreas tumor normal pancreas tissue DNA96792 (TAT239) lung tumor normal lung tissue DNA96792 (TAT239) stomach tumor normal stomach tissue DNA96792 (TAT239) esophagus tumor normal esophagus tissue DNA96792 (TAT239) breast tumor normal breast tissue DNA96792 (TAT239) uterus tumor normal uterus tissue DNA225793 (TAT223) ovarian tumor normal ovarian tissue DNA225793 (TAT223) kidney tumor normal kidney tissue DNA227611 (TAT175) prostate tumor normal prostate tissue DNA227611 (TAT175) colon tumor normal colon tissue DNA227611 (TAT175) breast tumor normal breast tissue DNA261021 (TAT208) breast tumor normal breast tissue DNA260655 (TAT209) lung tumor normal lung tissue DNA260655 (TAT209) colon tumor normal colon tissue DNA260655 (TAT209) breast tumor normal breast tissue DNA260655 (TAT209) liver tumor normal liver tissue DNA260655 (TAT209) ovarian tumor normal ovarian tissue DNA260655 (TAT209) skin tumor normal skin tissue DNA260655 (TAT209) spleen tumor normal spleen tissue DNA260655 (TAT209) myeloid tumor normal myeloid tissue DNA260655 (TAT209) muscle tumor normal muscle tissue DNA260655 (TAT209) bone tumor normal bone tissue DNA261001 (TAT181) bone tumor normal bone tissue DNA261001 (TAT181) lung tumor normal lung tissue DNA266928 (TAT182) bone tumor normal bone tissue DNA266928 (TAT182) lung tumor normal lung tissue DNA268035 (TAT222) breast tumor normal breast tissue DNA268035 (TAT222) colon tumor normal colon tissue DNA268035 (TAT222) ovarian tumor normal ovarian tissue DNA268035 (TAT222) uterine tumor normal uterine tissue DNA77509 (TAT177) colon tumor normal colon tissue DNA87993 (TAT235) breast tumor normal breast tissue DNA87993 (TAT235) pancreatic tumor normal pancreatic tissue DNA87993 (TAT235) lung tumor normal lung tissue DNA87993 (TAT235) colon tumor normal colon tissue DNA87993 (TAT235) rectum tumor normal rectum tissue DNA87993 (TAT235) gallbladder tumor normal gallbladder tissue DNA92980 (TAT234) bone tumor normal bone tissue DNA92980 (TAT234) breast tumor normal breast tissue DNA92980 (TAT234) cervical tumor normal cervical tissue DNA92980 (TAT234) colon tumor normal colon tissue DNA92980 (TAT234) rectum tumor normal rectum tissue DNA92980 (TAT234) endometrial normal endometrial tissue tumor DNA92980 (TAT234) liver tumor normal liver tissue DNA92980 (TAT234) lung tumor normal lung tissue DNA92980 (TAT234) ovarian tumor normal ovarian tissue DNA92980 (TAT234) pancreatic tumor normal pancreatic tissue DNA92980 (TAT234) skin tumor normal skin tissue DNA92980 (TAT234) soft tissue tumor normal soft tissue DNA92980 (TAT234) stomach tumor normal stomach tissue DNA92980 (TAT234) bladder tumor normal bladder tissue DNA92980 (TAT234) thyroid tumor normal thyroid tissue DNA105792 (TAT233) bone tumor normal bone tissue DNA105792 (TAT233) breast tumor normal breast tissue DNA105792 (TAT233) endometrial normal endometrial tissue tumor DNA105792 (TAT233) esophagus tumor normal esophagus tissue DNA105792 (TAT233) kidney tumor normal kidney tissue DNA105792 (TAT233) lung tumor normal lung tissue DNA105792 (TAT233) ovarian tumor normal ovarian tissue DNA105792 (TAT233) pancreatic tumor normal pancreatic tissue DNA105792 (TAT233) prostate tumor normal prostate tissue DNA105792 (TAT233) soft tissue tumor normal soft tissue DNA105792 (TAT233) stomach tumor normal stomach tissue DNA105792 (TAT233) thyroid tumor normal thyroid tissue DNA105792 (TAT233) bladder tumor normal bladder tissue DNA105792 (TAT233) brain tumor normal brain tissue DNA105792 (TAT233) Wilm's tumor normal associated tissue DNA119474 (TAT228) uterine tumor normal uterine tissue DNA119474 (TAT228) ovarian tumor normal ovarian tissue DNA280351 (TAT248) squamous cell normal squamous lung tumor cell lung tissue DNA280351 (TAT248) colon tumor normal colon tissue DNA150648 (TAT232) liver tumor normal liver tissue DNA150648 (TAT232) breast tumor normal breast tissue DNA150648 (TAT232) brain tumor normal brain tissue DNA150648 (TAT232) lung tumor normal lung tissue DNA150648 (TAT232) colon tumor normal colon tissue DNA150648 (TAT232) rectum tumor normal rectum tissue DNA150648 (TAT232) kidney tumor normal kidney tissue DNA150648 (TAT232) bladder tumor normal bladder tissue DNA179651 (TAT224) breast tumor normal breast tissue DNA179651 (TAT224) cervical tumor normal cervical tissue DNA179651 (TAT224) colon tumor normal colon tissue DNA179651 (TAT224) rectum tumor normal rectum tissue DNA179651 (TAT224) uterine tumor normal uterine tissue DNA179651 (TAT224) lung tumor normal lung tissue DNA179651 (TAT224) ovarian tumor normal ovarian tissue DNA207698 (TAT237) breast tumor normal breast tissue DNA207698 (TAT237) colon tumor normal colon tissue DNA207698 (TAT237) ovarian tumor normal ovarian tissue DNA207698 (TAT237) pancreatic tumor normal pancreatic tissue DNA207698 (TAT237) stomach tumor normal stomach tissue DNA225886 (TAT236) breast tumor normal breast tissue DNA225886 (TAT236) colon tumor normal colon tissue DNA225886 (TAT236) rectum tumor normal rectum tissue DNA225886 (TAT236) endometrial normal endometrial tissue tumor DNA225886 (TAT236) lung tumor normal lung tissue DNA225886 (TAT236) ovarian tumor normal ovarian tissue DNA225886 (TAT236) pancreas tumor normal pancreas tissue DNA225886 (TAT236) prostate tumor normal prostate tissue DNA225886 (TAT236) bladder tumor normal bladder tissue DNA226717 (TAT185) glioma normal glial tissue DNA226717 (TAT185) brain tumor normal brain tissue DNA227162 (TAT225) breast tumor normal breast tissue DNA227162 (TAT225) endometrial normal endometrial tissue tumor DNA227162 (TAT225) lung tumor normal lung tissue DNA227162 (TAT225) ovarian tumor normal ovarian tissue DNA277804 (TAT247) breast tumor normal breast tissue DNA277804 (TAT247) endometrial normal endometrial tissue tumor DNA277804 (TAT247) lung tumor normal lung tissue DNA277804 (TAT247) ovarian tumor normal ovarian tissue DNA233034 (TAT174) glioma normal glial tissue DNA233034 (TAT174) brain tumor normal brain tissue DNA266920 (TAT214) glioma normal glial tissue DNA266920 (TAT214) brain tumor normal brain tissue DNA266921 (TAT220) glioma normal glial tissue DNA266921 (TAT220) brain tumor normal brain tissue DNA266922 (TAT221) glioma normal glial tissue DNA266922 (TAT221) brain tumor normal brain tissue DNA234441 (TAT201) colon tumor normal colon tissue DNA234441 (TAT201) rectum tumor normal rectum tissue DNA234834 (TAT179) breast tumor normal breast tissue DNA234834 (TAT179) colon tumor normal colon tissue DNA234834 (TAT179) rectum tumor normal rectum tissue DNA234834 (TAT179) prostate tumor normal prostate tissue DNA234834 (TAT179) pancreatic tumor normal pancreatic tissue DNA234834 (TAT179) endometrial normal endometrial tissue tumor DNA234834 (TAT179) lung tumor normal lung tissue DNA234834 (TAT179) ovarian tumor normal ovarian tissue DNA247587 (TAT216) breast tumor normal breast tissue DNA247587 (TAT216) lung tumor normal lung tissue DNA247587 (TAT216) ovarian tumor normal ovarian tissue DNA247587 (TAT216) pancreatic tumor normal pancreatic tissue DNA247587 (TAT216) stomach tumor normal stomach tissue DNA247587 (TAT216) urinary tumor normal urinary tissue DNA255987 (TAT218) breast tumor normal breast tissue DNA56041 (TAT206) lymphoid tumor normal lymphoid tissue DNA257845 (TAT374) lymphoid tumor normal lymphoid tissue DNA247476 (TAT180) bone tumor normal bone tissue DNA247476 (TAT180) breast tumor normal breast tissue DNA247476 (TAT180) colon tumor normal colon tissue DNA247476 (TAT180) rectum tumor normal rectum tissue DNA247476 (TAT180) kidney tumor normal kidney tissue DNA247476 (TAT180) lung tumor normal lung tissue DNA247476 (TAT180) pancreatic tumor normal pancreatic tissue DNA247476 (TAT180) prostate tumor normal prostate tissue DNA247476 (TAT180) skin tumor normal skin tissue DNA247476 (TAT180) soft tissue tumor normal soft tissue DNA247476 (TAT180) stomach tumor normal stomach tissue DNA260990 (TAT375) bone tumor normal bone tissue DNA260990 (TAT375) breast tumor normal breast tissue DNA260990 (TAT375) colon tumor normal colon tissue DNA260990 (TAT375) rectum tumor normal rectum tissue DNA260990 (TAT375) kidney tumor normal kidney tissue DNA260990 (TAT375) lung tumor normal lung tissue DNA260990 (TAT375) pancreatic tumor normal pancreatic tissue DNA260990 (TAT375) prostate tumor normal prostate tissue DNA260990 (TAT375) skin tumor normal skin tissue DNA260990 (TAT375) soft tissue tumor normal soft tissue DNA260990 (TAT375) stomach tumor normal stomach tissue DNA261013 (TAT176) breast tumor normal breast tissue DNA261013 (TAT176) colon tumor normal colon tissue DNA261013 (TAT176) rectum tumor normal rectum tissue DNA261013 (TAT176) lung tumor normal lung tissue DNA261013 (TAT176) ovarian tumor normal ovarian tissue DNA261013 (TAT176) stomach tumor normal stomach tissue DNA262144 (TAT184) breast tumor normal breast tissue DNA262144 (TAT184) colon tumor normal colon tissue DNA262144 (TAT184) rectum tumor normal rectum tissue DNA262144 (TAT184) endometrial normal endometrial tissue tumor DNA262144 (TAT184) kidney tumor normal kidney tissue DNA262144 (TAT184) lung tumor normal lung tissue DNA262144 (TAT184) ovarian tumor normal ovarian tissue DNA267342 (TAT213)) stroma associated normal associated tissues, with the respectively following tumors: bone, breast, colon, rectum, lung, ovarian, pancreas, soft tissue, bladder DNA267626 (TAT217) breast tumor normal breast tissue DNA267626 (TAT217) colon tumor normal colon tissue DNA267626 (TAT217) rectum tumor normal rectum tissue DNA267626 (TAT217) endometrial normal endometrial tissue tumor DNA267626 (TAT217) lung tumor normal lung tissue DNA267626 (TAT217) pancreatic tumor normal pancreatic tissue DNA268334 (TAT202) kidney tumor normal kidney tissue DNA269238 (TAT215) kidney tumor normal kidney tissue DNA272578 (TAT238) liver tumor normal liver tissue DNA272578 (TAT238) lung tumor normal lung tissue DNA272578 (TAT238) ovarian tumor normal ovarian tissue DNA304853 (TAT376) breast tumor normal breast tissue DNA304853 (TAT376) colon tumor normal colon tissue DNA304853 (TAT376) rectum tumor normal rectum tissue DNA304853 (TAT376) prostate tumor normal prostate tissue DNA304853 (TAT376) pancreatic tumor normal pancreatic tissue DNA304853 (TAT376) endometrial normal endometrial tissue tumor DNA304853 (TAT376) lung tumor normal lung tissue DNA304853 (TAT376) ovarian tumor normal ovarian tissue DNA304854 (TAT377) breast tumor normal breast tissue DNA304854 (TAT377) colon tumor normal colon tissue DNA304854 (TAT377) rectum tumor normal rectum tissue DNA304854 (TAT377) prostate tumor normal prostate tissue DNA304854 (TAT377) pancreatic tumor normal pancreatic tissue DNA304854 (TAT377) endometrial normal endometrial tissue tumor DNA304854 (TAT377) lung tumor normal lung tissue DNA304854 (TAT377) ovarian tumor normal ovarian tissue DNA304855 (TAT378) breast tumor normal breast tissue DNA304855 (TAT378) colon tumor normal colon tissue DNA304855 (TAT378) rectum tumor normal rectum tissue DNA304855 (TAT378) prostate tumor normal prostate tissue DNA304855 (TAT378) pancreatic tumor normal pancreatic tissue DNA304855 (TAT378) endometrial normal endometrial tissue tumor DNA304855 (TAT378) lung tumor normal lung tissue DNA304855 (TAT378) ovarian tumor normal ovarian tissue DNA287971 (TAT379) bone tumor normal bone tissue DNA287971 (TAT379) breast tumor normal breast tissue
DNA287971 (TAT379) colon tumor normal colon tissue DNA287971 (TAT379) rectum tumor normal rectum tissue DNA287971 (TAT379) kidney tumor normal kidney tissue DNA287971 (TAT379) lung tumor normal lung tissue DNA287971 (TAT379) pancreatic tumor normal pancreatic tissue DNA287971 (TAT379) prostate tumor normal prostate tissue DNA287971 (TAT379) skin tumor normal skin tissue DNA287971 (TAT379) soft tissue tumor normal soft tissue DNA287971 (TAT379) stomach tumor normal stomach tissue
Example 2
Microarray Analysis to Detect Upregulation of TAT Polypeptides in Cancerous Tumors
[0846]Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sample is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified. The implication of this result is that an overexpressed protein in a diseased tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition.
[0847]The methodology of hybridization of nucleic acids and microarray technology is well known in the art. In one example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in PCT Patent Application Serial No. PCT/US01/10482, filed on Mar. 30, 2001 and which is herein incorporated by reference.
[0848]In the present example, cancerous tumors derived from various human tissues were studied for upregulated gene expression relative to cancerous tumors from different tissue types and/or non-cancerous human tissues in an attempt to identify those polypeptides which are overexpressed in a particular cancerous tumor(s). In certain experiments, cancerous human tumor tissue and non-cancerous human tumor tissue of the same tissue type (often from the same patient) were obtained and analyzed for TAT polypeptide expression. Additionally, cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a "universal" epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung. mRNA isolated from the pooled tissues represents a mixture of expressed gene products from these different tissues. Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:control detection) within each experiment. The normalized ratios from various experiments were then compared and used to identify clustering of gene expression. Thus, the pooled "universal control" sample not only allowed effective relative gene expression determinations in a simple 2-sample comparison, it also allowed multi-sample comparisons across several experiments.
[0849]In the present experiments, nucleic acid probes derived from the herein described TAT polypeptide-encoding nucleic acid sequences were used in the creation of the microarray and RNA from various tumor tissues were used for the hybridization thereto. Below is shown the results of these experiments, demonstrating that various TAT polypeptides of the present invention are significantly overexpressed in various human tumor tissues as compared to their normal counterpart tissue(s). Moreover, all of the molecules shown below are significantly overexpressed in their specific tumor tissue(s) as compared to in the "universal" epithelial control. As described above, these data demonstrate that the TAT polypeptides of the present invention are useful not only as diagnostic markers for the presence of one or more cancerous tumors, but also serve as therapeutic targets for the treatment of those tumors.
TABLE-US-00008 Molecule uprepulation of expression in: as compared to: DNA172500 renal cell carcinoma normal kidney (renal cell) (TAT219) tissue
Example 3
Quantitative Analysis of TAT mRNA Expression
[0850]In this assay, a 5' nuclease assay (for example, TaqMan®) and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System® (Perkin Elmer, Applied Biosystems Division, Foster City, Calif.)), were used to find genes that are significantly overexpressed in a cancerous tumor or tumors as compared to other cancerous tumors or normal non-cancerous tissue. The 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor gene expression in real time. Two oligonucleotide primers (whose sequences are based upon the gene or EST sequence of interest) are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
[0851]The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700® Sequence Detection. The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
[0852]The starting material for the screen was mRNA isolated from a variety of different cancerous tissues. The mRNA is quantitated precisely, e.g., fluorometrically. As a negative control, RNA was isolated from various normal tissues of the same tissue type as the cancerous tissues being tested.
[0853]5' nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The ΔCt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer mRNA results to normal human mRNA results. As one Ct unit corresponds to 1 PCR cycle or approximately a 2-fold relative increase relative to normal, two units corresponds to a 4-fold relative increase, 3 units corresponds to an 8-fold relative increase and so on, one can quantitatively measure the relative fold increase in mRNA expression between two or more different tissues. Using this technique, the molecules listed below have been identified as being significantly overexpressed in a particular tumor(s) as compared to their normal non-cancerous counterpart tissue(s) (from both the same and different tissue donors) and thus, represent excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
TABLE-US-00009 upregulation of Molecule expression in: as compared to: DNA261021 (TAT208) lung tumor normal lung tissue DNA77509 (TAT177) colon tumor normal colon tissue DNA119474 (TAT226) ovarian tumor normal ovarian tissue DNA179651 (TAT224) ovarian tumor normal ovarian tissue DNA226717 (TAT185) glioma normal glial/brain tissue DNA227162 (TAT225) ovarian tumor normal ovarian tissue DNA277804 (TAT247) ovarian tumor normal ovarian tissue DNA233034 (TAT174) glioma normal glial/brain tissue DNA266920 (TAT214) glioma normal glial/brain tissue DNA266921 (TAT220) glioma normal glial/brain tissue DNA266922 (TAT221) glioma normal glial/brain tissue DNA234441 (TAT201) colon tumor normal colon tissue DNA234834 (TAT179) colon tumor normal colon tissue DNA247587 (TAT216) squamous celll ung normal squamous tumor cell lung tissue DNA255987 (TAT218) breast tumor normal breast tissue DNA247476 (TAT180) colon tumor normal colon tissue DNA260990 (TAT375) colon tumor normal colon tissue DNA261013 (TAT176) breast tumor normal breast tissue DNA262144 (TAT184) kidney tumor normal kidney tissue DNA267342 (TAT213) breast tumor normal breast tissue DNA267626 (TAT217) breast tumor normal breast tissue DNA268334 (TAT202) kidney tumor normal kidney tissue DNA269238 (TAT215) kidney tumor normal kidney tissue DNA87993 (TAT235) lung tumor normal lung tissue DNA92980 (TAT234) ovarian tumor normal ovarian tissue DNA105792 (TAT233) lung tumor normal lung tissue DNA207698 (TAT237) colon tumor normal colon tissue DNA225886 (TAT236) colon tumor normal colon tissue DNA272578 (TAT238) ovarian tumor normal ovarian tissue DNA304853 (TAT376) colon tumor normal colon tissue DNA304854 (TAT377) colon tumor normal colon tissue DNA304855 (TAT378) colon tumor normal colon tissue DNA287971 (TAT379) colon tumor normal colon tissue
Example 4
In Situ Hybridization
[0854]In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping.
[0855]In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision 1:169-176 (1994), using PCR-generated 33P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., and further processed for in situ hybridization as described by Lu and Gillett, supra. A [33-P] UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55° C. overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
33P-Riboprobe Synthesis
[0856]6.0 μl (125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried 33P-UTP, the following ingredients were added:
[0857]2.0 μl 5× transcription buffer
[0858]1.0 μl DTT (100 mM)
[0859]2.0 μl NTP mix (2.5 mM: 10; each of 10 mM GTP, CTP & ATP+10 μl H2O)
[0860]1.0 μl UTP (50 μM)
[0861]1.0 μl Rnasin
[0862]1.0 μl DNA template (1 μg)
[0863]1.0 μl H2O
[0864]1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)
[0865]The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase were added, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE were added. 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II.
[0866]The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNA Mrk III were added to 3 μl of loading buffer. After heating on a 95° C. heat block for three minutes, the probe was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70° C. freezer one hour to overnight.
33P-Hybridization
[0867]A. Pretreatment of Frozen Sections
[0868]The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes. The trays were placed in 55° C. incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975 ml SQ H2O). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.
[0869]B. Pretreatment of Paraffin-Embedded Sections
[0870]The slides were deparaffinized, placed in SQ H2O, and rinsed twice in 2×SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNase buffer; 37° C., 15 minutes)--human embryo, or 8× proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30 minutes)--formalin tissues. Subsequent rinsing in 0.5×SSC and dehydration were performed as described above.
[0871]C. Prehybridization
[0872]The slides were laid out in a plastic box lined with Box buffer (4×SSC, 50% formamide)--saturated filter paper.
[0873]D. Hybridization
[0874]1.0×106 cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95° C. for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer were added per slide. After vortexing, 50 μl 33P mix were added to 50 μl prehybridization on slide. The slides were incubated overnight at 55° C.
[0875]E. Washes
[0876]Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400 ml 20×SSC+16 ml 0.25M EDTA, Vf=4 L), followed by RNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, Vf=4 L).
[0877]F. Oligonucleotides
[0878]In situ analysis was performed on a variety of DNA sequences disclosed herein. The oligonucleotides employed for these analyses were obtained so as to be complementary to the nucleic acids (or the complements thereof) as shown in the accompanying figures.
[0879]G. Results
[0880]In situ analysis was performed on a variety of DNA sequences disclosed herein. The results from these analyses are as follows.
(1) DNA119474 (TAT226)
[0881]Positive expression is observed in 2 of 3 non-small cell lung carcinomas, 2 of 3 pancreatic adenocarcinomas, 1 of 2 hepatocellular carcinomas and 2 of 3 endometrial adenocarcinomas. In a separate analysis, 10 of 16 ovarian adenocarcinomas are positive and 3 of 9 endometrial adenocarcinomas are positive. All normal tissues examined are negative for expression.
(2) DNA179651 (TAT224)
[0882]In one analysis, expression is seen in 5 of 7 uterine adenocarcinomas and in 7 of 16 ovarian adenocarcinomas. Two cases of dysgerminoma are positive as is one case of a Brenner's tumor.
[0883]In another analysis, 33 of 68 ovarian adenocarcinomas (serous, mucinous, endometrioid, clear cell) are positive for expression. Moderate to strong expression is seen in normal endometrium (no other normal tissues) and normal ovarian stroma is negative.
[0884]In yet another analysis, positive:expression is seen in 3/3 endometrial, 2/2 colorectal, 1/3 transitional cell, 3/3 lung and 1/2 ovarian cancers.
(3) DNA227162 (TAT225)
[0885]Expression is seen in the following tumors: 1 of 3 lung cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.
[0886]In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors.
[0887]With regard to expression in normal tissues, weak expression is seen in one core of urothelium (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression.
(4) DNA277804 (TAT247)
[0888]Expression is seen in the following tumors: 1 of 3 lung cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.
[0889]In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors.
[0890]With regard to expression in normal tissues, weak expression is seen in one core of urothelium (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression.
(5) DNA234441 (TAT201)
[0891]Weak (and inconsistent) expression is seen in normal kidney, normal colon mucosa and normal gallbladder. Weak to moderate, though somewhat inconsistent expression is seen in normal gastrointestinal mucosa (esophagus, stomach, small intestine, colon, anus). Significant expression in tumors is seen as follows: 11 of 12 colorectal adenocarcinomas, 4 of 4 gastric adenocarcinomas, 6 of 8 metastatic adenocarcinomas, 4 of 4 esophageal cancers and 1 of 2 pancreatic adenocarcinomas.
(6) DNA234834 (TAT179)
[0892]With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.
[0893]In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.
(7) DNA247587 (TAT216)
[0894]Expression is seen in 13 of 16 non-small cell lung carcinomas. Expression is also seen in benign bronchial mucosa and occasional activated pneumocytes. Moreover, 65 of 89 cases of invasive breast cancer are positive for expression. Strong expression is seen in normal skin and normal urothelium. Moderate expression is seen in normal mammary epithelium and trophoblasts of the placenta, weak expression in normal prostate and normal gall bladder epithelium and distal renal tubules.
(8) DNA56041 (TAT206)
[0895]In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone.
[0896]In four cases of Hodgkin's disease Reed-Sternberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six
cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells.
(9) DNA257845 (TAT374)
[0897]In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone.
[0898]In four cases of Hodgkin's disease Reed-Sternberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six
cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells.
(10) DNA247476 (TAT180)
[0899]With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.
[0900]In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression.
[0901]In yet another analysis, 8 of 11 breast cancers are positive for expression.
[0902]In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.
[0903]In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers.
[0904]In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed.
[0905]In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma-2 of 2 cases are positive; colorectal adenocarcinoma-12 of 14 cases are positive; gastric adenocarcinoma-6 of 8 cases are positive; esophageal carcinoma-2 of 3 cases are positive; cholangiocarcinoma-1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)-8 of 12 cases are positive.
(11) DNA260990 (TAT375)
[0906]With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.
[0907]In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression.
[0908]In yet another analysis, 8 of 11 breast cancers are positive for expression.
[0909]In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.
[0910]In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers.
[0911]In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed.
[0912]In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma-2 of 2 cases are positive; colorectal adenocarcinoma-12 of 14 cases are positive; gastric adenocarcinoma-6 of 8 cases are positive; esophageal carcinoma-2 of 3 cases are positive; cholangiocarcinoma-1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)-8 of 12 cases are positive.
(12) DNA261013 (TAT176)
[0913]With regard to normal tissues, prostate epithelium shows a weak positive signal. Also, one core of colonic mucosa shows a weak signal in mucosal epithelium. Two cores of a testicular neoplasm are positive.
[0914]In another analysis, 87 cases of infiltrating ductal breast cancer are available for review. 40 cases are positive for expression. Additionally, all tested cell lines (A549, SK-MES, SKBR3, MDA231, MDA453, MDA175, MCF7) are positive for expression.
[0915]In another analysis, there is no consistent expression in benign colon, small intestinal, liver, pancreatic, gastric or esophageal tissue. In malignant tumors expression is observed as follows: colorectal adenocarcinoma: 10 of 14 cases are positive, gastric adenocarcinoma: 4 of 8 cases are positive, esophageal carcinoma: 3 of 4 cases are positive and metastatic adenocarcinoma: 8 of 11 cases are positive.
(13) DNA262144 (TAT184)
[0916]Two of 4 cases of non-small cell lung carcinoma are positive for expression while no signal is observed in non-neoplastic lung. In a separate analysis, three cases of non-small cell lung carcinoma are positive
(14) DNA267342 (TAT213)
[0917]Expression is not observed in any of the normal adult tissues tested. Seventy four cases of breast cancer are available for review and 30 cases give a positive signal Expression localizes to tumor-associated stroma.
[0918]In a separate analysis, expression is seen in a minority of sarcomas; moderate and occasionally strong expression is seen in a case of a synovial sarcoma, angiosarcoma, fibrosarcoma, gliosarcoma and malignant fibrohistiocytoma. In most cases expression appears to localize to the malignant cell population.
(15) DNA267626 (TAT217)
[0919]Expression is seen in 6 of 9 invasive breast cancers. Expression is in most cases of moderate intensity, expression is also seen in benign mammary epithelium and fibroadenoma. The large sections included in this study show expression in 1 of 1 endometrial adenocarcinomas, in 2 of 3 invasive ductal breast cancers, in benign renal tubules, in normal breast epithelium and in epidermis. Sections of lung, brain, myometrium and eye are negative.
(16) DNA268334 (TAT202)
[0920]No expression is seen in any of the adult, normal tissues tested while expression is observed in 3 of 3 renal cell carcinomas.
(17) DNA269238 (TAT215)
[0921]Tumor-associated vasculature was strongly positive in all renal cell carcinomas tested (n=6), in all hepatocellular carcinomas tested (n=3), in all gastric adenocarcinomas tested (n=5), in all endometrial adenocarcinomas tested (n=3), in all malignant melanomas tested (n=3), in all malignant lymphomas tested (n=3), in all pancreatic adenocarcinomas tested (n=1), in all esophageal carcinomas tested (n=4), in all cholangiocarcinomas tested (n=2), in 93% of all non-small cell lung cancers tested (n=15), in 86% of all invasive ductal breast cancers tested (n=88), in 83% of all colorectal adenocarcinomas tested (n=12), in 67% of all metastatic adenocarcinomas tested (n=6), in 75% of all transitional cell carcinomas tested (n=4). While TAT215 expression is also observed in endothelial components of various normal non-cancerous tissues, the expression level is significantly lower in these non-cancerous tissues as compared to their cancerous counterparts and the expression pattern in the tumor tissues was distinct from that in the normal tissues, thereby providing a means for both therapy and diagnosis of the cancerous condition.
(18) DNA304853 (TAT376)
[0922]With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.
[0923]In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.
(19) DNA304854 (TAT377)
[0924]With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian
carcinomas and 2 of 3 malignant melanomas.
[0925]In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.
(20) DNA304855 (TAT378)
[0926]With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.
[0927]In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.
(21) DNA287971 (TAT379)
[0928]With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.
[0929]In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression.
[0930]In yet another analysis, 8 of 11 breast cancers are positive for expression.
[0931]In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.
[0932]In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers.
[0933]In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed.
[0934]In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma-2 of 2 cases are positive; colorectal adenocarcinoma-12 of 14 cases are positive; gastric adenocarcinoma-6 of 8 cases are positive; esophageal carcinoma-2 of 3 cases are positive; cholangiocarcinoma-1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)-8 of 12 cases are positive.
Example 5
Verification and Analysis of Differential TAT Polypeptide Expression by GEPIS
[0935]TAT polypeptides which may have been identified as a tumor antigen as described in one or more of the above Examples were analyzed and verified as follows. An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and interesting EST sequences were identified by GEPIS. Gene expression profiling in silico (GEPIS) is a bioinformatics tool developed at Genentech, Inc. that characterizes genes of interest for new cancer therapeutic targets. GEPIS takes advantage of large amounts of EST sequence and library information to determine gene expression profiles. GEPIS is capable of determining the expression profile of a gene based upon its proportional correlation with the number of its occurrences in EST databases, and it works by integrating the LIFESEQ® EST relational database and Genentech proprietary information in a stringent and statistically meaningful way. In this example, GEPIS is used to identify and cross-validate novel tumor antigens, although GEPIS can be configured to perform either very specific analyses or broad screening tasks. For the initial screen, GEPIS is used to identify EST sequences from the LIFESEQ® database that correlate to expression in a particular tissue or tissues of interest (often a tumor tissue of interest). The EST sequences identified in this initial screen (or consensus sequences obtained from aligning multiple related and overlapping EST sequences obtained from the initial screen) were then subjected to a screen intended to identify the presence of at least one transmembrane domain in the encoded protein. Finally, GEPIS was employed to generate a complete tissue expression profile for the various sequences of interest. Using this type of screening bioinformatics, various TAT polypeptides (and their encoding nucleic acid molecules) were identified as being significantly overexpressed in a particular type of cancer or certain cancers as compared to other cancers and/or normal non-cancerous tissues. The rating of GEPIS hits is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined by GEPIS evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
TABLE-US-00010 upregulation of Molecule expression in: as compared to: DNA67962 (TAT207) colon tumor normal colon tissue DNA67962 (TAT207) uterus tumor normal uterus tissue DNA67962 (TAT207) lung tumor normal lung tissue DNA67962 (TAT207) prostate tumor normal prostate tissue DNA67962 (TAT207) breast tumor normal breast tissue DNA96792 (TAT239) colon tumor normal colon tissue DNA96792 (TAT239) rectum tumor normal rectum tissue DNA96792 (TAT239) pancreas tumor normal pancreas tissue DNA96792 (TAT239) lung tumor normal lung tissue DNA96792 (TAT239) stomach tumor normal stomach tissue DNA96792 (TAT239) esophagus tumor normal esophagus tissue DNA96792 (TAT239) breast tumor normal breast tissue DNA96792 (TAT239) uterus tumor normal uterus tissue DNA96964 (TAT193) breast tumor normal breast tissue DNA96964 (TAT193) brain tumor normal brain tissue DNA142915 (TAT199) breast tumor normal breast tissue DNA142915 (TAT199) ovary tumor normal ovary tissue DNA142915 (TAT199) brain tumor normal brain tissue DNA208551 (TAT178) prostate tumor normal prostate tissue DNA208551 (TAT178) colon tumor normal colon tissue DNA210159 (TAT198) prostate tumor normal prostate tissue DNA210159 (TAT198) uterus tumor normal uterus tissue DNA210159 (TAT198) breast tumor normal breast tissue DNA210159 (TAT198) ovarian tumor normal ovarian tissue DNA225706 (TAT194) adrenal tumor normal adrenal tissue DNA225706 (TAT194) prostate tumor normal prostate tissue DNA225706 (TAT194) breast tumor normal breast tissue DNA225706 (TAT194) connective tissue normal connective tissue tumor DNA225793 (TAT223) ovarian tumor normal ovarian tissue DNA225793 (TAT223) fallopian tube normal fallopian tube tissue tumor DNA225793 (TAT223) kidney tumor normal kidney tissue DNA225796 (TAT196) breast tumor normal breast tissue DNA225943 (TAT195) liver tumor normal liver tissue DNA225943 (TAT195) lung tumor normal lung tissue DNA225943 (TAT195) breast tumor normal breast tissue DNA226283 (TAT203) uterine tumor normal uterine tissue DNA226283 (TAT203) breast tumor normal breast tissue DNA226283 (TAT203) squamous cell normal squamous lung tumor cell lung tissue DNA226283 (TAT203) colon tumor normal colon tissue DNA226283 (TAT203) ovarian tumor normal ovarian tissue DNA226589 (TAT200) brain tumor normal brain tissue DNA226589 (TAT200) colon tumor normal colon tissue DNA226589 (TAT200) breast tumor normal breast tissue DNA226589 (TAT200) prostate tumor normal prostate tissue DNA226622 (TAT205) squamous cell normal squamous lung tumor cell lung tissue DNA226622 (TAT205) kidney tumor normal kidney tissue DNA226622 (TAT205) uterine tumor normal uterine tissue DNA226622 (TAT205) breast tumor normal breast tissue DNA226622 (TAT205) colon tumor normal colon tissue DNA227545 (TAT197) breast tumor normal breast tissue DNA227611 (TAT175) prostate tumor normal prostate tissue DNA227611 (TAT175) colon tumor normal colon tissue DNA227611 (TAT175) breast tumor normal breast tissue DNA227611 (TAT175) uterine tumor normal uterine tissue DNA261021 (TAT208) prostate tumor normal prostate tissue DNA261021 (TAT208) colon tumor normal colon tissue DNA261021 (TAT208) breast tumor normal breast tissue DNA261021 (TAT208) uterine tumor normal uterine tissue DNA260655 (TAT209) lung tumor normal lung tissue DNA260655 (TAT209) colon tumor normal colon tissue DNA260655 (TAT209) breast tumor normal breast tissue DNA260655 (TAT209) liver tumor normal liver tissue DNA260655 (TAT209) ovarian tumor normal ovarian tissue DNA260655 (TAT209) skin tumor normal skin tissue DNA260655 (TAT209) spleen tumor normal spleen tissue DNA260655 (TAT209) myeloid tumor normal myeloid tissue DNA260655 (TAT209) muscle tumor normal muscle tissue DNA260655 (TAT209) bone tumor normal bone tissue DNA260945 (TAT192) brain tumor normal brain tissue DNA260945 (TAT192) breast tumor normal breast tissue DNA260945 (TAT192) colon tumor normal colon tissue DNA260945 (TAT192) ovarian tumor normal ovarian tissue DNA260945 (TAT192) pancreatic tumor normal pancreatic tissue DNA261001 (TAT181) bone tumor normal bone tissue DNA261001 (TAT181) lung tumor normal lung tissue DNA266928 (TAT182) bone tumor normal bone tissue DNA266928 (TAT182) lung tumor normal lung tissue DNA268035 (TAT222) ovarian tumor normal ovarian tissue DNA277797 (TAT212) breast tumor normal breast tissue DNA277797 (TAT212) pancreatic tumor normal pancreatic tissue DNA77509 (TAT177) colon tumor normal colon tissue DNA77509 (TAT177) testis tumor normal testis tissue DNA87993 (TAT235) breast tumor normal breast tissue DNA87993 (TAT235) prostate tumor normal prostate tissue DNA87993 (TAT235) colon tumor normal colon tissue DNA87993 (TAT235) ovarian tumor normal ovarian tissue DNA92980 (TAT234) bone tumor normal bone tissue DNA92980 (TAT234) breast tumor normal breast tissue DNA92980 (TAT234) cervical tumor normal cervical tissue DNA92980 (TAT234) colon tumor normal colon tissue DNA92980 (TAT234) rectum tumor normal rectum tissue DNA92980 (TAT234) endometrial normal endometrial tissue tumor DNA92980 (TAT234) liver tumor normal liver tissue DNA92980 (TAT234) lung tumor normal lung tissue DNA92980 (TAT234) ovarian tumor normal ovarian tissue DNA92980 (TAT234) pancreatic tumor normal pancreatic tissue DNA92980 (TAT234) skin tumor normal skin tissue DNA92980 (TAT234) soft tissue tumor normal soft tissue DNA92980 (TAT234) stomach tumor normal stomach tissue DNA92980 (TAT234) bladder tumor normal bladder tissue DNA92980 (TAT234) thyroid tumor normal thyroid tissue DNA92980 (TAT234) esophagus tumor normal esophagus tissue DNA92980 (TAT234) testis tumor normal testis tissue DNA105792 (TAT233) adrenal tumor normal adrenal tissue DNA105792 (TAT233) breast tumor normal breast tissue DNA105792 (TAT233) endometrial normal endometrial tissue tumor DNA105792 (TAT233) esophagus tumor normal esophagus tissue DNA105792 (TAT233) kidney tumor normal kidney tissue DNA105792 (TAT233) lung tumor normal lung tissue DNA105792 (TAT233) ovarian tumor normal ovarian tissue DNA105792 (TAT233) pancreatic tumor normal pancreatic tissue DNA105792 (TAT233) prostate tumor normal prostate tissue DNA105792 (TAT233) soft tissue tumor normal soft tissue DNA105792 (TAT233) myeloid tumor normal myeloid tissue DNA105792 (TAT233) thyroid tumor normal thyroid tissue DNA105792 (TAT233) bladder tumor normal bladder tissue DNA105792 (TAT233) brain tumor normal brain tissue DNA105792 (TAT233) testis tumor normal testis tissue DNA119474 (TAT226) kidney tumor normal kidney tissue DNA119474 (TAT226) adrenal tumor normal adrenal tissue DNA119474 (TAT226) uterine tumor normal uterine tissue DNA119474 (TAT226) ovarian tumor normal ovarian tissue DNA150491 (TAT204) squamous cell normal squamous lung tumor cell lung tissue DNA150491 (TAT204) colon tumor normal colon tissue DNA280351 (TAT248) squamous cell normal squamous lung tumor cell lung tissue DNA280351 (TAT248) colon tumor normal colon tissue DNA150648 (TAT232) liver tumor normal liver tissue DNA150648 (TAT232) breast tumor normal breast tissue DNA150648 (TAT232) brain tumor normal brain tissue DNA150648 (TAT232) lung tumor normal lung tissue DNA150648 (TAT232) colon tumor normal colon tissue DNA150648 (TAT232) rectum tumor normal rectum tissue DNA150648 (TAT232) kidney tumor normal kidney tissue DNA150648 (TAT232) bladder tumor normal bladder tissue DNA179651 (TAT224) colon tumor normal colon tissue DNA179651 (TAT224) uterine tumor normal uterine tissue DNA179651 (TAT224) lung tumor normal lung tissue DNA179651 (TAT224) kidney tumor normal kidney tissue DNA225886 (TAT236) breast tumor normal breast tissue DNA225886 (TAT236) colon tumor normal colon tissue DNA225886 (TAT236) rectum tumor normal rectum tissue DNA225886 (TAT236) ovarian tumor normal ovarian tissue DNA225886 (TAT236) pancreas tumor normal pancreas tissue DNA225886 (TAT236) prostate tumor normal prostate tissue DNA225886 (TAT236) bladder tumor normal bladder tissue DNA225886 (TAT236) testis tumor normal testis tissue DNA226717 (TAT185) glioma normal glial tissue DNA226717 (TAT185) brain tumor normal brain tissue DNA227162 (TAT225) myeloid tumor normal myeloid tissue DNA227162 (TAT225) uterine tumor normal uterine tissue DNA227162 (TAT225) prostate tumor normal prostate tissue DNA277804 (TAT247) myeloid tumor normal myeloid tissue DNA277804 (TAT247) uterine tumor normal uterine tissue DNA277804 (TAT247) prostate tumor normal prostate tissue DNA233034 (TAT174) glioma normal glial tissue DNA233034 (TAT174) brain tumor normal brain tissue DNA233034 (TAT174) kidney tumor normal kidney tissue DNA233034 (TAT174) adrenal tumor normal adrenal tissue DNA266920 (TAT214) glioma normal glial tissue DNA266920 (TAT214) brain tumor normal brain tissue DNA266920 (TAT214) kidney tumor normal kidney tissue DNA266920 (TAT214) adrenal tumor normal adrenal tissue DNA266921 (TAT220) glioma normal glial tissue DNA266921 (TAT220) brain tumor normal brain tissue DNA266921 (TAT220) kidney tumor normal kidney tissue DNA266921 (TAT220) adrenal tumor normal adrenal tissue DNA266922 (TAT221) glioma normal glial tissue DNA266922 (TAT221) brain tumor normal brain tissue DNA266922 (TAT221) kidney tumor normal kidney tissue DNA266922 (TAT221) adrenal tumor normal adrenal tissue DNA234834 (TAT179) colon tumor normal colon tissue DNA234834 (TAT179) uterine tumor normal uterine tissue DNA234834 (TAT179) breast tumor normal breast tissue DNA234834 (TAT179) prostate tumor normal prostate tissue DNA247587 (TAT216) breast tumor normal breast tissue DNA247587 (TAT216) prostate tumor normal prostate tissue DNA247587 (TAT216) bladder tumor normal bladder tissue DNA247587 (TAT216) lymphoid tumor normal lymphoid tissue DNA255987 (TAT218) brain tumor normal brain tissue DNA255987 (TAT218) breast tumor normal breast tissue DNA247476 (TAT180) prostate tumor normal prostate tissue DNA247476 (TAT180) pancreas tumor normal pancreas tissue DNA247476 (TAT180) brain tumor normal brain tissue DNA247476 (TAT180) stomach tumor normal stomach tissue DNA247476 (TAT180) bladder tumor normal bladder tissue DNA247476 (TAT180) soft tissue tumor normal soft tissue DNA247476 (TAT180) skin tumor normal skin tissue DNA247476 (TAT180) kidney tumor normal kidney tissue DNA260990 (TAT375) prostate tumor normal prostate tissue DNA260990 (TAT375) pancreas tumor normal pancreas tissue DNA260990 (TAT375) brain tumor normal brain tissue DNA260990 (TAT375) stomach tumor normal stomach tissue DNA260990 (TAT375) bladder tumor normal bladder tissue DNA260990 (TAT375) soft tissue tumor normal soft tissue DNA260990 (TAT375) skin tumor normal skin tissue DNA260990 (TAT375) kidney tumor normal kidney tissue DNA261013 (TAT176) prostate tumor normal prostate tissue DNA261013 (TAT176) colon tumor normal colon tissue DNA261013 (TAT176) small intestine normal small intestine tumor tissue DNA261013 (TAT176) pancreatic tumor normal pancreatic tissue DNA261013 (TAT176) uterine tumor normal uterine tissue DNA261013 (TAT176) ovarian tumor normal ovarian tissue DNA261013 (TAT176) bladder tumor normal bladder tissue DNA261013 (TAT176) stomach tumor normal stomach tissue DNA267342 (TAT213) breast tumor normal breast tissue DNA267342 (TAT213) uterine tumor normal uterine tissue DNA267342 (TAT213) colon tumor normal colon tissue DNA267342 (TAT213) kidney tumor normal kidney tissue DNA267342 (TAT213) bladder tumor normal bladder tissue DNA267342 (TAT213) bone tumor normal bone tissue DNA267342 (TAT213) ovarian tumor normal ovarian tissue DNA267342 (TAT213) pancreatic tumor normal pancreatic tissue DNA267626 (TAT217) breast tumor normal breast tissue DNA267626 (TAT217) colon tumor normal colon tissue DNA267626 (TAT217) pancreatic tumor normal pancreatic tissue DNA267626 (TAT217) ovarian tumor normal ovarian tissue DNA268334 (TAT202) kidney tumor normal kidney tissue DNA269238 (TAT215) colon tumor normal colon tissue DNA269238 (TAT215) kidney tumor normal kidney tissue DNA269238 (TAT215) adrenal tumor normal adrenal tissue DNA269238 (TAT215) bladder tumor normal bladder tissue DNA272578 (TAT238) adrenal tumor normal adrenal tissue DNA272578 (TAT238) lung tumor normal lung tissue DNA272578 (TAT238) ovarian tumor normal ovarian tissue DNA272578 (TAT238) uterine tumor normal uterine tissue DNA304853 (TAT376) colon tumor normal colon tissue DNA304853 (TAT376) uterine tumor normal uterine tissue
DNA304853 (TAT376) breast tumor normal breast tissue DNA304853 (TAT376) prostate tumor normal prostate tissue DNA304854 (TAT377) colon tumor normal colon tissue DNA304854 (TAT377) uterine tumor normal uterine tissue DNA304854 (TAT377) breast tumor normal breast tissue DNA304854 (TAT377) prostate tumor normal prostate tissue DNA304855 (TAT378) colon tumor normal colon tissue DNA304855 (TAT378) uterine tumor normal uterine tissue DNA304855 (TAT378) breast tumor normal breast tissue DNA304855 (TAT378) prostate tumor normal prostate tissue DNA287971 (TAT379) prostate tumor normal prostate tissue DNA287971 (TAT379) pancreas tumor normal pancreas tissue DNA287971 (TAT379) brain tumor normal brain tissue DNA287971 (TAT379) stomach tumor normal stomach tissue DNA287971 (TAT379) bladder tumor normal bladder tissue DNA287971 (TAT379) soft tissue tumor normal soft tissue DNA287971 (TAT379) skin tumor normal skin tissue DNA287971 (TAT379) kidney tumor normal kidney tissue
Example 6
Use of TAT as a Hybridization Probe
[0936]The following method describes use of a nucleotide sequence encoding TAT as a hybridization probe for, i.e., diagnosis of the presence of a tumor in a mammal.
[0937]DNA comprising the coding sequence of full-length or mature TAT as disclosed herein can also be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of TAT) in human tissue cDNA libraries or human tissue genomic libraries.
[0938]Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled TAT-derived probe to the filters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.
[0939]DNAs having a desired sequence identity with the DNA encoding full-length native sequence TAT can then be identified using standard techniques known in the art.
Example 7
Expression of TAT in E. coli
[0940]This example illustrates preparation of an unglycosylated form of TAT by recombinant expression in E. coli.
[0941]The DNA sequence encoding TAT is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the TAT coding region, lambda transcriptional terminator, and an argU gene.
[0942]The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
[0943]Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.
[0944]After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized TAT protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
[0945]TAT may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding TAT is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) cipP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2SO4, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO4) and grown for approximately 20-30 hours at 30° C. with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
[0946]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution is stirred overnight at 4° C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4° C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
[0947]The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4° C. for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros R1/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
[0948]Fractions containing the desired folded TAT polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.
[0949]Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).
Example 8
Expression of TAT in Mammalian Cells
[0950]This example illustrates preparation of a potentially glycosylated form of TAT by recombinant expression in mammalian cells.
[0951]The vector, pRK5 (see EP 307,247, published Mar. 15, 198 employed as the expression vector. Optionally, the TAT DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the TAT DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-TAT.
[0952]In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-TAT DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO4, and a precipitate is allowed to form for 10 minutes at 25° C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37° C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
[0953]Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml 35S-cysteine and 200 μCi/ml 35S methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of TAT polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
[0954]In an alternative technique, TAT may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-TAT DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed TAT can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
[0955]In another embodiment, TAT can be expressed in CHO cells. The pRK5-TAT can be transfected into CHO cells using known reagents such as CaPO4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35S-methionine. After determining the presence of TAT polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed TAT can then be concentrated and purified by any selected method.
[0956]Epitope-tagged TAT may also be expressed in host CHO cells. The TAT may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged TAT insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged TAT can then be concentrated and purified by any selected method, such as by Ni2+-chelate affinity chromatography.
[0957]TAT may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.
[0958]Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG1 constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
[0959]Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
[0960]Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim). The cells are grown as described in Lucas et al., supra. Approximately 3×107 cells are frozen in an ampule for further growth and production as described below.
[0961]The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3×105 cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3 L production spinner is seeded at 1.2×106 cells/mL. On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
[0962]For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4° C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80° C.
[0963]Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
[0964]Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).
Example 9
Expression of TAT in Yeast
[0965]The following method describes recombinant expression of TAT in yeast.
[0966]First, yeast expression vectors are constructed for intracellular production or secretion of TAT from the ADH2/GAPDH promoter. DNA encoding TAT and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of TAT. For secretion, DNA encoding TAT can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native TAT signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of TAT.
[0967]Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
[0968]Recombinant TAT can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing TAT may further be purified using selected column chromatography resins.
[0969]Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).
Example 10
Expression of TAT in Baculovirus-Infected Insect Cells
[0970]The following method describes recombinant expression of TAT in Baculovirus-infected insect cells.
[0971]The sequence coding for TAT is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding TAT or the desired portion of the coding sequence of TAT such as the sequence encoding an extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
[0972]Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold® virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28° C., the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
[0973]Expressed poly-his tagged TAT can then be purified, for example, by Ni2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His10-tagged TAT are pooled and dialyzed against loading buffer.
[0974]Alternatively, purification of the IgG tagged (or Fc tagged) TAT can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
[0975]Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).
Example 11
Preparation of Antibodies that Bind TAT
[0976]This example illustrates preparation of monoclonal antibodies which can specifically bind TAT.
[0977]Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified TAT, fusion proteins containing TAT, and cells expressing recombinant TAT on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
[0978]Mice, such as Balb/c, are immunized with the TAT immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-TAT antibodies.
[0979]After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of TAT. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0980]The hybridoma cells will be screened in an ELISA for reactivity against TAT. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against TAT is within the skill in the art.
[0981]The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-TAT monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.
Example 12
Purification of TAT Polypeptides Using Specific Antibodies
[0982]Native or recombinant TAT polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-TAT polypeptide, mature TAT polypeptide, or pre-TAT polypeptide is purified by immunoaffinity chromatography using antibodies specific for the TAT polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-TAT polypeptide antibody to an activated chromatographic resin.
[0983]Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE® (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
[0984]Such an immunoaffinity column is utilized in the purification of TAT polypeptide by preparing a fraction from cells containing TAT polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble TAT polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
[0985]A soluble TAT polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of TAT polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and TAT polypeptide is collected.
Example 13
In Vitro Tumor Cell Killing Assay
[0986]Mammalian cells expressing the TAT polypeptide of interest may be obtained using standard expression vector and cloning techniques. Alternatively, many tumor cell lines expressing TAT polypeptides of interest are publicly available, for example, through the ATCC and can be routinely identified using standard ELISA or FACS analysis. Anti-TAT polypeptide monoclonal antibodies (and toxin conjugated derivatives thereof) may then be employed in assays to determine the ability of the antibody to kill TAT polypeptide expressing cells in vitro.
[0987]For example, cells expressing the TAT polypeptide of interest are obtained as described above and plated into 96 well dishes. In one analysis, the antibody/toxin conjugate (or naked antibody) is included throughout the cell incubation for a period of 4 days. In a second independent analysis, the cells are incubated for 1 hour with the antibody/toxin conjugate (or naked antibody) and then washed and incubated in the absence of antibody/toxin conjugate for a period of 4 days. Cell viability is then measured using the CellTiter-Glo Luminescent Cell Viability Assay from Promega (Cat# G7571). Untreated cells serve as a negative control.
Example 14
In Vivo Tumor Cell Killing Assay
[0988]To test the efficacy of conjugated or unconjugated anti-TAT polypeptide monoclonal antibodies, anti-TAT antibody is injected intraperitoneally into nude mice 24 hours prior to receiving tumor promoting cells subcutaneously in the flank. Antibody injections continue twice per week for the remainder of the study. Tumor volume is then measured twice per week.
[0989]The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
[0990]The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
Sequence CWU
1
12013781DNAHomo Sapien 1ctccgggtcc ccaggggctg cgccgggccg gcctggcaag
ggggacgagt 50cagtggacac tccaggaaga gcggccccgc ggggggcgat
gaccgtgcgc 100tgaccctgac tcactccagg tccggaggcg ggggcccccg
gggcgactcg 150ggggcggacc gcggggcgga gctgccgccc gtgagtccgg
ccgagccacc 200tgagcccgag ccgcgggaca ccgtcgctcc tgctctccga
atgctgcgca 250ccgcgatggg cctgaggagc tggctcgccg ccccatgggg
cgcgctgccg 300cctcggccac cgctgctgct gctcctgctg ctgctgctcc
tgctgcagcc 350gccgcctccg acctgggcgc tcagcccccg gatcagcctg
cctctgggct 400ctgaagagcg gccattcctc agattcgaag ctgaacacat
ctccaactac 450acagcccttc tgctgagcag ggatggcagg accctgtacg
tgggtgctcg 500agaggccctc tttgcactca gtagcaacct cagcttcctg
ccaggcgggg 550agtaccagga gctgctttgg ggtgcagacg cagagaagaa
acagcagtgc 600agcttcaagg gcaaggaccc acagcgcgac tgtcaaaact
acatcaagat 650cctcctgccg ctcagcggca gtcacctgtt cacctgtggc
acagcagcct 700tcagccccat gtgtacctac atcaacatgg agaacttcac
cctggcaagg 750gacgagaagg ggaatgtcct cctggaagat ggcaagggcc
gttgtccctt 800cgacccgaat ttcaagtcca ctgccctggt ggttgatggc
gagctctaca 850ctggaacagt cagcagcttc caagggaatg acccggccat
ctcgcggagc 900caaagccttc gccccaccaa gaccgagagc tccctcaact
ggctgcaaga 950cccagctttt gtggcctcag cctacattcc tgagagcctg
ggcagcttgc 1000aaggcgatga tgacaagatc tactttttct tcagcgagac
tggccaggaa 1050tttgagttct ttgagaacac cattgtgtcc cgcattgccc
gcatctgcaa 1100gggcgatgag ggtggagagc gggtgctaca gcagcgctgg
acctccttcc 1150tcaaggccca gctgctgtgc tcacggcccg acgatggctt
ccccttcaac 1200gtgctgcagg atgtcttcac gctgagcccc agcccccagg
actggcgtga 1250cacccttttc tatggggtct tcacttccca gtggcacagg
ggaactacag 1300aaggctctgc cgtctgtgtc ttcacaatga aggatgtgca
gagagtcttc 1350agcggcctct acaaggaggt gaaccgtgag acacagcagt
ggtacaccgt 1400gacccacccg gtgcccacac cccggcctgg agcgtgcatc
accaacagtg 1450cccgggaaag gaagatcaac tcatccctgc agctcccaga
ccgcgtgctg 1500aacttcctca aggaccactt cctgatggac gggcaggtcc
gaagccgcat 1550gctgctgctg cagccccagg ctcgctacca gcgcgtggct
gtacaccgcg 1600tccctggcct gcaccacacc tacgatgtcc tcttcctggg
cactggtgac 1650ggccggctcc acaaggcagt gagcgtgggc ccccgggtgc
acatcattga 1700ggagctgcag atcttctcat cgggacagcc cgtgcagaat
ctgctcctgg 1750acacccacag ggggctgctg tatgcggcct cacactcggg
cgtagtccag 1800gtgcccatgg ccaactgcag cctgtaccgg agctgtgggg
actgcctcct 1850cgcccgggac ccctactgtg cttggagcgg ctccagctgc
aagcacgtca 1900gcctctacca gcctcagctg gccaccaggc cgtggatcca
ggacatcgag 1950ggagccagcg ccaaggacct ttgcagcgcg tcttcggttg
tgtccccgtc 2000ttttgtacca acaggggaga agccatgtga gcaagtccag
ttccagccca 2050acacagtgaa cactttggcc tgcccgctcc tctccaacct
ggcgacccga 2100ctctggctac gcaacggggc ccccgtcaat gcctcggcct
cctgccacgt 2150gctacccact ggggacctgc tgctggtggg cacccaacag
ctgggggagt 2200tccagtgctg gtcactagag gagggcttcc agcagctggt
agccagctac 2250tgcccagagg tggtggagga cggggtggca gaccaaacag
atgagggtgg 2300cagtgtaccc gtcattatca gcacatcgcg tgtgagtgca
ccagctggtg 2350gcaaggccag ctggggtgca gacaggtcct actggaagga
gttcctggtg 2400atgtgcacgc tctttgtgct ggccgtgctg ctcccagttt
tattcttgct 2450ctaccggcac cggaacagca tgaaagtctt cctgaagcag
ggggaatgtg 2500ccagcgtgca ccccaagacc tgccctgtgg tgctgccccc
tgagacccgc 2550ccactcaacg gcctagggcc ccctagcacc ccgctcgatc
accgagggta 2600ccagtccctg tcagacagcc ccccgggggc ccgagtcttc
actgagtcag 2650agaagaggcc actcagcatc caagacagct tcgtggaggt
atccccagtg 2700tgcccccggc cccgggtccg ccttggctcg gagatccgtg
actctgtggt 2750gtgagagctg acttccagag gacgctgccc tggcttcagg
ggctgtgaat 2800gctcggagag ggtcaactgg acctcccctc cgctctgctc
ttcgtggaac 2850acgaccgtgg tgcccggccc ttgggagcct tggagccagc
tggcctgctg 2900ctctccagtc aagtagcgaa gctcctacca cccagacacc
caaacagccg 2950tggccccaga ggtcctggcc aaatatgggg gcctgcctag
gttggtggaa 3000cagtgctcct tatgtaaact gagccctttg tttaaaaaac
aattccaaat 3050gtgaaactag aatgagaggg aagagatagc atggcatgca
gcacacacgg 3100ctgctccagt tcatggcctc ccaggggtgc tggggatgca
tccaaagtgg 3150ttgtctgaga cagagttgga aaccctcacc aactggcctc
ttcaccttcc 3200acattatccc gctgccaccg gctgccctgt ctcactgcag
attcaggacc 3250agcttgggct gcgtgcgttc tgccttgcca gtcagccgag
gatgtagttg 3300ttgctgccgt cgtcccacca cctcagggac cagagggcta
ggttggcact 3350gcggccctca ccaggtcctg ggctcggacc caactcctgg
acctttccag 3400cctgtatcag gctgtggcca cacgagagga cagcgcgagc
tcaggagaga 3450tttcgtgaca atgtacgcct ttccctcaga attcagggaa
gagactgtcg 3500cctgccttcc tccgttgttg cgtgagaacc cgtgtgcccc
ttcccaccat 3550atccaccctc gctccatctt tgaactcaaa cacgaggaac
taactgcacc 3600ctggtcctct ccccagtccc cagttcaccc tccatccctc
accttcctcc 3650actctaaggg atatcaacac tgcccagcac aggggccctg
aatttatgtg 3700gtttttatac attttttaat aagatgcact ttatgtcatt
ttttaataaa 3750gtctgaagaa ttactgttta aaaaaaaaaa a
378122010DNAHomo Sapien 2ggaaaggctg agtctccagc
tcaaggtcaa aacgtccaag gccgaaagcc 50ctccagtttc ccctggacgc
cttgctcctg cttctgctac gaccttctgg 100ggaaaacgaa tttctcattt
tcttcttaaa ttgccatttt cgctttagga 150gatgaatgtt ttcctttggc
tgttttggca atgactctga attaaagcga 200tgctaacgcc tcttttcccc
ctaattgtta aaagctatgg actgcaggaa 250gatggcccgc ttctcttaca
gtgtgatttg gatcatggcc atttctaaag 300tctttgaact gggattagtt
gccgggctgg gccatcagga atttgctcgt 350ccatctcggg gatacctggc
cttcagagat gacagcattt ggccccagga 400ggagcctgca attcggcctc
ggtcttccca gcgtgtgccg cccatgggga 450tacagcacag taaggagcta
aacagaacct gctgcctgaa tgggggaacc 500tgcatgctgg ggtccttttg
tgcctgccct ccctccttct acggacggaa 550ctgtgagcac gatgtgcgca
aagagaactg tgggtctgtg ccccatgaca 600cctggctgcc caagaagtgt
tccctgtgta aatgctggca cggtcagctc 650cgctgctttc ctcaggcatt
tctacccggc tgtgatggcc ttgtgatgga 700tgagcacctc gtggcttcca
ggactccaga actaccaccg tctgcacgta 750ctaccacttt tatgctagtt
ggcatctgcc tttctataca aagctactat 800taatcgacat tgacctattt
ccagaaatac aattttagat atcatgcaaa 850tttcatgacc agtaaaggct
gctgctacaa tgtcctaact gaaagatgat 900catttgtagt tgccttaaaa
taatgaatac atttccaaaa tggtctctaa 950catttcctta cagaactact
tcttacttct ttgccctgcc ctctcccaaa 1000aaactacttc ttttttcaaa
agaaagtcag ccatatctcc attgtgccta 1050agtccagtgt ttcttttttt
tttttttttg agacggagtc tcactctgtc 1100acccaggctg gactgcaatg
acgcgatctt ggttcactgc aacctccgca 1150tccggggttc aagccattct
cctgcctcag cctcccaagt aactgggatt 1200acaggcatgt gtcaccatgc
ccagctaatt tttttgtatt tttagtagag 1250atgggggttt caccatattg
gccagtctgg tctcgaactc ctgaccttgt 1300gatccactcg cctcagcctc
tcgaagtgct gagattacac acgtgagcaa 1350ctgtgcaagg cctggtgttt
cttgatacat gtaattctac caaggtcttc 1400ttaatatgtt cttttaaatg
attgaattat atgttcagat tattggagac 1450taattctaat gtggacctta
gaatacagtt ttgagtagag ttgatcaaaa 1500tcaattaaaa tagtctcttt
aaaaggaaag aaaacatctt taaggggagg 1550aaccagagtg ctgaaggaat
ggaagtccat ctgcgtgtgt gcagggagac 1600tgggtaggaa agaggaagca
aatagaagag agaggttgaa aaacaaaatg 1650ggttacttga ttggtgatta
ggtggtggta gagaagcaag taaaaaggct 1700aaatggaagg gcaagtttcc
atcatctata gaaagctata taagacaaga 1750actccccttt ttttcccaaa
ggcattataa aaagaatgaa gcctccttag 1800aaaaaaaatt atacctcaat
gtccccaaca agattgctta ataaattgtg 1850tttcctccaa gctattcaat
tcttttaact gttgtagaag acaaaatgtt 1900cacaatatat ttagttgtaa
accaagtgat caaactacat attgtaaagc 1950ccatttttaa aatacattgt
atatatgtgt atgcacagta aaaatggaaa 2000ctatattgaa
20103549DNAHomo Sapien
3gccaggaggg agagccttcc ccaagcaaac aatccagagc agctgtgcaa
50acaacggtgc ataaatgagg cctcctggac catgaagcga gtcctgagct
100gcgtcccgga gcccacggtg gtcatggctg ccagagcgct ctgcatgctg
150gggctggtcc tggccttgct gtcctccagc tctgctgagg agtacgtggg
200cctgtctgca aaccagtgtg ccgtgccagc caaggacagg gtggactgcg
250gctaccccca tgtcaccccc aaggagtgca acaaccgggg ctgctgcttt
300gactccagga tccctggagt gccttggtgt ttcaagcccc tgcaggaagc
350agaatgcacc ttctgaggca cctccagctg cccccggccg ggggatgcga
400ggctcggagc acccttgccc ggctgtgatt gctgccaggc actgttcatc
450tcagcttttc tgtccctttg ctcccggcaa gcgcttctgc tgaaagttca
500tatctggagc ctgatgtctt aacgaataaa ggtcccatgc tccacccga
54941424DNAHomo Sapien 4gaccagactc gtctcaggcc agttgcagcc ttctcagcca
aacgccgacc 50aaggaaaact cactaccatg agaattgcag tgatttgctt
ttgcctccta 100ggcatcacct gtgccatacc agttaaacag gctgattctg
gaagttctga 150ggaaaagcag ctttacaaca aatacccaga tgctgtggcc
acatggctaa 200accctgaccc atctcagaag cagaatctcc tagccccaca
gaatgctgtg 250tcctctgaag aaaccaatga ctttaaacaa gagacccttc
caagtaagtc 300caacgaaagc catgaccaca tggatgatat ggatgatgaa
gatgatgatg 350accatgtgga cagccaggac tccattgact cgaacgactc
tgatgatgta 400gatgacactg atgattctca ccagtctgat gagtctcacc
attctgatga 450atctgatgaa ctggtcactg attttcccac ggacctgcca
gcaaccgaag 500ttttcactcc agttgtcccc acagtagaca catatgatgg
ccgaggtgat 550agtgtggttt atggactgag gtcaaaatct aagaagtttc
gcagacctga 600catccagtac cctgatgcta cagacgagga catcacctca
cacatggaaa 650gcgaggagtt gaatggtgca tacaaggcca tccccgttgc
ccaggacctg 700aacgcgcctt ctgattggga cagccgtggg aaggacagtt
atgaaacgag 750tcagctggat gaccagagtg ctgaaaccca cagccacaag
cagtccagat 800tatataagcg gaaagccaat gatgagagca atgagcattc
cgatgtgatt 850gatagtcagg aactttccaa agtcagccgt gaattccaca
gccatgaatt 900tcacagccat gaagatatgc tggttgtaga ccccaaaagt
aaggaagaag 950ataaacacct gaaatttcgt atttctcatg aattagatag
tgcatcttct 1000gaggtcaatt aaaaggagaa aaaatacaat ttctcacttt
gcatttagtc 1050aaaagaaaaa atgctttata gcaaaatgaa agagaacatg
aaatgcttct 1100ttctcagttt attggttgaa tgtgtatcta tttgagtctg
gaaataacta 1150atgtgtttga taattagttt agtttgtggc ttcatggaaa
ctccctgtaa 1200actaaaagct tcagggttat gtctatgttc attctataga
agaaatgcaa 1250actatcactg tattttaata tttgttattc tctcatgaat
agaaatttat 1300gtagaagcaa acaaaatact tttacccact taaaaagaga
atataacatt 1350ttatgtcact ataatctttt gttttttaag ttagtgtata
ttttgttgtg 1400attatctttt tgtggtgtga ataa
142451166DNAHomo Sapienunsure721-761unknown base
5cggacgcgtg ggcggaggga agaggaccgc aaaccaaccc aggacccgct
50cagttccacg cgcggcagcc ctccgtgcgc gcaggctcgg tatgagccgc
100acagcctaca cggtgggagc cctgcttctc ctcttgggga ccctgctgcc
150ggctgctgaa gggaaaaaga aagggtccca aggtgccatc cccccgccag
200acaaggccca gcacaatgac tcagagcaga ctcagtcgcc ccagcagcct
250ggctccagga accgggggcg gggccaaggg cggggcactg ccatgcccgg
300ggaggaggtg ctggagtcca gccaagaggc cctgcatgtg acggagcgca
350aatacctgaa gcgagactgg tgcaaaaccc agccgcttaa gcagaccatc
400cacgaggaag gctgcaacag tcgcaccatc atcaaccgct tctgttacgg
450ccagtgcaac tctttctaca tccccaggca catccggaag gaggaaggtt
500cctttcagtc ctgctccttc tgcaagccca agaaattcac taccatgatg
550gtcacactca actgccctga actacagcca cctaccaaga agaagagagt
600cacacgtgtg aagcagtgtc gttgcatatc catcgatttg gattaagcca
650aatccaggtg cacccagcat gtcctaggaa tgcagcccca ggaagtccca
700gacctaaaac aaccagattc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
750nnnnnnnnnn nagacttacg atgcatgtat acaaacgaat agcagataat
800gatgactagt tcacacataa agtcctttta aggagaaaat ctaaaatgaa
850aagtggataa acagaacatt tataagtgat cagttaatgc ctaagagtga
900aagtagttct attgacattc ctcaagatat ttaatatcaa ctgcattatg
950tattatgtct gcttaaatca tttaaaaacg gcaaagaatt atatagacta
1000tgaggtacct tgctgtgtag gaggatgaaa ggggagttga tagtctcata
1050aaactaattt ggcttcaagt ttcatgaatc tgtaactaga atttaatttt
1100caccccaata atgttctata tagcctttgc taaagagcaa ctaataaatt
1150aaacctattc tttcaa
116662279DNAHomo Sapien 6cggacctgaa cccctaaaag cggaaccgcc tcccgccctc
gccatcgcgg 50agctgagtcg ccggcggcgg tggctgctgc cagacccgga
gtttcctctt 100tcactggatg gagctgaact ttgggcggcc agagcagcac
agctgtccgg 150ggatcgctgc atgctgagct ccctcggcaa gacccagcgg
cggctcggga 200tttttttggg ggggcgggga ccagccccgc gccggcacca
tgttcctggc 250gaccctgtac ttcgcgctgc cgctcttgga cttgctcctg
tcggccgaag 300tgagcggcgg agaccgcctg gattgcgtga aagccagtga
tcagtgcctg 350aaggagcaga gctgcagcac caagtaccgc acgctaaggc
agtgcgtggc 400gggcaaggag accaacttca gcctggcatc cggcctggag
gccaaggatg 450agtgccgcag cgccatggag gccctgaagc agaagtcgct
ctacaactgc 500cgctgcaagc ggggtatgaa gaaggagaag aactgcctgc
gcatttactg 550gagcatgtac cagagcctgc agggaaatga tctgctggag
gattccccat 600atgaaccagt taacagcaga ttgtcagata tattccgggt
ggtcccattc 650atatcagtgg agcacattcc caaagggaac aactgcctgg
atgcagcgaa 700ggcctgcaac ctcgacgaca tttgcaagaa gtacaggtcg
gcgtacatca 750ccccgtgcac caccagcgtg tccaatgatg tctgcaaccg
ccgcaagtgc 800cacaaggccc tccggcagtt ctttgacaag gtcccggcca
agcacagcta 850cggaatgctc ttctgctcct gccgggacat cgcctgcaca
gagcggaggc 900gacagaccat cgtgcctgtg tgctcctatg aagagaggga
gaagcccaac 950tgtttgaatt tgcaggactc ctgcaagacg aattacatct
gcagatctcg 1000ccttgcggat ttttttacca actgccagcc agagtcaagg
tctgtcagca 1050gctgtctaaa ggaaaactac gctgactgcc tcctcgccta
ctcggggctt 1100attggcacag tcatgacccc caactacata gactccagta
gcctcagtgt 1150ggccccatgg tgtgactgca gcaacagtgg gaacgaccta
gaagagtgct 1200tgaaattttt gaatttcttc aaggacaata catgtcttaa
aaatgcaatt 1250caagcctttg gcaatggctc cgatgtgacc gtgtggcagc
cagccttccc 1300agtacagacc accactgcca ctaccaccac tgccctccgg
gttaagaaca 1350agcccctggg gccagcaggg tctgagaatg aaattcccac
tcatgttttg 1400ccaccgtgtg caaatttaca ggcacagaag ctgaaatcca
atgtgtcggg 1450caatacacac ctctgtattt ccaatggtaa ttatgaaaaa
gaaggtctcg 1500gtgcttccag ccacataacc acaaaatcaa tggctgctcc
tccaagctgt 1550ggtctgagcc cactgctggt cctggtggta accgctctgt
ccaccctatt 1600atctttaaca gaaacatcat agctgcatta aaaaaataca
atatggacat 1650gtaaaaagac aaaaaccaag ttatctgttt cctgttctct
tgtatagctg 1700aaattccagt ttaggagctc agttgagaaa cagttccatt
caactggaac 1750attttttttt tttcctttta agaaagcttc ttgtgatcct
tcggggcttc 1800tgtgaaaaac ctgatgcagt gctccatcca aactcagaag
gctttgggat 1850atgctgtatt ttaaagggac agtttgtaac ttgggctgta
aagcaaactg 1900gggctgtgtt ttcgatgatg atgatgatca tgatgatgat
catcatgatc 1950atgatgatga tcatcatgat catgatgatg attttaacag
ttttacttct 2000ggcctttcct agctagagaa ggagttaata tttctaaggt
aactcccata 2050tctcctttaa tgacattgat ttctaatgat ataaatttca
gcctacattg 2100atgccaagct tttttgccac aaagaagatt cttaccaaga
gtgggctttg 2150tggaaacagc tggtactgat gttcaccttt atatatgtac
tagcattttc 2200cacgctgatg tttatgtact gtaaacagtt ctgcactctt
gtacaaaaga 2250aaaaacacct gtcacatcca aatataaaa
22797562DNAHomo Sapien 7atgcagcacc gaggcttcct
cctcctcacc ctcctcgccc tgctggcgct 50cacctccgcg gtcgccaaaa
agaaagataa ggtgaagaag ggcggcccgg 100ggagcgagtg cgctgagtgg
gcctgggggc cctgcacccc cagcagcaag 150gattgcggcg tgggtttccg
cgagggcacc tgcggggccc agacccagcg 200catccggtgc agggtgccct
gcaactggaa gaaggagttt ggagccgact 250gcaagtacaa gtttgagaac
tggggtgcgt gtgatggggg cacaggcacc 300aaagtccgcc aaggcaccct
gaagaaggcg cgctacaatg ctcagtgcca 350ggagaccatc cgcgtcacca
agccctgcac ccccaagacc aaagcaaagg 400ccaaagccaa gaaagggaag
ggaaaggact agacgccaag cctggatgcc 450aaggagcccc tggtgtcaca
tggggcctgg cccacgccct ccctctccca 500ggcccgagat gtgacccacc
agtgccttct gtctgctcgt tagctttaat 550caatcatgcc cc
56281524DNAHomo Sapien
8gcggcagcag cgcgggcccc agcagcctcg gcagccacag ccgctgcagc
50cggggcagcc tccgctgctg tcgcctcctc tgatgcgctt gccctctccc
100ggccccggga ctccgggaga atgtgggtcc taggcatcgc ggcaactttt
150tgcggattgt tcttgcttcc aggctttgcg ctgcaaatcc agtgctacca
200gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg
250tgaattgcac ggtgaacgtt caagacatgt gtcagaaaga agtgatggag
300caaagtgccg ggatcatgta ccgcaagtcc tgtgcatcat cagcggcctg
350tctcatcgcc tctgccgggt accagtcctt ctgctcccca gggaaactga
400actcagtttg catcagctgc tgcaacaccc ctctttgtaa cgggccaagg
450cccaagaaaa ggggaagttc tgcctcggcc ctcaggccag ggctccgcac
500caccatcctg ttcctcaaat tagccctctt ctcggcacac tgctgaagct
550gaaggagatg ccaccccctc ctgcattgtt cttccagccc tcgcccccaa
600ccccccacct ccctgagtga gtttcttctg ggtgtccttt tattctgggt
650agggagcggg agtccgtgtt ctcttttgtt cctgtgcaaa taatgaaaga
700gctcggtaaa gcattctgaa taaattcagc ctgactgaat tttcagtatg
750tacttgaagg aaggaggtgg agtgaaagtt cacccccatg tctgtgtaac
800cggagtcaag gccaggctgg cagagtcagt ccttagaagt cactgaggtg
850ggcatctgcc ttttgtaaag cctccagtgt ccattccatc cctgatgggg
900gcatagtttg agactgcaga gtgagagtga cgttttctta gggctggagg
950gccagttccc actcaaggct ccctcgcttg acattcaaac ttcatgctcc
1000tgaaaaccat tctctgcagc agaattggct ggtttcgcgc ctgagttggg
1050ctctagtgac tcgagactca atgactggga cttagactgg ggctcggcct
1100cgctctgaaa agtgcttaag aaaatcttct cagttctcct tgcagaggac
1150tggcgccggg acgcgaagag caacgggcgc tgcacaaagc gggcgctgtc
1200ggtggtggag tgcgcatgta cgcgcaggcg cttctcgtgg ttggcgtgct
1250gcagcgacag gcggcagcac agcacctgca cgaacacccg ccgaaactgc
1300tgcgaggaca ccgtgtacag gagcgggttg atgaccgagc tgaggtagaa
1350aaacgtctcc gagaagggga ggaggatcat gtacgcccgg aagtaggacc
1400tcgtccagtc gtgcttgggt ttggccgcag ccatgatcct ccgaatctgg
1450ttgggcatcc agcatacggc caatgtcaca acaatcagcc ctgggcagac
1500acgagcagga gggagagaca gaga
152491253DNAHomo Sapien 9caccctccgt ggcaaggcga ggccccgggg gcgggccggg
gtcaccacgc 50ctgccccagg gaaccgcaca gacggtactc acccttcttg
cgatgatgtg 100agatgataaa atgcctacat gatgagatga agtgagatga
aaaacatagg 150ccttgtgatg gaatgggaaa ttccagagat aatttgcacg
tgcgctaagc 200tgcggctacc cccgcaagca accttccaag tccttcgtgg
caatggtgct 250tccgtgggga ccgtgctcat gttccgctgc ccctccaacc
accagatggt 300ggggtctggg ctcctcacct gcacctggaa ggggagcatc
gctgagtggt 350cttcagggtc cccagtgtgc aaactggtgc caccacacga
gacctttggc 400ttcaaggtgg ccgtgatcgc ctccattgtg agctgtgcca
tcatcctgct 450catgtccatg gccttcctca cctgctgcct cctcaagtgc
gtgaagaaga 500gcaagcggcg gcgctccaac aggtcagccc agctgtggtc
ccagctgaaa 550gatgaggact tggagacggt gcaggccgca taccttggcc
tcaagcactt 600caacaaaccc gtgagcgggc ccagccaggc gcacgacaac
cacagcttca 650ccacagacca tggtgagagc accagcaagc tggccagtgt
gacccgcagc 700gtggacaagg accctgggat ccccagagct ctaagcctca
gtggctcctc 750cagctcaccc caagcccagg tgatggtgca catggcaaac
cccagacagc 800ccctgcctgc ctctgggctg gccacaggaa tgccacaaca
gcccgcagca 850tatgccctag ggtgaccacg cagtgaggct ggtgcccatg
ctccacactg 900ggaggccagg ctgaccccac cagccagtca gctacaactc
cacatcaact 950ccacatgcgc ccagctcgag actgatgagt ggaatcagct
tccaggtgta 1000gggacccctt gaggggccga gctgacatcc aaggctgagg
accccagtgg 1050ggagtgttct gttccggcat atcctggccg taacgatttt
tatagttatg 1100gactacttga aaccactact gagggtaatt tactagctgt
ggcctcccac 1150taactagcat tcctttaaag agactgggaa atgttttaag
caaatctagt 1200tttgtataat aaaataagaa aatagcaata aacttctttt
cagcaactac 1250aaa 1253105542DNAHomo Sapien 10ctgactgcac
tggtgatggt ccctggcaat ccaacctggc accatcgcag 50ttggagtact
atgcatcttc accagatgaa aaggctctag tagaagctgc 100tgcaaggatt
ggtattgtgt ttattggcaa ttctgaagaa actatggagg 150ttaaaactct
tggaaaactg gaacggtaca aactgcttca tattctggaa 200tttgattcag
atcgtaggag aatgagtgta attgttcagg caccttcagg 250tgagaagtta
ttatttgcta aaggagctga gtcatcaatt ctccctaaat 300gtataggtgg
agaaatagaa aaaaccagaa ttcatgtaga tgaatttgct 350ttgaaagggc
taagaactct gtgtatagca tatagaaaat ttacatcaaa 400agagtatgag
gaaatagata aacgcatatt tgaagccagg actgccttgc 450agcagcggga
agagaaattg gcagctgttt tccagttcat agagaaagac 500ctgatattac
ttggagccac agcagtagaa gacagactac aagataaagt 550tcgagaaact
attgaagcat tgagaatggc tggtatcaaa gtatgggtac 600ttactgggga
taaacatgaa acagctgtta gtgtgagttt atcatgtggc 650cattttcata
gaaccatgaa catccttgaa cttataaacc agaaatcaga 700cagcgagtgt
gctgaacaat tgaggcagct tgccagaaga attacagagg 750atcatgtgat
tcagcatggg ctggtagtgg atgggaccag cctatctctt 800gcactcaggg
agcatgaaaa actatttatg gaagtttgca gaaattgttc 850agctgtatta
tgctgtcgta tggctccact gcagaaagca aaagtaataa 900gactaataaa
aatatcacct gagaaaccta taacattggc tgttggtgat 950ggtgctaatg
acgtaagcat gatacaagaa gcccatgttg gcataggaat 1000catgggtaaa
gaaggaagac aggctgcaag aaacagtgac tatgcaatag 1050ccagatttaa
gttcctctcc aaattgcttt ttgttcatgg tcatttttat 1100tatattagaa
tagctaccct tgtacagtat tttttttata agaatgtgtg 1150ctttatcaca
ccccagtttt tatatcagtt ctactgtttg ttttctcagc 1200aaacattgta
tgacagcgtg tacctgactt tatacaatat ttgttttact 1250tccctaccta
ttctgatata tagtcttttg gaacagcatg tagaccctca 1300tgtgttacaa
aataagccca ccctttatcg agacattagt aaaaaccgcc 1350tcttaagtat
taaaacattt ctttattgga ccatcctggg cttcagtcat 1400gcctttattt
tcttttttgg atcctattta ctaataggga aagatacatc 1450tctgcttgga
aatggccaga tgtttggaaa ctggacattt ggcactttgg 1500tcttcacagt
catggttatt acagtcacag taaagatggc tctggaaact 1550catttttgga
cttggatcaa ccatctcgtt acctggggat ctattatatt 1600ttattttgta
ttttccttgt tttatggagg gattctctgg ccatttttgg 1650gctcccagaa
tatgtatttt gtgtttattc agctcctgtc aagtggttct 1700gcttggtttg
ccataatcct catggttgtt acatgtctat ttcttgatat 1750cataaagaag
gtctttgacc gacacctcca ccctacaagt actgaaaagg 1800cacagcttac
tgaaacaaat gcaggtatca agtgcttgga ctccatgtgc 1850tgtttcccgg
aaggagaagc agcgtgtgca tctgttggaa gaatgctgga 1900acgagttata
ggaagatgta gtccaaccca catcagcaga tcatggagtg 1950catcggatcc
tttctatacc aacgacagga gcatcttgac tctctccaca 2000atggactcat
ctacttgtta aaggggcagt agtactttgt gggagccagt 2050tcacctcctt
tcctaaaatt cagtgtgatc accctgttaa tggccacact 2100agctctgaaa
ttaatttcca aaatctttgt agtagttcat acccactcag 2150agttataatg
gcaaacaaac agaaagcatt agtacaagcc cctcccaaca 2200cccttaattt
gaatctgaac atgttaaaat ttgagaataa agagacattt 2250ttcatctctt
tgtctggttt gtcccttgtg cttatgggac tcctaatggc 2300atttcagtct
gttgctgagg ccattatatt ttaatataaa tgtagaaaaa 2350agagagaaat
cttagtaaag agtatttttt agtattagct tgattattga 2400ctcttctatt
taaatctgct tctgtaaatt atgctgaaag tttgccttga 2450gaactctatt
tttttattag agttatattt aaagcttttc atgggaaaag 2500ttaatgtgaa
tactgaggaa ttttggtccc tcagtgacct gtgttgttaa 2550ttcattaatg
cattctgagt tcacagagca aattaggaga atcatttcca 2600accattattt
actgcagtat ggggagtaaa tttataccaa ttcctctaac 2650tgtactgtaa
cacagcctgt aaagttagcc atataaatgc aagggtatat 2700catatataca
aatcaggaat caggtccgtt caccgaactt caaattgatg 2750tttactaata
tttttgtgac agagtataaa gaccctatag tgggtaaatt 2800agatactatt
agcatattat taatttaatg tctttatcat tggatctttt 2850gcatgcttta
atctggttaa catatttaaa tttgcttttt ttctctttac 2900ctgaaggctc
tgtgtatagt atttcatgac atcgttgtac agtttaacta 2950tcaataaaaa
gtttggacag tatttaaata ttgcaaatat gtttaattat 3000acaaatcaga
atagtatggg taattaaatg aatacaaaaa gaagagcctc 3050tttctgcagc
cgacttagac atgctcttcc ctttctataa gctagatttt 3100agaataaagg
gtttcagtta ataatcttat tttcaggtta tgtcatctaa 3150cttatagcaa
actaccacaa tacagtgagt tctgccagtg tcccagtaca 3200aggcatattt
caggtgtggc tgtggaatgt aaaaatgctc aacttgtatc 3250aggtaatgtt
agcaataaat taaatgctaa gaatgattaa tcgggtacat 3300gttactgtaa
ttaactcatt gcacttcaaa acctaacttc catcctgaat 3350ttatcaagta
gttcagtatt gtcatttgtt tttgttttat tgaaaagtaa 3400tgttgtctta
agatttagaa gtgattatta gcttgagaac tattacccag 3450ctctaagcaa
ataatgattg tatacatatt aagataatgg ttaaatgcgg 3500ttttaccaag
ttttcccttg aaaatgtaat tcctttatgg agatttattg 3550tgcagcccta
agcttccttc ccatttcatg aatataaggc ttctagaatt 3600ggactggcag
gggaaagaat ggtagagaca gaaattaaga ctttatcctt 3650gtttgcttgt
aaactattat tttcttgcta atgtaacatt tgtctgttcc 3700agtgatgtaa
ggatattaag ttattaagct aaatattaat tttcaaaaat 3750agtccttctt
taacttagat atttcatagc tggatttagg aagatctgtt 3800attctggaag
tactaaaaag aataatacaa cgtacaatgt ctgcattcac 3850taattcatgt
tccagaagag gaaataatga agatatactc agtagagtac 3900taggtgggag
gatatggaaa tttgctcata aaatctctta taaaacgtgc 3950atataacaaa
atgacaccca gtaggcctgc attacattta catgaccgtg 4000tttatttgcc
atcaaataaa ctgagtactg acaccagaca aagactccaa 4050agtcataaaa
tagcctatga ccaactgcag caagacagga ggtcagctcg 4100cctataatgg
tgcttaaagt gtgattgatg taattttctg tactcaccat 4150ttgaagttag
ttaaggagaa ctttattttt ttaaaaaaag taaatggcaa 4200ccactagtgt
gctcatcctg aactgttact ccaaatccac tccgttttta 4250aagcaaaatt
atcttgtgat tttaagaaaa gagttttcta tttatttaag 4300aaagtaacaa
tgcagtctgc aagctttcag tagttttcta gtgctatatt 4350catcctgtaa
aactcttact acgtaaccag taatcacaag gaaagtgtcc 4400cctttgcata
tttctttaaa attctttctt tggaaagtat gatgttgata 4450attaacttac
ccttatctgc caaaaccaga gcaaaatgct aaatacgtta 4500ttgctaatca
gtggtctcaa atcgatttgc ctccctttgc ctcgtctgag 4550ggctgtaagc
ctgaagatag tggcaagcac caagtcagtt tccaaaattg 4600cccctcagct
gctttaagtg actcagcacc ctgcctcagc ttcagcaggc 4650gtaggctcac
cctgggcgga gcaaagtatg ggccagggag aactacagct 4700acgaagacct
gctgtcgagt tgagaaaagg ggagaattta tggtctgaat 4750tttctaactg
tcctctttct tgggtctaaa gctcataata cacaaaggct 4800tccagacctg
agccacaccc aggccctatc ctgaacagga gactaaacag 4850aggcaaatca
accctaggaa atacttgcat tctgccctac ggttagtacc 4900aggactgagg
tcatttctac tggaaaagat tgtgagattg aacttatctg 4950atcgcttgag
actcctaata ggcaggagtc aaggccacta gaaaattgac 5000agttaagagc
caaaagtttt taaaatatgc tactctgaaa aatctcgtga 5050aggctgtagg
aaaagggaga atcttccatg ttggtgtttt tcctgtaaag 5100atcagtttgg
ggtatgatat aagcaggtat taataaaaat aacacaccaa 5150agagttacgt
aaaacatgtt ttattaattt tggtccccac gtacagacat 5200tttatttcta
ttttgaaatg agttatctat tttcataaaa gtaaaacact 5250attaaagtgc
tgttttatgt gaaataactt gaatgttgtt cctataaaaa 5300atagatcata
actcatgata tgtttgtaat catggtaatt tagattttta 5350tgaggaatga
gtatctggaa atattgtagc aatacttggt ttaaaatttt 5400ggacctgaga
cactgtggct gtctaatgta atcctttaaa aattctctgc 5450attgtcagta
aatgtagtat attattgtac agctactcat aattttttaa 5500agtttatgaa
gttatattta tcaaataaaa actttcctat at
5542116155DNAHomo Sapien 11atgtgggaag aagaagacat tgctattctg ttcaataaag
aaccaggaaa 50aacagagaat attgaaaata atctaagttc caaccataga
agaagctgca 100gaagaagtga agaaagtgat gatgatttgg attttgatat
tggtttagaa 150aacacaggag gagaccctca aattctgaga tttatttcag
acttccttgc 200ttttttggtt ctctacaatt tcatcattcc aatttcatta
tatgtgacag 250tcgaaatgca gaaatttctt ggatcatttt ttattggctg
ggatcttgat 300ctgtatcatg aagaatcaga tcagaaagct caagtcaata
cttccgatct 350gaatgaagag cttggacagg tagagtacgt gtttacagat
aaaactggta 400cactgacaga aaatgagatg cagtttcggg aatgttcaat
taatggcatg 450aaataccaag aaattaatgg tagacttgta cccgaaggac
caacaccaga 500ctcttcagaa ggaaacttat cttatcttag tagtttatcc
catcttaaca 550acttatccca tcttacaacc agttcctctt tcagaaccag
tcctgaaaat 600gaaactgaac taattaaaga acatgatctc ttctttaaag
cagtcagtct 650ctgtcacact gtacagatta gcaatgttca aactgactgc
actggtgatg 700gtccctggca atccaacctg gcaccatcgc agttggagta
ctatgcatct 750tcaccagatg aaaaggctct agtagaagct gctgcaaggt
acaaactgct 800tcatattctg gaatttgatt cagatcgtag gagaatgagt
gtaattgttc 850aggcaccttc aggtgagaag ttattatttg ctaaaggagc
tgagtcatca 900attctcccta aatgtatagg tggagaaata gaaaaaacca
gaattcatgt 950agatgaattt gctttgaaag ggctaagaac tctgtgtata
gcatatagaa 1000aatttacatc aaaagagtat gaggaaatag ataaacgcat
atttgaagcc 1050aggactgcct tgcagcagcg ggaagagaaa ttggcagctg
ttttccagtt 1100catagagaaa gacctgatat tacttggagc cacagcagta
gaagacagac 1150tacaagataa agttcgagaa actattgaag cattgagaat
ggctggtatc 1200aaagtatggg tacttactgg ggataaacat gaaacagctg
ttagtgtgag 1250tttatcatgt ggccattttc atagaaccat gaacatcctt
gaacttataa 1300accagaaatc agacagcgag tgtgctgaac aattgaggca
gcttgccaga 1350agaattacag aggatcatgt gattcagcat gggctggtag
tggatgggac 1400cagcctatct cttgcactca gggagcatga aaaactattt
atggaagttt 1450gcagaaattg ttcagctgta ttatgctgtc gtatggctcc
actgcagaaa 1500gcaaaagtaa taagactaat aaaaatatca cctgagaaac
ctataacatt 1550ggctgttggt gatggtgcta atgacgtaag catgatacaa
gaagcccatg 1600ttggcatagg aatcatgggt aaagaaggaa gacaggctgc
aagaaacagt 1650gactatgcaa tagccagatt taagttcctc tccaaattgc
tttttgttca 1700tggtcatttt tattatatta gaatagctac ccttgtacag
tatttttttt 1750ataagaatgt gtgctttatc acaccccagt ttttatatca
gttctactgt 1800ttgttttctc agcaaacatt gtatgacagc gtgtacctga
ctttatacaa 1850tatttgtttt acttccctac ctattctgat atatagtctt
ttggaacagc 1900atgtagaccc tcatgtgtta caaaataagc ccacccttta
tcgagacatt 1950agtaaaaacc gcctcttaag tattaaaaca tttctttatt
ggaccatcct 2000gggcttcagt catgccttta ttttcttttt tggatcctat
ttactaatag 2050ggaaagatac atctctgctt ggaaatggcc agatgtttgg
aaactggaca 2100tttggcactt tggtcttcac agtcatggtt attacagtca
cagtaaagat 2150ggctctggaa actcattttt ggacttggat caaccatctc
gttacctggg 2200gatctattat attttatttt gtattttcct tgttttatgg
agggattctc 2250tggccatttt tgggctccca gaatatgtat tttgtgttta
ttcagctcct 2300gtcaagtggt tctgcttggt ttgccataat cctcatggtt
gttacatgtc 2350tatttcttga tatcataaag aaggtctttg accgacacct
ccaccctaca 2400agtactgaaa aggcacagct tactgaaaca aatgcaggta
tcaagtgctt 2450ggactccatg tgctgtttcc cggaaggaga agcagcgtgt
gcatctgttg 2500gaagaatgct ggaacgagtt ataggaagat gtagtccaac
ccacatcagc 2550agatcatgga gtgcatcgga tcctttctat accaacgaca
ggagcatctt 2600gactctctcc acaatggact catctacttg ttaaaggggc
agtagtactt 2650tgtgggagcc agttcacctc ctttcctaaa attcagtgtg
atcaccctgt 2700taatggccac actagctctg aaattaattt ccaaaatctt
tgtagtagtt 2750catacccact cagagttata atggcaaaca aacagaaagc
attagtacaa 2800gcccctccca acacccttaa tttgaatctg aacatgttaa
aatttgagaa 2850taaagagaca tttttcatct ctttgtctgg tttgtccctt
gtgcttatgg 2900gactcctaat ggcatttcag tctgttgctg aggccattat
attttaatat 2950aaatgtagaa aaaagagaga aatcttagta aagagtattt
tttagtatta 3000gcttgattat tgactcttct atttaaatct gcttctgtaa
attatgctga 3050aagtttgcct tgagaactct atttttttat tagagttata
tttaaagctt 3100ttcatgggaa aagttaatgt gaatactgag gaattttggt
ccctcagtga 3150cctgtgttgt taattcatta atgcattctg agttcacaga
gcaaattagg 3200agaatcattt ccaaccatta tttactgcag tatggggagt
aaatttatac 3250caattcctct aactgtactg taacacagcc tgtaaagtta
gccatataaa 3300tgcaagggta tatcatatat acaaatcagg aatcaggtcc
gttcaccgaa 3350cttcaaattg atgtttacta atatttttgt gacagagtat
aaagacccta 3400tagtgggtaa attagatact attagcatat tattaattta
atgtctttat 3450cattggatct tttgcatgct ttaatctggt taacatattt
aaatttgctt 3500tttttctctt tacctgaagg ctctgtgtat agtatttcat
gacatcgttg 3550tacagtttaa ctatcaataa aaagtttgga cagtatttaa
atattgcaaa 3600tatgtttaat tatacaaatc agaatagtat gggtaattaa
atgaatacaa 3650aaagaagagc ctctttctgc agccgactta gacatgctct
tccctttcta 3700taagctagat tttagaataa agggtttcag ttaataatct
tattttcagg 3750ttatgtcatc taacttatag caaactacca caatacagtg
agttctgcca 3800gtgtcccagt acaaggcata tttcaggtgt ggctgtggaa
tgtaaaaatg 3850ctcaacttgt atcaggtaat gttagcaata aattaaatgc
taagaatgat 3900taatcgggta catgttactg taattaactc attgcacttc
aaaacctaac 3950ttccatcctg aatttatcaa gtagttcagt attgtcattt
gtttttgttt 4000tattgaaaag taatgttgtc ttaagattta gaagtgatta
ttagcttgag 4050aactattacc cagctctaag caaataatga ttgtatacat
attaagataa 4100tggttaaatg cggttttacc aagttttccc ttgaaaatgt
aattccttta 4150tggagattta ttgtgcagcc ctaagcttcc ttcccatttc
atgaatataa 4200ggcttctaga attggactgg caggggaaag aatggtagag
acagaaatta 4250agactttatc cttgtttgct tgtaaactat tattttcttg
ctaatgtaac 4300atttgtctgt tccagtgatg taaggatatt aagttattaa
gctaaatatt 4350aattttcaaa aatagtcctt ctttaactta gatatttcat
agctggattt 4400aggaagatct gttattctgg aagtactaaa aagaataata
caacgtacaa 4450tgtctgcatt cactaattca tgttccagaa gaggaaataa
tgaagatata 4500ctcagtagag tactaggtgg gaggatatgg aaatttgctc
ataaaatctc 4550ttataaaacg tgcatataac aaaatgacac ccagtaggcc
tgcattacat 4600ttacatgacc gtgtttattt gccatcaaat aaactgagta
ctgacaccag 4650acaaagactc caaagtcata aaatagccta tgaccaactg
cagcaagaca 4700ggaggtcagc tcgcctataa tggtgcttaa agtgtgattg
atgtaatttt 4750ctgtactcac catttgaagt tagttaagga gaactttatt
tttttaaaaa 4800aagtaaatgg caaccactag tgtgctcatc ctgaactgtt
actccaaatc 4850cactccgttt ttaaagcaaa attatcttgt gattttaaga
aaagagtttt 4900ctatttattt aagaaagtaa caatgcagtc tgcaagcttt
cagtagtttt 4950ctagtgctat attcatcctg taaaactctt actacgtaac
cagtaatcac 5000aaggaaagtg tcccctttgc atatttcttt aaaattcttt
ctttggaaag 5050tatgatgttg ataattaact tacccttatc tgccaaaacc
agagcaaaat 5100gctaaatacg ttattgctaa tcagtggtct caaatcgatt
tgcctccctt 5150tgcctcgtct gagggctgta agcctgaaga tagtggcaag
caccaagtca 5200gtttccaaaa ttgcccctca gctgctttaa gtgactcagc
accctgcctc 5250agcttcagca ggcgtaggct caccctgggc ggagcaaagt
atgggccagg 5300gagaactaca gctacgaaga cctgctgtcg agttgagaaa
aggggagaat 5350ttatggtctg aattttctaa ctgtcctctt tcttgggtct
aaagctcata 5400atacacaaag gcttccagac ctgagccaca cccaggccct
atcctgaaca 5450ggagactaaa cagaggcaaa tcaaccctag gaaatacttg
cattctgccc 5500tacggttagt accaggactg aggtcatttc tactggaaaa
gattgtgaga 5550ttgaacttat ctgatcgctt gagactccta ataggcagga
gtcaaggcca 5600ctagaaaatt gacagttaag agccaaaagt ttttaaaata
tgctactctg 5650aaaaatctcg tgaaggctgt aggaaaaggg agaatcttcc
atgttggtgt 5700ttttcctgta aagatcagtt tggggtatga tataagcagg
tattaataaa 5750aataacacac caaagagtta cgtaaaacat gttttattaa
ttttggtccc 5800cacgtacaga cattttattt ctattttgaa atgagttatc
tattttcata 5850aaagtaaaac actattaaag tgctgtttta tgtgaaataa
cttgaatgtt 5900gttcctataa aaaatagatc ataactcatg atatgtttgt
aatcatggta 5950atttagattt ttatgaggaa tgagtatctg gaaatattgt
agcaatactt 6000ggtttaaaat tttggacctg agacactgtg gctgtctaat
gtaatccttt 6050aaaaattctc tgcattgtca gtaaatgtag tatattattg
tacagctact 6100cataattttt taaagtttat gaagttatat ttatcaaata
aaaactttcc 6150tatat
6155121372DNAHomo Sapien 12gcacgagggc gcttttgtct
ccggtgagtt ttgtggcggg aagcttctgc 50gctggtgctt agtaaccgac
tttcctccgg actcctgcac gacctgctcc 100tacagccggc gatccactcc
cggctgttcc cccggagggt ccagaggcct 150ttcagaagga gaaggcagct
ctgtttctct gcagaggagt agggtccttt 200cagccatgaa gcatgtgttg
aacctctacc tgttaggtgt ggtactgacc 250ctactctcca tcttcgttag
agtgatggag tccctagaag gcttactaga 300gagcccatcg cctgggacct
cctggaccac cagaagccaa ctagccaaca 350cagagcccac caagggcctt
ccagaccatc catccagaag catgtgataa 400gacctccttc catactggcc
atattttgga acactgacct agacatgtcc 450agatgggagt cccattccta
gcagacaagc tgagcaccgt tgtaaccaga 500gaactattac taggccttga
agaacctgtc taactggatg ctcattgcct 550gggcaaggcc tgtttaggcc
ggttgcggtg gctcatgcct gtaatcctag 600cactttggga ggctgaggtg
ggtggatcac ctgaggtcag gagttcgaga 650ccagcctcgc caacatggcg
aaaccccatc tctactaaaa atacaaaagt 700tagctgggtg tggtggcaga
ggcctgtaat cccagttcct tgggaggctg 750aggcgggaga attgcttgaa
cccggggacg gaggttgcag tgaaccgaga 800tcgcactgct gtacccagcc
tgggccacag tgcaagactc catctcaaaa 850aaaaaaagaa aagaaaaagc
ctgtttaatg cacaggtgtg agtggattgc 900ttatggctat gagataggtt
gatctcgccc ttaccccggg gtctggtgta 950tgctgtgctt tcctcagcag
tatggctctg acatctctta gatgtcccaa 1000cttcagctgt tgggagatgg
tgatattttc aaccctactt cctaaacatc 1050tgtctggggt tcctttagtc
ttgaatgtct tatgctcaat tatttggtgt 1100tgagcctctc ttccacaaga
gctcctccat gtttggatag cagttgaaga 1150ggttgtgtgg gtgggctgtt
gggagtgagg atggagtgtt cagtgcccat 1200ttctcatttt acattttaaa
gtcgttcctc caacatagtg tgtattggtc 1250tgaagggggt ggtgggatgc
caaagcctgc tcaagttatg gacattgtgg 1300ccaccatgtg gcttaaatga
ttttttctaa ctaataaagt ggaatatata 1350tttcaaaaaa aaaaaaaaaa
aa 137213770DNAHomo
Sapienunsure45, 611, 715unknown base 13atacgactca ctatagggcg aattgggtac
cgggcccccc ctcgngtcga 50cggtatcgat aagcttgata tcgaattcgg
ccacactggc cggatcctct 100agagatccct cgacctcgac ccacgcgtcc
gcccacgcgt ccgatgtgcc 150tctgggcaaa gaagcagagc taacgaggaa
agggatttaa agagtttttc 200ttgggtgttt gtcaaacttt tattccctgt
ctgtgtgcag aggggattca 250acttcaattt ttctgcagtg gctctgagtc
cagcccctta cttaaagatc 300tggaaagcat gaagactggg ctttttttcc
tatgtctctt gggaactgca 350gctgcaatcc cgacaaatgc aagattatta
tctgatcatt ccaaaccaac 400tgctgaaacg gtagcacccg acaacactgc
aatccccagt ttaagggctg 450aagatgaaga aaatgaaaaa gaaacagcag
tatccacaga agacgattcc 500caccataagg ctgaaaaatc atcagtacta
aagtcaaaag aggaaagcca 550tgaacagtca gcagaacagg gcaagagttc
tagccaagag ctgggattga 600aggatcaaga ngacagtgat ggtgacttaa
gtgtgaattt ggagtatgca 650ccaactgaag gtacattgga cataaaagaa
gatatgagtg agcctcagga 700gaaaaactct caganacact gattttttgg
ctcctggggt agttccttcc 750agattctacc acagaagttt
770141187DNAHomo Sapien 14cgcgggccat
ggctccctgg gcggaggccg agcactcggc gctgaacccg 50ctgcgcgcgg
tgtggctcac gctgaccgcc gccttcctgc tgaccctact 100gctgcagctc
ctgccgcccg gcctgctccc gggctgcgcg atcttccagg 150acctgatccg
ctatgggaaa accaagtgtg gggagccgtc gcgccccgcc 200gcctgccgag
cctttgatgt ccccaagaga tatttttccc acttttatat 250catctcagtg
ctgtggaatg gcttcctgct ttggtgcctt actcaatctc 300tgttcctggg
agcacctttt ccaagctggc ttcatggttt gctcagaatt 350ctcggggcgg
cacagttcca gggaggggag ctggcactgt ctgcattctt 400agtgctagta
tttctgtggc tgcacagctt acgaagactc ttcgagtgcc 450tctacgtcag
tgtcttctcc aatgtcatga ttcacgtcgt gcagtactgt 500tttggacttg
tctattatgt ccttgttggc ctaactgtgc tgagccaagt 550gccaatggat
ggcaggaatg cctacataac agggaaaaat ctattgatgc 600aagcacggtg
gttccatatt cttgggatga tgatgttcat ctggtcatct 650gcccatcagt
ataagtgcca tgttattctc ggcaatctca ggaaaaataa 700agcaggagtg
gtcattcact gtaaccacag gatcccattt ggagactggt 750ttgaatatgt
ttcttcccct aactacttag cagagctgat gatctacgtt 800tccatggccg
tcacctttgg gttccacaac ttaacttggt ggctagtggt 850gacaaatgtc
ttctttaatc aggccctgtc tgcctttctc agccaccaat 900tctacaaaag
caaatttgtc tcttacccga agcataggaa agctttccta 950ccatttttgt
tttaagttaa cctcagtcat gaagaatgca aaccaggtga 1000tggtttcaat
gcctaaggac agtgaagtct ggagcccaaa gtacagtttc 1050agcaaagctg
tttgaaactc tccattccat ttctataccc cacaagtttt 1100cactgaatga
gcatggcagt gccactcaat aaaatgaatc tccaaagtat 1150cttcaaagaa
taaatactaa tggcaaaaaa aaaaaaa
1187151840DNAHomo Sapien 15tccacacaca caaaaaacct gcgcgtgagg ggggaggaaa
agcagggcct 50ttaaaaaggc aatcacaaca acttttgctg ccaggatgcc
cttgctttgg 100ctgagaggat ttctgttggc aagttgctgg attatagtga
ggagttcccc 150caccccagga tccgaggggc acagcgcggc ccccgactgt
ccgtcctgtg 200cgctggccgc cctcccaaag gatgtaccca actctcagcc
agagatggtg 250gaggccgtca agaagcacat tttaaacatg ctgcacttga
agaagagacc 300cgatgtcacc cagccggtac ccaaggcggc gcttctgaac
gcgatcagaa 350agcttcatgt gggcaaagtc ggggagaacg ggtatgtgga
gatagaggat 400gacattggaa ggagggcaga aatgaatgaa cttatggagc
agacctcgga 450gatcatcacg tttgccgagt caggaacagc caggaagacg
ctgcacttcg 500agatttccaa ggaaggcagt gacctgtcag tggtggagcg
tgcagaagtc 550tggctcttcc taaaagtccc caaggccaac aggaccagga
ccaaagtcac 600catccgcctc ttccagcagc agaagcaccc gcagggcagc
ttggacacag 650gggaagaggc cgaggaagtg ggcttaaagg gggagaggag
tgaactgttg 700ctctctgaaa aagtagtaga cgctcggaag agcacctggc
atgtcttccc 750tgtctccagc agcatccagc ggttgctgga ccagggcaag
agctccctgg 800acgttcggat tgcctgtgag cagtgccagg agagtggcgc
cagcttggtt 850ctcctgggca agaagaagaa gaaagaagag gagggggaag
ggaaaaagaa 900gggcggaggt gaaggtgggg caggagcaga tgaggaaaag
gagcagtcgc 950acagaccttt cctcatgctg caggcccggc agtctgaaga
ccaccctcat 1000cgccggcgtc ggcggggctt ggagtgtgat ggcaaggtca
acatctgctg 1050taagaaacag ttctttgtca gtttcaagga catcggctgg
aatgactgga 1100tcattgctcc ctctggctat catgccaact actgcgaggg
tgagtgcccg 1150agccatatag caggcacgtc cgggtcctca ctgtccttcc
actcaacagt 1200catcaaccac taccgcatgc ggggccatag cccctttgcc
aacctcaaat 1250cgtgctgtgt gcccaccaag ctgagaccca tgtccatgtt
gtactatgat 1300gatggtcaaa acatcatcaa aaaggacatt cagaacatga
tcgtggagga 1350gtgtgggtgc tcatagagtt gcccagccca gggggaaagg
gagcaagagt 1400tgtccagaga agacagtggc aaaatgaaga aatttttaag
gtttctgagt 1450taaccagaaa aatagaaatt aaaaacaaaa caaaacaaaa
aaaaaaacaa 1500aaaaaaacaa aagtaaatta aaaacaaacc tgatgaaaca
gatgaaacag 1550atgaaggaag atgtggaaat cttagcctgc cttagccagg
gctcagagat 1600gaagcagtga agagacagat tgggagggaa agggagaatg
gtgtaccctt 1650tatttcttct gaaatcacac tgatgacatc agttgtttaa
acggggtatt 1700gtcctttccc cccttgaggt tcccttgtga gcttgaatca
accaatctga 1750tctgcagtag tgtggactag aacaacccaa atagcatcta
gaaagccatg 1800agtttgaaag ggcccatcac aggcactttc ctagcctaat
1840161771DNAHomo Sapien 16gcggagaagc cgggagcgcg
gggctcagtc ggggggcggc ggcggcggcg 50gctccgggga tggcggcggc
tccgctgctg ctgctgctgc tgctcgtgcc 100cgtgccgctg ctgccgctgc
tggcccaagg gcccggaggg gcgctgggaa 150accggcatgc ggtgtactgg
aacagctcca accagcacct gcggcgagag 200ggctacaccg tgcaggtgaa
cgtgaacgac tatctggata tttactgccc 250gcactacaac agctcggggg
tgggccccgg ggcgggaccg gggcccggag 300gcggggcaga gcagtacgtg
ctgtacatgg tgagccgcaa cggctaccgc 350acctgcaacg ccagccaggg
cttcaagcgc tgggagtgca accggccgca 400cgccccgcac agccccatca
agttctcgga gaagttccag cgctacagcg 450ccttctctct gggctacgag
ttccacgccg gccacgagta ctactacatc 500tccacgccca ctcacaacct
gcactggaag tgtctgagga tgaaggtgtt 550cgtctgctgc gcctccacat
cgcactccgg ggagaagccg gtccccactc 600tcccccagtt caccatgggc
cccaatgtga agatcaacgt gctggaagac 650tttgagggag agaaccctca
ggtgcccaag cttgagaaga gcatcagcgg 700gaccagcccc aaacgggaac
acctgcccct ggccgtgggc atcgccttct 750tcctcatgac gttcttggcc
tcctagctct gccccctccc ctgggggggg 800agagatgggg cggggcttgg
aaggagcagg gagcctttgg cctctccaag 850ggaagcctag tgggcctaga
cccctcctcc catggctaga agtggggcct 900gcaccataca tctgtgtccg
ccccctctac cccttccccc cacgtagggc 950actgtagtgg accaagcacg
gggacagcca tgggtcccgg gcggccttgt 1000ggctctggta atgtttggta
ccaaacttgg gggccaaaaa gggcagtgct 1050caggactccc tggcccctgg
tacctttccc tgactcctgg tgccctctcc 1100ctttgtcccc ccagagagac
atatgccccc agagagagca aatcgaagcg 1150tgggaggcac ccccattgct
ctcctccagg ggcagaacat ggggagggga 1200ctagatgggc aaggggcagc
actgcctgct gcttccttcc cctgtttaca 1250gcaataaagc acgtcctcct
cccccactcc cacttccagg attgtggttt 1300ggattgaaac caagtttaca
agtagacacc cctggggggg cgggcagtgg 1350acaaggatgg caaggggtgg
gcattggggt gccaggcagg catgtacaga 1400ctctatatct ctatatataa
tgtacagaca gacagagtcc cttccctctt 1450taaccccctg acctttcttg
acttcccctt cagcttcaga ccccttcccc 1500accaggctta ggccccccca
caccttgggg ggacccccct ggcccctctt 1550ttgtcttctg tgaagacagg
acctatgcaa cgcacagaca cttttggaga 1600ccgtaaaaca acagcgcccc
ctcccttcca gccctgagcc gggaaccatc 1650tcccaggacc ttgccctgct
caccctatgt ggtcccacct atcctcctgg 1700gcctttttca agtgctttgg
ctgtgacttt catactctgc tcttagtcta 1750aaaaaaataa actggagata a
1771174126DNAHomo sapien
17cgctcgccat gggccactcc ccacctgtcc tgcctttgtg tgcctctgtg
50tctttgctgg gtggcctgac ctttggttat gaactggcag tcatatcagg
100tgccctgctg ccactgcagc ttgactttgg gctaagctgc ttggagcagg
150agttcctggt gggcagcctg ctcctggggg ctctcctcgc ctccctggtt
200ggtggcttcc tcattgactg ctatggcagg aagcaagcca tcctcgggag
250caacttggtg ctgctggcag gcagcctgac cctgggcctg gctggttccc
300tggcctggct ggtcctgggc cgcgctgtgg ttggcttcgc catttccctc
350tcctccatgg cttgctgtat ctacgtgtca gagctggtgg ggccacggca
400gcggggagtg ctggtgtccc tctatgaggc aggcatcacc gtgggcatcc
450tgctctccta tgccctcaac tatgcactgg ctggtacccc ctggggatgg
500aggcacatgt tcggctgggc cactgcacct gctgtcctgc aatccctcag
550cctcctcttc ctccctgctg gtacagatga gactgcaaca cacaaggacc
600tcatcccact ccagggaggt gaggccccca agctgggccc ggggaggcca
650cggtactcct ttctggacct cttcagggca cgcgataaca tgcgaggccg
700gaccacagtg ggcctggggc tggtgctctt ccagcaacta acagggcagc
750ccaacgtgct gtgctatgcc tccaccatct tcagctccgt tggtttccat
800gggggatcct cagccgtgct ggcctctgtg gggcttggcg cagtgaaggt
850ggcagctacc ctgaccgcca tggggctggt ggaccgtgca ggccgcaggg
900ctctgttgct agctggctgt gccctcatgg ccctgtccgt cagtggcata
950ggcctcgtca gctttgccgt gcccatggac tcaggcccaa gctgtctggc
1000tgtgcccaat gccaccgggc agacaggcct ccctggagac tctggcctgc
1050tgcaggactc ctctctacct cccattccaa ggaccaatga ggaccaaagg
1100gagccaatct tgtccactgc taagaaaacc aagccccatc ccagatctgg
1150agacccctca gcccctcctc ggctggccct gagctctgcc ctccctgggc
1200cccctctgcc cgctcggggg catgcactgc tgcgctggac cgcactgctg
1250tgcctgatgg tctttgtcag tgccttctcc tttgggtttg ggccagtgac
1300ctggcttgtc ctcagcgaga tctaccctgt ggagatacga ggaagagcct
1350tcgccttctg caacagcttc aactgggcgg ccaacctctt catcagcctc
1400tccttcctcg atctcattgg caccatcggc ttgtcctgga ccttcctgct
1450ctacggactg accgctgtcc tcggcctggg cttcatctat ttatttgttc
1500ctgaaacaaa aggccagtcg ttggcagaga tagaccagca gttccagaag
1550agacggttca ccctgagctt tggccacagg cagaactcca ctggcatccc
1600gtacagccgc atcgagatct ctgcggcctc ctgaggaatc cgtctgcctg
1650gaaattctgg aactgtggct ttggcagacc atctccagca tcctgcttcc
1700taggccccag agcacaagtt ccagctggtc ttttgggagt ggcccctgcc
1750cccaaaggtg gtctgctttt gctggggtaa aaaggatgaa agtctgagaa
1800tgcccaactc ttcattttga gtctcaggcc ctgaaggttc ctgaggatct
1850agcttcatgc ctcagtttcc ccattgactt gcacatctct gcagtattta
1900taagaagaat attctatgaa gtctttgttg caccatggac ttttctcaaa
1950gaatctcaag ggtaccaatc ctggcaggaa gtctctcccg atatcacccc
2000taaatccaaa tgaggatatc atcttttcta atctcttttt tcaactggct
2050gggacatttt cggaaggggg aagtctcttt ttttactctt atcatttttt
2100ttttttgagg tggagtctca ttctgttgcc caggctggcc tgatcttggc
2150tcactgcaac ctccacctcc tgagttcaag cgattcttgt gcctcagcct
2200cctaagcagc tgggactaca ggcgcatgca accataccca gctaatttat
2250ttttagcaga gatggggttt cactgtgttg gccaggctgg tcgtgaactc
2300ctgagctcaa gtgatccacc cacctcagcc tcccagagtg ctaggattac
2350aggccttttg actcttttat ctgagtttta ttgacccctc taattctctt
2400acccagaata tttatccttc accagcaact ctgactcttt gacgggaggc
2450ctcagttcta gtccttggtc tgctggtgtc attgctgtag gaatgaccac
2500gggcctcagt ttccccattt gtataatggg aagcctgtac caggtcattc
2550ttaagatttc tcctgactcc agtgagctgg aattctaaat gctggtctag
2600gagctgtctc caggatggtg caggatggct ttgcggaaag gagatgggtt
2650tggaggccaa caaacctgct tgtcaatatt gcctttgcct cttggcagcc
2700cttgaacttg agtaaataac aactccctga acctcagttt cctcatctgc
2750agaatgggga taattatgtc ccaggggtat atttagaccc tgtttccttt
2800caggagggtc cccagctggt ccagggcctg ggaaatttct acttatcctc
2850attacccagg tccctccttt ggaccctgta aagggtcagg gtgaatcaga
2900tgggggactg agcaagtagc tatgactgca gatcatgtaa ggaagggact
2950gacaagaagc tcccagatgc tggggagaat gaagagctaa aatagatcct
3000aggtgctgga tgctttgtca tccatgcgtg cacatatggg tgctggcaga
3050gcccccaagg actctggcct ctcgagttct cctatcttct ccattctaga
3100tgcttccctt gtatccagtg atgtgctgga gctggctttg ccaagcttgt
3150gagagctggt tgctacattt tcaggatttt tacaagttgg taaacacagc
3200cattataaaa aattaaatga tttaaattta taattaagta aattacatta
3250aaacaaaaaa attatactca aaattcatta cttaatttta ctacctgtta
3300ctattatctg tgcttttgag gctatttcta catagtaact cttatggaga
3350cctaggggag acaccgcgca tctcttcctg attccccact caatgacatc
3400atgttagtct ttggttgctt aactggctgt ggggagtgtt tttgtatcac
3450aaagattaga gaggactaca catcagggct tgatttattg tttgttgatt
3500ttctagactt cagaacatgc tggataaaat gtcagtaatg caaattaaac
3550tttaaagtat gtcttgtttg tagccaatac atggtgtata gcaccaaaaa
3600atggagggat tattcttcca gtagttgaac actgtcatcc gtttcagctg
3650acagctgctc aaatcattta agaaggagtt ctgacattca ttttcattgt
3700tttacttttg tcttcctcac tagtgtaaac aaaaatttca accagcattc
3750atgccgaacc tatacccatt cttcagtgcc tagctgtaca gttatcaggg
3800atttttattt gtagtctaat tttgtcaaat catggccaaa tcgcagtgat
3850agttgacttt ggatacaagg tttggcaaaa aaaaaaatat taacaaaata
3900ttctgtaaga atcaattgtc tatatggaat ttaggataaa gaatatttac
3950aataaagaat atttacaata aagagtttat tattatttgt aagttgtgtg
4000caacaaacat accctttatc tctgtaaaat ttatacacac aaaaattaac
4050aaaagattct gtaagaatta attggctata tggaatttag gatagaatat
4100ttacaataaa gagtatttac aataaa
4126185615DNAHomo Sapienunsure429unknown base 18gcttcagtcc cgcgaccgaa
gcagggcgcg cagcagcgct gagtgccccg 50gaacgtgcgt cgcgccccca
gtgtccgtcg cgtccgccgc gccccgggcg 100gggatggggc ggccagactg
agcgccgcac ccgccatcca gacccgccgg 150ccctagccgc agtccctcca
gccgtggccc cagcgcgcac gggcgatggc 200gaaggcgacg tccggtgccg
cggggctgcg tctgctgttg ctgctgctgc 250tgccgctgct aggcaaagtg
gcattgggcc tctacttctc gagggatgct 300tactgggaga agctgtatgt
ggaccaggcg gccggcacgc ccttgctgta 350cgtccatgcc ctgcgggacg
cccctgagga ggtgcccagc ttccgcctgg 400gccagcatct ctacggcacg
taccgcacnc ggctgcatga gaacaactgg 450atctgcatcc aggaggacac
cggcctcctc taccttaacc ggagcctgga 500ccatagctcc tgggagaagc
tcagtgtccg caaccgcggc tttcccctgc 550tcaccgtcta cctcaaggtc
ttcctgtcac ccacatccct tcgtgagggc 600gagtgccagt ggccaggctg
tgcccgcgta tacttctcct tcttcaacac 650ctcctttcca gcctgcagct
ccctcaagcc ccgggagctc tgcttcccag 700agacaaggcc ctccttccgc
attcgggaga accgaccccc aggcaccttc 750caccagttcc gcctgctgcc
tgtgcagttc ttgtgcccca acatcagcgt 800ggcctacagg ctcctggagg
gtgagggtct gcccttccgc tgcgccccgg 850acagcctgga ggtgagcacg
cgctgggccc tggaccgcga gcagcgggag 900aagtacgagc tggtggccgt
gtgcaccgtg cacgccggcg cgcgcgagga 950ggtggtgatg gtgcccttcc
cggtgaccgt gtacgacgag gacgactcgg 1000cgcccacctt ccccgcgggc
gtcgacaccg ccagcgccgt ggtggagttc 1050aagcggaagg aggacaccgt
ggtggccacg ctgcgtgtct tcgatgcaga 1100cgtggtacct gcatcagggg
agctggtgag gcggtacaca agcacgctgc 1150tccccgggga cacctgggcc
cagcagacct tccgggtgga acactggccc 1200aacgagacct cggtccaggc
caacggcagc ttcgtgcggg cgaccgtaca 1250tgactatagg ctggttctca
accggaacct ctccatctcg gagaaccgca 1300ccatgcagct ggcggtgctg
gtcaatgact cagacttcca gggcccagga 1350gcgggcgtcc tcttgctcca
cttcaacgtg tcggtgctgc cggtcagcct 1400gcacctgccc agtacctact
ccctctccgt gagcaggagg gctcgccgat 1450ttgcccagat cgggaaagtc
tgtgtggaaa actgccaggc gttcagtggc 1500atcaacgtcc agtacaagct
gcattcctct ggtgccaact gcagcacgct 1550aggggtggtc acctcagccg
aggacacctc ggggatcctg tttgtgaatg 1600acaccaaggc cctgcggcgg
cccaagtgtg ccgaacttca ctacatggtg 1650gtggccaccg accagcagac
ctctaggcag gcccaggccc agctgcttgt 1700aacagtggag gggtcatatg
tggccgagga ggcgggctgc cccctgtcct 1750gtgcagtcag caagagacgg
ctggagtgtg aggagtgtgg cggcctgggc 1800tccccaacag gcaggtgtga
gtggaggcaa ggagatggca aagggatcac 1850caggaacttc tccacctgct
ctcccagcac caagacctgc cccgacggcc 1900actgcgatgt tgtggagacc
caagacatca acatttgccc tcaggactgc 1950ctccggggca gcattgttgg
gggacacgag cctggggagc cccgggggat 2000taaagctggc tatggcacct
gcaactgctt ccctgaggag gagaagtgct 2050tctgcgagcc cgaagacatc
caggatccac tgtgcgacga gctgtgccgc 2100acggtgatcg cagccgctgt
cctcttctcc ttcatcgtct cggtgctgct 2150gtctgccttc tgcatccact
gctaccacaa gtttgcccac aagccaccca 2200tctcctcagc tgagatgacc
ttccggaggc ccgcccaggc cttcccggtc 2250agctactcct cttccggtgc
ccgccggccc tcgctggact ccatggagaa 2300ccaggtctcc gtggatgcct
tcaagatcct ggaggatcca aagtgggaat 2350tccctcggaa gaacttggtt
cttggaaaaa ctctaggaga aggcgaattt 2400ggaaaagtgg tcaaggcaac
ggccttccat ctgaaaggca gagcagggta 2450caccacggtg gccgtgaaga
tgctgaaaga gaacgcctcc ccgagtgagc 2500ttcgagacct gctgtcagag
ttcaacgtcc tgaagcaggt caaccaccca 2550catgtcatca aattgtatgg
ggcctgcagc caggatggcc cgctcctcct 2600catcgtggag tacgccaaat
acggctccct gcggggcttc ctccgcgaga 2650gccgcaaagt ggggcctggc
tacctgggca gtggaggcag ccgcaactcc 2700agctccctgg accacccgga
tgagcgggcc ctcaccatgg gcgacctcat 2750ctcatttgcc tggcagatct
cacaggggat gcagtatctg gccgagatga 2800agctcgttca tcgggacttg
gcagccagaa acatcctggt agctgagggg 2850cggaagatga agatttcgga
tttcggcttg tcccgagatg tttatgaaga 2900ggattcctac gtgaagagga
gccagggtcg gattccagtt aaatggatgg 2950caattgaatc cctttttgat
catatctaca ccacgcaaag tgatgtatgg 3000tcttttggtg tcctgctgtg
ggagatcgtg accctagggg gaaaccccta 3050tcctgggatt cctcctgagc
ggctcttcaa ccttctgaag accggccacc 3100ggatggagag gccagacaac
tgcagcgagg agatgtaccg cctgatgctg 3150caatgctgga agcaggagcc
ggacaaaagg ccggtgtttg cggacatcag 3200caaagacctg gagaagatga
tggttaagag gagagactac ttggaccttg 3250cggcgtccac tccatctgac
tccctgattt atgacgacgg cctctcagag 3300gaggagacac cgctggtgga
ctgtaataat gcccccctcc ctcgagccct 3350cccttccaca tggattgaaa
acaaactcta tggcatgtca gacccgaact 3400ggcctggaga gagtcctgta
ccactcacga gagctgatgg cactaacact 3450gggtttccaa gatatccaaa
tgatagtgta tatgctaact ggatgctttc 3500accctcagcg gcaaaattaa
tggacacgtt tgatagttaa catttctttg 3550tgaaaggtaa tggactcaca
aggggaagaa acatgctgag aatggaaagt 3600ctaccggccc tttctttgtg
aacgtcacat tggccgagcc gtgttcagtt 3650cccaggtggc agactcgttt
ttggtagttt gttttaactt ccaaggtggt 3700tttacttctg atagccggtg
attttccctc ctagcagaca tgccacaccg 3750ggtaagagct ctgagtctta
gtggttaagc attcctttct cttcagtgcc 3800cagcagcacc cagtgttggt
ctgtgtccat cagtgaccac caacattctg 3850tgttcacatg tgtgggtcca
acacttacta cctggtgtat gaaattggac 3900ctgaactgtt ggatttttct
agttgccgcc aaacaaggca aaaaaattta 3950aacatgaagc acacacacaa
aaaaggcagt aggaaaaatg ctggccctga 4000tgacctgtcc ttattcagaa
tgagagactg cggggggggc ctgggggtag 4050tgtcaatgcc cctccagggc
tggaggggaa gaggggcccc gaggatgggc 4100ctgggctcag cattcgagat
cttgagaatg atttttttta aatcatgcaa 4150cctttcctta ggaagacatt
tggttttcat catgattaag atgattccta 4200gatttagcac aatggagaga
ttccatgcca tctttactat gtggatggtg 4250gtatcaggga agagggctca
caagacacat ttgtcccccg ggcccaccac 4300atcatcctca cgtgttcggt
actgagcagc cactacccct gatgagaaca 4350gtatgaagaa agggggctgt
tggagtccca gaattgctga cagcagaggc 4400tttgctgctg tgaatcccac
ctgccaccag cctgcagcac accccacagc 4450caagtagagg cgaaacgagt
ggctcatcct acctgttagg agcaggtagg 4500gcttgtactc actttaattt
gaatcttatc aacttactca taaagggaca 4550ggctagctag ctgtgtcaga
agtagcaatg acaatgacca aggactgcta 4600cacctctgat tacaattctg
atgtgaaaaa gatggtgttt ggctcttata 4650gagcctgtgt gaaaggccca
tggatcagct cttcctgtgt ttgtaattta 4700atgctgctac aagatgtttc
tgtttcttag attctgacca tgactcataa 4750gcttcttgtc attcttcatt
gcttgtttgt ggtcacagat gcacaacact 4800cctccagtct tgtgggggca
gcttttggga agtctcagca gctcttctgg 4850ctgtgttgtc agcactgtaa
cttcgcagaa aagagtcgga ttaccaaaac 4900actgcctgct cttcagactt
aaagcactga taggacttaa aatagtctca 4950ttcaaatact gtattttata
taggcatttc acaaaaacag caaaattgtg 5000gcattttgtg aggccaaggc
ttggatgcgt gtgtaataga gccttatggt 5050gtgtgcgcac acacccagag
gagagtttga aaaatgctta ttggacacgt 5100aacctggctc taatttgggc
tgtttttcag atacactgtg ataagttctt 5150ttacaaatat ctatagacat
ggtaaacttt tggttttcag atatgcttaa 5200tgatagtctt actaaatgca
gaaataagaa taaactttct caaattatta 5250aaaatgccta cacagtaagt
gtgaattgct gcaacaggtt tgttctcagg 5300agggtaagaa ctccaggtct
aaacagctga cccagtgatg gggaatttat 5350ccttgaccaa tttatccttg
accaataacc taattgtcta ttcctgagtt 5400ataaaggtcc ccatccttat
tagctctact ggaattttca tacacgtaaa 5450tgcagaagtt actaagtatt
aagtattact gagtattaag tagtaatctg 5500tcagttatta aaatttgtaa
aatctattta tgaaaggtca ttaaaccaga 5550tcatgttcct ttttttgtaa
tcaaggtgac taagaaaatc agttgtgtaa 5600ataaaatcat gtatc
5615194315DNAHomo Sapien
19tgagagccaa gcaaagaaca ttaaggaagg aaggaggaat gaggctggat
50acggtgcagt gaaaaaggca cttccaagag tggggcactc actacgcaca
100gactcgacgg tgccatcagc atgagaactt accgctactt cttgctgctc
150ttttgggtgg gccagcccta cccaactctc tcaactccac tatcaaagag
200gactagtggt ttcccagcaa agaaaagggc cctggagctc tctggaaaca
250gcaaaaatga gctgaaccgt tcaaaaagga gctggatgtg gaatcagttc
300tttctcctgg aggaatacac aggatccgat tatcagtatg tgggcaagtt
350acattcagac caggatagag gagatggatc acttaaatat atcctttcag
400gagatggagc aggagatctc ttcattatta atgaaaacac aggcgacata
450caggccacca agaggctgga cagggaagaa aaacccgttt acatccttcg
500agctcaagct ataaacagaa ggacagggag acccgtggag cccgagtctg
550aattcatcat caagatccat gacatcaatg acaatgaacc aatattcacc
600aaggaggttt acacagccac tgtccctgaa atgtctgatg tcggtacatt
650tgttgtccaa gtcactgcga cggatgcaga tgatccaaca tatgggaaca
700gtgctaaagt tgtctacagt attctacagg gacagcccta tttttcagtt
750gaatcagaaa caggtattat caagacagct ttgctcaaca tggatcgaga
800aaacagggag cagtaccaag tggtgattca agccaaggat atgggcggcc
850agatgggagg attatctggg accaccaccg tgaacatcac actgactgat
900gtcaacgaca accctccccg attcccccag agtacatacc agtttaaaac
950tcctgaatct tctccaccgg ggacaccaat tggcagaatc aaagccagcg
1000acgctgatgt gggagaaaat gctgaaattg agtacagcat cacagacggt
1050gaggggctgg atatgtttga tgtcatcacc gaccaggaaa cccaggaagg
1100gattataact gtcaaaaagc tcttggactt tgaaaagaag aaagtgtata
1150cccttaaagt ggaagcctcc aatccttatg ttgagccacg atttctctac
1200ttggggcctt tcaaagattc agccacggtt agaattgtgg tggaggatgt
1250agatgagcca cctgtcttca gcaaactggc ctacatctta caaataagag
1300aagatgctca gataaacacc acaataggct ccgtcacagc ccaagatcca
1350gatgctgcca ggaatcctgt caagtactct gtagatcgac acacagatat
1400ggacagaata ttcaacattg attctggaaa tggttcgatt tttacatcga
1450aacttcttga ccgagaaaca ctgctatggc acaacattac agtgatagca
1500acagagatca ataatccaaa gcaaagtagt cgagtacctc tatatattaa
1550agttctagat gtcaatgaca acgccccaga atttgctgag ttctatgaaa
1600cttttgtctg tgaaaaagca aaggcagatc agttgattca gaccctgcat
1650gctgttgaca aggatgaccc ttatagtgga caccaatttt cgttttcctt
1700ggcccctgaa gcagccagtg gctcaaactt taccattcaa gacaacaaag
1750acaacacggc gggaatctta actcggaaaa atggctataa tagacacgag
1800atgagcacct atctcttgcc tgtggtcatt tcagacaacg actacccagt
1850tcaaagcagc actgggacag tgactgtccg ggtctgtgca tgtgaccacc
1900acgggaacat gcaatcctgc catgcggagg cgctcatcca ccccacggga
1950ctgagcacgg gggctctggt tgccatcctt ctgtgcatcg tgatcctact
2000agtgacagtg gtgctgtttg cagctctgag gcggcagcga aaaaaagagc
2050ctttgatcat ttccaaagag gacatcagag ataacattgt cagttacaac
2100gacgaaggtg gtggagagga ggacacccag gcttttgata tcggcaccct
2150gaggaatcct gaagccatag aggacaacaa attacgaagg gacattgtgc
2200ccgaagccct tttcctaccc cgacggactc caacagctcg cgacaacacc
2250gatgtcagag atttcattaa ccaaaggtta aaggaaaatg acacggaccc
2300cactgccccg ccatacgact ccttggccac ttacgcctat gaaggcactg
2350gctccgtggc ggattccctg agctcgctgg agtcagtgac cacggatgca
2400gatcaagact atgattacct tagtgactgg ggacctcgat tcaaaaagct
2450tgcagatatg tatggaggag tggacagtga caaagactcc taatctgttg
2500cctttttcat tttccaatac gacactgaaa tatgtgaagt ggctatttct
2550ttatatttat ccactactcc gtgaaggctt ctctgttcta cccgttccaa
2600aagccaatgg ctgcagtccg tgtggatcca atgttagaga cttttttcta
2650gtacactttt atgagcttcc aaggggcaaa tttttatttt ttagtgcatc
2700cagttaacca agtcagccca acaggcaggt gccggagggg aggacaggga
2750acagtatttc cacttgttct cagggcagcg tgcccgcttc cgctgtcctg
2800gtgttttact acactccatg tcaggtcagc caactgccct aactgtacat
2850ttcacaggct aatgggataa aggactgtgc tttaaagata aaaatatcat
2900catagtaaaa gaaatgaggg catatcggct cacaaagaga taaactacat
2950aggggtgttt atttgtgtca caaagaattt aaaataacac ttgcccatgc
3000tatttgttct tcaagaactt tctctgccat caactactat tcaaaacctc
3050aaatccaccc atatgttaaa attctcatta ctcttaagga atagaagcaa
3100attaaacggt aacatccaaa agcaaccaca aacctagtac gacttcattc
3150cttccactaa ctcatagttt gttatatcct agactagaca tgcgaaagtt
3200tgcctttgta ccatataaag ggggagggaa atagctaata atgttaacca
3250aggaaatata ttttaccata catttaaagt tttggccacc acatgtatca
3300cgggtcactt gaaattcttt cagctatcag taggctaatg tcaaaattgt
3350ttaaaaattc ttgaaagaat tttcctgaga caaattttaa cttcttgtct
3400atagttgtca gtattattct actatactgt acatgaaagt agcagtgtga
3450agtacaataa ttcatattct tcatatcctt cttacacgac taagttgaat
3500tagtaaagtt agattaaata aaacttaaat ctcactctag gagttcagtg
3550gagaggttag agccagccac acttgaacct aataccctgc ccttgacatc
3600tggaaacctc tacatattta tataacgtga tacatttgga taaacaacat
3650tgagattatg atgaaaacct acatattcca tgtttggaag acccttggaa
3700gaggaaaatt ggattccctt aaacaaaagt gtttaagatt gtaattaaaa
3750tgatagttga ttttcaaaag cattaatttt ttttcattgt ttttaacttt
3800gctttcatga ccatcctgcc atccttgact ttgaactaat gataaagtaa
3850tgatctcaaa ctatgacaga aaagtaatgt aaaatccatc caatctatta
3900tttctctaat tatgcaatta gcctcatagt tattatccag aggacccaac
3950tgaactgaac taatccttct ggcagattca aatcgtttat ttcacacgct
4000gttctaatgg cacttatcat tagaatctta ccttgtgcag tcatcagaaa
4050ttccagcgta ctataatgaa aacatccttg ttttgaaaac ctaaaagaca
4100ggctctgtat atatatatac ttaagaatat gctgacttca cttattagtc
4150ttagggattt attttcaatt aatattaatt ttctacaaat aattttagtg
4200tcatttccat ttggggatat tgtcatatca gcacatattt tctgtttgga
4250aacacactgt tgtttagtta agttttaaat aggtgtatta cccaagaagt
4300aaagatggaa acgtt
4315202521DNAHomo Sapien 20cggtggaggc cacagacacc tcaaacctgg attccacaat
tctacgttaa 50gtgttggagt ttttattact ctgctgtagg aaagcctttg
ccaatgctta 100caaggaactg tttatccctg cttctctggg ttctgtttga
tggaggtctc 150ctaacaccac tacaaccaca gccacagcag actttagcca
cagagccaag 200agaaaatgtt atccatctgc caggacaacg gtcacatttc
caacgtgtta 250aacgtggctg ggtatggaat caattttttg tgctggaaga
atacgtgggc 300tccgagcctc agtatgtggg aaagctccat tccgacttag
acaagggaga 350gggcactgtg aaatacaccc tctcaggaga tggcgctggc
accgttttta 400ccattgatga aaccacaggg gacattcatg caataaggag
cctagataga 450gaagagaaac ctttctacac tcttcgtgct caggctgtgg
acatagaaac 500cagaaagccc ctggagcctg aatcagaatt catcatcaaa
gtgcaggata 550ttaatgataa tgagccaaag tttttggatg gaccttatgt
tgctactgtt 600ccagaaatgt ctcctgtggg tgcatatgta ctccaggtca
aggccacaga 650tgcagatgac ccgacctatg gaaacagtgc cagagtcgtt
tacagcattc 700ttcagggaca accttatttc tctattgatc ccaagacagg
tgttattaga 750acagctttgc caaacatgga cagagaagtc aaagaacaat
atcaagtact 800catccaagcc aaggatatgg gaggacagct tggaggatta
gccggaacaa 850caatagtcaa catcactctc accgatgtca atgacaatcc
acctcgattc 900cccaaaagca tcttccactt gaaagttcct gagtcttccc
ctattggttc 950agctattgga agaataagag ctgtggatcc tgattttgga
caaaatgcag 1000aaattgaata caatattgtt ccaggagatg ggggaaattt
gtttgacatc 1050gtcacagatg aggatacaca agagggagtc atcaaattga
aaaagccttt 1100agattttgaa acaaagaagg catacacttt caaagttgag
gcttccaacc 1150ttcaccttga ccaccggttt cactcggcgg gccctttcaa
agacacagct 1200acggtgaaga tcagcgtgct ggacgtagat gagccaccgg
ttttcagcaa 1250gccgctctac accatggagg tttatgaaga cactccggta
gggaccatca 1300ttggcgctgt cactgctcaa gacctggatg taggcagcgg
tgctgttagg 1350tacttcatag attggaagag tgatggggac agctacttta
caatagatgg 1400aaatgaagga accatcgcca ctaatgaatt actagacaga
gaaagcactg 1450cgcagtataa tttctccata attgcgagta aagttagtaa
ccctttattg 1500accagcaaag tcaatatact gattaatgtc ttagatgtaa
atgaatttcc 1550tccagaaata tctgtgccat atgagacagc cgtgtgtgaa
aatgccaagc 1600caggacagat aattcagata gtcagtgctg cagaccgaga
tctttcacct 1650gctgggcaac aattctcctt tagattatca cctgaggctg
ctatcaaacc 1700aaattttaca gttcgtgact tcagaaacaa cacagcgggg
attgaaaccc 1750gaagaaatgg atacagccgc aggcagcaag agttgtattt
cctccctgtt 1800gtaatagaag acagcagcta ccctgtccag agcagcacaa
acacaatgac 1850tattcgagtc tgtagatgtg actctgatgg caccatcctg
tcttgtaatg 1900tggaagcaat ttttctacct gtaggactta gcactggggc
gttgattgca 1950attctactat gcattgttat actcttagcc atagttgtac
tgtatgtagc 2000actgcgaagg cagaagaaaa agcacaccct gatgacctct
aaagaagaca 2050tcagagacaa cgtcatccat tacgatgatg aaggaggtgg
ggaggaagat 2100acccaggctt tcgacatcgg ggctctgaga aacccaaaag
tgattgagga 2150gaacaaaatt cgcagggata taaaaccaga ctctctctgt
ttacctcgtc 2200agagaccacc catggaagat aacacagaca taagggattt
cattcatcaa 2250aggctacagg aaaatgatgt agatccaact gccccaccaa
tcgattcact 2300ggccacatat gcctacgaag ggagtgggtc cgtggcagag
tccctcagct 2350ctatagactc tctcaccaca gaagccgacc aggactatga
ctatctgaca 2400gactggggac cccgctttaa agtcttggca gacatgtttg
gcgaagaaga 2450gagttataac cctgataaag tcacttaagg gagtcgtgga
ggctaaaata 2500caaccgagag gggagatttt t
252121736DNAHomo Sapien 21ggctctcacc ctcctctcct
gcagctccag ctctgtgctc tgcctctgag 50gagaccatgg cccggcctct
gtgtaccctg ctactcctga tggctaccct 100ggctggggct ctggcctcga
gctccaagga ggagaatagg ataatcccag 150gtggcatcta tgatgcagac
ctcaatgatg agtgggtaca gcgtgccctt 200cacttcgcca tcagcgagta
caacaaggcc accgaagatg agtactacag 250acgcccgctg caggtgctgc
gagccaggga gcagaccttt gggggggtga 300attacttctt cgacgtagag
gtgggccgca ccatatgtac caagtcccag 350cccaacttgg acacctgtgc
cttccatgaa cagccagaac tgcagaagaa 400acagttatgc tctttcgaga
tctacgaagt tccctgggag gacagaatgt 450ccctggtgaa ttccaggtgt
caagaagcct aggggtctgt gccaggccag 500tcacaccgac caccacccac
tcccaccccc tgtagtgctc ccacccctgg 550actggtggcc cccaccctgc
gggaggcctc cccatgtgcc tgtgccaaga 600gacagacaga gaaggctgca
ggagtccttt gttgctcagc agggcgctct 650gccctccctc cttccttctt
gcttctaata gacctggtac atggtacaca 700cacccccacc tcctgcaatt
aaacagtagc atcgcc 736222025DNAHomo Sapien
22ggcagcggtg gcaggggctg caggagcaag tgaccaggag caggactggg
50gacaggcctg atcgcccctg cacgaaccag acccttcgcc gccctcacga
100tgactacctc tccgatcctg cagctgctgc tgcggctctc actgtgcggg
150ctgctgctcc agagggcgga gacaggctct aaggggcaga cggcggggga
200gctgtaccag cgctgggaac ggtaccgcag ggagtgccag gagaccttgg
250cagccgcgga accgccttca ggcctcgcct gtaacgggtc cttcgatatg
300tacgtctgct gggactatgc tgcacccaat gccactgccc gtgcgtcctg
350cccctggtac ctgccctggc accaccatgt ggctgcaggt ttcgtcctcc
400gccagtgtgg cagtgatggc caatggggac tttggagaga ccatacacaa
450tgtgagaacc cagagaagaa tgaggccttt ctggaccaaa ggctcatctt
500ggagcggttg caggtcatgt acactgtcgg ctactccctg tctctcgcca
550cactgctgct agccctgctc atcttgagtt tgttcaggcg gctacattgc
600actagaaact atatccacat caacctgttc acgtctttca tgctgcgagc
650tgcggccatt ctcagccgag accgtctgct acctcgacct ggcccctacc
700ttggggacca ggcccttgcg ctgtggaacc aggccctcgc tgcctgccgc
750acggcccaga tcgtgaccca gtactgcgtg ggtgccaact acacgtggct
800gctggtggag ggcgtctacc tgcacagtct cctggtgctc gtgggaggct
850ccgaggaggg ccacttccgc tactacctgc tcctcggctg gggggccccc
900gcgcttttcg tcattccctg ggtgatcgtc aggtacctgt acgagaacac
950gcagtgctgg gagcgcaacg aagtcaaggc catttggtgg attatacgga
1000cccccatcct catgaccatc ttgattaatt tcctcatttt tatccgcatt
1050cttggcattc tcctgtccaa gctgaggaca cggcaaatgc gctgccggga
1100ttaccggctg aggctggctc gctccacgct gacgctggtg cccctgctgg
1150gtgtccacga ggtggtgttt gctcccgtga cagaggaaca ggcccggggc
1200gccctgcgct tcgccaagct cggctttgag atcttcctca gctccttcca
1250gggcttcctg gtcagcgtcc tctactgctt catcaacaag gaggtgcagt
1300cggagatccg ccgtggctgg caccactgcc gcctgcgccg cagcctgggc
1350gaggagcaac gccagctccc ggagcgcgcc ttccgggccc tgccctccgg
1400ctccggcccg ggcgaggtcc ccaccagccg cggcttgtcc tcggggaccc
1450tcccagggcc tgggaatgag gccagccggg agttggaaag ttactgctag
1500ggggcgggat ccccgtgtct gttcagttag catggattta ttgagtgcca
1550actgcgtgcc aggcccagta cggaggacgc tggggaaatg gtgaaggaaa
1600cagaaaaaag gtccctgccc ttctggagat gacaactgag tggggaaaac
1650agaccgtgaa cacaaaacat caagttccac acacgctatg gaatggttat
1700gaagggaagc gagaaggggg cctagggtgg tctgggaggc gtctccaagg
1750aggtgacact taagccatcc ccgaaagagg tgaaagagat cactttgggg
1800agagctggag aacaggattc taggcggaag cgatagcata ggcaaaggcc
1850cttgggcagg aaggcgctca gccttggctg gagtagaatt aagtcagagc
1900caacaggttg gggagagaca gagaagtggg caggggcacc caagttggga
1950tttcatttca ggtgcattgg agattcttag gagtgtctct tgggggtaat
2000attttatttt ttaaaaaatg aggat
2025233168DNAHomo Sapien 23gccagagcgt gagccgcgac ctccgcgcag gtggtcgcgc
cggtctccgc 50ggaaatgttg tccaaagttc ttccagtcct cctaggcatc
ttattgatcc 100tccagtcgag ggtcgaggga cctcagactg aatcaaagaa
tgaagcctct 150tcccgtgatg ttgtctatgg cccccagccc cagcctctgg
aaaatcagct 200cctctctgag gaaacaaagt caactgagac tgagactggg
agcagagttg 250gcaaactgcc agaagcctct cgcatcctga acactatcct
gagtaattat 300gaccacaaac tgcgccctgg cattggagag aagcccactg
tggtcactgt 350tgagatcgcc gtcaacagcc ttggtcctct ctctatccta
gacatggaat 400acaccattga catcatcttc tcccagacct ggtacgacga
acgcctctgt 450tacaacgaca cctttgagtc tcttgttctg aatggcaatg
tggtgagcca 500gctatggatc ccggacacct tttttaggaa ttctaagagg
acccacgagc 550atgagatcac catgcccaac cagatggtcc gcatctacaa
ggatggcaag 600gtgttgtaca caattaggat gaccattgat gccggatgct
cactccacat 650gctcagattt ccaatggatt ctcactcttg ccctctatct
ttctctagct 700tttcctatcc tgagaatgag atgatctaca agtgggaaaa
tttcaagctt 750gaaatcaatg agaagaactc ctggaagctc ttccagtttg
attttacagg 800agtgagcaac aaaactgaaa taatcacaac cccagttggt
gacttcatgg 850tcatgacgat tttcttcaat gtgagcaggc ggtttggcta
tgttgccttt 900caaaactatg tcccttcttc cgtgaccacg atgctctcct
gggtttcctt 950ttggatcaag acagagtctg ctccagcccg gacctctcta
gggatcacct 1000ctgttctgac catgaccacg ttgggcacct tttctcgtaa
gaatttcccg 1050cgtgtctcct atatcacagc cttggatttc tatatcgcca
tctgcttcgt 1100cttctgcttc tgcgctctgt tggagtttgc tgtgctcaac
ttcctgatct 1150acaaccagac aaaagcccat gcttctccta aactccgcca
tcctcgtatc 1200aatagccgtg cccatgcccg tacccgtgca cgttcccgag
cctgtgcccg 1250ccaacatcag gaagcttttg tgtgccagat tgtcaccact
gagggaagtg 1300atggagagga gcgcccgtct tgctcagccc agcagccccc
tagcccaggt 1350agccctgagg gtccccgcag cctctgctcc aagctggcct
gctgtgagtg 1400gtgcaagcgt tttaagaagt acttctgcat ggtccccgat
tgtgagggca 1450gtacctggca gcagggccgc ctctgcatcc atgtctaccg
cctggataac 1500tactcgagag ttgttttccc agtgactttc ttcttcttca
atgtgctcta 1550ctggcttgtt tgccttaact tgtaggtacc agctggtacc
ctgtggggca 1600acctctccag ttccccagga ggtccaagcc ccttgccaag
ggagttgggg 1650gaaagcagca gcagcagcag gagcgactag agtttttcct
gccccattcc 1700ccaaacagaa gcttgcagag ggtttgtctt tgctgcccct
ctcccctacc 1750tggcccattc actgagtctt ctcagcagac catttcaaat
tattaataaa 1800tgggccacct ccctcttctt caaggagcat ccgtgatgct
cagtgttcaa 1850aaccacagcc acttagtgat cagctcccta aaaccatgcc
taagtacagg 1900cggattagct atcttccaac aatgctgacc accagacaat
tactgcattt 1950ttccagaagc ccactattgc ctttgtagtg ctttcggccc
agttctggcc 2000tcagcctcaa agtgcaccga ctagttgctt gcctatacct
ggcacctcat 2050taagatgctg ggcagcagta taacaggagg aagagatccc
tctcctttgg 2100tcagattatt atgttctcag ttctctctcc ctgctacccc
tttctctgca 2150gatagataga cactggcatt atccctttag gaagaggggg
gggcagcaag 2200agagcctatt tgggacagca ttcctctctc tctgctgctg
tgacatctcc 2250ctctccttgc tggctccatc tttcgtctgc actaccaatt
caatgccctt 2300catccaatgg gtatctattt ttgtgtgtga ttatagtaac
tactccctgc 2350tttatatgcc accctcttcc ttctctttga cccctgtgac
tctttctgta 2400actttcccag tgacttcccc tagccctgac ccaggcacta
ggccttggtg 2450acttcctggg gccaagaaac taaggaaact cggctttgca
acaggcatta 2500ctcgccattg attggtgccc acccagggca cactgtcgga
gttctatcac 2550ttgcttgacc cctggaccca taaaccagtc cactgttata
cccggggcac 2600tctaaccatc acaatcaatc aatcaaattc ccttaaattt
gtatggcact 2650ggaactttgg caaagcactt ttgacaagtt gtgtctgatt
ggagcttcat 2700gatagccttg tgacatcttt agggcaggat tcttatcccc
attttgcaga 2750tgaaaaccct gagtcacaga tttctgtggg actgtggatc
tcactggaag 2800ctatccaaga gcccactgtc accttctaga ccacatgata
gggctagaca 2850gctcagttca ccatgattct cttctgtcac ctctgctggc
acaccagtgg 2900caaggcccag aatggcgacc tctctttagc tcaatttctg
ggcctgaggt 2950gctcagactg cccccaagat caaatctctc ctggctgtag
taacccagtg 3000gaatgaattt ggacatgccc caatgcttct atatgctaag
tgaaatctgt 3050gtctgtaatt tgttgggggg tggatagggt ggggtctcca
tctacttttt 3100gtcaccatca tctgaaatgg ggaaatatgt aaataaatat
atcagcaaag 3150caaaaagaaa aaaaaaaa
3168242837DNAHomo Sapien 24tatcacagga ttgtctactt
cagcaatagc aactaatgga tttgtaagag 50gaggaggagc atattattta
atatctagaa gtctagggcc agaatttggt 100ggtgcaattg gtctaatctt
cgcctttgcc aacgctgttg cagttgctat 150gtatgtggtt ggatttgcag
aaaccgtggt ggagttgctt aaggaacatt 200ccatacttat gatagatgaa
atcaatgata tccgaattat tggagccatt 250acagtcgtga ttcttttagg
tatctcagta gctggaatgg agtgggaagc 300aaaagctcag attgttcttt
tggtgatcct acttcttgct attggtgatt 350tcgtcatagg aacatttatc
ccactggaga gcaagaagcc aaaagggttt 400tttggttata aatctgaaat
atttaatgag aactttgggc ccgattttcg 450agaggaagag actttctttt
ctgtatttgc catctttttt cctgctgcaa 500ctggtattct ggctggagca
aatatctcag gtgatcttgc agatcctcag 550tcagccatac ccaaaggaac
actcctagcc attttaatta ctacattggt 600ttacgtagga attgcagtat
ctgtaggttc ttgtgttgtt cgagatgcca 650ctggaaacgt taatgacact
atcgtaacag agctaacaaa ctgtacttct 700gcagcctgca aattaaactt
tgatttttca tcttgtgaaa gcagtccttg 750ttcctatggc ctaatgaaca
acttccaggt aatgagtatg gtgtcaggat 800ttacaccact aatttctgca
ggtatatttt cagccactct ttcttcagca 850ttagcatccc tagtgagtgc
tcccaaaata tttcaggctc tatgtaagga 900caacatctac ccagctttcc
agatgtttgc taaaggttat gggaaaaata 950atgaacctct tcgtggctac
atcttaacat tcttaattgc acttggattc 1000atcttaattg ctgaactgaa
tgttattgca ccaattatct caaacttctt 1050ccttgcatca tatgcattga
tcaatttttc agtattccat gcatcacttg 1100caaaatctcc aggatggcgt
cctgcattca aatactacaa catgtggata 1150tcacttcttg gagcaattct
ttgttgcata gtaatgttcg tcattaactg 1200gtgggctgca ttgctaacat
atgtgatagt ccttgggctg tatatttatg 1250ttacctacaa aaaaccagat
gtgaattggg gatcctctac acaagccctg 1300acttacctga atgcactgca
gcattcaatt cgtctttctg gagtggaaga 1350ccacgtgaaa aactttaggc
cacagtgtct tgttatgaca ggtgctccaa 1400actcacgtcc agctttactt
catcttgttc atgatttcac aaaaaatgtt 1450ggtttgatga tctgtggcca
tgtacatatg ggtcctcgaa gacaagccat 1500gaaagagatg tccatcgatc
aagccaaata tcagcgatgg cttattaaga 1550acaaaatgaa ggcattttat
gctccagtac atgcagatga cttgagagaa 1600ggtgcacagt atttgatgca
ggctgctggt cttggtcgta tgaagccaaa 1650cacacttgtc cttggattta
agaaagattg gttgcaagca gatatgaggg 1700atgtggatat gtatataaac
ttatttcatg atgcttttga catacaatat 1750ggagtagtgg ttattcgcct
aaaagaaggt ctggatatat ctcatcttca 1800aggacaagaa gaattattgt
catcacaaga gaaatctcct ggcaccaagg 1850atgtggtagt aagtgtggaa
tatagtaaaa agtccgattt agatacttcc 1900aaaccactca gtgaaaaacc
aattacacac aaagttgagg aagaggatgg 1950caagactgca actcaaccac
tgttgaaaaa agaatccaaa ggccctattg 2000tgcctttaaa tgtagctgac
caaaagcttc ttgaagctag tacacagttt 2050cagaaaaaac aaggaaagaa
tactattgat gtctggtggc tttttgatga 2100tggaggtttg accttattga
taccttacct tctgacgacc aagaaaaaat 2150ggaaagactg taagatcaga
gtattcattg gtggaaagat aaacagaata 2200gaccatgacc ggagagcgat
ggctactttg cttagcaagt tccggataga 2250cttttctgat atcatggttc
taggagatat caataccaaa ccaaagaaag 2300aaaatattat agcttttgag
gaaatcattg agccatacag acttcatgaa 2350gatgataaag agcaagatat
tgcagataaa atgaaagaag atgaaccatg 2400gcgaataaca gataatgagc
ttgaacttta taagaccaag acataccggc 2450agatcaggtt aaatgagtta
ttaaaggaac attcaagcac agctaatatt 2500attgtcatga gtctcccagt
tgcacgaaaa ggtgctgtgt ctagtgctct 2550ctacatggca tggttagaag
ctctatctaa ggacctacca ccaatcctcc 2600tagttcgtgg gaatcatcag
agtgtcctta ccttctattc ataaatgttc 2650tatacagtgg acagccctcc
agaatggtac ttcagtgcct agtgtagtaa 2700cctgaaatct tcaatgacac
attaacatca caatggcgaa tggtgacttt 2750tctttcacga tttcattaat
ttgaaagcac acaggaaagc ttgctccatt 2800gataacgtgt atggagactt
cggttttagt caattcc 2837254709DNAHomo Sapien
25gagcttgtcc agacgaagcc tcgcagggat gggttggagc ctgggccgtg
50cttcgctcag gcagcgtttg aggcagaccc agcagggtcc tcctggggcc
100ttcctgcctt tgaactgcgg tggcgggcgg gcgcacggtc tcctgtacgc
150cctagactag gggccgccat ctccatggcc acggccgtga gccggccctg
200cgccggcagg tcgcgggaca tactgtggcg cgttttgggc tggaggatag
250ttgcaagtat tgtttggtca gtgctatttc tacccatctg caccacagta
300tttataattt tcagcaggat tgatttgttt catcctatac agtggctgtc
350tgattctttc agtgacctgt atagttccta tgtaatcttt tacttcctgc
400tgctgtcagt ggtaataata ataataagta ttttcaatgt ggagttctat
450gcagttgtgc cttctattcc ttgctccaga ctagctctga tagggaagat
500cattcatcct cagcaactca tgcactcatt tattcatgct gcaatgggaa
550tggtgatggc ctggtgtgct gcagtgataa cccagggcca gtacagcttt
600cttgtggttc cctgcactgg tactaacagc tttggtagcc ctgctgcgca
650aacctgctta aatgaatatc atcttttttt cctactgact ggagcattta
700tgggctatag ctatagcctc ctgtattttg ttaacaacat gaactatctt
750ccatttccca tcatacagca atacaagttc ttgcgtttta ggagatctct
800gctcttatta gttaaacaca gttgtgtgga atcactgttc ctggttagaa
850atttctgcat tttatattat tttcttggct atattcccaa agcttggatt
900agcactgcta tgaaccttca catagatgag caggttcata ggccacttga
950cacagtgagt ggcctcttaa atctctcgtt actctaccat gtctggctgt
1000gtggtgtctt tctcctgacg acttggtatg tctcatggat actcttcaaa
1050atctatgcca cagaggctca tgtgtttcct gttcaaccac catttgcaga
1100agggtcagat gagtgccttc caaaagtgtt aaatagcaat cctcccccca
1150tcataaagta tttagccttg caggacctga tgttgctttc tcaatattct
1200ccttcacgaa gacaagaagt tttcagcctc agccaaccag gtggacatcc
1250ccacaattgg acagccattt caagggagtg tttgaatctt ttaaatggta
1300tgactcagaa actgattctc tatcaagaag ctgctgctac gaatgggaga
1350gtgtcttcat cttacccagt ggaacctaag aaattaaatt ctccagaaga
1400aactgctttt cagacaccaa aatctagcca gatgcctcgg ccttcagtgc
1450caccattagt taaaacatca ctgttttctt caaaattatc tacacctgat
1500gttgtgagcc catttgggac cccatttggc tctagtgtaa tgaatcggat
1550ggctggaatt tttgatgtaa acacctgcta tgggtcaccg caaagtcctc
1600agctaataag aagggggcca agattgtgga catcagcttc tgatcagcaa
1650atgactgaat tttctaatcc ttctccatct acctctatta gtgctgaggg
1700taagacaatg agacaaccca gtgtgattta ttcatggatt cagaataaac
1750gtgaacagat taagaatttc ttgtcaaaac gggtgctgat aatgtatttt
1800ttcagtaagc acccagaggc ctccattcag gctgtttttt cagatgccca
1850aatgcatatt tgggcattag aaggtctgtc gcacttagta gcagcatcat
1900ttacagagga tagatttgga gttgtccaga cgacactacc agctatcctt
1950aatactttgt tgacactgca agaggcagtc gacaagtact ttaagcttcc
2000tcatgcttcc agtaaaccac cccggatttc aggaagcctt gtggacactt
2050catataaaac attaagattt gcattcagag catcactgaa aactgccatc
2100tatcgaataa ctactacatt tggtgaacat ctgaatgctg tgcaagcatc
2150tgcagaacat cagaaaagac ttcaacagtt cttggagttc aaagaatagt
2200taagtaatat aaactgtgtt cattacactg ctgatacaac tacagatggg
2250acagtaaatg ttcagcattc ttggatcaga agaaaacgga ctaattagat
2300gcttcctttg tcgtggtggt tgctttgaaa actatacttt aatgggagaa
2350atcatggaaa gaaattctca acagaataac tgaaaactgc cttttctgta
2400ccgattgctt tttgtgtgtg tggtataata aaatctttat tcaattttac
2450agaagcattg atggcagtcg aaatgtctct agctcatata acttaatagt
2500aataactaaa aaacttttag aatttacttt tgaaaggagg gaagccagtt
2550ctgaaatgag tataggttga tttcatagtc ttcttaatta agagtttagc
2600tctttgtaaa ctcaaaatac ataaactttt taagtgtagt ttcatttact
2650gaaggataaa aatggtaaca gtgcagcaat attcacaaaa aatattgtct
2700aacggacata ttttgttaat ctgttaggtt gggtttttgt ttccagggac
2750aaattaaatt tgtatgatta cccaaaaaag ggtctcagtt tacagatgct
2800aactctatat aaaggaatgt ggaaaaactc agttcttaag ttacaagatt
2850aaaaattcac atttggtctt taagaaacaa ttgactgaca tctatgaatt
2900tattttgtat catgctagta aacacgaagt attaatgtat gggtattttc
2950ccagctagtt ttgctttctt tttctggagc aaaacattaa gtgattgcag
3000agtttttcaa gcaagagaaa aaggtttgca aaaaaaccca ggaaatgttc
3050ccttttttcc ccaccattca tcttcattag atcaaattct gtgaaacttg
3100tctggtctct caaagggagc agcctctgta gtgttaaatg gctaattaaa
3150ataggaagat ctttatagcc agaaacaact tagtcatcaa atagcaagtg
3200aaaccaaaac gtcagaggga ttactgtact tggaagtatg ttgtgtgtcc
3250caaatgtgaa cgaagtattg ttagaattta ttagatcagc ttctttggag
3300atcaaagatt ggaaatccta gtcatagata ttcactggac tggctttgga
3350ctgaaatgct cctttgtaat tcttttccta ttgtcttttc cttctagtgt
3400cccaaaatat tttctttaaa gtcagcacag tactgtatat gaatctttaa
3450tgtggtatca tatatgtcta cttttgtctg attcatcgat gtattatatc
3500tttataattg aatattttag ctccgggtcc tgttgcccct tcaagcagta
3550catgccaaat tataaatagg tgctactggc cttgagcata tcactgtggg
3600acagttcccc aattgtcaag tgtttagata tgtagactat tgccatttgt
3650ttttttgttt tggttttgct ttgtgtctga agctgaattg atttcttttt
3700tttgaatgtg aaagttgaat ttcaaacgta gtcatttctt acagatggcc
3750aagacagaaa attgtggcta ggttgactga gaactgttgt cttccatgta
3800ttaacacaat taagcttttt atattccact ctctgtgctg accctggctg
3850aggcattttg ggagacaagg actctgaatc ttctgcttcc attaaagaag
3900aactgtgata ttcaacattg gatttctgag aataaagata ggatgattcc
3950tttgaacttt gacttacttg tataaaatgt ccagctaggt taggtttttg
4000ccatttccta tatactttgg gtaaagctac atttgatgag caatgtgaat
4050gtttctgaga atgttcattc ctgttttctc ttaagagaat gtgctgtgta
4100ctaaatacag gccacatagt gtctgcctgt tgaagatctg gaaactgcct
4150ccccagatct gtattgtatt tggtaggtaa gggggtcagt ttctttttct
4200cattgtgtgt tgataatcta cacaccatct gttggaacca gggtgttatt
4250atggggaact cctcctgtgt actaggagga ggaccttagg gagaccaaga
4300ggagagaagc atttcctttg atgaagtcac atcctgtcta tgagcccact
4350aatgctgtaa cattggcctg aaagagagtg ttctttaaaa gcctttctcg
4400gctgttagta taaaaacatg atggtatcag ctcttagcat gtttgcttga
4450cccttatgga aggtataaat ccacagaact tccttcccag agaactggga
4500aattgtccta gaaataaacc ttgtacagtt gagtggacat ggataagcaa
4550caatttgtta ctttgcagga tttgttcctt ggtaattgtt tggtgtgtca
4600tcctgtaaat attcatgata gtctgtttat atccttttgt atatcgttga
4650tactggattg ggtagaaaaa taaattggca atttaaaaaa atggaacagt
4700taattgaaa
4709266310DNAHomo Sapien 26gatgggggcc ccgtttgtct gggccttggg ccttttgatg
ctgcagatgc 50tgctctttgt ggctggggaa cagggcacac aggatatcac
cgatgccagc 100gaaagggggc tccacatgca gaagctgggg tctgggtcag
tgcaggctgc 150gctggcggag ctggtggccc tgccctgtct ctttaccctg
cagccacggc 200caagcgcagc ccgagatgcc cctcggataa agtggaccaa
ggtgcggact 250gcgtcgggcc agcgacagga cttgcccatc ctggtggcca
aggacaatgt 300cgtgagggtg gccaaaagct ggcagggacg agtgtcactg
ccttcctacc 350cccggcgccg agccaacgcc acgctacttc tggggccact
gagggccagt 400gactctgggc tgtaccgctg ccaggtggtg aggggcatcg
aggatgagca 450ggacctggtg cccttggagg tgacaggtgt tgtgttccac
taccgatcag 500cccgggaccg ctatgcactg accttcgctg aggcccagga
ggcctgccgt 550ctcagctcag ccatcattgc agcccctcgg catctacagg
ctgcctttga 600ggatggcttt gacaactgtg atgctggctg gctctctgac
cgcactgttc 650ggtatcctat cacccagtcc cgtcctggtt gctatggcga
ccgtagcagc 700cttccagggg ttcggagcta tgggaggcgc aacccacagg
aactctacga 750tgtgtattgc tttgcccggg agctgggggg cgaggtcttc
tacgtgggcc 800cggcccgccg cctgacactg gccggcgcgc gtgcacagtg
ccgccgccag 850ggtgccgcgc tggcctcggt gggacagctg cacctggcct
ggcatgaggg 900cctggaccag tgcgacccgg gctggctggc cgacggcagc
gtgcgctacc 950cgatccagac gccgcgccgg cgctgcgggg gcccagcccc
gggcgtgcgc 1000accgtctacc gcttcgctaa ccggaccggc ttcccctcac
ccgccgagcg 1050cttcgacgcc tactgcttcc gagctcatca ccccacgtca
caacatggag 1100acctagagac cccatcctct ggggatgagg gggagattct
gtcagcagag 1150gggcccccag ttagagaact ggagcccacc ctggaggagg
aagaggtggt 1200cacccctgac ttccaggagc ctctggtgtc cagtggggaa
gaagaaaccc 1250tgattttgga ggagaagcag gagtctcaac agaccctcag
ccctacccct 1300ggggacccca tgctggcctc atggcccact ggggaagtgt
ggctaagcac 1350ggtggccccc agccctagcg acatgggggc aggcactgca
gcaagttcac 1400acacggaggt ggccccaact gaccctatgc ctaggagaag
ggggcgcttc 1450aaagggttga atgggcgcta cttccagcag caggaaccgg
agccggggct 1500gcaagggggg atggaggcca gcgcccagcc ccccacctca
gaggctgcag 1550tgaaccaaat ggagcctccg ttggccatgg cagtcacaga
gatgttgggc 1600agtggccaga gccggagccc ctgggctgat ctgaccaatg
aggtggatat 1650gcctggagct ggttctgctg gtggcaagag ctccccagag
ccctggctgt 1700ggccccctac catggtccca cccagcatct caggccacag
cagggcccct 1750gtcctggagc tagagaaagc cgagggcccc agtgccaggc
cagccacccc 1800agacctgttt tggtccccct tggaggccac tgtctcagct
cccagccctg 1850ccccctggga ggcattccct gtggccacct ccccagatct
ccctatgatg 1900gccatgctgc gtggtcccaa agagtggatg ctaccacacc
ccacccccat 1950ctccaccgag gccaatagag ttgaggcaca tggtgaggcc
accgccacgg 2000ctccaccctc ccctgctgca gagaccaagg tgtattccct
gcctctctct 2050ttgaccccaa caggacaggg tggagaggcc atgcccacaa
cacctgagtc 2100ccccagggca gacttcagag aaactgggga gaccagccct
gctcaggtca 2150acaaagctga gcactccagc tccagcccat ggccttctgt
aaacaggaat 2200gtggctgtag gttttgtccc cactgagact gccactgagc
caacgggcct 2250caggggtatc ccggggtctg agtctggggt cttcgacaca
gcagaaagcc 2300ccacttctgg cttgcaggcc actgtagatg aggtgcagga
cccctggccc 2350tcagtgtaca gcaaagggct ggatgcaagt tccccatctg
cccccctggg 2400gagccctgga gtcttcttgg tacccaaagt caccccaaat
ttggagcctt 2450gggttgctac agatgaagga cccactgtga atcccatgga
ttccacagtc 2500acgccggccc ccagtgatgc tagtggaatt tgggaacctg
gatcccaggt 2550gtttgaagaa gccgaaagca ccaccttgag ccctcaggtg
gccctggata 2600caagcattgt gacgcccctc acgaccctgg agcaggggga
caaggttgga 2650gttccagcca tgtctacact gggctcctca agctcccaac
cccacccaga 2700gccagaggat caggtggaga cccagggaac atcaggagct
tcagtgcctc 2750cgcatcagag cagtccccta gggaaaccgg ctgttcctcc
tgggacaccg 2800actgcagcca gtgtgggcga gtctgcctca gtttcctcag
gggagcctac 2850ggtaccgtgg gacccctcca gcaccctgct gcctgtcacc
ctgggcatag 2900aggacttcga actggaggtc ctggcaggga gcccgggtgt
agagagcttc 2950tgggaggagg tggcaagtgg agaggagcca gccctgccag
ggacccctat 3000gaatgcaggt gcggaggagg tgcactcaga tccctgtgag
aacaaccctt 3050gtcttcatgg agggacatgt aatgccaatg gcaccatgta
tggctgtagc 3100tgtgatcagg gcttcgccgg ggagaactgt gagattgaca
ttgatgactg 3150cctctgcagc ccctgtgaga atggaggcac ctgtattgat
gaggtcaatg 3200gctttgtctg cctttgcctc cccagctatg ggggcagctt
ttgtgagaaa 3250gacaccgagg gctgtgaccg cggctggcat aagttccagg
gccactgtta 3300ccgctatttt gcccaccgga gggcatggga agatgccgag
aaggactgcc 3350gccgccgctc cggccacctg accagcgtcc actcaccgga
ggaacacagc 3400ttcattaata gctttgggca tgaaaacacg tggatcggcc
tgaacgacag 3450gatcgtggag agagatttcc agtggacgga caacaccggg
ctgcaatttg 3500agaactggcg agagaaccag ccggacaatt tcttcgcggg
tggcgaggac 3550tgtgtggtga tggtggcgca tgaaagcggg cgctggaacg
atgtcccctg 3600caactacaac ctaccctatg tctgcaagaa gggcacagtg
ctctgtggtc 3650cccctccggc agtggagaat gcctcactca tcggtgcccg
caaggccaag 3700aacaatgtcc atgccactgt aaggtaccag tgcaatgaag
gatttgccca 3750gcaccatgtg gtcaccattc gatgccggag caatggcaag
tgggacaggc 3800cccaaattgt ctgcaccaaa cccagacgtt cacatcggat
gcggggacac 3850caccaccacc accaacacca ccaccagcat caccaccaca
aatcccgcaa 3900ggagcgcaga aaacacaaga aacacccaac ggaggactgg
gagaaggacg 3950aagggaattt ttgctgaaga accagaaaaa agaaagcaca
acacctttcc 4000catgcctcct ctggagcctt cgcctgggga gacagaaccc
agagagaaac 4050aagagagtcc agaagtccct gaaccccaaa ctgttctcgc
aaaaaaaata 4100ttcctttgaa caaaggtctt cttttccttt ttttacatac
acaagatctt 4150cttggcaggt ggagccaggt gtctgaaaag ttcattctcg
tctggctgaa 4200ctctgggagt gtgtcccagc tgagggaagc acaagtagca
aagctcattg 4250gtctggtctc ttgtttgcca ggctgattga agcaggcctt
gatgagggtg 4300catgagtgta tgtttgcatt cacatgaagg aattgctttt
cacaccagaa 4350attcagactt agtcaatgtt ggctgaattc ctaaatccag
gaagaagcct 4400ggacgtaggg tcattagctt tgggaataga aggctacaca
gaagcacact 4450gtttttgaac ttgacaacag ctctcccttt accctggact
tcagcccaag 4500ttccgtcttt ggtcttggtg gataaacaca cagtgtggag
atcccacgta 4550ctgcatttta gggatgtttt taggacaacc tccctccatg
ccttcagagt 4600taggagtgag aatgatcaaa gcaatatgta ggtgatggag
ggagagtgta 4650ttgctaaccc ttccaggtct agtccagcgc tgagatttgg
tggttctgca 4700tgtgtgatga atctctttca cacaaataga cgagaggata
tttagggcta 4750gatgagccca gatttcttcc ccctccatct ctcagggaga
caaagaacct 4800ccttcctgga ccaaggaggt gctgccaagt tttctagccc
agtgcacata 4850cccagtcctt aagcagacat tggtagtgcc cctgccctgg
gtcccactcc 4900tgccccaccc cacccttgtc cctggccatt gcctggtggt
ctagaaacac 4950ttaaaacttg aagtagtgac acctacctgc ggtcatattg
tagagagatg 5000ctcagtgtta aaactgaaac acacaaacac acacacacac
acatttttct 5050cttgtagatt ttaatttttt aagtgggaaa gaactcacct
tgccttcctc 5100ccccaaatgt gcaacctgta aaaggtctct ccacaccagg
ggccaggatc 5150cagttccctc atctctggca ggaaagatcc acagcttttc
ctccatgtct 5200gttactcact ttcagcagtc cgggtaaaat ctgtggatca
gggttaaaaa 5250agcaccgtgg agaatggccc tcttcaggaa agaaaaataa
gcaaatgaat 5300ggtccaccta ggggttcagt aaagaaagaa atgtgttaac
tgagcctgaa 5350tcccttctgg gaagtaataa tgaccattga caactaagaa
gtagacacca 5400tgctaaagac ttacatacaa tctccttgaa tcttctcaat
agcccattga 5450cttagaaact gttactttcc cattttacac acagtgaaac
tgaggctcag 5500atataaagga aaggtactgg cttgaagtca caaccacgac
aggagtaagg 5550atttggaata aggatttggt cctgttttct ggaccaaatc
cttactctgg 5600ctctgcttac actttctctc catcaccaaa tccttactcc
aaatccagaa 5650gtcagagcca actcccatct tggttctgac ccaaatcctg
ctctggactc 5700tggagaggag attgaaatat aattgcaccc tcatacacat
ttaggaaatg 5750gttaagaagt gtaaactgaa cccttatcct tgtcttcaat
cttcctccct 5800gtagacatct atcttattat ggttattatt cagaaaaccc
agggatacag 5850gtttgtcttc ttactttgat aactcttctt agtttaaaat
aataataata 5900acacatcttt ggtcatctat gtcacacaaa aattttcctt
tgtttgcggg 5950gggctgggga tgcagtgttt tttggggggt cttggtttat
gctccctgcc 6000cttgagcccc tcagccgttt gccctgcccc cacctcggct
ccatggtggg 6050agggggctct ggtcttttct aaagtgggcg gtttgtcttt
tgatctttcc 6100cttttggatg tgcgtgtgtg tctgcgtgtg ccatgtgcgt
ggcacgcata 6150tgagtgtgtg tgcgtgtgaa cggctttggg tcctgctggt
tttgctgtga 6200gctgcagtgt tctgtgggtc tgtggtatct gacactgtgg
acattaatgt 6250acttcttgga cattttaata aattttttaa cagttcaaaa
aaaaaaaaaa 6300aaaaaaaaaa
6310274577DNAHomo Sapien 27actagagatg gcgggcgggc
tgctctgaag agacctcggc ggcggcggag 50gaggagagaa gcgcagcgcc
gcgccgcgcc ggggcccatg tggggaggag 100tcggagtcgc tgttgccgcc
gccgcctgta gctgctggac ccgagtggga 150gtgaggggga aacggcagga
tgaagttcgc cgagcacctc tccgcgcaca 200tcactcccga gtggaggaag
caatacatcc agtatgaggc tttcaaggat 250atgctgtatt cagctcagga
ccaggcacct tctgtggaag ttacagatga 300ggacacagta aagaggtatt
ttgccaagtt tgaagagaag tttttccaaa 350cctgtgaaaa agaacttgcc
aaaatcaaca cattttattc agagaagctc 400gcagaggctc agcgcaggtt
tgctacactt cagaatgagc ttcagtcatc 450actggatgca cagaaagaaa
gcactggtgt tactacgctg cgacaacgca 500gaaagccagt cttccacttg
tcccatgagg aacgtgtcca acatagaaat 550attaaagacc ttaaactggc
cttcagtgag ttctacctca gtctaatcct 600gctgcagaac tatcagaatc
tgaattttac agggtttcga aaaatcctga 650aaaagcatga caagatcctg
gaaacatctc gtggagcaga ttggcgagtg 700gctcacgtag aggtggcccc
attttataca tgcaagaaaa tcaaccagct 750tatctctgaa actgaggctg
tagtgaccaa tgaacttgaa gatggtgaca 800gacaaaaggc tatgaagcgt
ttacgtgtcc cccctttggg agctgctcag 850cctgcaccag catggactac
ttttagagtt ggcctatttt gtggaatatt 900cattgtactg aatattaccc
ttgtgcttgc cgctgtattt aaacttgaaa 950cagatagaag tatatggccc
ttgataagaa tctatcgggg tggctttctt 1000ctgattgaat tcctttttct
actgggcatc aacacgtatg gttggagaca 1050ggctggagta aaccatgtac
tcatctttga acttaatccg agaagcaatt 1100tgtctcatca acatctcttt
gagattgctg gattcctcgg gatattgtgg 1150tgcctgagcc ttctggcatg
cttctttgct ccaattagtg tcatccccac 1200atatgtgtat ccacttgccc
tttatggatt tatggttttc ttccttatca 1250accccaccaa aactttctac
tataaatccc ggttttggct gcttaaactg 1300ctgtttcgag tatttacagc
ccccttccat aaggtaggct ttgctgattt 1350ctggctggcg gatcagctga
acagcctgtc agtgatactg atggacctgg 1400aatatatgat ctgcttctac
agtttggagc tcaaatggga tgaaagtaag 1450ggcctgttgc caaataattc
agaagaatca ggaatttgcc acaaatatac 1500atatggtgtg cgggccattg
ttcagtgcat tcctgcttgg cttcgcttca 1550tccagtgcct gcgccgatat
cgagacacaa aaagggcctt tcctcattta 1600gttaatgctg gcaagtactc
cacaactttc ttcatggtgg cgtttgcagc 1650cctttacagc actcacaaag
aacgaggtca ctcggacact atggtgttct 1700tttacctgtg gattgtcttt
tatatcatca gttcctgcta taccctcatc 1750tgggatctca agatggactg
gggtctcttc gataagaatg ctggagagaa 1800cactttcctc cgggaagaga
ttgtataccc ccaaaaagcc tactactact 1850gtgccataat agaggatgtg
attctgcgct ttgcttggac tatccaaatc 1900tcgattacct ctacaacttt
gttgcctcat tctggggaca tcattgctac 1950tgtctttgcc ccacttgagg
ttttccggcg atttgtgtgg aacttcttcc 2000gcctggagaa tgaacatctg
aataactgtg gtgaattccg tgctgtgcgg 2050gacatctctg tggcccccct
gaacgcagat gatcagactc tcctagaaca 2100gatgatggac caggatgatg
gggtacgaaa ccgccagaag aatcggtcat 2150ggaagtacaa ccagagcata
tccctgcgcc ggcctcgcct cgcttctcaa 2200tccaaggctc gtgacactaa
ggtattgata gaagacacag atgatgaagc 2250taacacttga attttctgaa
gtctagctta acatctttgg ttttcctact 2300ctacaatcct ttcctcgacc
aacgcaacct ctagtacctt tccagccgaa 2350aacaggagaa aacacataac
acattttccg agctcttccg gatcggatcc 2400tatggactcc aaacaagctc
actgtgtttc ttttcttttc ttctggttta 2450attttaattt tctattttca
aaacaagtat ttacttcatt tgccaatcag 2500aggatgtttt aagaaacaaa
acatagtatc ttatggattg tttacaatca 2550caaggacata gatacctatc
aggatgaaga acaggcattg caaggaccct 2600ctgatgggac ggtactgaga
tatctcggct tccgctcagc ccggttttga 2650atggttgaaa ccggacattg
gtttttaaat tttttgtcag tttatgtgga 2700gaattttttt ctttccttca
tacccagcgc aaaggcactg gccgcacttg 2750caggaaaagt gcaacttaaa
gcagtacctt cattcatgaa gctacttttt 2800aatttgatgt aacttttctt
attttgggaa gggttgctgg gtgggtggga 2850aatatgatgt atttgttaca
catagttttc tcattattta tgaaacttaa 2900ccatacagaa tgatataact
cctgtgcaat gaaggtgata acagtaaaag 2950tgatataact cctgtgcaat
gaaggtgata acagtaaaag aaggcagggg 3000aaacttacgt tggatgacat
ttatgagggt cagtcccaca tacctctttc 3050aggagacaac ttgcaccagt
ttgacctttt cttttctttg tttttatttt 3100aagccaaagt ttcattgcta
acttcttaag ttgctgctgc tttagagtcc 3150tgagcatatc tctcataaca
aggaatccca cacttcacac caccggctga 3200atttcatgga agaggttctg
ataatttttt taacttttta aggaacagat 3250gtggaataca ctggcccata
tttcaacctt aacagctgaa gctatgcctt 3300attatgcatc cacatgtatg
gtccctgtag cgtgaccttt actagctctg 3350aatcagaaga cagagctatt
tcagaggctc tgtgtgccct cactagatag 3400tttttcttct gggttcaacc
actttagcca gaatttgatc aaattaaaag 3450tctgtcatgg ggaaactata
tttttgagca catggaacaa attatacttc 3500ctcattcata ttatgttgat
acaaaagacc ttggcagcca tttctcccag 3550cagttttaaa ggatgaacat
tggatttcat gccatcccat agaaaacctg 3600ttttaaaatt ttagggatct
ttacttggtc atacatgaaa agtacactgc 3650ttagaaatta tagactatta
tgatctgtcc acagtgccca ttgtcacttc 3700tttgtctcat ttcttccctt
tgttccttag tcatccaaat aagcctgaaa 3750accataagag atattacttt
attgaatatg gttggcatta aatttagcat 3800ttcattatct aacaaaatta
atataaattc caggacatgg taaaatgtgt 3850tttaataacc cccagaccca
aatgaaaatt tcaaagtcaa taccagcaga 3900ttcatgaaag taaatttagt
cctataattt tcagcttaat tataaacaaa 3950ggaacaaata agtggaaggg
cagctattac cattcgctta gtcaaaacat 4000tcggttactg ccctttaata
cactcctatc atcagcactt ccaccatgta 4050ttacaagtct tgacccatcc
ctgtcgtaac tccagtaaaa gttactgtta 4100ctagaaaatt tttatcaatt
aactgacaaa tagtttcttt ttaaagtagt 4150ttcttccatc tttattctga
ctagcttcca aaatgtgttc cctttttgaa 4200tcgaggtttt tttgttttgt
tttgttttct gaaaaaatca tacaactttg 4250tgcttctatt gcttttttgt
gttttgttaa gcatgtccct tggcccaaat 4300ggaagaggaa atgtttaatt
aatgcttttt agtttaaata aattgaatca 4350tttataataa tcagtgttaa
caatttagtg acccttggta ggttaaaggt 4400tgcattattt atacttgaga
tttttttccc ctaactattc tgttttttgt 4450actttaaaac tatgggggaa
atatcactgg tctgtcaaga aacagcagta 4500attattactg agttaaattg
aaaagtccag tggaccaggc atttcttata 4550taaataaaat tggtggtact
aatgtgt 4577282203DNAHomo Sapien
28cccttgctgg acccgagtgg gagtgagggg gaaacggcag gatgaagttc
50gccgagcacc tctccgcgca catcactccc gagtggagga agcaatacat
100ccagtatgag gctttcaagg atatgctgta ttcagctcag gaccaggcac
150cttctgtgga agttacagat gaggacacag taaagaggta ttttgccaag
200tttgaagaga agtttttcca aacctgtgaa aaagaacttg ccaaaatcaa
250cacattttat tcagagaagc tcgcagaggc tcagcgcagg tttgctacac
300ttcagaatga gcttcagtca tcactggatg cacagaaaga aagcactggt
350gttactacgc tgcgacaacg cagaaagcca gtcttccact tgtcccatga
400ggaacgtgtc caacatagaa atattaaaga ccttaaactg gccttcagtg
450agttctacct cagtctaatc ctgctgcaga actatcagaa tctgaatttt
500acagggtttc gaaaaatcct gaaaaagcat gacaagatcc tggaaacatc
550tcgtggagca gattggcgag tggctcacgt agaggtggcc ccattttata
600catgcaagaa aatcaaccag cttatctctg aaactgaggc tgtagtgacc
650aatgaacttg aagatggtga cagacaaaag gctatgaagc gtttacgtgt
700cccccctttg ggagctgctc agcctgcacc agcatggact acttttagag
750ttggcctatt ttgtggaata ttcattgtac tgaatattac ccttgtgctt
800gccgctgtat ttaaacttga aacagataga agtatatggc ccttgataag
850aatctatcgg ggtggctttc ttctgattga attccttttt ctactgggca
900tcaacacgta tggttggaga caggctggag taaaccatgt actcatcttt
950gaacttaatc cgagaagcaa tttgtctcat caacatctct ttgagattgc
1000tggattcctc gggatattgt ggtgcctgag ccttctggca tgcttctttg
1050ctccaattag tgtcatcccc acatatgtgt atccacttgc cctttatgga
1100tttatggttt tcttccttat caaccccacc aaaactttct actataaatc
1150ccggttttgg ctgcttaaac tgctgtttcg agtatttaca gcccccttcc
1200ataaggtagg ctttgctgat ttctggctgg cggatcagct gaacagcctg
1250tcagtgatac tgatggacct ggaatatatg atctgcttct acagtttgga
1300gctcaaatgg gatgaaagta agggcctgtt gccaaataat tcagaagaat
1350caggaatttg ccacaaatat acatatggtg tgcgggccat tgttcagtgc
1400attcctgctt ggcttcgctt catccagtgc ctgcgccgat atcgagacac
1450aaaaagggcc tttcctcatt tagttaatgc tggcaaatac tccacaactt
1500tcttcatggt gacgtttgca gccctttaca gcactcacaa agaacgaggt
1550cactcggaca ctatggtgtt cttttacctg tggattgtct tttatatcat
1600cagttcctgc tataccctca tctgggatct caagatggac tggggtctct
1650tcgataagaa tgctggagag aacactttcc tccgggaaga gattgtatac
1700ccccaaaaag cctactacta ctgtgccata atagaggatg tgattctgcg
1750ctttgcttgg actatccaaa tctcgattac ctctacaact ttgttgcctc
1800attctgggga catcattgct actgtctttg ccccacttga ggttttccgg
1850cgatttgtgt ggaacttctt ccgcctggag aatgaacatc tgaataactg
1900tggtgaattc cgtgctgtgc gggacatctc tgtggccccc ctgaacgcag
1950atgatcagac tctcctagaa cagatgatgg accaggatga tggggtacga
2000aaccgccaga agaatcggtc atggaagtac aaccagagca tatccctgcg
2050ccggcctcgc ctcgcttctc aatccaaggc tcgtgacact aaggtattga
2100tagaagacac agatgatgaa gctaacactt gaattttctg aagtctagct
2150taacatcttt ggttttccta ctctacaatc ctttcctcga ccaacgcaag
2200ggc 2203293162DNAHomo Sapien 29gcgccctagc cctctttcgg ggatactggc
cgaccccctc ttccttttcc 50cctttagtga aggcctcccc cgtcgccgcg
cggcttcccg gagccgactg 100cagactccct cagcccggtg ttccccgcgt
ccggacgccg aggtcgcggc 150ttcgcagaaa ctcgggcccc tccatccgcc
ctcagaaaag ggagcgatgt 200tgatctcagg aagcacaaag ggaccttcct
agctctgact gaaccacgga 250gctcaccctg gacagtatca ctccgtggag
gaagactgtg agactgtggc 300tggaagccag attgtagcca cacatccgcc
cctgccctac cccagagccc 350tggagcagca actggctgca gatcacagac
acagtgagga tatgagtgta 400ggggtgagca cctcagcccc tctttcccca
acctcgggca caagcgtggg 450catgtctacc ttctccatca tggactatgt
ggtgttcgtc ctgctgctgg 500ttctctctct tgccattggg ctctaccatg
cttgtcgtgg ctggggccgg 550catactgttg gtgagctgct gatggcggac
cgcaaaatgg gctgccttcc 600ggtggcactg tccctgctgg ccaccttcca
gtcagccgtg gccatcctgg 650gtgtgccgtc agagatctac cgatttggga
cccaatattg gttcctgggc 700tgctgctact ttctggggct gctgatacct
gcacacatct tcatccccgt 750tttctaccgc ctgcatctca ccagtgccta
tgagtacctg gagcttcgat 800tcaataaaac tgtgcgagtg tgtggaactg
tgaccttcat ctttcagatg 850gtgatctaca tgggagttgt gctctatgct
ccgtcattgg ctctcaatgc 900agtgactggc tttgatctgt ggctgtccgt
gctggccctg ggcattgtct 950gtaccgtcta tacagctctg ggtgggctga
aggccgtcat ctggacagat 1000gtgttccaga cactggtcat gttcctcggg
cagctggcag ttatcatcgt 1050ggggtcagcc aaggtgggcg gcttggggcg
tgtgtgggcc gtggcttccc 1100agcacggccg catctctggg tttgagctgg
atccagaccc ctttgtgcgg 1150cacaccttct ggaccttggc cttcgggggt
gtcttcatga tgctctcctt 1200atacggggtg aaccaggctc aggtgcagcg
gtacctcagt tcccgcacgg 1250agaaggctgc tgtgctctcc tgttatgcag
tgttcccctt ccagcaggtg 1300tccctctgcg tgggctgcct cattggcctg
gtcatgttcg cgtattacca 1350ggagtatccc atgagcattc agcaggctca
ggcagcccca gaccagttcg 1400tcctgtactt tgtgatggat ctcctgaagg
gcctgccagg cctgccaggg 1450ctcttcattg cctgcctctt cagcggctct
ctcagcacta tatcctctgc 1500ttttaattca ttggcaactg ttacgatgga
agacctgatt cgaccttggt 1550tccctgagtt ctctgaagcc cgggccatca
tgctttccag aggccttgcc 1600tttggctatg ggctgctttg tctaggaatg
gcctatattt cctcccagat 1650gggacctgtg ctgcaggcag caatcagcat
ctttggcatg gttgggggac 1700cgctgctggg actcttctgc cttggaatgt
tctttccatg tgctaaccct 1750cctggtgctg ttgtgggcct gttggctggg
ctcgtcatgg ccttctggat 1800tggcatcggg agcatcgtga ccagcatggg
cttcagcatg ccaccctctc 1850cctctaatgg gtccagcttc tccctgccca
ccaatctaac cgttgccact 1900gtgaccacac tgatgccctt gactaccttc
tccaagccca cagggctgca 1950gcggttctat tccttgtctt acttatggta
cagtgctcac aactccacca 2000cagtgattgt ggtgggcctg attgtcagtc
tactcactgg gagaatgcga 2050ggccggtccc tgaaccctgc aaccatttac
ccagtgttgc caaagctcct 2100gtccctcctt ccgttgtcct gtcagaagcg
gctccactgc aggagctacg 2150gccaggacca cctcgacact ggcctgtttc
ctgagaagcc gaggaatggt 2200gtgctggggg acagcagaga caaggaggcc
atggccctgg atggcacagc 2250ctatcagggg agcagctcca cctgcatcct
ccaggagacc tccctgtgat 2300gttgactcag gaccccgcct ctgtcctcac
tgtgccaggc catagccaga 2350ggccaccctg tagtacaggg atgagtcttg
gtgtgttctg cagggacagg 2400cctggatgat ctagctcata ccaaaggacc
ttgttctgag aggttcttgc 2450ctgcaggaga agctgtcaca tctcaagcat
gtgaggcacc gtttttctcg 2500tcgcttgcca atctgttttt taaaggatca
ggctcgtagg gagcaggatc 2550atgccagaaa tagggatgga agtgcatcct
ctgggaaaaa gataatggct 2600tctgattcaa catagccata gtcctttgaa
gtaagtggct agaaacagca 2650ctctggttat aattgcccca gggcctgatt
caggactgac tctccaccat 2700aaaactggaa gctgcttccc ctgtagtccc
catttcagta ccagttctgc 2750cagccacagt gagcccctat tattactttc
agattgtctg tgacactcaa 2800gcccctctca tttttatctg tctacctcca
ttctgaagag ggaggttttg 2850gtgtccctgg tcctctggga atagaagatc
catttgtctt tgtgtagagc 2900aagcacgttt tccacctcac tgtctccatc
ctccacctct gagatggaca 2950cttaagagac ggggcaaatg tggatccaag
aaaccagggc catgaccagg 3000tccactgtgg agcagccatc tatctacctg
actcctgagc caggctgccg 3050tggtgtcatt tctgtcatcc gtgctctgtt
tccttttgga gtttcttctc 3100cacattatct ttgttcctgg ggaataaaaa
ctaccattgg acctaaaaaa 3150aaaaaaaaaa aa
3162301432DNAHomo Sapien 30gcgggcgccc
agtgcaccgg aggaggtgag cgccaggtcg ccttcgcggc 50ccggggacac
aggcagggac gcgggagctg atgcggctgg accggccggg 100gaaacagtat
tttctggaag ggggcccctc tgaagcggtc caggatcctg 150cacatggcgc
tgaccggggc ctcagacccc tctgcagagg cagaggccaa 200cggggagaag
ccctttctgc tgcgggcatt gcagatcgcg ctggtggtct 250ccctctactg
ggtcacctcc atctccatgg tgttccttaa taagtacctg 300ctggacagcc
cctccctgcg gctggacacc cccatcttcg tcaccttcta 350ccagtgcctg
gtgaccacgc tgctgtgcaa aggcctcagc gctctggccg 400cctgctgccc
tggtgccgtg gacttcccca gcttgcgcct ggacctcagg 450gtggcccgca
gcgtcctgcc cctgtcggtg gtcttcatcg gcatgatcac 500cttcaataac
ctctgcctca agtacgtcgg tgtggccttc tacaatgtgg 550gccgctcact
caccaccgtc ttcaacgtgc tgctctccta cctgctgctc 600aagcagacca
cctccttcta tgccctgctc acctgcggta tcatcatcgg 650gggcttctgg
cttggtgtgg accaggaggg ggcagaaggc accctgtcgt 700ggctgggcac
cgtcttcggc gtgctggcta gcctctgtgt ctcgctcaac 750gccatctaca
ccacgaaggt gctcccggcg gtggacggca gcatctggcg 800cctgactttc
tacaacaacg tcaacgcctg catcctcttc ctgcccctgc 850tcctgctgct
cggggagctt caggccctgc gtgaccttgc ccagctgggc 900agtgcccact
tctgggggat gatgacgctg ggcggcctgt ttggctttgc 950catcggctac
gtgacaggac tgcagatcaa gttcaccagt ccgctgaccc 1000acaatgtgtc
gggcacggcc aaggcctgtg cccagacagt gctggccgtg 1050ctctactacg
aggagaccaa gagcttcctc tggtggacga gcaacatgat 1100ggtgctgggc
ggctcctccg cctacacctg ggtcaggggc tgggagatga 1150agaagactcc
ggaggagccc agccccaaag acagcgagaa gagcgccatg 1200ggggtgtgag
caccacaggc accctggatg gcccggcccc ggggcccgta 1250cacaggcggg
gccagcacag tagtgaaggc ggtctcctgg accccagaag 1300cgtgctgtgg
tgtggactgg gtgctactta tagacccaat cagaatacgg 1350tggttgagaa
ggaaccagtg tttacaagta atatcagaaa gttgaaggaa 1400ccagtgttta
caagtaatac cagaaagttg cc
1432311094DNAHomo Sapien 31gcccttatcc tgcacatggc gctgaccggg gcctcagacc
cctctgcaga 50ggcagaggcc aacggggaga agccctttct gctgcgggca
ttgcagatcg 100cgctggtggt ctccctctac tgggtcacct ccatctccat
ggtgttcctt 150aataagtacc tgctggacag cccctccctg cggctggaca
cccccatctt 200cgtcaccttc taccagtgcc tggtgaccac gctgctgtgc
aaaggcctca 250gcgctctggc cgcctgctgc cctggtgccg tggacttccc
cagcttgcgc 300ctggacctca gggtggcccg cagcgtcctg cccctgtcgg
tggtcttcat 350cggcatgatc accttcaata acctctgcct caagtacgtc
ggtgtggcct 400tctacaatgt gggccgctca ctcaccaccg tcttcaacgt
gctgctctcc 450tacctgctgc tcaagcagac cacctccttc tatgccctgc
tcacctgcgg 500tatcatcatc gggggcttct ggcttggtgt ggaccaggag
ggggcagaag 550gcaccctgtc gtggctgggc accgtcttcg gcgtgctggc
tagcctctgt 600gtctcgctca acgccatcta caccacgaag gtgctcccgg
cggtggacgg 650cagcatctgg cgcctgactt tctacaacaa cgtcaacgcc
tgcatcctct 700tcctgcccct gctcctgctg ctcggggagc ttcaggccct
gcgtgacttt 750gcccagctgg gcagtgccca cttctggggg atgatgacgc
tgggcggcct 800gtttggcttt gccatcggct acgtgacagg actgcagatc
aagttcacca 850gtccgctgac ccacaatgtg tcgggcacgg ccaaggcctg
tgcccagaca 900gtgctggccg tgctctacta cgaggagacc aagagcttcc
tctggtggac 950gagcaacatg atggtgctgg gcggctcctc cgcctacacc
tgggtcaggg 1000gctgggagat gaagaagact ccggaggagc ccagccccaa
agacagcgag 1050aagagcgcca tgggggtgtg agcaccacag gcaccctgaa
gggc 109432900DNAHomo Sapien 32ccgagcgcgg ggcaccgggg
gcctcctgta taggcgggca ccatgggctc 50ctgctccggc cgctgcgcgc
tcgtcgtcct ctgcgctttt cagctggtcg 100ccgccctgga gaggcaggtg
tttgacttcc tgggctacca gtgggcgccc 150atcctggcca actttgtcca
catcatcatc gtcatcctgg gactcttcgg 200caccatccag taccggctgc
gctacgtcat ggtgtacacg ctgtgggcag 250ccgtctgggt cacctggaac
gtcttcatca tctgcttcta cctggaagtc 300ggtggcctct tacaggacag
cgagctactg accttcagcc tctcccggca 350tcgctcctgg tggcgtgagc
gctggccagg ctgtctgcat gaggaggtgc 400cagcagtggg cctcggggcc
ccccatggcc aggccctggt gtcaggtgct 450ggctgtgccc tggagcccag
ctatgtggag gccctacaca gtggcctgca 500gatcctgatc gcgcttctgg
gctttgtctg tggctgccag gtggtcagcg 550tgtttacgga ggaagaggac
agctttgatt tcattggtgg atttgatcca 600tttcctctct accatgtcaa
tgaaaagcca tccagtctct tgtccaagca 650ggtgtacttg cctgcgtaag
tgaggaaaca gctgatcctg ctcctgtggc 700ctccagcctc agcgaccgac
cagtgacaat gacaggagct cccaggcctt 750gggacgcgcc cccacccagc
accccccagg cggccggcag cacctgccct 800gggttctaag tactggacac
cagccagggc ggcagggcag tgccacggct 850ggctgcagcg tcaagagagt
ttgtaatttc ctttctctta aaaaaaaaaa 90033666DNAHomo Sapien
33ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc
50gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt
100tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca
150tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc
200tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt
250cttcatcatc tgcttctacc tggaagtcgg tggcctctta aaggacagcg
300agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc
350tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc
400ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct
450atgtggaggc cctacacagt tgcctgcaga tcctgatcgc gcttctgggc
500tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag
550ctttgatttc attggtggat ttgatccatt tcctctctac catgtcaatg
600aaaagccatc cagtctcttg tccaagcagg tgtacttgcc tgcgtaagtg
650aggaaacagc tgatcc
66634582DNAHomo sapien 34ctcctgtata ggcgggcacc atgggctcct gctccggccg
ctgcgcgctc 50gtcgtcctct gcgcttttca gctggtcgcc gccctggaga
ggcaggtgtt 100tgacttcctg ggctaccagt gggcgcccat cctggccaac
tttgtccaca 150tcatcatcgt catcctggga ctcttcggca ccatccagta
ccggctgcgc 200tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca
cctggaacgt 250cttcatcatc tgcttctacc tggaagtcgg tggcctctta
aaggacagcg 300agctactgac cttcagcctc tcccggcatc gctcctggtg
gcgtgagcgc 350tggccaggct gtctgcatga ggaggtgcca gcagtgggcc
tcggggcccc 400ccatggccag gccctggtgt caggtgctgg ctgtgccctg
gagcccagct 450atgtggaggc cctacacagt tgcctgcaga tcctgatcgc
gcttctgggc 500tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg
aagaggacag 550ctgcctgcgt aagtgaggaa acagctgatc ca
58235582DNAHomo Sapien 35ctcctgtata ggcgggcacc
atgggctcct gctccggccg ctgcgcgctc 50gtcgtcctct gcgcttttca
gctggtcgcc gccctggaga ggcaggtgtt 100tgacttcctg ggctaccagt
gggcgcccat cctggccaac tttgtccaca 150tcatcatcgt catcctggga
ctcttcggca ccatccagta ccggctgcgc 200tacgtcatgg tgtacacgct
gtgggcagcc gtctgggtca cctggaacgt 250cttcatcatc tgcttctacc
tggaagtcgg tggcctctta caggacagcg 300agctactgac cttcagcctc
tcccggcatc gctcctggtg gcgtgagcgc 350tggccaggct gtctgcatga
ggaggtgcca gcagtgggcc tcggggcccc 400ccatggccag gccctggtgt
caggtgctgg ctgtgccctg gagcccagct 450atgtggaggc cctacacagt
ggcctgcaga tcctgatcgc gcttctgggc 500tttgtctgtg gctgccaggt
ggtcagcgtg tttacggagg aagaggacag 550ctgcctgcgt aagtgaggaa
acagctgatc ca 582361546DNAHomo sapien
36gcatggaaag tctttatttg agccccttag ctgatgtgga atcagaagag
50caaaaaggtc atcttcagag tggcctgggc tgggtccttt tctctccagg
100atagaaaagt ggtggtcact ttatccctag tagacatgct gctgggcttt
150atcgccccag cattcccatc ccctccagag ccccttgtca ctccagacca
200gcgagtgtgg gcctttatct ggactctgct tcctccctgg ggacaccagg
250tcttggagca agagaacttg gcaggctctc cccatggcag tcttattcct
300cctcctgttc ctatgtggaa ctccccaggc tgcagacaac atgcaggcca
350tctatgtggc cttgggggag gcagtagagc tgccatgtcc ctcaccacct
400actctacatg gggacgaaca cctgtcatgg ttctgcagcc ctgcagcagg
450ctccttcacc accctggtag cccaagtcca agtgggcagg ccagccccag
500accctggaaa accaggaagg gaatccaggc tcagactgct ggggaactat
550tctttgtggt tggagggatc caaagaggaa gatgccgggc ggtactggtg
600cgctgtgcta ggtcagcacc acaactacca gaactggagg gtgtacgacg
650tcttggtgct caaaggatcc cagttatctg caagggctgc agatggatcc
700ccctgcaatg tcctcctgtg ctctgtggtc cccagcagac gcatggactc
750tgtgacctgg caggaaggga agggtcccgt gaggggccgt gttcagtcct
800tctggggcag tgaggctgcc ctgctcttgg tgtgtcctgg ggaggggctt
850tctgagccca ggagccgaag accaagaatc atccgctgcc tcatgactca
900caacaaaggg gtcagcttta gcctggcagc ctccatcgat gcttctcctg
950ccctctgtgc cccttccacg ggctgggaca tgccttggat tctgatgctg
1000ctgctcacaa tgggccaggg agttgtcatc ctggccctca gcatcgtgct
1050ctggaggcag agggtccgtg gggctccagg cagaggaaac cgaatgcggt
1100gctacaactg tggtggaagc cccagcagtt cttgcaaaga ggccgtgacc
1150acctgtggcg agggcagacc ccagccaggc ctggaacaga tcaagctacc
1200tggaaacccc ccagtgacct tgattcacca acatccagcc tgcgtcgcag
1250cccatcattg caatcaagtg gagacagagt cggtgggaga cgtgacttat
1300ccagcccaca gggactgcta cctgggagac ctgtgcaaca gcgccgtggc
1350aagccatgtg gcccctgcag gcattttggc tgcagcagct accgccctga
1400cctgtctctt gccaggactg tggagcggat agggggagta ggagtagaga
1450agggaacaag ggagcaaggg aacaagggac atctgaacat ctaatgtgag
1500aagagaaaca tccttctgtg agtcattaaa atctatgaac cactct
1546374619DNAHomo Sapien 37ctttagagaa aggaagggcc aaaactacga cttggctttc
tgaaacggaa 50gcataaatgt tcttttcctc catttgtctg gatctgagaa
cctgcatttg 100gtattagcta gtggaagcag tatgtatggt tgaagtgcat
tgctgcagct 150ggtagcatga gtggtggcca ccagctgcag ctggctgccc
tctggccctg 200gctgctgatg gctaccctgc aggcaggctt tggacgcaca
ggactggtac 250tggcagcagc ggtggagtct gaaagatcag cagaacagaa
agctgttatc 300agagtgatcc ccttgaaaat ggaccccaca ggaaaactga
atctcacttt 350ggaaggtgtg tttgctggtg ttgctgaaat aactccagca
gaaggaaaat 400taatgcagtc ccacccactg tacctgtgca atgccagtga
tgacgacaat 450ctggagcctg gattcatcag catcgtcaag ctggagagtc
ctcgacgggc 500cccccgcccc tgcctgtcac tggctagcaa ggctcggatg
gcgggtgagc 550gaggagccag tgctgtcctc tttgacatca ctgaggatcg
agctgctgct 600gagcagctgc agcagccgct ggggctgacc tggccagtgg
tgttgatctg 650gggtaatgac gctgagaagc tgatggagtt tgtgtacaag
aaccaaaagg 700cccatgtgag gattgagctg aaggagcccc cggcctggcc
agattatgat 750gtgtggatcc taatgacagt ggtgggcacc atctttgtga
tcatcctggc 800ttcggtgctg cgcatccggt gccgcccccg ccacagcagg
ccggatccgc 850ttcagcagag aacagcctgg gccatcagcc agctggccac
caggaggtac 900caggccagct gcaggcaggc ccggggtgag tggccagact
cagggagcag 950ctgcagctca gcccctgtgt gtgccatctg tctggaggag
ttctctgagg 1000ggcaggagct acgggtcatt tcctgcctcc atgagttcca
tcgtaactgt 1050gtggacccct ggttacatca gcatcggact tgccccctct
gcgtgttcaa 1100catcacagag ggagattcat tttcccagtc cctgggaccc
tctcgatctt 1150accaagaacc aggtcgaaga ctccacctca ttcgccagca
tcccggccat 1200gcccactacc acctccctgc tgcctacctg ttgggccctt
cccggagtgc 1250agtggctcgg cccccacgac ctggtccctt cctgccatcc
caggagccag 1300gcatgggccc tcggcatcac cgcttcccca gagctgcaca
tccccgggct 1350ccaggagagc agcagcgcct ggcaggagcc cagcacccct
atgcacaagg 1400ctggggaatg agccacctcc aatccacctc acagcaccct
gctgcttgcc 1450cagtgcccct acgccgggcc aggccccctg acagcagtgg
atctggagaa 1500agctattgca cagaacgcag tgggtacctg gcagatgggc
cagccagtga 1550ctccagctca gggccctgtc atggctcttc cagtgactct
gtggtcaact 1600gcacggacat cagcctacag ggggtccatg gcagcagttc
tactttctgc 1650agctccctaa gcagtgactt tgacccccta gtgtactgca
gccctaaagg 1700ggatccccag cgagtggaca tgcagcctag tgtgacctct
cggcctcgtt 1750ccttggactc ggtggtgccc acaggggaaa cccaggtttc
cagccatgtc 1800cactaccacc gccaccggca ccaccactac aaaaagcggt
tccagtggca 1850tggcaggaag cctggcccag aaaccggagt cccccagtcc
aggcctccta 1900ttcctcggac acagccccag ccagagccac cttctcctga
tcagcaagtc 1950accggatcca actcagcagc cccttcgggg cggctctcta
acccacagtg 2000ccccagggcc ctccctgagc cagcccctgg cccagttgac
gcctccagca 2050tctgccccag taccagcagt ctgttcaact tgcaaaaatc
cagcctctct 2100gcccgacacc cacagaggaa aaggcggggg ggtccctccg
agcccacccc 2150tggctctcgg ccccaggatg caactgtgca cccagcttgc
cagatttttc 2200cccattacac ccccagtgtg gcatatcctt ggtccccaga
ggcacacccc 2250ttgatctgtg gacctccagg cctggacaag aggctgctac
cagaaacccc 2300aggcccctgt tactcaaatt cacagccagt gtggttgtgc
ctgactcctc 2350gccagcccct ggaaccacat ccacctgggg aggggccttc
tgaatggagt 2400tctgacaccg cagagggcag gccatgccct tatccgcact
gccaggtgct 2450gtcggcccag cctggctcag aggaggaact cgaggagctg
tgtgaacagg 2500ctgtgtgaga tgttcaggcc tagctccaac caagagtgtg
ctccagatgt 2550gtttgggccc tacctggcac agagtcctgc tcctgggaaa
ggaaaggacc 2600acagcaaaca ccattctttt tgccgtactt cctagaagca
ctggaagagg 2650actggtgatg gtggagggtg agagggtgcc gtttcctgct
ccagctccag 2700accttgtctg cagaaaacat ctgcagtgca gcaaatccat
gtccagccag 2750gcaaccagct gctgcctgtg gcgtgtgtgg gctggatccc
ttgaaggctg 2800agtttttgag ggcagaaagc tagctatggg tagccaggtg
ttacaaaggt 2850gctgctcctt ctccaacccc tacttggttt ccctcacccc
aagcctcatg 2900ttcataccag ccagtgggtt cagcagaacg catgacacct
tatcacctcc 2950ctccttgggt gagctctgaa caccagcttt ggcccctcca
cagtaaggct 3000gctacatcag gggcaaccct ggctctatca ttttcctttt
ttgccaaaag 3050gaccagtagc ataggtgagc cctgagcact aaaaggaggg
gtccctgaag 3100ctttcccact atagtgtgga gttctgtccc tgaggtgggt
acagcagcct 3150tggttcctct gggggttgag aataagaata gtggggaggg
aaaaactcct 3200ccttgaagat ttcctgtctc agagtcccag agaggtagaa
aggaggaatt 3250tctgctggac ttcatctggg cagaggaagg atggaatgaa
ggtagaaaag 3300gcagaattac agctgagcgg ggacaacaaa gagttcttct
ctgggaaaag 3350ttttgtctta gagcaaggat ggaaaatggg gacaacaaag
gaaaagcaaa 3400gtgtgaccct tgggtttgga cagcccagag gcccagctcc
ccagtataag 3450ccatacaggc cagggaccca caggagagtg gattagagca
caagtctggc 3500ctcactgagt ggacaagagc tgatgggcct catcagggtg
acattcaccc 3550cagggcagcc tgaccactct tggcccctca ggcattatcc
catttggaat 3600gtgaatgtgg tggcaaagtg ggcagaggac cccacctggg
aacctttttc 3650cctcagttag tggggagact agcacctagg tacccacatg
ggtatttata 3700tctgaaccag acagacgctt gaatcaggca ctatgttaag
aaatatattt 3750atttgctaat atatttatcc acaaatgtgg tctggtcttg
tggttttgtt 3800ctgtcgtgac tgtcactcag ggtaacaacg tcatctcttt
ctacatcaag 3850agaagtaaat tatttatgtt atcagaggct aggctccgat
tcatgaaagg 3900atagggtaga gtagagggct tggcaataag aactggtttg
taagccccta 3950aaagtgtggc ttagtgagat cagggaagga gaaagcatga
ctggattctt 4000actgtgcttc agtcattatt attatactgt tcacttcaca
cattatcata 4050cttcagtgac tyagaccttg ggcaaatact ctgtgcctcg
ctttttcagt 4100ccataaaatg ggcctactta atagttgttg caggacttac
atgagataat 4150agagtgtaga aaatatgttc caaagtggaa agttttattc
agtgatagaa 4200aacatccaaa cctgtcacag agcccatctg aacacagcat
gggaccgcca 4250acaagaagaa agcccgcccg gaagcagctc aatcaggagg
ctgggctgga 4300atgacagcgc agcggggcct gaaactattt atatcccaaa
gctcctctca 4350gataaacaca aatgactgcg ttctgcctgc actcgggcta
ttgcgaggac 4400agagagctgg tgctccattg gcgtgaagtc tccagggcca
gaaggggcct 4450ttgtcgcttc ctcacaaggc acaagttccc cttctgcttc
cccgagaaag 4500gtttggtagg ggtggtggtt tagtgcctat agaacaaggc
atttcgcttc 4550ctagacggtg aaatgaaagg gaaaaaaagg acacctaatc
tcctacaaat 4600ggtctttagt aaaggaacc
4619383510DNAHomo Sapien 38gcagctctgg gggagctcgg
agctcccgat cacggcttct tgggggtagc 50tacggctggg tgtgtagaac
ggggccgggg ctggggctgg gtcccctagt 100ggagacccaa gtgcgagagg
caagaactct gcagcttcct gccttctggg 150tcagttcctt attcaagtct
gcagccggct cccagggaga tctcggtgga 200acttcagaaa cgctgggcag
tctgcctttc aaccatgccc ctgtccctgg 250gagccgagat gtgggggcct
gaggcctggc tgctgctgct gctactgctg 300gcatcattta caggccggtg
ccccgcgggt gagctggaga cctcagacgt 350ggtaactgtg gtgctgggcc
aggacgcaaa actgccctgc ttctaccgag 400gggactccgg cgagcaagtg
gggcaagtgg catgggctcg ggtggacgcg 450ggcgaaggcg cccaggaact
agcgctactg cactccaaat acgggcttca 500tgtgagcccg gcttacgagg
gccgcgtgga gcagccgccg cccccacgca 550accccctgga cggctcagtg
ctcctgcgca acgcagtgca ggcggatgag 600ggcgagtacg agtgccgggt
cagcaccttc cccgccggca gcttccaggc 650gcggctgcgg ctccgagtgc
tggtgcctcc cctgccctca ctgaatcctg 700gtccagcact agaagagggc
cagggcctga ccctggcagc ctcctgcaca 750gctgagggca gcccagcccc
cagcgtgacc tgggacacgg aggtcaaagg 800cacaacgtcc agccgttcct
tcaagcactc ccgctctgct gccgtcacct 850cagagttcca cttggtgcct
agccgcagca tgaatgggca gccactgact 900tgtgtggtgt cccatcctgg
cctgctccag gaccaaagga tcacccacat 950cctccacgtg tccttccttg
ctgaggcctc tgtgaggggc cttgaagacc 1000aaaatctgtg gcacattggc
agagaaggag ctatgctcaa gtgcctgagt 1050gaagggcagc cccctccctc
atacaactgg acacggctgg atgggcctct 1100gcccagtggg gtacgagtgg
atggggacac tttgggcttt cccccactga 1150ccactgagca cagcggcatc
tacgtctgcc atgtcagcaa tgagttctcc 1200tcaagggatt ctcaggtcac
tgtggatgtt cttgaccccc aggaagactc 1250tgggaagcag gtggacctag
tgtcagcctc ggtggtggtg gtgggtgtga 1300tcgccgcact cttgttctgc
cttctggtgg tggtggtggt gctcatgtcc 1350cgataccatc ggcgcaaggc
ccagcagatg acccagaaat atgaggagga 1400gctgaccctg accagggaga
actccatccg gaggctgcat tcccatcaca 1450cggaccccag gagccagccg
gaggagagtg tagggctgag agccgagggc 1500caccctgata gtctcaagga
caacagtagc tgctctgtga tgagtgaaga 1550gcccgagggc cgcagttact
ccacgctgac cacggtgagg gagatagaaa 1600cacagactga actgctgtct
ccaggctctg ggcgggccga ggaggaggaa 1650gatcaggatg aaggcatcaa
acaggccatg aaccattttg ttcaggagaa 1700tgggacccta cgggccaagc
ccacgggcaa tggcatctac atcaatgggc 1750ggggacacct ggtctgaccc
aggcctgcct cccttcccta ggcctggctc 1800cttctgttga catgggagat
tttagctcat cttgggggcc tccttaaaca 1850cccccatttc ttgcggaaga
tgctccccat cccactgact gcttgacctt 1900tacctccaac ccttctgttc
atcgggaggg ctccaccaat tgagtctctc 1950ccaccatgca tgcaggtcac
tgtgtgtgtg catgtgtgcc tgtgtgagtg 2000ttgactgact gtgtgtgtgt
ggaggggtga ctgtccgtgg aggggtgact 2050gtgtccgtgg tgtgtattat
gctgtcatat cagagtcaag tgaactgtgg 2100tgtatgtgcc acgggatttg
agtggttgcg tgggcaacac tgtcagggtt 2150tggcgtgtgt gtcatgtggc
tgtgtgtgac ctctgcctga aaaagcaggt 2200attttctcag accccagagc
agtattaatg atgcagaggt tggaggagag 2250aggtggagac tgtggctcag
acccaggtgt gcgggcatag ctggagctgg 2300aatctgcctc cggtgtgagg
gaacctgtct cctaccactt cggagccatg 2350ggggcaagtg tgaagcagcc
agtccctggg tcagccagag gcttgaactg 2400ttacagaagc cctctgccct
ctggtggcct ctgggcctgc tgcatgtaca 2450tattttctgt aaatatacat
gcgccgggag cttcttgcag gaatactgct 2500ccgaatcact tttaattttt
ttcttttttt tttcttgccc tttccattag 2550ttgtattttt tatttatttt
tatttttatt tttttttaga gatggagtct 2600cactatgttg ctcaggctgg
ccttgaactc ctgggctcaa gcaatcctcc 2650tgcctcagcc tccctagtag
ctgggacttt aagtgtacac cactgtgcct 2700gctttgaatc ctttacgaag
agaaaaaaaa aattaaagaa agcctttaga 2750tttatccaat gtttactact
gggattgctt aaagtgaggc ccctccaaca 2800ccagggggtt aattcctgtg
attgtgaaag gggctacttc caaggcatct 2850tcatgcaggc agccccttgg
gagggcacct gagagctggt agagtctgaa 2900attagggatg tgagcctcgt
ggttactgag taaggtaaaa ttgcatccac 2950cattgtttgt gataccttag
ggaattgctt ggacctggtg acaagggctc 3000ctgttcaata gtggtgttgg
ggagagagag agcagtgatt atagaccgag 3050agagtaggag ttgaggtgag
gtgaaggagg tgctgggggt gagaatgtcg 3100cctttccccc tgggttttgg
atcactaatt caaggctctt ctggatgttt 3150ctctgggttg gggctggagt
tcaatgaggt ttatttttag ctggcccacc 3200cagatacact cagccagaat
acctagattt agtacccaaa ctcttcttag 3250tctgaaatct gctggatttc
tggcctaagg gagaggctcc catccttcgt 3300tccccagcca gcctaggact
tcgaatgtgg agcctgaaga tctaagatcc 3350taacatgtac attttatgta
aatatgtgca tatttgtaca taaaatgata 3400ttctgttttt aaataaacag
acaaaacttg aaaaaaaaaa aaaaaaaaaa 3450aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3500aaaaaaaaaa
3510392211DNAHomo sapien
39ttgggggttt attctcttcc cttctaactt gacagggtct tgctctgtca
50ttcaggcaag agtgcagtag tgtgatcact tcttactgcc gcctcaagct
100tccagcctca actcaagcaa tcctcccacc tcagccaccc aagtggctgg
150gactacagat taagaatgac ccaaaataaa ttaaagcttt gttccaaagc
200caatgtgtat actgaagtgc ctgatggagg atggggctgg gcggtagctg
250tttcattttt cttcgttgaa gtcttcacct acggcatcat caagacattt
300ggtgtcttct ttaatgactt aatggacagt tttaatgaat ccaatagcag
350gatctcatgg ataatctcaa tctgtgtgtt tgtcttaaca ttttcagctc
400ccctcgccac agtcctgagc aatcgtttcg gacaccgtct ggtagtgatg
450ttgggggggc tacttgtcag caccgggatg gtggccgcct ccttctcaca
500agaggtttct catatgtacg tcgccatcgg catcatctct ggtctgggat
550actgctttag ttttctccca actgtaacca tcctatcaca atattttggc
600aaaagacgtt ccatagtcac tgcagttgct tccacaggag aatgtttcgc
650tgtgtttgct ttcgcaccag caatcatggc tctgaaggag cgcattggct
700ggagatacag cctcctcttc gtgggcctac tacagttaaa cattgtcatc
750ttcggagcac tgctcagacc catcattatc agaggaccag cgtcaccgaa
800aatagtcatc caggaaaatc ggaaagaagc gcagtatatg cttgaaaatg
850agaaaacacg aacctcaata gactccattg actcaggagt agaactaact
900acctcaccta aaaatgtgcc tactcacact aacctggaac tggagccgaa
950ggccgacatg cagcaggtcc tggtgaagac cagccccagg ccaagcgaaa
1000agaaagcccc gctattagac ttctccattt tgaaagagaa aagttttatt
1050tgttatgcat tatttggtct ctttgcaaca ctgggattct ttgcaccttc
1100cttgtacatc attcctctgg gcattagtct gggcattgac caggaccgcg
1150ctgctttttt attatctacg atggccattg cagaagtttt cggaaggatc
1200ggagctggtt ttgtcctcaa cagggagccc attcgtaaga tttacattga
1250gctcatctgc gtcatcttat tgactgtgtc tctgtttgcc tttacttttg
1300ctactgaatt ctggggtcta atgtcatgca gcatattttt tgggtttatg
1350gttggaacaa taggaggact cacattccac tgcttgctga agatgatgtc
1400gtgggcattg cagaagatgt cttctgcagc tggggtctac atcttcattc
1450agagcatagc aggactggct ggaccgcccc ttgcaggttt gttggtggac
1500caaagtaaga tctacagcag ggccttctac tcctgcgcag ctggcatggc
1550cctggctgct gtgtgcctcg ccctggtgag accgtgtaag atgggactgt
1600gccagcgtca tcactcaggt gaaacaaagg tagtgagcca tcgtgggaag
1650actttacagg acatacctga agactttctg gaaatggatc ttgcaaaaaa
1700tgagcacaga gttcacgtgc aaatggagcc ggtatgacac actttcttac
1750aacaacagcc actgtgttgg ctggagaggg atggggtggg cccaacgggg
1800acacaaggag gcagaggagc taacccctct actccacttt caaaactaca
1850ttttaaaggg aatgtgtatg tgaagagcac taccaacatc gcttttgttt
1900tgttttgttt tgttttaagc tttttttttt tgcttgtttt taaagccaaa
1950acaaaaaaca accaagcact cttccatata taaatctggc tgtattcagt
2000agcaatacaa gagatatgta gaaagactct ttggttcaca ttccgatatt
2050aaaatagtga catgaactgg caaagtggtt ttaaaagctt tcacgtggga
2100taaatgattt tctttttttc ttttctttct tcctatggtc ttgtctgaat
2150aaactactct cctgaataaa acaacatcca acccaggtca ttgaaatgaa
2200attggccagt c
221140685DNAHomo Sapien 40gatgtgctcc ttggagctgg tgtgcagtgt cctgactgta
agatcaagtc 50caaacctgtt ttggaattga ggaaacttct cttttgatct
cagcccttgg 100tggtccaggt cttcatgctg ctgtgggtga tattactggt
cctggctcct 150gtcagtggac agtttgcaag gacacccagg cccattattt
tcctccagcc 200tccatggacc acagtcttcc aaggagagag agtgaccctc
acttgcaagg 250gatttcgctt ctactcacca cagaaaacaa aatggtacca
tcggtacctt 300gggaaagaaa tactaagaga aaccccagac aatatccttg
aggttcagga 350atctggagag tacagatgcc aggcccaggg ctcccctctc
agtagccctg 400tgcacttgga tttttcttca gagatgggat ttcctcatgc
tgcccaggct 450aatgttgaac tcctgggctc aagtgatctg ctcacctagg
cctctcaaag 500cgctgggatt acagcttcgc tgatcctgca agctccactt
tctgtgtttg 550aaggagactc tgtggttctg aggtgccggg caaaggcgga
agtaacactg 600aataatacta tttacaagaa tgataatgtc ctggcattcc
ttaataaaag 650aactgacttc caaaaaaaaa aaaaaaaaaa aaaaa
685415392DNAHomo Sapien 41aattcactaa tgcattctgc
tctttttgag agcacagctt ctcagatgtg 50ctccttggag ctggtgtgca
gtgtcctgac tgtaagatca agtccaaacc 100tgttttggaa ttgaggaaac
ttctcttttg atctcagccc ttggtggtcc 150aggtcttcat gctgctgtgg
gtgatattac tggtcctggc tcctgtcagt 200ggacagtttg caaggacacc
caggcccatt attttcctcc agcctccatg 250gaccacagtc ttccaaggag
agagagtgac cctcacttgc aagggatttc 300gcttctactc accacagaaa
acaaaatggt accatcggta cctcgggaaa 350gaaatactaa gagaaacccc
agacaatatc cttgaggttc aggaatctgg 400agagtacaga tgccaggccc
agggctcccc tctcagtagc cctgtgcact 450tggatttttc ttcagcttcg
ctgatcctgc aagctccact ttctgtgttt 500gaaggagact ctgtggttct
gaggtgccgg gcaaaggcgg aagtaacact 550gaataatact atttacaaga
atgataatgt cctggcattc cttaataaaa 600gaactgactt ccatattcct
catgcatgtc tcaaggacaa tggtgcatat 650cgctgtactg gatataagga
aagttgttgc cctgtttctt ccaatacagt 700caaaatccaa gtccaagagc
catttacacg tccagtgctg agagccagct 750ccttccagcc catcagcggg
aacccagtga ccctgacctg tgagacccag 800ctctctctag agaggtcaga
tgtcccgctc cggttccgct tcttcagaga 850tgaccagacc ctgggattag
gctggagtct ctccccgaat ttccagatta 900ctgccatgtg gagtaaagat
tcagggttct actggtgtaa ggcagcaaca 950atgcctcaca gcgtcatatc
tgacagcccg agatcctgga tacaggtgca 1000gatccctgca tctcatcctg
tcctcactct cagccctgaa aaggctctga 1050attttgaggg aaccaaggtg
acacttcact gtgaaaccca ggaagattct 1100ctgcgcactt tgtacaggtt
ttatcatgag ggtgtccccc tgaggcacaa 1150gtcagtccgc tgtgaaaggg
gagcatccat cagcttctca ctgactacag 1200agaattcagg gaactactac
tgcacagctg acaatggcct tggcgccaag 1250cccagtaagg ctgtgagcct
ctcagtcact gttcccgtgt ctcatcctgt 1300cctcaacctc agctctcctg
aggacctgat ttttgaggga gccaaggtga 1350cacttcactg tgaagcccag
agaggttcac tccccatcct gtaccagttt 1400catcatgagg atgctgccct
ggagcgtagg tcggccaact ctgcaggagg 1450agtggccatc agcttctctc
tgactgcaga gcattcaggg aactactact 1500gcacagctga caatggcttt
ggcccccagc gcagtaaggc ggtgagcctc 1550tccatcactg tccctgtgtc
tcatcctgtc ctcaccctca gctctgctga 1600ggccctgact tttgaaggag
ccactgtgac acttcactgt gaagtccaga 1650gaggttcccc acaaatccta
taccagtttt atcatgagga catgcccctg 1700tggagcagct caacaccctc
tgtgggaaga gtgtccttca gcttctctct 1750gactgaagga cattcaggga
attactactg cacagctgac aatggctttg 1800gtccccagcg cagtgaagtg
gtgagccttt ttgtcactgt tccagtgtct 1850cgccccatcc tcaccctcag
ggttcccagg gcccaggctg tggtggggga 1900cctgctggag cttcactgtg
aggccccgag aggctctccc ccaatcctgt 1950actggtttta tcatgaggat
gtcaccctgg ggagcagctc agccccctct 2000ggaggagaag cttctttcaa
cctctctctg actgcagaac attctggaaa 2050ctactcatgt gaggccaaca
atggcctagt ggcccagcac agtgacacaa 2100tatcactcag tgttatagtt
ccagtatctc gtcccatcct caccttcagg 2150gctcccaggg cccaggctgt
ggtgggggac ctgctggagc ttcactgtga 2200ggccctgaga ggctcctccc
caatcctgta ctggttttat catgaagatg 2250tcaccctggg taagatctca
gccccctctg gaggaggggc ctccttcaac 2300ctctctctga ctacagaaca
ttctggaatc tactcctgtg aggcagacaa 2350tggtccggag gcccagcgca
gtgagatggt gacactgaaa gttgcagttc 2400cggtgtctcg cccggtcctc
accctcaggg ctcccgggac ccatgctgcg 2450gtgggggacc tgctggagct
tcactgtgag gccctgagag gctctcccct 2500gatcctgtac cggttttttc
atgaggatgt caccctagga aataggtcgt 2550ccccctctgg aggagcgtcc
ttaaacctct ctctgactgc agagcactct 2600ggaaactact cctgtgaggc
cgacaatggc ctcggggccc agcgcagtga 2650gacagtgaca ctttatatca
cagggctgac cgcgaacaga agtggccctt 2700ttgccacagg agtcgccggg
ggcctgctca gcatagcagg ccttgctgcg 2750ggggcactgc tgctctactg
ctggctctcg agaaaagcag ggagaaagcc 2800tgcctctgac cccgccagga
gccctccaga ctcggactcc caagagccca 2850cctatcacaa tgtaccagcc
tgggaagagc tgcaaccagt gtacactaat 2900gcaaatccta gaggagaaaa
tgtggtttac tcagaagtac ggatcatcca 2950agagaaaaag aaacatgcag
tggcctctga ccccaggcat ctcaggaaca 3000agggttcccc tatcatctac
tctgaagtta aggtggcgtc aaccccggtt 3050tccggatccc tgttcttggc
ttcctcagct cctcacagat gagtccacac 3100gtctctccaa ctgctgtttc
agcctctgca ccccaaagtt ccccttgggg 3150gagaagcagc attgaagtgg
gaagatttag gctgccccag accatatcta 3200ctggcctttg tttcacatgt
cctcattctc agtctgacca gaatgcaggg 3250ccctgctgga ctgtcacctg
tttcccagtt aaagccctga ctggcaggtt 3300ttttaatcca gtggcaaggt
gctcccactc cagggcccag cacatctcct 3350ggattcctta gtgggcttca
gctgtgattg ctgttctgag tactgctctc 3400atcacacccc cacagagggg
gtcttaccac acaaagggag agtgggcctt 3450caggagatgc cgggctggcc
taacagctca ggtgctccta aactccgaca 3500cagagttcct gctttgggtg
gatgcatttc tcaattgtca tcagcctggt 3550ggggctactg cagtgtgctg
ccaaatggga cagcacacag cctgtgcaca 3600tgggacatgt gatgggtctc
cccacggggg ctgcatttca cactcctcca 3650cctgtctcaa actctaaggt
cggcacttga caccaaggta acttctctcc 3700tgctcatgtg tcagtgtcta
cctgcccaag taagtggctt tcatacacca 3750agtcccaagt tcttcccatc
ctaacagaag taacccagca agtcaaggcc 3800aggaggacca ggggtgcaga
cagaacacat actggaacac aggaggtgct 3850caattactat ttgactgact
gactgaatga atgaatgaat gaggaagaaa 3900actgtgggta atcaaactgg
cataaaatcc agtgcactcc ctaggaaatc 3950cgggaggtat tctggcttcc
ctaagaaaca acggaagaga aggagcttgg 4000atgaggaaac tgttcagcaa
gaggaagggc ttctcacact ttcatgtgct 4050tgtggatcac ctgaggatcc
tgtgaaaata cagatactga ttcagtgggt 4100ctgtgtagag cctgagactg
ccattctaac atgttcccag gggatgctga 4150tgctgctggc cctgggactg
cactgcatgc atgtgaagcc ctataggtct 4200cagcagaggc ccatggagag
ggaatgtgtg gctctggctg cccagggccc 4250aactcggttc acacggatcg
tgctgctccc tggccagcct ttggccacag 4300caccaccagc tgctgttgct
gagagagctt cttctctgtg acatgttggc 4350tttcatcagc caccctggga
agcggaaagt agctgccact atctttgttt 4400ccccacctca ggcctcacac
tttcccatga aaagggtgaa tgtatataac 4450ctgagccctc tccattcaga
gttgttctcc catctctgag caatgggatg 4500ttctgttccg cttttatgat
atccatcaca tcttatcttg atctttgctc 4550ccagtggatt gtacagtgat
gacttttaag ccccacggcc ctgaaataaa 4600atccttccaa gggcattgga
agctctctcc acctgaacca tggcttttca 4650tgcttccaag tgtcagggcc
ttgcccagat agacagggct gactctgctg 4700ccccaacctt tcaaggagga
aaccagacac ctgagacagg agcctgtatg 4750cagcccagtg cagccttgca
gaggacaagg ctggaggcat ttgtcatcac 4800tacagatatg caactaaaat
agacgtggag caagagaaat gcattcccac 4850cgaggccgct tttttaggcc
tagttgaaag tcaagaagga cagcagcaag 4900cataggctca ggattaaaga
aaaaaatctg ctcacagttt gttctggagg 4950tcacatcacc aacaaagctc
acgccctatg cagttctgag aaggtggagg 5000caccaggctc aaaagaggaa
atttagaatt tctcattggg agagtaaggt 5050acccccatcc cagaatgata
actgcacagt ggcagaacaa actccaccct 5100aatgtgggtg gaccccatcc
agtctgttga aggcctgagt gtaacaaaag 5150ggcttattct tcctcaagta
agggggaact cctgctttgg gctgggacat 5200aagtttttct gctttcagac
gcaaactgaa aaatggctct tcttgggtct 5250tgagcttgct ggcatatgga
ctgaaagaaa ctatgctatt ggatctcctg 5300gatctccagc ttgctgactg
cagatcttga gatatgtcag cctctacagt 5350cacaagagct aattcattct
aataaaccaa tctttctgta aa 539242626DNAHomo Sapien
42ggacctggga aggagcatag gacagggcaa ggcgggataa ggaggggcac
50cacagccctt aaggcacgag ggaacctcac tgcgcatgct cctttggtgc
100ccacctcagt gcgcatgttc actgggcgtc ttcccatcgg ccccttcgcc
150agtgtgggga acgcggcgga gctgtgagcc ggcgactcgg gtccctgagg
200tctggattct ttctccgcta ctgagacacg gcggacacac acaaacacag
250aaccacacag ccagtcccag gagcccagta atggagagcc ccaaaaagaa
300gaaccagcag ctgaaagtcg ggatcctaca cctgggcagc agacagaaga
350agatcaggat acagctgaga tcccagtgcg cgacatggaa ggtgatctgc
400aagagctgca tcagtcaaac accggggata aatctggatt tgggttccgg
450cgtcaaggtg aagataatac ctaaagagga acactgtaaa atgccagaag
500caggtgaaga gcaaccacaa gtttaaatga agacaagctg aaacaacgca
550agctggtttt atattagata tttgacttaa actatctcaa taaagttttg
600cagctttcac caaaaaaaaa aaaaaa
626431505DNAHomo Sapien 43agcggctggc gagccggcgc cggccgagct gcgggagccg
cggagagcac 50cagctgtcgc cgcgggagct gctccggccg caccatgcgg
gagctggcca 100ttgagatcgg ggtgcgagcc ctgctcttcg gagtcttcgt
ttttacagag 150tttttggatc cgttccagag agtcatccag ccagaagaga
tctggctcta 200taaaaatcct ttggtgcaat cagataacat acctacccgc
ctcatgtttg 250caatttcttt cctcacaccc ctggctgtta tttgtgtggt
gaaaattatc 300cggcgaacag acaagactga aattaaggaa gccttcttag
cggtgtcctt 350ggctcttgct ttgaatggag tctgcacaaa cactattaaa
ttaatagtgg 400gaagacctcg cgccgatttc ttttaccgct gctttccaga
tggagtgatg 450aactcggaaa tgcattgcac aggtgacccc gatctggtgt
ccgagggccg 500caaaagcttc cccagcatcc attcctcctt tgccttttcg
ggccttggct 550tcacgacgtt ctacttggcg ggcaagctgc actgcttcac
cgagagtggg 600cggggaaaga gctggcggct ctgtgctgcc atcctgccct
tgtactgcgc 650catgatgatt gccctgtccc gcatgtgcga ctacaagcat
cactggcaag 700attcctttgt gggtggagtc atcgcgctca tttttgcata
catttgctac 750agacagcact atcctcctct gggccaacac agcttgccat
aaaccctacg 800ttagtctgcg agtttgccat aaaccctacg ttagtctgcg
agtcccagcc 850tcactgaaga aagaggagag gcccacagct gacagcgcac
ccagcttgcc 900tctggagggg atcaccgaag gcccggtatg accagtgtcc
tgggaggatg 950gacactaagc cctgggcaca tctgccaccc tgacatcata
acacaataga 1000aatggttttc tgtagtgtat ttttcatcag ttgtttctca
aagtcatcgt 1050acttctgctt ctgtttcact gatggtgttc ctgctacttt
aaatgtctac 1100ttccaacatc cttgaatttg caagtgaagg acaacaatct
ctgagagacg 1150tgtggaagag gctgcgaagg tggggtttgg ggagcttcgc
cgattcgtct 1200atctgaaatg tttgctgtaa cagccacctt cctatgtttt
catggttagt 1250aaacataata aaacctccca tcgggaaaaa atacaaaatt
cattgattta 1300ggaatatata tataatattc acatgtgtaa ttccccccct
ccctttagtg 1350agggtaattc aagatccttc tcaactgctt tgtgcgactt
agactttatg 1400ttgcagcaga cttttttatt ttacttatag cgcggaatcc
gtgtttcctc 1450agaatcaggg aatccgcccg aaaatctgtt acaaaggccg
ccaagtgaca 1500taact
1505441850DNAHomo Sapien 44tccttgggtt cgggtgaaag
cgcctggggg ttcgtggcca tgatccccga 50gctgctggag aactgaaggc
ggacagtctc ctgcgaaacc aggcaatggc 100ggagctggag tttgttcaga
tcatcatcat cgtggtggtg atgatggtga 150tggtggtggt gatcacgtgc
ctgctgagcc actacaagct gtctgcacgg 200tccttcatca gccggcacag
ccaggggcgg aggagagaag atgccctgtc 250ctcagaagga tgcctgtggc
cctcggagag cacagtgtca ggcaacggaa 300tcccagagcc gcaggtctac
gccccgcctc ggcccaccga ccgcctggcc 350gtgccgccct tcgcccagcg
ggagcgcttc caccgcttcc agcccaccta 400tccgtacctg cagcacgaga
tcgacctgcc acccaccatc tcgctgtcag 450acggggagga gcccccaccc
taccagggcc cctgcaccct ccagcttcgg 500gaccccgagc agcagctgga
actgaaccgg gagtcggtgc gcgcaccccc 550aaacagaacc atcttcgaca
gtgacctgat ggatagtgcc aggctgggcg 600gcccctgccc ccccagcagt
aactcgggca tcagcgccac gtgctacggc 650agcggcgggc gcatggaggg
gccgccgccc acctacagcg aggtcatcgg 700ccactacccg gggtcctcct
tccagcacca gcagagcagt gggccgccct 750ccttgctgga ggggacccgg
ctccaccaca cacacatcgc gcccctagag 800agcgcagcca tctggagcaa
agagaaggat aaacagaaag gacaccctct 850ctagggtccc caggggggcc
gggctggggc tgcgtaggtg aaaaggcaga 900acactccgcg cttcttagaa
gaggagtgag aggaaggcgg ggggcgcagc 950aacgcatcgt gtggccctcc
cctcccacct ccctgtgtat aaatatttac 1000atgtgatgtc tggtctgaat
gcacaagcta agagagcttg caaaaaaaaa 1050aagaaaaaag aaaaaaaaaa
accacgtttc tttgttgagc tgtgtcttga 1100aggcaaaaga aaaaaaattt
ctacagtagt ctttcttgtt tctagttgag 1150ctgcgtgcgt gaatgcttat
tttcttttgt ttatgataat ttcacttaac 1200tttaaagaca tatttgcaca
aaacctttgt ttaaagatct gcaatattat 1250atatataaat atatataaga
taagagaaac tgtatgtgcg agggcaggag 1300tatttttgta ttagaagagg
cctattaaaa aaaaaagttg ttttctgaac 1350tagaagagga aaaaaatggc
aatttttgag tgccaagtca gaaagtgtgt 1400attaccttgt aaagaaaaaa
attacaaagc aggggtttag agttatttat 1450ataaatgttg agattttgca
ctatttttta atataaatat gtcagtgctt 1500gcttgatgga aacttctctt
gtgtctgttg agactttaag ggagaaatgt 1550cggaatttca gagtcgcctg
acggcagagg gtgagccccc gtggagtctg 1600cagagaggcc ttggccagga
gcggcgggct ttcccgaggg gccactgtcc 1650ctgcagagtg gatgcttctg
cctagtgaca ggttatcacc acgttatata 1700ttccctaccg aaggagacac
cttttccccc ctgacccaga acagccttta 1750aatcacaagc aaaataggaa
agttaaccac ggaggcaccg agttccaggt 1800agtggttttg cctttcccaa
aaatgaaaat aaactgttac cgaaggaatt 185045806DNAHomo Sapien
45gcccttcgga cagtctcctg cgaaaccagg caatggcgga gctggagttt
50gttcagatca tcatcatcgt ggtggtgatg atggtgatgg tggtggtgat
100cacgtgcctg ctgagccact acaagctgtc tgcacggtcc ttcatcagcc
150ggcacagcca ggggcggagg agagaagatg ccctgtcctc agaaggatgc
200ctgtggccct cggagagcac agtgtcaggc aacggaatcc cagagccgca
250ggtctacgcc ccgcctcggc ccaccgaccg cctggccgtg ccgcccttcg
300cccagcggga gcgcttccac cgcttccagc ccacctatcc gtacctgcag
350cacgagatcg acctgccgcc caccatctcg ctgtcagacg gggaggagcc
400cccaccctac cagggcccct gcaccctcca gcttcgggac cccgagcagc
450agctggaact gaaccgggag tcggtgcgcg cacccccaaa cagaaccatc
500ttcgacagtg acctgatgga tagtgccagg ctgggcggcc cctgcccccc
550cagcagtaac tcgggcatca gcgccacgtg ctacggcagc ggcgggcgca
600tggaggggcc gccgcccacc tacagcgagg tcatcggcca ctacccgggg
650tcctccttcc agcaccagca gagcagtggg ccgccctcct tgctggaggg
700gacccggctc caccacacac acatcgcgcc cctagagagc gcagccatct
750ggagcaaaga gaaggataaa cagaaaggac accctctcta gggtccccag
800aagggc
806461982DNAHomo Sapien 46ggcgagaggc gggctgaggc ggcccagcgg cggcaggtga
ggcggaacca 50accctcctgg ccatgggagg ggccgtggtg gacgagggcc
ccacaggcgt 100caaggcccct gacggcggct ggggctgggc cgtgctcttc
ggctgtttcg 150tcatcactgg cttctcctac gccttcccca aggccgtcag
tgtcttcttc 200aaggagctca tacaggagtt tgggatcggc tacagcgaca
cagcctggat 250ctcctccatc ctgctggcca tgctctacgg gacaggtccg
ctctgcagtg 300tgtgcgtgaa ccgctttggc tgccggcccg tcatgcttgt
ggggggtctc 350tttgcgtcgc tgggcatggt ggctgcgtcc ttttgccgga
gcatcatcca 400ggtctacctc accactgggg tcatcacggg gttgggtttg
gcactcaact 450tccagccctc gctcatcatg ctgaaccgct acttcagcaa
gcggcgcccc 500atggccaacg ggctggcggc agcaggtagc cctgtcttcc
tgtgtgccct 550gagcccgctg gggcagctgc tgcaggaccg ctacggctgg
cggggcggct 600tcctcatcct gggcggcctg ctgctcaact gctgcgtgtg
tgccgcactc 650atgaggcccc tggtggtcac ggcccagccg ggctcggggc
cgccgcgacc 700ctcccggcgc ctgctagacc tgagcgtctt ccgggaccgc
ggctttgtgc 750tttacgccgt ggccgcctcg gtcatggtgc tggggctctt
cgtcccgccc 800gtgttcgtgg tgagctacgc caaggacctg ggcgtgcccg
acaccaaggc 850cgccttcctg ctcaccatcc tgggcttcat tgacatcttc
gcgcggccgg 900ccgcgggctt cgtggcgggg cttgggaagg tgcggcccta
ctccgtctac 950ctcttcagct tctccatgtt cttcaacggc ctcgcggacc
tggcgggctc 1000tacggcgggc gactacggcg gcctcgtggt cttctgcatc
ttctttggca 1050tctcctacgg catggtgggg gccctgcagt tcgaggtgct
catggccatc 1100gtgggcaccc acaagttctc cagtgccatt ggcctggtgc
tgctgatgga 1150ggcggtggcc gtgctcgtcg ggcccccttc gggaggcaaa
ctcctggatg 1200cgacccacgt ctacatgtac gtgttcatcc tggcgggggc
cgaggtgctc 1250acctcctccc tgattttgct gctgggcaac ttcttctgca
ttaggaagaa 1300gcccaaagag ccacagcctg aggtggcggc cgcggaggag
gagaagctcc 1350acaagcctcc tgcagactcg ggggtggact tgcgggaggt
ggagcatttc 1400ctgaaggctg agcctgagaa aaacggggag gtggttcaca
ccccggaaac 1450aagtgtctga gtggctgggc ggggccggca ggcacaggga
ggaggtacag 1500aagccggcaa cgcttgctat ttattttaca aactggactg
gctcaggcag 1550ggccacggct gggctccagc tgccggccca gcggatcgtc
gcccgatcag 1600tgttttgagg gggaaggtgg cggggtggga accgtgtcat
tccagagtgg 1650atctgcggtg aagccaagcc gcaaggttac aaggcatcct
caccaggggc 1700cccgcctgct gctcccaggt ggcctgcggc cactgctatg
ctcaaggacc 1750tggaaaccca tgcttcgaga caacgtgact ttaatgggag
ggtgggtggg 1800ccgcagacag gctggcaggg caggtgctgc gtggggccct
ctccagcccg 1850tcctaccctg ggctcacatg gggcctgtgc ccacccctct
tgagtgtctt 1900ggggacagct ctttccaccc ctggaagatg gaaataaacc
tgcgtgtggg 1950tggagtgttc tcgtgccgaa ttcaaaaagc tt
1982472171DNAHomo Sapien 47cccacgcgtc cgcccacgcg
tccgccgggt cctgcgcgct ccggactgag 50gtggcgtccc tgggccggac
ggcggtgtcc cggcgtggcg ggaagccggc 100actggagcgg gagcgcactg
ggcgcgggac cgggaggcgc agggaccgga 150cggctcccga gtcgcccacc
tgacggtacc gagagggcgg cgcccctccg 200agcagagccg tcccggccac
tcccctggga tctgacttgg ctcttgcggt 250cgcgggcacc gtgaagccct
ggggtgtgcg tggctcctcc tggtaggcgc 300cctttcccgg cgtccggctt
ggggtggtgg tggcgttgac tccagccccg 350cctctccctg gagaggaggg
ctccactcgc tccttcggcc tcctcccctg 400gggccgcagc gactcgggcc
ggcttcctgc ttccctgcct gccggcggtc 450ccgctggcta gaagaagtct
tcacttccca ggagagccaa agcgtgtctg 500gccctaggtg ggaaaagaac
tggctgtgac ctttgccctg acctggaagg 550gcccagcctt gggctgaatg
gcagcaccca cgcccgcccg tccggtgctg 600acccacctgc tggtggctct
cttcggcatg ggctcctggg ctgcggtcaa 650tgggatctgg gtggagctac
ctgtggtggt caaagagctt ccagagggtt 700ggagcctccc ctcttacgtc
tctgtgcttg tggctctggg gaacctgggt 750ctgctggtgg tgaccctctg
gaggaggctg gccccaggaa aggacgagca 800ggtccccatc cgggtggtgc
aggtgctggg catggtgggc acagccctgc 850tggcctctct gtggcaccat
gtggccccag tggcaggaca gttgcattct 900gtggccttct tagcactggc
ctttgtgctg gcactggcat gctgtgcctc 950gaatgtcact ttcctgccct
tcttgagcca cctgccacct cgcttcttac 1000ggtcattctt cctgggtcaa
ggcctgagtg ccctgctgcc ctgcgtgctg 1050gccctagtgc agggtgtggg
ccgcctcgag tgcccgccag cccccatcaa 1100cggcacccct ggccccccgc
tcgacttcct tgagcgtttt cccgccagca 1150ccttcttctg ggcactgact
gcccttctgg tcgcttcagc tgctgccttc 1200cagggtcttc tgctgctgtt
gccgccacca ccatctgtac ccacagggga 1250gttaggatca ggcctccagg
tgggagcccc aggagcagag gaagaggtgg 1300aagagtcctc accactgcaa
gagccaccaa gccaggcagc aggcaccacc 1350cctggtccag accctaaggc
ctatcagctt ctatcagccc gcagtgcctg 1400cctgctgggc ctgttggccg
ccaccaacgc gctgaccaat ggcgtgctgc 1450ctgccgtgca gagcttttcc
tgcttaccct acgggcgtct ggcctaccac 1500ctggctgtgg tgctgggcag
tgctgccaat cccctggcct gcttcctggc 1550catgggtgtg ctgtgcaggt
ccttggcagg gctgggcggc ctctctctgc 1600tgggcgtgtt ctgtgggggc
tacctgatgg cgctggcagt cctgagcccc 1650tgcccgcccc tggtgggcac
ctcggcgggg gtggtcctcg tggtgctgtc 1700gtgggtgctg tgtcttggcg
tgttctccta cgtgaaggtg gcagccagct 1750ccctgctgca tggcgggggc
cggccggcat tgctggcagc cggcgtggcc 1800atccaggtgg gctctctgct
cggcgctgtt gctatgttcc ccccgaccag 1850catctatcac gtgttccaca
gcagaaagga ctgtgcagac ccctgtgact 1900cctgagcctg ggcaggtggg
gaccccgctc cccaacacct gtctttccct 1950caatgctgcc accatgcctg
agtgcctgca gcccaggagg cccgcacacc 2000ggtacactcg tggacaccta
cacactccat aggagatcct ggctttccag 2050ggtgggcaag ggcaaggagc
aggcttggag ccagggacca gtgggggctg 2100tagggtaagc ccctgagcct
gggacctaca tgtggtttgc gtaataaaac 2150atttgtattt aaaaaaaaaa a
2171481617DNAHomo Sapien
48gccagcacag ctgccctctg gaccctgcgg accccagccg agccccttcc
50tgagttccac aggcgcagcc cccgggcggt cgggcggagg ggtccccggg
100gcggtgccag gcgcaatcct ggagggcggc cgggaggagg aggtgcgcgc
150ggccatgcac accgtggcta cgtccggacc caacgcgtcc tggggggcac
200cggccaacgc ctccggctgc ccgggctgtg gcgccaacgc ctcggacggc
250ccagtccctt cgccgcgggc cgtggacgcc tggctcgtgc cgctcttctt
300cgcggcgctg atgctgctgg gcctggtggg gaactcgctg gtcatctacg
350tcatctgccg ccacaagccg atgcggaccg tgaccaactt ctacatcgcc
400aacctggcgg ccacggacgt gaccttcctc ctgtgctgcg tccccttcac
450ggccctgctg tacccgctgc ccggctgggt gctgggcgac ttcatgtgca
500agttcgtcaa ctacatccag caggtctcgg tgcaggccac gtgtgccact
550ctgaccgcca tgagtgtgga ccgctggtac gtgacggtgt tcccgttgcg
600cgccctgcac cgccgcacgc cccgcctggc gctggctgtc agcctcagca
650tctgggtagg ctctgcggcg gtgtctgcgc cggtgctcgc cctgcaccgc
700ctgtcacccg ggccgcgcgc ctactgcagt gaggccttcc ccagccgcgc
750cctggagcgc gccttcgcac tgtacaacct gctggcgctg tacctgctgc
800cgctgctcgc cacctgcgcc tgctatgcgg ccatgctgcg ccacctgggc
850cgggtcgccg tgcgccccgc gcccgccgat agcgccctgc aggggcaggt
900gctggcagag cgcgcaggcg ccgtgcgggc caaggtctcg cggctggtgg
950cggccgtggt cctgctcttc gccgcctgct ggggccccat ccagctgttc
1000ctggtgctgc aggcgctggg ccccgcgggc tcctggcacc cacgcagcta
1050cgccgcctac gcgcttaaga cctgggctca ctgcatgtcc tacagcaact
1100ccgcgctgaa cccgctgctc tacgccttcc tgggctcgca cttccgacag
1150gccttccgcc gcgtctgccc ctgcgcgccg cgccgccccc gccgcccccg
1200ccggcccgga ccctcggacc ccgcagcccc acacgcggag ctgcaccgcc
1250tggggtccca cccggccccc gccagggcgc agaagccagg gagcagtggg
1300ctggccgcgc gcgggctgtg cgtcctgggg gaggacaacg cccctctttg
1350agcggacccg gtgggaatcc gagcggctcc ctcgggagcg gggactgctg
1400gaacagcggc tattcttctg ttattagtat tttttttact gtccaagatc
1450aactgtggaa atattttggt ctcttgtgac gttcggtgca gtttcgttgt
1500gaagtttgct attgatattg aaattatgac ttctgtgttt cctgaaatta
1550aacatgtgtc aacacaggac tttttggatc attccagaaa gtgtcagacg
1600tttaaaaaaa aaaaaaa
1617493095DNAHomo Sapien 49ggcgcggggc gccatggcac accgagcggc tccgtcttct
gctcctcaga 50gagcccggct ggcggcctgg gatgacaaga tgtctggact
gcaatcctgc 100acagttttga gagggagatg acttgagtgg ttggctttta
tctccacaac 150aatgtccatg aacaattcca aacagctagt gtctcctgca
gctgcgcttc 200tttcaaacac aacctgccag acggaaaacc ggctttccgt
atttttttca 250gtaatcttca tgacagtggg aatcttgtca aacagccttg
ccatcgccat 300tctcatgaag gcatatcaga gatttagaca gaagtccaag
gcatcgtttc 350tgcttttggc cagcggcctg gtaatcactg atttctttgg
ccatctcatc 400aatggagcca tagcagtatt tgtatatgct tctgataaag
aatggatccg 450ctttgaccaa tcaaatgtcc tttgcagtat ttttggtatc
tgcatggtgt 500tttctggtct gtgcccactt cttctaggca gtgtgatggc
cattgagcgg 550tgtattggag tcacaaaacc aatatttcat tctacgaaaa
ttacatccaa 600acatgtgaaa atgatgttaa gtggtgtgtg cttgtttgct
gttttcatag 650ctttgctgcc catccttgga catcgagact ataaaattca
ggcgtcgagg 700acctggtgtt tctacaacac agaagacatc aaagactggg
aagatagatt 750ttatcttcta cttttttctt ttctggggct cttagccctt
ggtgtttcat 800tgttgtgcaa tgcaatcaca ggaattacac ttttaagagt
taaatttaaa 850agtcagcagc acagacaagg cagatctcat catttggaaa
tggtaatcca 900gctcctggcg ataatgtgtg tctcctgtat ttgttggagc
ccatttctgg 950ttacaatggc caacattgga ataaatggaa atcattctct
ggaaacctgt 1000gaaacaacac tttttgctct ccgaatggca acatggaatc
aaatcttaga 1050tccttgggta tatattcttc tacgaaaggc tgtccttaag
aatctctata 1100agcttgccag tcaatgctgt ggagtgcatg tcatcagctt
acatatttgg 1150gagcttagtt ccattaaaaa ttccttaaag gttgctgcta
tttctgagtc 1200accagttgca gagaaatcag caagcaccta gcttaatagg
acagtaaatc 1250tgtgtggggc tagaacaaaa attaagacat gtttggcaat
atttcagtta 1300gttaaatacc tgtagcctaa ctggaaaatt caggcttcat
catgtagttt 1350gaagatacta ttgtcagatt caggttttga aatttgtcaa
ataaacagga 1400taactgtaca ttttcaactt gtttttgcca atgggaggta
gacacaataa 1450aataatgcca tgggagtcac actgaaagca attttgagct
tatctgtctt 1500atttatgctt tgagtgaatc atctgttgag gtctaatgcc
tctacttggc 1550ctatttgcca gagaacatct taatgcagcc tgcatagtga
aatggttatt 1600ttgagatcac cgctctgtag ctaaccctta taaactaggc
tcagtaaaat 1650aaagcactct tattttttga tctggcctat tttgcccctc
attgtgtagc 1700ctcaattaac acatgcatgg tcatgacacc cagaattcat
gatggtttgt 1750tataacaacc tctgcatatt ccaggtctgg cagacaggtt
gcctgaccct 1800gcaatcctat ctagaatggg cccattcttg tcacatttga
caaataggac 1850tgcctacatt tattattatg aaggtcgatt gttgttggaa
gtgttttttc 1900atgtcataga ttagcaattt tcaaataatt attttttctc
tgaaaatttt 1950gtgtgtgatt gcacaataaa taatttttag agaaacaaag
gctctttctc 2000agcacattga tgggcaacta gaattacagc agtttcaaac
tctaccatgg 2050ataatgcaaa caaaccgaag ctacatgcca atgataggtg
caaagaatat 2100tggcaaaagg tgctttacct tgagccatta tttgtgtcag
agaacaaaag 2150aaacagaatc aatatataaa ttcaaagact atctgcagct
agtgtgtttc 2200ttctttacac acatatacac acagacatca gaaaattctg
ttgagagcag 2250gttcattaaa tttgtaagat ggcatattct aaagcctgtg
ctaccagtac 2300taagagggga agactggcaa tttgccaagc acttggggat
tattataaca 2350attaactagg agatcaagag ataataatct ctccccaaat
tttccaataa 2400taattgagac tttttctttg cttgtttgtg taattcaacc
aaaagaattt 2450caatacccat tcaaattgtc ctaggtctat cagaaattag
ggaaggtagt 2500cctgctttat aataggaaaa tgtatttctg tataagattt
ctttgctttc 2550attaaaaatg ggattcattt aaaaattaat ctttccctgt
taggctgatt 2600tcagattctc taggaaatct ggtgaagtaa ccagaagact
ttcagatggt 2650ttatttgctt tcagcagaga atttatttca tacagttact
taagagtgtt 2700gatgtcttgt gaacagagat ataaggaacc attctccatc
cttccttatc 2750atgctgggta caatgcttct atgaatattt ccatgtattt
tgactgggga 2800gaggcatgga gaagaaactc tcattcaggg gctccaggat
ccttctcctt 2850gaggcttcta aataaatggc agaattcttg ctgtattgcc
atgatgtcac 2900cctggccatg tgtactgact tgaggagatc ttgcaacatg
gccatgtgca 2950aggctttaag gagtgagaga gatgtgtaca tatcttagga
gggttatcta 3000tgttatctga gtatatgttt gggtaaccaa attggtctta
aaaatgatgt 3050taacccaaga agtagacatc aaaaattaaa aaaaaaaaaa
aaaaa 3095506476DNAHomo Sapien 50atgtcacgca tgagccggca
tccagacaag gacctggccc agggtccctt 50caacacctgc tgtggctgca
ccttaatggc tagtcctgct aatctccctc 100cgaacactca agcagctgca
gaaagggccc tttcccagag caggtggaag 150agggtgcaag tgcccgcccc
ggcatccctg tcccctttcc cactggccat 200ggcttcagtt gccttctgga
tcagcatcct gattggctgc gaggaacaga 250ctctctgcag aggctggcgt
agcccagtcg gggatggctg tgctcatgtg 300cctccccagg agcgagcgac
cgcagaggca gaccctccag ggcggtgcag 350cacctccacg gcgtcgtcta
ccatctgtgg cctgtggcat ttgtccccac 400ggctgcagct cctcccacct
ctgcattcca ggcagggaga agagtcgggc 450aaaactgaga aggtgcttct
ctggggaaga gagggcctcc atgtgtggaa 500acccggagtc ctgcagcccg
atgtccacgg cacctccaac ctggggaact 550gctccttcct gcacggcctg
gttacggctc cctcttgtcc acggcgggcg 600ggcgccgagc tgctgaattc
tttaggaagt cagtttgcca ttagcctttt 650tgaagttcag agtggaactg
agcccagcat tacaggtgtg gccacgtcag 700ggcagtgcag ggctatgcca
ctgaagcatt atctcctttt gctggtgggc 750tgccaagcct ggggtgcagg
gttggcctac catggctgcc ctagcgagtg 800tacctgctcc agggcctccc
aggtggagtg caccggggca cgcattgtgg 850cggtgcccac ccctctgccc
tggaacgcca tgagcctgca gatcctcaac 900acgcacatca ctgaactcaa
tgagtccccg ttcctcaata tttcagccct 950catcgccctg aggattgaga
agaatgagct gtcgcgcatc acgcctgggg 1000ccttccgaaa cctgggctcg
ctgcgctatc tcagcctcgc caacaacaag 1050ctgcaggttc tgcccatcgg
cctcttccag ggcctggaca gccttgagtc 1100tctccttctg tccagtaacc
agctgttgca gatccagccg gcccacttct 1150cccagtgcag caacctcaag
gagctgcagt tgcacggcaa ccacctggaa 1200tacatccctg acggagcctt
cgaccacctg gtaggactca cgaagctcaa 1250tctgggcaag aatagcctca
cccacatctc acccagggtc ttccagcacc 1300tgggcaatct ccaggtcctc
cggctgtatg agaacaggct cacggatatc 1350cccatgggca cttttgatgg
gcttgttaac ctgcaggaac tggctctaca 1400gcagaaccag attggactgc
tctcccctgg tctcttccac aacaaccaca 1450acctccagag actctacctg
tccaacaacc acatctccca gctgccaccc 1500agcatcttca tgcagctgcc
ccagctcaac cgtcttactc tctttgggaa 1550ttccctgaag gagctctctc
tggggatctt cgggcccatg cccaacctgc 1600gggagctttg gctctatgac
aaccacatct cttctctacc cgacaatgtc 1650ttcagcaacc tccgccagtt
gcaggtcctg attcttagcc gcaatcagat 1700cagcttcatc tccccgggtg
ccttcaacgg gctaacggag cttcgggagc 1750tgtccctcca caccaacgca
ctgcaggacc tggacgggaa tgtcttccgc 1800atgttggcca acctgcagaa
catctccctg cagaacaatc gcctcagaca 1850gctcccaggg aatatcttcg
ccaacgtcaa tggcctcatg gccatccagc 1900tgcagaacaa ccagctggag
aacttgcccc tcggcatctt cgatcacctg 1950gggaaactgt gtgagctgcg
gctgtatgac aatccctgga ggtgtgactc 2000agacatcctt ccgctccgca
actggctcct gctcaaccag cctaggttag 2050ggacggacac tgtacctgtg
tgtttcagcc cagccaatgt ccgaggccag 2100tccctcatta tcatcaatgt
caacgttgct gttccaagcg tccatgtacc 2150tgaggtgcct agttacccag
aaacaccatg gtacccagac acacccagtt 2200accctgacac cacatccgtc
tcttctacca ctgagctaac cagccctgtg 2250gaagactaca ctgatctgac
taccattcag gtcactgatg accgcagcgt 2300ttggggcatg acccatgccc
atagcgggct ggccattgcc gccattgtaa 2350ttggcattgt cgccctggcc
tgctccctgg ctgcctgcgt cggctgttgc 2400tgctgcaaga agaggagcca
agctgtcctg atgcagatga aggcacccaa 2450tgagtgttaa agaggcaggc
tggagcaggg ctggggaatg atgggactgg 2500aggacctggg aatttcatct
ttctgcctcc acccctgggt ccatggagct 2550ttcccgtgat tgctctttct
ggccctagat aaaggtgtgc ctacctcttc 2600ctgacttgcc tgattctccc
gtagagaagc aggtcgtgcc ggaccttcct 2650acaatcagga agatagatcc
aactggccat ggcaaaagcc ctggggattt 2700ccgattcata cccctgggct
tccttcgaga gggctcttcc tccaaatcct 2750ccccacctgt cctccaagaa
cagccttccc tgcgcccagg ccccctccgg 2800gcctctgtag actcagttag
tccacagcct gctcacttcg tgggaatagt 2850tctccgctga gatagcccct
ctcgcctaag tattatgtaa gttgatttcc 2900cttcttttgt ttctcttgtt
tgtgctatgg cttgacccag catgtcccct 2950caaatgaaag ttctcccctt
gattttctgc tcctgaaggc agggtgagtt 3000ctctcctcaa agaagacttc
aaaccattta actggtttct taagagccgt 3050caatcagcct ggttttgggg
atgctatgaa agagagaagg aaaatcatgc 3100cgctcagttc ctggagacag
aagagccgtc atcagtgtct cacttgtgat 3150ttttatctgg aaaaggaaga
aacaccccag cacagcaagc tcagcctttt 3200agagaaggat atttccaaac
tgcaaacttt gctttgaaaa gtttagccct 3250ttaaggaatg aaatcatgta
gaattttgga cttctaaaaa cattaaaatc 3300agcttattaa tacgggatag
agaaagaaat ctggtgcctg ggggtccctg 3350tgttcacccc tagagtttgt
tttaaaattt ttaattgaag catgtgaagt 3400gtacctgcag aaaagtggga
acatgatagt gtatggcttg gtggattttc 3450acaaactgaa catacctgtg
taatcagcat ctagacccag acccagagcg 3500tcacaaatat cccccatcct
gggcttttcc cagaggagat gggggcttct 3550gaagatggac ttacctggga
cctgcccccc atgagccagg acggtccccc 3600cacagtcagc ctgtgcaaag
gccccgtggc caggggtgga ggagaatatg 3650tgggtgtgga caggatggga
gactgtggcc tgaacaggag attttattat 3700atctggagac cctgagagac
cctgagacct ggggcaccct ggctggccag 3750gtcagaagca tcctgactgc
agaggtccgt gcagccacac cctcttccct 3800gccagcaagc tgtctgcggc
tcatcggagg cccctccgcc tggagccttc 3850tatggacgtg atatgcctgt
atctgttttt aattttcatt cttcacttag 3900gggaagtgaa atcgctcaga
gatgagatcc tttaattgaa aacgaagtgt 3950aacggaatct agtgtctttc
taatgtggta aaattctcca tcaacatcac 4000agtcagctgg cagctgaact
tcagaatctc acttacagca ggcgacacgg 4050gggtacaccg atgggtcaca
ctgggtctgg gggctccctg gagctcctcc 4100tgcgtgtggt ctggttagga
gttgagttgt ttgctccagg gttattctcc 4150tcctcgagtc acagtcacac
gaatacctgc cttctctggc tttcctgcta 4200tacacatatt cacatggcgc
tcaagaagtt aggctcatgg caacgtgtgt 4250ctttctctgg acaactggcc
cagtttacag tgaaatggag aatttcaggt 4300ctccacgtct gcccaggaaa
gaacttcagc tgactccacg gggatctgga 4350aatccacgac caatcccgat
cggctcttat tagctccccg ctccacaaga 4400cacctgtgct ttggaaatcc
accaccaatc ccgatcggct cttattagct 4450ccccgctcca caagacacct
gtgatctgga aatctaccac caatcccgat 4500cggctcttat tagctccccg
ctccacaaga cacctgtgac atcctccagg 4550gccacaggag cacgtgctga
ccagttttcc cttccagttc ctgcacaaaa 4600agtgtccaga gggctgtttg
caaacactag tgcactttgt agcttttcac 4650cctctgtccc agggaatcta
ggagagatga ggcccgtcag agtcaagaga 4700tgtcatcccc ccagggtctc
caaggcattt ccacactatt ggtggcacct 4750ggaggacatg caccaaggct
tgccagagcc aacaggaagt gagcccagag 4800catggcacat gagcatcacc
cgctgatggt ggcctgctgt gcctggtgcc 4850aacaggggca tcccggccca
tacccctcca gacaggaagc atgggtttgc 4900ccacagacct gtcgggtgct
cctgtgagtg gcctccagat gtctttgtgc 4950ataggcacaa gtgggccagg
gctggaggga ggtgggaaac ctcatcatcc 5000ggtgggccct gccaatctta
acccagaacc cttaggtatt cctggcagta 5050gccatgacat tggagcacct
tcctctccag ccagaggctg acctgagggc 5100cactgtcctc agatgacacc
acccaggagc accctaggtg aggggtgagg 5150gcccccttat gtgaacctct
tgcctcttcc tttctcccat cagagtggtt 5200ggatggagcc attggcctcc
ttttcttcag cgggcccttc aacctctctg 5250caccatgttg tctggctgag
gagctactag aaaagctgag tggagtctcc 5300tttccaacag gatgatgcat
ttgctcaatt ctcagggctg gaatgagccg 5350gctggtcccc cagaaagctg
gagtggggta cagagttcag ttttcctctc 5400tgtttacagc tccttgacag
tcccacgccc atctggagtg ggagctggga 5450gtcagtgttg gagaagaaac
aacaaaagcc aattagaacc actattttta 5500aaaagtgctt actgtgcaca
gatactcttc aagcactgga cgtggattct 5550ctctctagcc ctcagcaccc
ctgcggtagg agtgccgcct ctacccactt 5600gtgatggggt acagaggcac
ttgctcttct gcatggtgtt caataggctg 5650ggagttttat ttatctcttc
aaactttgta caagagctca tggcttgtct 5700tgggctttcg tcattaaacc
aaaggaaatg gaagccattc ccctgttgct 5750ctccttagtc ttggtcatca
gaacctcact tggtaccata tagatcaaaa 5800gctttgtaac cacaggaaaa
aataaactct tccatccctt aaagaataga 5850atagtttgtc cctctcatgg
gaattgggct gtatgtatat tgttcttcct 5900ccttagaatt tagagataca
agagttctac ttagaacttt tcatggacac 5950aatttccaca acctttcaga
tgctgatgta gagctattgg gaaagaactt 6000ccaaactcag gaagtttgca
gagagcagac agctagagat aactcgggac 6050ccagagttgg tcgacagatg
ttagatgtat cctagctttt agctataaac 6100cactcaaaga ttcagccccc
agatcccaca gtcagaactg aatctgcgtt 6150gttgggaagc cagcagtggc
cttgggaagg aagccatggc tgtggttcag 6200agagggtggg ctggcaagcc
acttccgggg aaaactcctt ccgccccagg 6250tttcttcttc tcttaaggag
agattattct caccaacccg ctgccttcat 6300gctgccttca aagctagatc
atgtttgcct tgcttagaga attactgcaa 6350atcagcccca gtgcttggcg
atgcatttac agatttctag gccctcaggg 6400ttttgtagag tgtgagccct
ggtgggcagg gttggggggt ctgtcttctg 6450ctggatgctg cttgtaatcc
atttgg 64765111389DNAHomo sapien
51atggcgccgc cgccgccgcc cgtgctgccc gtgctgctgc tcctggccgc
50cgccgccgcc ctgccggcga tggggctgcg agcggccgcc tgggagccgc
100gcgtacccgg cgggacccgc gccttcgccc tccggcccgg ctgtacctac
150gcggtgggcg ccgcttgcac gccccgggcg ccgcgggagc tgctggacgt
200gggccgcgat gggcggctgg caggacgtcg gcgcgtctcg ggcgcggggc
250gcccgctgcc gctgcaagtc cgcttggtgg cccgcagtgc cccgacggcg
300ctgagccgcc gcctgcgggc gcgcacgcac cttcccggct gcggagcccg
350tgcccggctc tgcggaaccg gtgcccggct ctgcggggcg ctctgcttcc
400ccgtccccgg cggctgcgcg gccgcgcagc attcggcgct cgcagctccg
450accaccttac ccgcctgccg ctgcccgccg cgccccaggc cccgctgtcc
500cggccgtccc atctgcctgc cgccgggcgg ctcggtccgc ctgcgtctgc
550tgtgcgccct gcggcgcgcg gctggcgccg tccgggtggg actggcgctg
600gaggccgcca ccgcggggac gccctccgcg tcgccatccc catcgccgcc
650cctgccgccg aacttgcccg aagcccgggc ggggccggcg cgacgggccc
700ggcggggcac gagcggcaga gggagcctga agtttccgat gcccaactac
750caggtggcgt tgtttgagaa cgaaccggcg ggcaccctca tcctccagct
800gcacgcgcac tacaccatcg agggcgagga ggagcgcgtg agctattaca
850tggaggggct gttcgacgag cgctcccggg gctacttccg aatcgactct
900gccacgggcg ccgtgagcac ggacagcgta ctggaccgcg agaccaagga
950gacgcacgtc ctcagggtga aagccgtgga ctacagtacg ccgccgcgct
1000cggccaccac ctacatcact gtcttggtca aagacaccaa cgaccacagc
1050ccggtcttcg agcagtcgga gtaccgcgag cgcgtgcggg agaacctgga
1100ggtgggctac gaggtgctga ccatccgcgc cagcgaccgc gactcgccca
1150tcaacgccaa cttgcgttac cgcgtgttgg ggggcgcgtg ggacgtcttc
1200cagctcaacg agagctctgg cgtggtgagc acacgggcgg tgctggaccg
1250ggaggaggcg gccgagtacc agctcctggt ggaggccaac gaccaggggc
1300gcaatccggg cccgctcagt gccacggcca ccgtgtacat cgaggtggag
1350gacgagaacg acaactaccc ccagttcagc gagcagaact acgtggtcca
1400ggtgcccgag gacgtggggc tcaacacggc tgtgctgcga gtgcaggcca
1450cggaccggga ccagggccag aacgcggcca ttcactacag catcctcagc
1500gggaacgtgg ccggccagtt ctacctgcac tcgctgagcg ggatcctgga
1550tgtgatcaac cccttggatt tcgaggatgt ccagaaatac tcgctgagca
1600ttaaggccca ggatgggggc cggcccccgc tcatcaattc ttcaggggtg
1650gtgtctgtgc aggtgctgga tgtcaacgac aacgagccta tctttgtgag
1700cagccccttc caggccacgg tgctggagaa tgtgcccctg ggctaccccg
1750tggtgcacat tcaggcggtg gacgcggact ctggagagaa cgcccggctg
1800cactatcgcc tggtggacac ggcctccacc tttctggggg gcggcagcgc
1850tgggcctaag aatcctgccc ccacccctga cttccccttc cagatccaca
1900acagctccgg ttggatcaca gtgtgtgccg agctggaccg cgaggaggtg
1950gagcactaca gcttcggggt ggaggcggtg gaccacggct cgccccccat
2000gagctcctcc accagcgtgt ccatcacggt gctggacgtg aatgacaacg
2050acccggtgtt cacgcagccc acctacgagc ttcgtctgaa tgaggatgcg
2100gccgtgggga gcagcgtgct gaccctgcag gcccgcgacc gtgacgccaa
2150cagtgtgatt acctaccagc tcacaggcgg caacacccgg aaccgctttg
2200cactcagcag ccagagaggg ggcggcctca tcaccctggc gctacctctg
2250gactacaagc aggagcagca gtacgtgctg gcggtgacag catccgacgg
2300cacacggtcg cacactgcgc atgtcctaat caacgtcact gatgccaaca
2350cccacaggcc tgtctttcag agctcccatt acacagtgag tgtcagtgag
2400gacaggcctg tgggcacctc cattgctacc ctcagtgcca acgatgagga
2450cacaggagag aatgcccgca tcacctacgt gattcaggac cccgtgccgc
2500agttccgcat tgaccccgac agtggcacca tgtacaccat gatggagctg
2550gactatgaga accaggtcgc ctacacgctg accatcatgg cccaggacaa
2600cggcatcccg cagaaatcag acaccaccac cctagagatc ctcatcctcg
2650atgccaatga caatgcaccc cagttcctgt gggatttcta ccagggttcc
2700atctttgagg atgctccacc ctcgaccagc atcctccagg tctctgccac
2750ggaccgggac tcaggtccca atgggcgtct gctgtacacc ttccagggtg
2800gggacgacgg cgatggggac ttctacatcg agcccacgtc cggtgtgatt
2850cgcacccagc gccggctgga ccgggagaat gtggccgtgt acaacctttg
2900ggctctggct gtggatcggg gcagtcccac tccccttagc gcctcggtag
2950aaatccaggt gaccatcttg gacattaatg acaatgcccc catgtttgag
3000aaggacgaac tggagctgtt tgttgaggag aacaacccag tggggtcggt
3050ggtggcaaag attcgtgcta acgaccctga tgaaggccct aatgcccaga
3100tcatgtatca gattgtggaa ggggacatgc ggcatttctt ccagctggac
3150ctgctcaacg gggacctgcg tgccatggtg gagctggact ttgaggtccg
3200gcgggagtat gtgctggtgg tgcaggccac gtcggctccg ctggtgagcc
3250gagccacggt gcacatcctt ctcgtggacc agaatgacaa cccgcctgtg
3300ctgcccgact tccagatcct cttcaacaac tatgtcacca acaagtccaa
3350cagtttcccc accggcgtga tcggctgcat cccggcccat gaccccgacg
3400tgtcagacag cctcaactac accttcgtgc agggcaacga gctgcgcctg
3450ttgctgctgg accccgccac gggcgaactg cagctcagcc gcgacctgga
3500caacaaccgg ccgctggagg cgctcatgga ggtgtctgtg tctgatggca
3550tccacagcgt cacggccttc tgcaccctgc gtgtcaccat catcacggac
3600gacatgctga ccaacagcat cactgtccgc ctggagaaca tgtcccagga
3650gaagttcctg tccccgctgc tggccctctt cgtggagggg gtggccgccg
3700tgctgtccac caccaaggac gacgtcttcg tcttcaacgt ccagaacgac
3750accgacgtca gctccaacat cctgaacgtg accttctcgg cgctgctgcc
3800tggcggcgtc cgcggccagt tcttcccgtc ggaggacctg caggagcaga
3850tctacctgaa tcggacgctg ctgaccacca tctccacgca gcgcgtgctg
3900cccttcgacg acaacatctg cctgcgcgag ccctgcgaga actacatgaa
3950gtgcgtgtcc gttctgcgat tcgacagctc cgcgcccttc ctcagctcca
4000ccaccgtgct cttccggccc atccacccca tcaacggcct gcgctgccgc
4050tgcccgcccg gcttcaccgg cgactactgc gagacggaga tcgacctctg
4100ctactccgac ccgtgcggcg ccaacggccg ctgccgcagc cgcgagggcg
4150gctacacctg cgagtgcttc gaggacttca ctggagagca ctgtgaggtg
4200gatgcccgct caggccgctg tgccaacggg gtgtgcaaga acgggggcac
4250ctgcgtgaac ctgctcatcg gcggcttcca ctgcgtgtgt cctcctggcg
4300agtatgagag gccctactgt gaggtgacca ccaggagctt cccgccccag
4350tccttcgtca ccttccgggg cctgagacag cgcttccact tcaccatctc
4400cctcacgttt gccactcagg aaaggaacgg cttgcttctc tacaacggcc
4450gcttcaatga gaagcacgac ttcatcgccc tggagatcgt ggacgagcag
4500gtgcagctca ccttctctgc aggcgagaca acaacgaccg tggcaccgaa
4550ggttcccagt ggtgtgagtg acgggcggtg gcactctgtg caggtgcagt
4600actacaacaa gcccaatatt ggccacctgg gcctgcccca tgggccgtcc
4650ggggaaaaga tggccgtggt gacagtggat gattgtgaca caaccatggc
4700tgtgcgcttt ggaaaggaca tcgggaacta cagctgcgct gcccagggca
4750ctcagaccgg ctccaagaag tccctggatc tgaccggccc tctactcctg
4800gggggtgtcc ccaacctgcc agaagacttc ccagtgcaca accggcagtt
4850cgtgggctgc atgcggaacc tgtcagtcga cggcaaaaat gtggacatgg
4900ccggattcat cgccaacaat ggcacccggg aaggctgcgc tgctcggagg
4950aacttctgcg atgggaggcg gtgtcagaat ggaggcacct gtgtcaacag
5000gtggaatatg tatctgtgtg agtgtccact ccgattcggc gggaagaact
5050gtgagcaagc catgcctcac ccccagctct tcagcggtga gagcgtcgtg
5100tcctggagtg acctgaacat catcatctct gtgccctggt acctggggct
5150catgttccgg acccggaagg aggacagcgt tctgatggag gccaccagtg
5200gtgggcccac cagctttcgc ctccagatcc tgaacaacta cctccagttt
5250gaggtgtccc acggcccctc cgatgtggag tccgtgatgc tgtccgggtt
5300gcgggtgacc gacggggagt ggcaccacct gctgatcgag ctgaagaatg
5350ttaaggagga cagtgagatg aagcacctgg tcaccatgac cttggactat
5400gggatggacc agaacaaggc agatatcggg ggcatgcttc ccgggctgac
5450ggtaaggagc gtggtggtcg gaggcgcctc tgaagacaag gtctccgtgc
5500gccgtggatt ccgaggctgc atgcagggag tgaggatggg ggggacgccc
5550accaacgtcg ccaccctgaa catgaacaac gcactcaagg tcagggtgaa
5600ggacggctgt gatgtggacg acccctgtac ctcgagcccc tgtcccccca
5650atagccgctg ccacgacgcc tgggaggact acagctgcgt ctgtgacaaa
5700gggtaccttg gaataaactg tgtggatgcc tgtcacctga acccctgcga
5750gaacatgggg gcctgcgtgc gctcccccgg ctccccgcag ggctacgtgt
5800gcgagtgtgg gcccagtcac tacgggccgt actgtgagaa caaactcgac
5850cttccgtgcc ccagaggctg gtgggggaac cccgtctgtg gaccctgcca
5900ctgtgccgtc agcaaaggct ttgatcccga ctgtaataag accaacggcc
5950agtgccaatg caaggagaat tactacaagc tcctagccca ggacacctgt
6000ctgccctgcg actgcttccc ccatggctcc cacagccgca cttgcgacat
6050ggccaccggg cagtgtgcct gcaagcccgg cgtcatcggc cgccagtgca
6100accgctgcga caacccgttt gccgaggtca ccacgctcgg ctgtgaagtg
6150atctacaatg gctgtcccaa agcatttgag gccggcatct ggtggccaca
6200gaccaagttc gggcagccgg ctgcggtgcc atgccctaag ggatccgttg
6250gaaatgcggt ccgacactgc agcggggaga agggctggct gcccccagag
6300ctctttaact gtaccaccat ctccttcgtg gacctcaggg ccatgaatga
6350gaagctgagc cgcaatgaga cgcaggtgga cggcgccagg gccctgcagc
6400tggtgagggc gctgcgcagt gctacacagc acacgggcac gctctttggc
6450aatgacgtgc gcacggccta ccagctgctg ggccacgtcc ttcagcacga
6500gagctggcag cagggcttcg acctggcagc cacgcaggac gccgactttc
6550acgaggacgt catccactcg ggcagcgccc tcctggcccc agccaccagg
6600gcggcgtggg agcagatcca gcggagcgag ggcggcacgg cacagctgct
6650ccggcgcctc gagggctact tcagcaacgt ggcacgcaac gtgcggcgga
6700cgtacctgcg gcccttcgtc atcgtcaccg ccaacatgat tcttgctgtc
6750gacatctttg acaagttcaa ctttacggga gccagggtcc cgcgattcga
6800caccatccat gaagagttcc ccagggagct ggagtcctcc gtctccttcc
6850cagccgactt cttcagacca cctgaagaaa aagaaggccc cctgctgagg
6900ccggctggcc ggaggaccac cccgcagacc acgcgcccgg ggcctggcac
6950cgagagggag gccccgatca gcaggcggag gcgacaccct gatgacgctg
7000gccagttcgc cgtcgctctg gtcatcattt accgcaccct ggggcagctc
7050ctgcccgagc gctacgaccc cgaccgtcgc agcctccggt tgcctcaccg
7100gcccatcatt aataccccga tggtgagcac gctggtgtac agcgaggggg
7150ctccgctccc gagacccctg gagaggcccg tcctggtgga gttcgccctg
7200ctggaggtgg aggagcgaac caagcctgtc tgcgtgttct ggaaccactc
7250cctggccgtt ggtgggacgg gagggtggtc tgcccggggc tgcgagctcc
7300tgtccaggaa ccggacacat gtcgcctgcc agtgcagcca cacagccagc
7350tttgcggtgc tcatggatat ctccaggcgt gagaacgggg aggtcctgcc
7400tctgaagatt gtcacctatg ccgctgtgtc cttgtcactg gcagccctgc
7450tggtggcctt cgtcctcctg agcctggtcc gcatgctgcg ctccaacctg
7500cacagcattc acaagcacct cgccgtggcg ctcttcctct ctcagctggt
7550gttcgtgatt gggatcaacc agacggaaaa cccgtttctg tgcacagtgg
7600ttgccatcct cctccactac atctacatga gcacctttgc ctggaccctc
7650gtggagagcc tgcatgtcta ccgcatgctg accgaggtgc gcaacatcga
7700cacggggccc atgcggttct actacgtcgt gggctggggc atcccggcca
7750ttgtcacagg actggcggtc ggcctggacc cccagggcta cgggaacccc
7800gacttctgct ggctgtcgct tcaagacacc ctgatttgga gctttgcggg
7850gcccatcgga gctgttataa tcatcaacac agtcacttct gtcctatctg
7900caaaggtttc ctgccaaaga aagcaccatt attatgggaa aaaagggatc
7950gtctccctgc tgaggaccgc attcctcctg ctgctgctca tcagcgccac
8000ctggctgctg gggctgctgg ctgtgaaccg cgatgcactg agctttcact
8050acctcttcgc catcttcagc ggcttacagg gccccttcgt cctccttttc
8100cactgcgtgc tcaaccagga ggtccggaag cacctgaagg gcgtgctcgg
8150cgggaggaag ctgcacctgg aggactccgc caccaccagg gccaccctgc
8200tgacgcgctc cctcaactgc aacaccacct tcggtgacgg gcctgacatg
8250ctgcgcacag acttgggcga gtccaccgcc tcgctggaca gcatcgtcag
8300ggatgaaggg atccagaagc tcggcgtgtc ctctgggctg gtgaggggca
8350gccacggaga gccagacgcg tccctcatgc ccaggagctg caaggatccc
8400cctggccacg attccgactc agatagcgag ctgtccctgg atgagcagag
8450cagctcttac gcctcctcac actcgtcaga cagcgaggac gatggggtgg
8500gagctgagga aaaatgggac ccggccaggg gcgccgtcca cagcaccccc
8550aaaggggacg ctgtggccaa ccacgttccg gccggctggc ccgaccagag
8600cctggctgag agtgacagtg aggaccccag cggcaagccc cgcctgaagg
8650tggagaccaa ggtcagcgtg gagctgcacc gcgaggagca gggcagtcac
8700cgtggagagt accccccgga ccaggagagc gggggcgcag ccaggcttgc
8750tagcagccag cccccagagc agaggaaagg catcttgaaa aataaagtca
8800cctacccgcc gccgctgacg ctgacggagc agacgctgaa gggccggctc
8850cgggagaagc tggccgactg tgagcagagc cccacatcct cgcgcacgtc
8900ttccctgggc tctggcggcc ccgactgcgc catcacagtc aagagccctg
8950ggagggagcc ggggcgtgac cacctcaacg gggtggccat gaatgtgcgc
9000actgggagcg cccaggccga tggctccgac tctgagaaac cgtgaggcaa
9050gcccgtcacc ccacacaggc tgcggcatca ccctcagacc ttggagccca
9100aggggccact gcccttgaag tggagtgggc ccagagtgtg gcggtcccca
9150tggtggcagc cccccgactg atcatccaga cacaaaggtc ttggttctcc
9200caggagctca gggcctgtca gacctggtga caagtgccaa aggccacagg
9250catgagggag gcgtggacca ctgggccagc accgctgagt cctaagactg
9300cagtcaaagc cagaactgag aggggacccc agactgggcc cagaggctgg
9350ccagagttca ggaacgccgg gcacagacca aagaccgcgg tccagccccg
9400cccaggcggg catctcatgg cagtgcggac ccgtggctgg cagcccgggc
9450agtcctttgc aaaggcaccc cttgtcttaa aatcacttcg ctatgtggga
9500aaggtggaga tacttttata tatttgtatg ggactctgag gaggtgcaac
9550ctgtatatat attgcattcg tgctgacttt gttatcccga gagatccatg
9600caatgatctc ttgctgtctt ctctgtcaag attgcacagt tgtacttgaa
9650tctggcatgt gttgacgaaa ctggtgcccc agcagatcaa aggtgggaaa
9700tacgtcagca gtggggctaa aaccaagcgg ctagaagccc tacagctgcc
9750ttcggccagg aagtgaggat ggtgtgggcc ctccccgccg gccccctggg
9800tccccagtgt tcgctgtgtg tgcgtttgtc ctctgctgcc atctgccccg
9850gctgtgtgaa ttcaagacag ggcagtgcag cactaggcag gtgtgaggag
9900ccctgctgag gtcactgtgg ggcacggttg ccacacggct gtcatttttc
9950acctggtcat tctgtgacca ccaccccctc ccctcaccgc ctcccaggtg
10000gcccgggagc tgcaggtggg gatggctttg tcctttgctc ctgctccccg
10050tgggacctgg gaccttaaag cgttgcaggt tcctgatttg gacagaggtg
10100tggggccttc caggccgtta catacctcct gccaattctc taactctctg
10150agactgcgag gatctccagg cagggttctc ccctctggag tctgaccaat
10200tacttcattt tgcttcaaat ggccaattgt gcagagggac aaagccacag
10250ccacactctt caacggttac caaactgttt ttggaaattc acaccaaggt
10300cgggcccact gcaggcagct ggcacagcgt ggcccgaggg gctgtggaac
10350gggtcccgga actgtcagac atgtttgatt ttagcgtttc ctttgttctt
10400caaatcaggt gcccaaataa gtgatcagca cagctgcttc caaataggag
10450aaaccataaa ataggatgaa aatcaagtaa aatgcaaaga tgtccacact
10500gttttaaact tgaccctgat gaaaatgtga gcactgttag cagatgccta
10550tgggagagga aaagcgtatc tgaaaatggt ccaggacagg aggatgaaat
10600gagatcccag agtcctcaca cctgaatgaa ttatacatgt gccttaccag
10650gtgagtggtc tttcgaagat aaaaaactct agtcccttta aacgtttgcc
10700cctggcgttt cctaagtacg aaaaggtttt taagtcttcg aacagtctcc
10750tttcatgact ttaacaggat tctgccccct gaggtgtaat ttttttgttc
10800tatttttttc cacgtactcc acagccaaca tcacgaggtg taatttttaa
10850tttgatcaga actgttacca aaaaacaact gtcagtttta ttgagatggg
10900aaaaatgtaa acctattttt attacttaag actttatggg agagattaga
10950cactggaggt ttttaacaga acgtgtattt attaatgttc aaaacactgg
11000aattacaaat gagaagagtc tacaataaat taagattttt gaatttgtac
11050ttctgcggtg ctggtttttc tccacaaaca cccccgcccc tccccatgcc
11100cagggtggcc gtggaaggga cggtttacgg acgtgcagct gagctgtccg
11150tgtcccatgc tccctcagcc agtggaacgt gccggaactt tttgtccatt
11200ccctagtagg cctgccacag cctagatggg cagtttttgt ctttcaccaa
11250atttgaggac tttttttttt tgccattatt tcttcagttt tcttttcttg
11300cactgatctt tctcctctcc ttctgtgact ccagtgactc agacgttaga
11350cctcttgatg ttttcccact ggtccctgag gctctgttc
11389521107DNAHomo Sapienunsure170-208unknown base 52cggcctaagg
tagcgacggg actggccggg ggcggcagga cccgaaggcg 50ctaggcggat
tcaccggatg ggagttgaat cgcgtcccgg tctttctagc 100tgtgcccgga
aatcgggcgt gcgggcagct acagcagaga atcggacaag 150gagggaagaa
agagatggtn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 200nnnnnnnnga
agtgagtgca agaggagccg gcttagcatc taaactgatt 250ctaccatcag
aaaagaggcc aaacttctat catcatggtg gatgtgaagt 300gtctgagtga
ctgtaaattg cagaaccaac ttgagaagct tggattttca 350cctggcccaa
tactaccttc caccagaaag ttgtatgaaa aaaagttagt 400acagttgttg
gtctcacctc cctgtgcacc acctgtgatg aatggaccca 450gagagctgga
tggagcgcag gacagtgatg acagcgaaga gcttaatatc 500attttgcaag
gaaatatcat actctcaaca gaaaaaagca agaaactcaa 550aaaatggcct
gaggcttcca ccactaaacg caaagctgta gatacctatt 600gcttggatta
taagccttcc aagggaagaa ggtgggctgc aagagcacca 650agcaccagaa
tcacatatgg gactatcacc aaagagagag actactgcgc 700ggaagaccag
actatcgaga gctggagaga agaaggtttc ccagtgggct 750tgaagcttgc
tgtgcttggt attttcatca ttgtggtgtt tgtctacctg 800actgtggaaa
ataagtcgct gtttggttaa gtaatttagg agcaaagcaa 850tgctccaagc
gaggcctcct gcttcaggaa agaaccaaaa cactaccctg 900aagggccagc
ctagcctgca gccctccctt gcagggagcc ttcccttgca 950ctgtgctgct
ctcacagatc ggtgtctggg ctcagccagg tggaaggaac 1000ctgcctaacc
aggcacctgt gttaagagca tgatggttag gaaatccccc 1050aagtcatgtc
aactctcatt aaaggtgctt ccatatttga gcaggcgtca 1100aacaagg
1107533946DNAHomo
Sapien 53accgctccgg agcgggaggg gaggcttcgc ggaacgctct cggcgccagg
50actcgcgtgc aaagcccagg cccgggcggc cagaccaaga gggaagaagc
100acagaattcc tcaactccca gtgtgcccat gagtaagagc aaatgctccg
150tgggactcat gtcttccgtg gtggccccgg ctaaggagcc caatgccgtg
200ggcccgaagg aggtggagct catccttgtc aaggagcaga acggagtgca
250gctcaccagc tccaccctca ccaacccgcg gcagagcccc gtggaggccc
300aggatcggga gacctggggc aagaagatcg actttctcct gtccgtcatt
350ggctttgctg tggacctggc caacgtctgg cggttcccct acctgtgcta
400caaaaatggt ggcggtgcct tcctggtccc ctacctgctc ttcatggtca
450ttgctgggat gccacttttc tacatggagc tggccctcgg ccagttcaac
500agggaagggg ccgctggtgt ctggaagatc tgccccatac tgaaaggtgt
550gggcttcacg gtcatcctca tctcactgta tgtcggcttc ttctacaacg
600tcatcatcgc ctgggcgctg cactatctct tctcctcctt caccacggag
650ctcccctgga tccactgcaa caactcctgg aacagcccca actgctcgga
700tgcccatcct ggtgactcca gtggagacag ctcgggcctc aacgacactt
750ttgggaccac acctgctgcc gagtactttg aacgtggcgt gctgcacctc
800caccagagcc atggcatcga cgacctgggg cctccgcggt ggcagctcac
850agcctgcctg gtgctggtca tcgtgctgct ctacttcagc ctctggaagg
900gcgtgaagac ctcagggaag gtggtatgga tcacagccac catgccatac
950gtggtcctca ctgccctgct cctgcgtggg gtcaccctcc ctggagccat
1000agacggcatc agagcatacc tgagcgttga cttctaccgg ctctgcgagg
1050cgtctgtttg gattgacgcg gccacccagg tgtgcttctc cctgggcgtg
1100gggttcgggg tgctgatcgc cttctccagc tacaacaagt tcaccaacaa
1150ctgctacagg gacgcgattg tcaccacctc catcaactcc ctgacgagct
1200tctcctccgg cttcgtcgtc ttctccttcc tggggtacat ggcacagaag
1250cacagtgtgc ccatcgggga cgtggccaag gacgggccag ggctgatctt
1300catcatctac ccggaagcca tcgccacgct ccctctgtcc tcagcctggg
1350ccgtggtctt cttcatcatg ctgctcaccc tgggtatcga cagcgccatg
1400ggtggtatgg agtcagtgat caccgggctc atcgatgagt tccagctgct
1450gcacagacac cgtgagctct tcacgctctt catcgtcctg gcgaccttcc
1500tcctgtccct gttctgcgtc accaacggtg gcatctacgt cttcacgctc
1550ctggaccatt ttgcagccgg cacgtccatc ctctttggag tgctcatcga
1600agccatcgga gtggcctggt tctatggtgt tgggcagttc agcgacgaca
1650tccagcagat gaccgggcag cggcccagcc tgtactggcg gctgtgctgg
1700aagctggtca gcccctgctt tctcctgttc gtggtcgtgg tcagcattgt
1750gaccttcaga cccccccact acggagccta catcttcccc gactgggcca
1800acgcgctggg ctgggtcatc gccacatcct ccatggccat ggtgcccatc
1850tatgcggcct acaagttctg cagcctgcct gggtcctttc gagagaaact
1900ggcctacgcc attgcacccg agaaggaccg tgagctggtg gacagagggg
1950aggtgcgcca gttcacgctc cgccactggc tcaaggtgta gagggagcag
2000agacgaagac cccaggaagt catcctgcaa tgggagagac acgaacaaac
2050caaggaaatc taagtttcga gagaaaggag ggcaacttct actcttcaac
2100ctctactgaa aacacaaaca acaaagcaga agactcctct cttctgactg
2150tttacacctt tccgtgccgg gagcgcacct cgccgtgtct tgtgttgctg
2200taataacgac gtagatctgt gcagcgaggt ccaccccgtt gttgtccctg
2250cagggcagaa aaacgtctaa cttcatgctg tctgtgtgag gctccctccc
2300tccctgctcc ctgctcccgg ctctgaggct gccccagggg cactgtgttc
2350tcaggcgggg atcacgatcc ttgtagacgc acctgctgag aatccccgtg
2400ctcacagtag cttcctagac catttacttt gcccatatta aaaagccaag
2450tgtcctgctt ggtttagctg tgcagaaggt gaaatggagg aaaccacaaa
2500ttcatgcaaa gtcctttccc gatgcgtggc tcccagcaga ggccgtaaat
2550tgagcgttca gttgacacat tgcacacaca gtctgttcag aggcattgga
2600ggatgggggt cctggtatgt ctcaccagga aattctgttt atgttcttgc
2650agcagagaga aataaaactc cttgaaacca gctcaggcta ctgccactca
2700ggcagcctgt gggtccttgt ggtgtaggga acggcctgag aggagcgtgt
2750cctatccccg gacgcatgca gggcccccac aggagcgtgt cctatccccg
2800gacgcatgca gggcccccac aggagcatgt cctatccctg gacgcatgca
2850gggcccccac aggagcgtgt actaccccag aacgcatgca gggcccccac
2900aggagcgtgt actaccccag gacgcatgca gggcccccac tggagcgtgt
2950actaccccag gacgcatgca gggcccccac aggagcgtgt cctatccccg
3000gaccggacgc atgcagggcc cccacaggag cgtgtactac cccaggacgc
3050atgcagggcc cccacaggag cgtgtactac cccaggatgc atgcagggcc
3100cccacaggag cgtgtactac cccaggacgc atgcagggcc cccatgcagg
3150cagcctgcag accaacactc tgcctggcct tgagccgtga cctccaggaa
3200gggaccccac tggaatttta tttctctcag gtgcgtgcca catcaataac
3250aacagttttt atgtttgcga atggcttttt aaaatcatat ttacctgtga
3300atcaaaacaa attcaagaat gcagtatccg cgagcctgct tgctgatatt
3350gcagtttttg tttacaagaa taattagcaa tactgagtga aggatgttgg
3400ccaaaagctg ctttccatgg cacactgccc tctgccactg acaggaaagt
3450ggatgccata gtttgaattc atgcctcaag tcggtgggcc tgcctacgtg
3500ctgcccgagg gcaggggccg tgcagggcca gtcatggctg tcccctgcaa
3550gtggacgtgg gctccaggga ctggagtgta atgctcggtg ggagccgtca
3600gcctgtgaac tgccaggcag ctgcagttag cacagaggat ggcttcccca
3650ttgccttctg gggagggaca cagaggacgg cttccccatc gccttctggc
3700cgctgcagtc agcacagaga gcggcttccc cattgccttc tggggaggga
3750cacagaggac agtttcccca tcgccttctg gttgttgaag acagcacaga
3800gagcggcttc cccatcgcct tctggggagg ggctccgtgt agcaacccag
3850gtgttgtccg tgtctgttga ccaatctcta ttcagcatcg tgtgggtccc
3900taagcacaat aaaagacatc cacaatggaa aaaaaaaaag gaattc
3946542317DNAHomo Sapien 54cggacgcgtg ggtgagcagg gacggtgcac cggacggcgg
gatcgagcaa 50atgggtctgg ccatggagca cggagggtcc tacgctcggg
cggggggcag 100ctctcggggc tgctggtatt acctgcgcta cttcttcctc
ttcgtctccc 150tcatccaatt cctcatcatc ctggggctcg tgctcttcat
ggtctatggc 200aacgtgcacg tgagcacaga gtccaacctg caggccaccg
agcgccgagc 250cgagggccta tacagtcagc tcctagggct cacggcctcc
cagtccaact 300tgaccaagga gctcaacttc accacccgcg ccaaggatgc
catcatgcag 350atgtggctga atgctcgccg cgacctggac cgcatcaatg
ccagcttccg 400ccagtgccag ggtgaccggg tcatctacac gaacaatcag
aggtacatgg 450ctgccatcat cttgagtgag aagcaatgca gagatcaatt
caaggacatg 500aacaagagct gcgatgcctt gctcttcatg ctgaatcaga
aggtgaagac 550gctggaggtg gagatagcca aggagaagac catttgcact
aaggataagg 600aaagcgtgct gctgaacaaa cgcgtggcgg aggaacagct
ggttgaatgc 650gtgaaaaccc gggagctgca gcaccaagag cgccagctgg
ccaaggagca 700actgcaaaag gtgcaagccc tctgcctgcc cctggacaag
gacaagtttg 750agatggacct tcgtaacctg tggagggact ccattatccc
acgcagcctg 800gacaacctgg gttacaacct ctaccatccc ctgggctcgg
aattggcctc 850catccgcaga gcctgcgacc acatgcccag cctcatgagc
tccaaggtgg 900aggagctggc ccggagcctc cgggcggata tcgaacgcgt
ggcccgcgag 950aactcagacc tccaacgcca gaagctggaa gcccagcagg
gcctgcgggc 1000cagtcaggag gcgaaacaga aggtggagaa ggaggctcag
gcccgggagg 1050ccaagctcca agctgaatgc tcccggcaga cccagctagc
gctggaggag 1100aaggcggtgc tgcggaagga acgagacaac ctggccaagg
agctggaaga 1150gaagaagagg gaggcggagc agctcaggat ggagctggcc
atcagaaact 1200cagccctgga cacctgcatc aagaccaagt cgcagccgat
gatgccagtg 1250tcaaggccca tgggccctgt ccccaacccc cagcccatcg
acccagctag 1300cctggaggag ttcaagagga agatcctgga gtcccagagg
ccccctgcag 1350gcatccctgt agccccatcc agtggctgag gaggctccag
gcctgaggac 1400caagggatgg cccgactcgg cggtttgcgg aggatgcagg
gatatgctca 1450cagcgcccga cacaaccccc tcccgccgcc cccaaccacc
cagggccacc 1500atcagacaac tccctgcatg caaaccccta gtaccctctc
acacccgcac 1550ccgcgcctca cgatccctca cccagagcac acggccgcgg
agatgacgtc 1600acgcaagcaa cggcgctgac gtcacatatc accgtggtga
tggcgtcacg 1650tggccatgta gacgtcacga agagatatag cgatggcgtc
gtgcagatgc 1700agcacgtcgc acacagacat ggggaacttg gcatgacgtc
acaccgagat 1750gcagcaacga cgtcacgggc catgtcgacg tcacacatat
taatgtcaca 1800cagacgcggc gatggcatca cacagacggt gatgatgtca
cacacagaca 1850cagtgacaac acacaccatg acaacgacac ctatagatat
ggcaccaaca 1900tcacatgcac gcatgccctt tcacacacac tttctaccca
attctcacct 1950agtgtcacgt tcccccgacc ctggcacacg ggccaaggta
cccacaggat 2000cccatcccct cccgcacagc cctgggcccc agcacctccc
ctcctccagc 2050ttcctggcct cccagccact tcctcacccc cagtgcctgg
acccggaggt 2100gagaacagga agccattcac ctccgctcct tgagcgtgag
tgtttccagg 2150accccctcgg ggccctgagc cgggggtgag ggtcacctgt
tgtcgggagg 2200ggagccactc cttctccccc aactcccagc cctgcctgtg
gcccgttgaa 2250atgttggtgg cacttaataa atattagtaa atccttaaaa
aaaaaaaaaa 2300aaaaaaaaaa aaaaaaa
231755756DNAHomo Sapien 55cggacttggc ttgttagaag
gctgaaagat gatggcagga atgaaaatcc 50agcttgtatg catgctactc
ctggctttca gctcctggag tctgtgctca 100gattcagaag aggaaatgaa
agcattagaa gcagatttct tgaccaatat 150gcatacatca aagattagta
aagcacatgt tccctcttgg aagatgactc 200tgctaaatgt ttgcagtctt
gtaaataatt tgaacagccc agctgaggaa 250acaggagaag ttcatgaaga
ggagcttgtt gcaagaagga aacttcctac 300tgctttagat ggctttagct
tggaagcaat gttgacaata taccagctcc 350acaaaatctg tcacagcagg
gcttttcaac actgggagtt aatccaggaa 400gatattcttg atactggaaa
tgacaaaaat ggaaaggaag aagtcataaa 450gagaaaaatt ccttatattc
tgaaacggca gctgtatgag aataaaccca 500gaagacccta catactcaaa
agagattctt actattactg agagaataaa 550tcatttattt acatgtgatt
gtgattcatc atcccttaat taaatatcaa 600attatatttg tgtgaaaatg
tgacaaacac acttatctgt ctcttctaca 650attgtggttt attgaatgtg
tttttctgca ctaatagaaa ttagactaag 700tgttttcaaa taaatctaaa
tcttcaaaaa aaaaaaaaaa aaatggggcc 750gcaatt
756563722DNAHomo Sapien
56cgcggggcgc ggagtcggcg gggcctcgcg ggacgcgggc agtgcggaga
50ccgcggcgct gaggacgcgg gagccgggag cgcacgcgcg gggtggagtt
100cagcctactc tttcttagat gtgaaaggaa aggaagatca tttcatgcct
150tgttgataaa ggttcagact tctgctgatt cataaccatt tggctctgag
200ctatgacaag agaggaaaca aaaagttaaa cttacaagcc tgccataagt
250gagaagcaaa cttccttgat aacatgcttt tgcgaagtgc aggaaaatta
300aatgtgggca ccaagaaaga ggatggtgag agtacagccc ccaccccccg
350tccaaaggtc ttgcgttgta aatgccacca ccattgtcca gaagactcag
400tcaacaatat ttgcagcaca gacggatatt gtttcacgat gatagaagag
450gatgactctg ggttgcctgt ggtcacttct ggttgcctag gactagaagg
500ctcagatttt cagtgtcggg acactcccat tcctcatcaa agaagatcaa
550ttgaatgctg cacagaaagg aacgaatgta ataaagacct acaccctaca
600ctgcctccat tgaaaaacag agattttgtt gatggaccta tacaccacag
650ggctttactt atatctgtga ctgtctgtag tttgctcttg gtccttatca
700tattattttg ttacttccgg tataaaagac aagaaaccag acctcgatac
750agcattgggt tagaacagga tgaaacttac attcctcctg gagaatccct
800gagagactta attgagcagt ctcagagctc aggaagtgga tcaggcctcc
850ctctgctggt ccaaaggact atagctaagc agattcagat ggtgaaacag
900attggaaaag gtcgctatgg ggaagtttgg atgggaaagt ggcgtggcga
950aaaggtagct gtgaaagtgt tcttcaccac agaggaagcc agctggttca
1000gagagacaga aatatatcag acagtgttga tgaggcatga aaacattttg
1050ggtttcattg ctgcagatat caaagggaca gggtcctgga cccagttgta
1100cctaatcaca gactatcatg aaaatggttc cctttatgat tatctgaagt
1150ccaccaccct agacgctaaa tcaatgctga agttagccta ctcttctgtc
1200agtggcttat gtcatttaca cacagaaatc tttagtactc aaggcaaacc
1250agcaattgcc catcgagatc tgaaaagtaa aaacattctg gtgaagaaaa
1300atggaacttg ctgtattgct gacctgggcc tggctgttaa atttattagt
1350gatacaaatg aagttgacat accacctaac actcgagttg gcaccaaacg
1400ctatatgcct ccagaagtgt tggacgagag cttgaacaga aatcacttcc
1450agtcttacat catggctgac atgtatagtt ttggcctcat cctttgggag
1500gttgctagga gatgtgtatc aggaggtata gtggaagaat accagcttcc
1550ttatcatgac ctagtgccca gtgacccctc ttatgaggac atgagggaga
1600ttgtgtgcat caagaagtta cgcccctcat tcccaaaccg gtggagcagt
1650gatgagtgtc taaggcagat gggaaaactc atgacagaat gctgggctca
1700caatcctgca tcaaggctga cagccctgcg ggttaagaaa acacttgcca
1750aaatgtcaga gtcccaggac attaaactct gataggagag gaaaagtaag
1800catctctgca gaaagccaac aggtactctt ctgtttgtgg gcagagcaaa
1850agacatcaaa taagcatcca cagtacaagc cttgaacatc gtcctgcttc
1900ccagtgggtt cagacctcac ctttcaggga gcgacctggg caaagacaga
1950gaagctccca gaaggagaga ttgatccatg tctgtttgta ggacggagaa
2000accgcttggg taacttgttc aagatatgat gcatgttgct ttctaagaaa
2050gccctgtatt ttgtgattgc cttttttttt ttttaagatg ctttcatttt
2100gccaaaataa aacagataat gtggatggtt taagggttat agtattatag
2150tttaaataat aacaacaaaa ttcttcccag gaactctgct ggaaggtaaa
2200ttaaaatact tgtttttcca ttggtaaaat attgttgcac tctgtgaacc
2250aaaagacagt ctaagttgga ggacatagaa cggaactcat cttaaacata
2300ctccccaccc cgtcttggcc tcctcagacc actttggcca tccctgcatt
2350tggggccgct atggtaatgt gaatgcactg ggtacaaaca ccgcctgtct
2400aggaccacat ttggaattcc tgcaggtggc cttttgcagc ttcaggcaat
2450atggaacaaa tgaaggttta tgtgactcta atagaagtaa ttgttgatag
2500gtgtttttca gatccacttc tgtttctgat tgagttaggc atctctttca
2550tggtaaaacc cttttcatta aacacaaaaa aagctttttt tttttttttt
2600tttttttttt ttttttaatg tgcagaggat tgacctgtgc atgcttttga
2650tctctcattc aaaggatcaa tattaaataa aattgtcatg agctgtgttg
2700aagacagggt gctttcaaat agaggtaatt tgctcttgtg ttgtaagagg
2750aacatgtcaa caaagatagg aaatgagggt gatcgtgcag atggcttgta
2800tcttatatat gcaaaggagc caatctcaga agcacaaaga aaaaagtgtg
2850cataccttat tttgtacaga taaagatgat gtctttttgt tattgtctgt
2900ctgttttgta tgtgtctgag ataagggata gagaggaaac atccgtcagg
2950ctaatttaac tacattttat tttaaaaata gagaaacata acctctagat
3000gggacagcag aggacagtta gtagaggcca caaactgtta tgggctgctg
3050tgttttgttc taaaatcaat atggttggag catgtatatc ttaggtgatc
3100atttcacatc ttaggaatgc ctactcattt tattttattc tagtgatgct
3150caattcacta tttaatttat tatattttct cttctgtggc acttatacaa
3200aatatctctt cacctactta gttctacagg gttttaactt tggagcaaca
3250tgaataaaat catcgagaag gccaatattg tttagcaaca tgaatacaat
3300acagtttaaa gttgtacaca tcctgctcaa ctttattcat atacatttcc
3350tttctgtggt tttcttttgc ttcttagaaa ttctgttagt ggttagtaaa
3400gaatttgaaa gtactttctc cttgctgttt tttttttttt ttaagacatt
3450cctcccagaa tactccaggg ggcagtgttt tataacacat tttccccact
3500gggtgattga aggatggagg atttttgaaa atttgacagc tacatgaaac
3550atgagaaaac attttcctca cttctgaagt cggtttgcag ctggtaactt
3600gttcatccag aaaacattct aaagcaatga gactttgtga gctgtgctta
3650cagtttggga gaatcatgaa gattctttct atattttgca tttacttccc
3700agtgcttcat agctgcattt tg
372257837PRTHomo Sapien 57Met Leu Arg Thr Ala Met Gly Leu Arg Ser Trp Leu
Ala Ala Pro 1 5 10 15Trp
Gly Ala Leu Pro Pro Arg Pro Pro Leu Leu Leu Leu Leu Leu 20
25 30Leu Leu Leu Leu Leu Gln Pro Pro Pro
Pro Thr Trp Ala Leu Ser 35 40
45Pro Arg Ile Ser Leu Pro Leu Gly Ser Glu Glu Arg Pro Phe Leu50
55 60Arg Phe Glu Ala Glu His Ile Ser Asn Tyr Thr
Ala Leu Leu Leu65 70 75Ser Arg Asp Gly
Arg Thr Leu Tyr Val Gly Ala Arg Glu Ala Leu80 85
90Phe Ala Leu Ser Ser Asn Leu Ser Phe Leu Pro Gly Gly Glu Tyr95
100 105Gln Glu Leu Leu Trp Gly Ala Asp Ala
Glu Lys Lys Gln Gln Cys110 115 120Ser Phe
Lys Gly Lys Asp Pro Gln Arg Asp Cys Gln Asn Tyr Ile125
130 135Lys Ile Leu Leu Pro Leu Ser Gly Ser His Leu Phe
Thr Cys Gly140 145 150Thr Ala Ala Phe Ser
Pro Met Cys Thr Tyr Ile Asn Met Glu Asn155 160
165Phe Thr Leu Ala Arg Asp Glu Lys Gly Asn Val Leu Leu Glu Asp170
175 180Gly Lys Gly Arg Cys Pro Phe Asp Pro
Asn Phe Lys Ser Thr Ala185 190 195Leu Val
Val Asp Gly Glu Leu Tyr Thr Gly Thr Val Ser Ser Phe200
205 210Gln Gly Asn Asp Pro Ala Ile Ser Arg Ser Gln Ser
Leu Arg Pro215 220 225Thr Lys Thr Glu Ser
Ser Leu Asn Trp Leu Gln Asp Pro Ala Phe230 235
240Val Ala Ser Ala Tyr Ile Pro Glu Ser Leu Gly Ser Leu Gln Gly245
250 255Asp Asp Asp Lys Ile Tyr Phe Phe Phe
Ser Glu Thr Gly Gln Glu260 265 270Phe Glu
Phe Phe Glu Asn Thr Ile Val Ser Arg Ile Ala Arg Ile275
280 285Cys Lys Gly Asp Glu Gly Gly Glu Arg Val Leu Gln
Gln Arg Trp290 295 300Thr Ser Phe Leu Lys
Ala Gln Leu Leu Cys Ser Arg Pro Asp Asp305 310
315Gly Phe Pro Phe Asn Val Leu Gln Asp Val Phe Thr Leu Ser Pro320
325 330Ser Pro Gln Asp Trp Arg Asp Thr Leu
Phe Tyr Gly Val Phe Thr335 340 345Ser Gln
Trp His Arg Gly Thr Thr Glu Gly Ser Ala Val Cys Val350
355 360Phe Thr Met Lys Asp Val Gln Arg Val Phe Ser Gly
Leu Tyr Lys365 370 375Glu Val Asn Arg Glu
Thr Gln Gln Trp Tyr Thr Val Thr His Pro380 385
390Val Pro Thr Pro Arg Pro Gly Ala Cys Ile Thr Asn Ser Ala Arg395
400 405Glu Arg Lys Ile Asn Ser Ser Leu Gln
Leu Pro Asp Arg Val Leu410 415 420Asn Phe
Leu Lys Asp His Phe Leu Met Asp Gly Gln Val Arg Ser425
430 435Arg Met Leu Leu Leu Gln Pro Gln Ala Arg Tyr Gln
Arg Val Ala440 445 450Val His Arg Val Pro
Gly Leu His His Thr Tyr Asp Val Leu Phe455 460
465Leu Gly Thr Gly Asp Gly Arg Leu His Lys Ala Val Ser Val Gly470
475 480Pro Arg Val His Ile Ile Glu Glu Leu
Gln Ile Phe Ser Ser Gly485 490 495Gln Pro
Val Gln Asn Leu Leu Leu Asp Thr His Arg Gly Leu Leu500
505 510Tyr Ala Ala Ser His Ser Gly Val Val Gln Val Pro
Met Ala Asn515 520 525Cys Ser Leu Tyr Arg
Ser Cys Gly Asp Cys Leu Leu Ala Arg Asp530 535
540Pro Tyr Cys Ala Trp Ser Gly Ser Ser Cys Lys His Val Ser Leu545
550 555Tyr Gln Pro Gln Leu Ala Thr Arg Pro
Trp Ile Gln Asp Ile Glu560 565 570Gly Ala
Ser Ala Lys Asp Leu Cys Ser Ala Ser Ser Val Val Ser575
580 585Pro Ser Phe Val Pro Thr Gly Glu Lys Pro Cys Glu
Gln Val Gln590 595 600Phe Gln Pro Asn Thr
Val Asn Thr Leu Ala Cys Pro Leu Leu Ser605 610
615Asn Leu Ala Thr Arg Leu Trp Leu Arg Asn Gly Ala Pro Val Asn620
625 630Ala Ser Ala Ser Cys His Val Leu Pro
Thr Gly Asp Leu Leu Leu635 640 645Val Gly
Thr Gln Gln Leu Gly Glu Phe Gln Cys Trp Ser Leu Glu650
655 660Glu Gly Phe Gln Gln Leu Val Ala Ser Tyr Cys Pro
Glu Val Val665 670 675Glu Asp Gly Val Ala
Asp Gln Thr Asp Glu Gly Gly Ser Val Pro680 685
690Val Ile Ile Ser Thr Ser Arg Val Ser Ala Pro Ala Gly Gly Lys695
700 705Ala Ser Trp Gly Ala Asp Arg Ser Tyr
Trp Lys Glu Phe Leu Val710 715 720Met Cys
Thr Leu Phe Val Leu Ala Val Leu Leu Pro Val Leu Phe725
730 735Leu Leu Tyr Arg His Arg Asn Ser Met Lys Val Phe
Leu Lys Gln740 745 750Gly Glu Cys Ala Ser
Val His Pro Lys Thr Cys Pro Val Val Leu755 760
765Pro Pro Glu Thr Arg Pro Leu Asn Gly Leu Gly Pro Pro Ser Thr770
775 780Pro Leu Asp His Arg Gly Tyr Gln Ser
Leu Ser Asp Ser Pro Pro785 790 795Gly Ala
Arg Val Phe Thr Glu Ser Glu Lys Arg Pro Leu Ser Ile800
805 810Gln Asp Ser Phe Val Glu Val Ser Pro Val Cys Pro
Arg Pro Arg815 820 825Val Arg Leu Gly Ser
Glu Ile Arg Asp Ser Val Val830 83558188PRTHomo Sapien
58Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp1
5 10 15Ile Met Ala Ile Ser Lys Val
Phe Glu Leu Gly Leu Val Ala Gly20 25
30Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala35
40 45Phe Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu
Pro Ala Ile Arg50 55 60Pro Arg Ser Ser
Gln Arg Val Pro Pro Met Gly Ile Gln His Ser65 70
75Lys Glu Leu Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met80
85 90Leu Gly Ser Phe Cys Ala Cys Pro Pro
Ser Phe Tyr Gly Arg Asn95 100 105Cys Glu
His Asp Val Arg Lys Glu Asn Cys Gly Ser Val Pro His110
115 120Asp Thr Trp Leu Pro Lys Lys Cys Ser Leu Cys Lys
Cys Trp His125 130 135Gly Gln Leu Arg Cys
Phe Pro Gln Ala Phe Leu Pro Gly Cys Asp140 145
150Gly Leu Val Met Asp Glu His Leu Val Ala Ser Arg Thr Pro Glu155
160 165Leu Pro Pro Ser Ala Arg Thr Thr Thr
Phe Met Leu Val Gly Ile170 175 180Cys Leu
Ser Ile Gln Ser Tyr Tyr1855980PRTHomo Sapien 59Met Ala Ala Arg Ala Leu
Cys Met Leu Gly Leu Val Leu Ala Leu1 5 10
15Leu Ser Ser Ser Ser Ala Glu Glu Tyr Val Gly Leu Ser Ala
Asn20 25 30Gln Cys Ala Val Pro Ala Lys
Asp Arg Val Asp Cys Gly Tyr Pro35 40
45His Val Thr Pro Lys Glu Cys Asn Asn Arg Gly Cys Cys Phe Asp50
55 60Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys
Pro Leu Gln Glu65 70 75Ala Glu Cys Thr
Phe8060314PRTHomo Sapien 60Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu
Gly Ile Thr Cys1 5 10
15Ala Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys20
25 30Gln Leu Tyr Asn Lys Tyr Pro Asp Ala Val Ala
Thr Trp Leu Asn35 40 45Pro Asp Pro Ser
Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala50 55
60Val Ser Ser Glu Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro65
70 75Ser Lys Ser Asn Glu Ser His Asp His
Met Asp Asp Met Asp Asp80 85 90Glu Asp
Asp Asp Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser95 100
105Asn Asp Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His
Gln Ser110 115 120Asp Glu Ser His His Ser
Asp Glu Ser Asp Glu Leu Val Thr Asp125 130
135Phe Pro Thr Asp Leu Pro Ala Thr Glu Val Phe Thr Pro Val Val140
145 150Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly
Asp Ser Val Val Tyr155 160 165Gly Leu Arg
Ser Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln170 175
180Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His Met Glu
Ser185 190 195Glu Glu Leu Asn Gly Ala Tyr
Lys Ala Ile Pro Val Ala Gln Asp200 205
210Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser Tyr215
220 225Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala
Glu Thr His Ser His230 235 240Lys Gln Ser
Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn245 250
255Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val
Ser260 265 270Arg Glu Phe His Ser His Glu
Phe His Ser His Glu Asp Met Leu275 280
285Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe290
295 300Arg Ile Ser His Glu Leu Asp Ser Ala Ser
Ser Glu Val Asn305 31061184PRTHomo Sapien 61Met Ser Arg
Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu Leu Leu1 5
10 15Gly Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys
Lys Gly Ser Gln20 25 30Gly Ala Ile Pro
Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu35 40
45Gln Thr Gln Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg Gly Arg50
55 60Gly Gln Gly Arg Gly Thr Ala Met Pro
Gly Glu Glu Val Leu Glu65 70 75Ser Ser
Gln Glu Ala Leu His Val Thr Glu Arg Lys Tyr Leu Lys80 85
90Arg Asp Trp Cys Lys Thr Gln Pro Leu Lys Gln Thr Ile
His Glu95 100 105Glu Gly Cys Asn Ser Arg
Thr Ile Ile Asn Arg Phe Cys Tyr Gly110 115
120Gln Cys Asn Ser Phe Tyr Ile Pro Arg His Ile Arg Lys Glu Glu125
130 135Gly Ser Phe Gln Ser Cys Ser Phe Cys Lys
Pro Lys Lys Phe Thr140 145 150Thr Met Met
Val Thr Leu Asn Cys Pro Glu Leu Gln Pro Pro Thr155 160
165Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys Arg Cys Ile
Ser170 175 180Ile Asp Leu Asp62460PRTHomo
Sapien 62Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu1
5 10 15Leu Leu Ser Ala Glu
Val Ser Gly Gly Asp Arg Leu Asp Cys Val20 25
30Lys Ala Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys35
40 45Tyr Arg Thr Leu Arg Gln Cys Val Ala Gly
Lys Glu Thr Asn Phe50 55 60Ser Leu Ala
Ser Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala65 70
75Met Glu Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg Cys
Lys80 85 90Arg Gly Met Lys Lys Glu Lys
Asn Cys Leu Arg Ile Tyr Trp Ser95 100
105Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro110
115 120Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp
Ile Phe Arg Val Val125 130 135Pro Phe Ile
Ser Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu140 145
150Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Ile Cys Lys Lys
Tyr155 160 165Arg Ser Ala Tyr Ile Thr Pro
Cys Thr Thr Ser Val Ser Asn Asp170 175
180Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe185
190 195Asp Lys Val Pro Ala Lys His Ser Tyr Gly
Met Leu Phe Cys Ser200 205 210Cys Arg Asp
Ile Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val215 220
225Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu
Asn230 235 240Leu Gln Asp Ser Cys Lys Thr
Asn Tyr Ile Cys Arg Ser Arg Leu245 250
255Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg Ser Val Ser260
265 270Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys
Leu Leu Ala Tyr Ser275 280 285Gly Leu Ile
Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser Ser290 295
300Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly
Asn305 310 315Asp Leu Glu Glu Cys Leu Lys
Phe Leu Asn Phe Phe Lys Asp Asn320 325
330Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp335
340 345Val Thr Val Trp Gln Pro Ala Phe Pro Val
Gln Thr Thr Thr Ala350 355 360Thr Thr Thr
Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro365 370
375Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro
Cys380 385 390Ala Asn Leu Gln Ala Gln Lys
Leu Lys Ser Asn Val Ser Gly Asn395 400
405Thr His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu410
415 420Gly Ala Ser Ser His Ile Thr Thr Lys Ser
Met Ala Ala Pro Pro425 430 435Ser Cys Gly
Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu440 445
450Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser455
46063143PRTHomo Sapien 63Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu Leu
Ala Leu Leu1 5 10 15Ala
Leu Thr Ser Ala Val Ala Lys Lys Lys Asp Lys Val Lys Lys20
25 30Gly Gly Pro Gly Ser Glu Cys Ala Glu Trp Ala Trp
Gly Pro Cys35 40 45Thr Pro Ser Ser Lys
Asp Cys Gly Val Gly Phe Arg Glu Gly Thr50 55
60Cys Gly Ala Gln Thr Gln Arg Ile Arg Cys Arg Val Pro Cys Asn65
70 75Trp Lys Lys Glu Phe Gly Ala Asp Cys Lys
Tyr Lys Phe Glu Asn80 85 90Trp Gly Ala
Cys Asp Gly Gly Thr Gly Thr Lys Val Arg Gln Gly95 100
105Thr Leu Lys Lys Ala Arg Tyr Asn Ala Gln Cys Gln Glu Thr
Ile110 115 120Arg Val Thr Lys Pro Cys Thr
Pro Lys Thr Lys Ala Lys Ala Lys125 130
135Ala Lys Lys Gly Lys Gly Lys Asp14064141PRTHomo sapien 64Met Trp Val
Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu1 5
10 15Leu Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr
Gln Cys Glu Glu20 25 30Phe Gln Leu Asn
Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn35 40
45Cys Thr Val Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu50
55 60Gln Ser Ala Gly Ile Met Tyr Arg Lys
Ser Cys Ala Ser Ser Ala65 70 75Ala Cys
Leu Ile Ala Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro80 85
90Gly Lys Leu Asn Ser Val Cys Ile Ser Cys Cys Asn Thr
Pro Leu95 100 105Cys Asn Gly Pro Arg Pro
Lys Lys Arg Gly Ser Ser Ala Ser Ala110 115
120Leu Arg Pro Gly Leu Arg Thr Thr Ile Leu Phe Leu Lys Leu Ala125
130 135Leu Phe Ser Ala His Cys14065242PRTHomo
Sapien 65Met Lys Asn Ile Gly Leu Val Met Glu Trp Glu Ile Pro Glu Ile1
5 10 15Ile Cys Thr Cys Ala
Lys Leu Arg Leu Pro Pro Gln Ala Thr Phe20 25
30Gln Val Leu Arg Gly Asn Gly Ala Ser Val Gly Thr Val Leu Met35
40 45Phe Arg Cys Pro Ser Asn His Gln Met Val
Gly Ser Gly Leu Leu50 55 60Thr Cys Thr
Trp Lys Gly Ser Ile Ala Glu Trp Ser Ser Gly Ser65 70
75Pro Val Cys Lys Leu Val Pro Pro His Glu Thr Phe Gly Phe
Lys80 85 90Val Ala Val Ile Ala Ser Ile
Val Ser Cys Ala Ile Ile Leu Leu95 100
105Met Ser Met Ala Phe Leu Thr Cys Cys Leu Leu Lys Cys Val Lys110
115 120Lys Ser Lys Arg Arg Arg Ser Asn Arg Ser
Ala Gln Leu Trp Ser125 130 135Gln Leu Lys
Asp Glu Asp Leu Glu Thr Val Gln Ala Ala Tyr Leu140 145
150Gly Leu Lys His Phe Asn Lys Pro Val Ser Gly Pro Ser Gln
Ala155 160 165His Asp Asn His Ser Phe Thr
Thr Asp His Gly Glu Ser Thr Ser170 175
180Lys Leu Ala Ser Val Thr Arg Ser Val Asp Lys Asp Pro Gly Ile185
190 195Pro Arg Ala Leu Ser Leu Ser Gly Ser Ser
Ser Ser Pro Gln Ala200 205 210Gln Val Met
Val His Met Ala Asn Pro Arg Gln Pro Leu Pro Ala215 220
225Ser Gly Leu Ala Thr Gly Met Pro Gln Gln Pro Ala Ala Tyr
Ala230 235 240Leu Gly66672PRTHomo sapien
66Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu Ala Pro Ser1
5 10 15Gln Leu Glu Tyr Tyr Ala Ser
Ser Pro Asp Glu Lys Ala Leu Val20 25
30Glu Ala Ala Ala Arg Ile Gly Ile Val Phe Ile Gly Asn Ser Glu35
40 45Glu Thr Met Glu Val Lys Thr Leu Gly Lys Leu
Glu Arg Tyr Lys50 55 60Leu Leu His Ile
Leu Glu Phe Asp Ser Asp Arg Arg Arg Met Ser65 70
75Val Ile Val Gln Ala Pro Ser Gly Glu Lys Leu Leu Phe Ala Lys80
85 90Gly Ala Glu Ser Ser Ile Leu Pro Lys
Cys Ile Gly Gly Glu Ile95 100 105Glu Lys
Thr Arg Ile His Val Asp Glu Phe Ala Leu Lys Gly Leu110
115 120Arg Thr Leu Cys Ile Ala Tyr Arg Lys Phe Thr Ser
Lys Glu Tyr125 130 135Glu Glu Ile Asp Lys
Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln140 145
150Gln Arg Glu Glu Lys Leu Ala Ala Val Phe Gln Phe Ile Glu Lys155
160 165Asp Leu Ile Leu Leu Gly Ala Thr Ala
Val Glu Asp Arg Leu Gln170 175 180Asp Lys
Val Arg Glu Thr Ile Glu Ala Leu Arg Met Ala Gly Ile185
190 195Lys Val Trp Val Leu Thr Gly Asp Lys His Glu Thr
Ala Val Ser200 205 210Val Ser Leu Ser Cys
Gly His Phe His Arg Thr Met Asn Ile Leu215 220
225Glu Leu Ile Asn Gln Lys Ser Asp Ser Glu Cys Ala Glu Gln Leu230
235 240Arg Gln Leu Ala Arg Arg Ile Thr Glu
Asp His Val Ile Gln His245 250 255Gly Leu
Val Val Asp Gly Thr Ser Leu Ser Leu Ala Leu Arg Glu260
265 270His Glu Lys Leu Phe Met Glu Val Cys Arg Asn Cys
Ser Ala Val275 280 285Leu Cys Cys Arg Met
Ala Pro Leu Gln Lys Ala Lys Val Ile Arg290 295
300Leu Ile Lys Ile Ser Pro Glu Lys Pro Ile Thr Leu Ala Val Gly305
310 315Asp Gly Ala Asn Asp Val Ser Met Ile
Gln Glu Ala His Val Gly320 325 330Ile Gly
Ile Met Gly Lys Glu Gly Arg Gln Ala Ala Arg Asn Ser335
340 345Asp Tyr Ala Ile Ala Arg Phe Lys Phe Leu Ser Lys
Leu Leu Phe350 355 360Val His Gly His Phe
Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln365 370
375Tyr Phe Phe Tyr Lys Asn Val Cys Phe Ile Thr Pro Gln Phe Leu380
385 390Tyr Gln Phe Tyr Cys Leu Phe Ser Gln
Gln Thr Leu Tyr Asp Ser395 400 405Val Tyr
Leu Thr Leu Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile410
415 420Leu Ile Tyr Ser Leu Leu Glu Gln His Val Asp Pro
His Val Leu425 430 435Gln Asn Lys Pro Thr
Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu440 445
450Leu Ser Ile Lys Thr Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser455
460 465His Ala Phe Ile Phe Phe Phe Gly Ser
Tyr Leu Leu Ile Gly Lys470 475 480Asp Thr
Ser Leu Leu Gly Asn Gly Gln Met Phe Gly Asn Trp Thr485
490 495Phe Gly Thr Leu Val Phe Thr Val Met Val Ile Thr
Val Thr Val500 505 510Lys Met Ala Leu Glu
Thr His Phe Trp Thr Trp Ile Asn His Leu515 520
525Val Thr Trp Gly Ser Ile Ile Phe Tyr Phe Val Phe Ser Leu Phe530
535 540Tyr Gly Gly Ile Leu Trp Pro Phe Leu
Gly Ser Gln Asn Met Tyr545 550 555Phe Val
Phe Ile Gln Leu Leu Ser Ser Gly Ser Ala Trp Phe Ala560
565 570Ile Ile Leu Met Val Val Thr Cys Leu Phe Leu Asp
Ile Ile Lys575 580 585Lys Val Phe Asp Arg
His Leu His Pro Thr Ser Thr Glu Lys Ala590 595
600Gln Leu Thr Glu Thr Asn Ala Gly Ile Lys Cys Leu Asp Ser Met605
610 615Cys Cys Phe Pro Glu Gly Glu Ala Ala
Cys Ala Ser Val Gly Arg620 625 630Met Leu
Glu Arg Val Ile Gly Arg Cys Ser Pro Thr His Ile Ser635
640 645Arg Ser Trp Ser Ala Ser Asp Pro Phe Tyr Thr Asn
Asp Arg Ser650 655 660Ile Leu Thr Leu Ser
Thr Met Asp Ser Ser Thr Cys665 67067877PRTHomo Sapien
67Met Trp Glu Glu Glu Asp Ile Ala Ile Leu Phe Asn Lys Glu Pro1
5 10 15Gly Lys Thr Glu Asn Ile Glu
Asn Asn Leu Ser Ser Asn His Arg20 25
30Arg Ser Cys Arg Arg Ser Glu Glu Ser Asp Asp Asp Leu Asp Phe35
40 45Asp Ile Gly Leu Glu Asn Thr Gly Gly Asp Pro
Gln Ile Leu Arg50 55 60Phe Ile Ser Asp
Phe Leu Ala Phe Leu Val Leu Tyr Asn Phe Ile65 70
75Ile Pro Ile Ser Leu Tyr Val Thr Val Glu Met Gln Lys Phe Leu80
85 90Gly Ser Phe Phe Ile Gly Trp Asp Leu
Asp Leu Tyr His Glu Glu95 100 105Ser Asp
Gln Lys Ala Gln Val Asn Thr Ser Asp Leu Asn Glu Glu110
115 120Leu Gly Gln Val Glu Tyr Val Phe Thr Asp Lys Thr
Gly Thr Leu125 130 135Thr Glu Asn Glu Met
Gln Phe Arg Glu Cys Ser Ile Asn Gly Met140 145
150Lys Tyr Gln Glu Ile Asn Gly Arg Leu Val Pro Glu Gly Pro Thr155
160 165Pro Asp Ser Ser Glu Gly Asn Leu Ser
Tyr Leu Ser Ser Leu Ser170 175 180His Leu
Asn Asn Leu Ser His Leu Thr Thr Ser Ser Ser Phe Arg185
190 195Thr Ser Pro Glu Asn Glu Thr Glu Leu Ile Lys Glu
His Asp Leu200 205 210Phe Phe Lys Ala Val
Ser Leu Cys His Thr Val Gln Ile Ser Asn215 220
225Val Gln Thr Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu230
235 240Ala Pro Ser Gln Leu Glu Tyr Tyr Ala
Ser Ser Pro Asp Glu Lys245 250 255Ala Leu
Val Glu Ala Ala Ala Arg Tyr Lys Leu Leu His Ile Leu260
265 270Glu Phe Asp Ser Asp Arg Arg Arg Met Ser Val Ile
Val Gln Ala275 280 285Pro Ser Gly Glu Lys
Leu Leu Phe Ala Lys Gly Ala Glu Ser Ser290 295
300Ile Leu Pro Lys Cys Ile Gly Gly Glu Ile Glu Lys Thr Arg Ile305
310 315His Val Asp Glu Phe Ala Leu Lys Gly
Leu Arg Thr Leu Cys Ile320 325 330Ala Tyr
Arg Lys Phe Thr Ser Lys Glu Tyr Glu Glu Ile Asp Lys335
340 345Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln Gln Arg
Glu Glu Lys350 355 360Leu Ala Ala Val Phe
Gln Phe Ile Glu Lys Asp Leu Ile Leu Leu365 370
375Gly Ala Thr Ala Val Glu Asp Arg Leu Gln Asp Lys Val Arg Glu380
385 390Thr Ile Glu Ala Leu Arg Met Ala Gly
Ile Lys Val Trp Val Leu395 400 405Thr Gly
Asp Lys His Glu Thr Ala Val Ser Val Ser Leu Ser Cys410
415 420Gly His Phe His Arg Thr Met Asn Ile Leu Glu Leu
Ile Asn Gln425 430 435Lys Ser Asp Ser Glu
Cys Ala Glu Gln Leu Arg Gln Leu Ala Arg440 445
450Arg Ile Thr Glu Asp His Val Ile Gln His Gly Leu Val Val Asp455
460 465Gly Thr Ser Leu Ser Leu Ala Leu Arg
Glu His Glu Lys Leu Phe470 475 480Met Glu
Val Cys Arg Asn Cys Ser Ala Val Leu Cys Cys Arg Met485
490 495Ala Pro Leu Gln Lys Ala Lys Val Ile Arg Leu Ile
Lys Ile Ser500 505 510Pro Glu Lys Pro Ile
Thr Leu Ala Val Gly Asp Gly Ala Asn Asp515 520
525Val Ser Met Ile Gln Glu Ala His Val Gly Ile Gly Ile Met Gly530
535 540Lys Glu Gly Arg Gln Ala Ala Arg Asn
Ser Asp Tyr Ala Ile Ala545 550 555Arg Phe
Lys Phe Leu Ser Lys Leu Leu Phe Val His Gly His Phe560
565 570Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln Tyr Phe
Phe Tyr Lys575 580 585Asn Val Cys Phe Ile
Thr Pro Gln Phe Leu Tyr Gln Phe Tyr Cys590 595
600Leu Phe Ser Gln Gln Thr Leu Tyr Asp Ser Val Tyr Leu Thr Leu605
610 615Tyr Asn Ile Cys Phe Thr Ser Leu Pro
Ile Leu Ile Tyr Ser Leu620 625 630Leu Glu
Gln His Val Asp Pro His Val Leu Gln Asn Lys Pro Thr635
640 645Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu Leu Ser
Ile Lys Thr650 655 660Phe Leu Tyr Trp Thr
Ile Leu Gly Phe Ser His Ala Phe Ile Phe665 670
675Phe Phe Gly Ser Tyr Leu Leu Ile Gly Lys Asp Thr Ser Leu Leu680
685 690Gly Asn Gly Gln Met Phe Gly Asn Trp
Thr Phe Gly Thr Leu Val695 700 705Phe Thr
Val Met Val Ile Thr Val Thr Val Lys Met Ala Leu Glu710
715 720Thr His Phe Trp Thr Trp Ile Asn His Leu Val Thr
Trp Gly Ser725 730 735Ile Ile Phe Tyr Phe
Val Phe Ser Leu Phe Tyr Gly Gly Ile Leu740 745
750Trp Pro Phe Leu Gly Ser Gln Asn Met Tyr Phe Val Phe Ile Gln755
760 765Leu Leu Ser Ser Gly Ser Ala Trp Phe
Ala Ile Ile Leu Met Val770 775 780Val Thr
Cys Leu Phe Leu Asp Ile Ile Lys Lys Val Phe Asp Arg785
790 795His Leu His Pro Thr Ser Thr Glu Lys Ala Gln Leu
Thr Glu Thr800 805 810Asn Ala Gly Ile Lys
Cys Leu Asp Ser Met Cys Cys Phe Pro Glu815 820
825Gly Glu Ala Ala Cys Ala Ser Val Gly Arg Met Leu Glu Arg Val830
835 840Ile Gly Arg Cys Ser Pro Thr His Ile
Ser Arg Ser Trp Ser Ala845 850 855Ser Asp
Pro Phe Tyr Thr Asn Asp Arg Ser Ile Leu Thr Leu Ser860
865 870Thr Met Asp Ser Ser Thr Cys8756863PRTHomo Sapien
68Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr1
5 10 15Leu Leu Ser Ile Phe Val Arg
Val Met Glu Ser Leu Glu Gly Leu20 25
30Leu Glu Ser Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln35
40 45Leu Ala Asn Thr Glu Pro Thr Lys Gly Leu Pro
Asp His Pro Ser50 55 60Arg Ser
Met69137PRTHomo Sapienunsure101, 136unknown amino acid 69Met Lys Thr Gly
Leu Phe Phe Leu Cys Leu Leu Gly Thr Ala Ala1 5
10 15Ala Ile Pro Thr Asn Ala Arg Leu Leu Ser Asp His
Ser Lys Pro20 25 30Thr Ala Glu Thr Val
Ala Pro Asp Asn Thr Ala Ile Pro Ser Leu35 40
45Arg Ala Glu Asp Glu Glu Asn Glu Lys Glu Thr Ala Val Ser Thr50
55 60Glu Asp Asp Ser His His Lys Ala Glu Lys
Ser Ser Val Leu Lys65 70 75Ser Lys Glu
Glu Ser His Glu Gln Ser Ala Glu Gln Gly Lys Ser80 85
90Ser Ser Gln Glu Leu Gly Leu Lys Asp Gln Xaa Asp Ser Asp
Gly95 100 105Asp Leu Ser Val Asn Leu Glu
Tyr Ala Pro Thr Glu Gly Thr Leu110 115
120Asp Ile Lys Glu Asp Met Ser Glu Pro Gln Glu Lys Asn Ser Gln125
130 135Xaa His70318PRTHomo Sapien 70Met Ala Pro
Trp Ala Glu Ala Glu His Ser Ala Leu Asn Pro Leu1 5
10 15Arg Ala Val Trp Leu Thr Leu Thr Ala Ala Phe
Leu Leu Thr Leu20 25 30Leu Leu Gln Leu
Leu Pro Pro Gly Leu Leu Pro Gly Cys Ala Ile35 40
45Phe Gln Asp Leu Ile Arg Tyr Gly Lys Thr Lys Cys Gly Glu Pro50
55 60Ser Arg Pro Ala Ala Cys Arg Ala Phe
Asp Val Pro Lys Arg Tyr65 70 75Phe Ser
His Phe Tyr Ile Ile Ser Val Leu Trp Asn Gly Phe Leu80 85
90Leu Trp Cys Leu Thr Gln Ser Leu Phe Leu Gly Ala Pro
Phe Pro95 100 105Ser Trp Leu His Gly Leu
Leu Arg Ile Leu Gly Ala Ala Gln Phe110 115
120Gln Gly Gly Glu Leu Ala Leu Ser Ala Phe Leu Val Leu Val Phe125
130 135Leu Trp Leu His Ser Leu Arg Arg Leu Phe
Glu Cys Leu Tyr Val140 145 150Ser Val Phe
Ser Asn Val Met Ile His Val Val Gln Tyr Cys Phe155 160
165Gly Leu Val Tyr Tyr Val Leu Val Gly Leu Thr Val Leu Ser
Gln170 175 180Val Pro Met Asp Gly Arg Asn
Ala Tyr Ile Thr Gly Lys Asn Leu185 190
195Leu Met Gln Ala Arg Trp Phe His Ile Leu Gly Met Met Met Phe200
205 210Ile Trp Ser Ser Ala His Gln Tyr Lys Cys
His Val Ile Leu Gly215 220 225Asn Leu Arg
Lys Asn Lys Ala Gly Val Val Ile His Cys Asn His230 235
240Arg Ile Pro Phe Gly Asp Trp Phe Glu Tyr Val Ser Ser Pro
Asn245 250 255Tyr Leu Ala Glu Leu Met Ile
Tyr Val Ser Met Ala Val Thr Phe260 265
270Gly Phe His Asn Leu Thr Trp Trp Leu Val Val Thr Asn Val Phe275
280 285Phe Asn Gln Ala Leu Ser Ala Phe Leu Ser
His Gln Phe Tyr Lys290 295 300Ser Lys Phe
Val Ser Tyr Pro Lys His Arg Lys Ala Phe Leu Pro305 310
315Phe Leu Phe71426PRTHomo sapien 71Met Pro Leu Leu Trp Leu
Arg Gly Phe Leu Leu Ala Ser Cys Trp1 5 10
15Ile Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His
Ser20 25 30Ala Ala Pro Asp Cys Pro Ser
Cys Ala Leu Ala Ala Leu Pro Lys35 40
45Asp Val Pro Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys50
55 60His Ile Leu Asn Met Leu His Leu Lys Lys Arg
Pro Asp Val Thr65 70 75Gln Pro Val Pro
Lys Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu80 85
90His Val Gly Lys Val Gly Glu Asn Gly Tyr Val Glu Ile Glu Asp95
100 105Asp Ile Gly Arg Arg Ala Glu Met Asn
Glu Leu Met Glu Gln Thr110 115 120Ser Glu
Ile Ile Thr Phe Ala Glu Ser Gly Thr Ala Arg Lys Thr125
130 135Leu His Phe Glu Ile Ser Lys Glu Gly Ser Asp Leu
Ser Val Val140 145 150Glu Arg Ala Glu Val
Trp Leu Phe Leu Lys Val Pro Lys Ala Asn155 160
165Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe Gln Gln Gln Lys170
175 180His Pro Gln Gly Ser Leu Asp Thr Gly
Glu Glu Ala Glu Glu Val185 190 195Gly Leu
Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val200
205 210Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro
Val Ser Ser215 220 225Ser Ile Gln Arg Leu
Leu Asp Gln Gly Lys Ser Ser Leu Asp Val230 235
240Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val245
250 255Leu Leu Gly Lys Lys Lys Lys Lys Glu
Glu Glu Gly Glu Gly Lys260 265 270Lys Lys
Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys275
280 285Glu Gln Ser His Arg Pro Phe Leu Met Leu Gln Ala
Arg Gln Ser290 295 300Glu Asp His Pro His
Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp305 310
315Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe320
325 330Lys Asp Ile Gly Trp Asn Asp Trp Ile
Ile Ala Pro Ser Gly Tyr335 340 345His Ala
Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile Ala Gly350
355 360Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val
Ile Asn His365 370 375Tyr Arg Met Arg Gly
His Ser Pro Phe Ala Asn Leu Lys Ser Cys380 385
390Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp395
400 405Asp Gly Gln Asn Ile Ile Lys Lys Asp
Ile Gln Asn Met Ile Val410 415 420Glu Glu
Cys Gly Cys Ser42572238PRTHomo Sapien 72Met Ala Ala Ala Pro Leu Leu Leu
Leu Leu Leu Leu Val Pro Val1 5 10
15Pro Leu Leu Pro Leu Leu Ala Gln Gly Pro Gly Gly Ala Leu Gly20
25 30Asn Arg His Ala Val Tyr Trp Asn Ser
Ser Asn Gln His Leu Arg35 40 45Arg Glu
Gly Tyr Thr Val Gln Val Asn Val Asn Asp Tyr Leu Asp50 55
60Ile Tyr Cys Pro His Tyr Asn Ser Ser Gly Val Gly Pro
Gly Ala65 70 75Gly Pro Gly Pro Gly Gly
Gly Ala Glu Gln Tyr Val Leu Tyr Met80 85
90Val Ser Arg Asn Gly Tyr Arg Thr Cys Asn Ala Ser Gln Gly Phe95
100 105Lys Arg Trp Glu Cys Asn Arg Pro His Ala Pro
His Ser Pro Ile110 115 120Lys Phe Ser Glu
Lys Phe Gln Arg Tyr Ser Ala Phe Ser Leu Gly125 130
135Tyr Glu Phe His Ala Gly His Glu Tyr Tyr Tyr Ile Ser Thr
Pro140 145 150Thr His Asn Leu His Trp Lys
Cys Leu Arg Met Lys Val Phe Val155 160
165Cys Cys Ala Ser Thr Ser His Ser Gly Glu Lys Pro Val Pro Thr170
175 180Leu Pro Gln Phe Thr Met Gly Pro Asn Val
Lys Ile Asn Val Leu185 190 195Glu Asp Phe
Glu Gly Glu Asn Pro Gln Val Pro Lys Leu Glu Lys200 205
210Ser Ile Ser Gly Thr Ser Pro Lys Arg Glu His Leu Pro Leu
Ala215 220 225Val Gly Ile Ala Phe Phe Leu
Met Thr Phe Leu Ala Ser230 23573541PRTHomo Sapien 73Met
Gly His Ser Pro Pro Val Leu Pro Leu Cys Ala Ser Val Ser1 5
10 15Leu Leu Gly Gly Leu Thr Phe Gly Tyr
Glu Leu Ala Val Ile Ser20 25 30Gly Ala
Leu Leu Pro Leu Gln Leu Asp Phe Gly Leu Ser Cys Leu35 40
45Glu Gln Glu Phe Leu Val Gly Ser Leu Leu Leu Gly Ala
Leu Leu50 55 60Ala Ser Leu Val Gly Gly
Phe Leu Ile Asp Cys Tyr Gly Arg Lys65 70
75Gln Ala Ile Leu Gly Ser Asn Leu Val Leu Leu Ala Gly Ser Leu80
85 90Thr Leu Gly Leu Ala Gly Ser Leu Ala Trp Leu
Val Leu Gly Arg95 100 105Ala Val Val Gly
Phe Ala Ile Ser Leu Ser Ser Met Ala Cys Cys110 115
120Ile Tyr Val Ser Glu Leu Val Gly Pro Arg Gln Arg Gly Val
Leu125 130 135Val Ser Leu Tyr Glu Ala Gly
Ile Thr Val Gly Ile Leu Leu Ser140 145
150Tyr Ala Leu Asn Tyr Ala Leu Ala Gly Thr Pro Trp Gly Trp Arg155
160 165His Met Phe Gly Trp Ala Thr Ala Pro Ala
Val Leu Gln Ser Leu170 175 180Ser Leu Leu
Phe Leu Pro Ala Gly Thr Asp Glu Thr Ala Thr His185 190
195Lys Asp Leu Ile Pro Leu Gln Gly Gly Glu Ala Pro Lys Leu
Gly200 205 210Pro Gly Arg Pro Arg Tyr Ser
Phe Leu Asp Leu Phe Arg Ala Arg215 220
225Asp Asn Met Arg Gly Arg Thr Thr Val Gly Leu Gly Leu Val Leu230
235 240Phe Gln Gln Leu Thr Gly Gln Pro Asn Val
Leu Cys Tyr Ala Ser245 250 255Thr Ile Phe
Ser Ser Val Gly Phe His Gly Gly Ser Ser Ala Val260 265
270Leu Ala Ser Val Gly Leu Gly Ala Val Lys Val Ala Ala Thr
Leu275 280 285Thr Ala Met Gly Leu Val Asp
Arg Ala Gly Arg Arg Ala Leu Leu290 295
300Leu Ala Gly Cys Ala Leu Met Ala Leu Ser Val Ser Gly Ile Gly305
310 315Leu Val Ser Phe Ala Val Pro Met Asp Ser
Gly Pro Ser Cys Leu320 325 330Ala Val Pro
Asn Ala Thr Gly Gln Thr Gly Leu Pro Gly Asp Ser335 340
345Gly Leu Leu Gln Asp Ser Ser Leu Pro Pro Ile Pro Arg Thr
Asn350 355 360Glu Asp Gln Arg Glu Pro Ile
Leu Ser Thr Ala Lys Lys Thr Lys365 370
375Pro His Pro Arg Ser Gly Asp Pro Ser Ala Pro Pro Arg Leu Ala380
385 390Leu Ser Ser Ala Leu Pro Gly Pro Pro Leu
Pro Ala Arg Gly His395 400 405Ala Leu Leu
Arg Trp Thr Ala Leu Leu Cys Leu Met Val Phe Val410 415
420Ser Ala Phe Ser Phe Gly Phe Gly Pro Val Thr Trp Leu Val
Leu425 430 435Ser Glu Ile Tyr Pro Val Glu
Ile Arg Gly Arg Ala Phe Ala Phe440 445
450Cys Asn Ser Phe Asn Trp Ala Ala Asn Leu Phe Ile Ser Leu Ser455
460 465Phe Leu Asp Leu Ile Gly Thr Ile Gly Leu
Ser Trp Thr Phe Leu470 475 480Leu Tyr Gly
Leu Thr Ala Val Leu Gly Leu Gly Phe Ile Tyr Leu485 490
495Phe Val Pro Glu Thr Lys Gly Gln Ser Leu Ala Glu Ile Asp
Gln500 505 510Gln Phe Gln Lys Arg Arg Phe
Thr Leu Ser Phe Gly His Arg Gln515 520
525Asn Ser Thr Gly Ile Pro Tyr Ser Arg Ile Glu Ile Ser Ala Ala530
535 540Ser741114PRTHomo Sapien 74Met Ala Lys Ala
Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu1 5
10 15Leu Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu
Gly Leu Tyr20 25 30Phe Ser Arg Asp Ala
Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala35 40
45Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro50
55 60Glu Glu Val Pro Ser Phe Arg Leu Gly Gln
His Leu Tyr Gly Thr65 70 75Tyr Arg Thr
Arg Leu His Glu Asn Asn Trp Ile Cys Ile Gln Glu80 85
90Asp Thr Gly Leu Leu Tyr Leu Asn Arg Ser Leu Asp His Ser
Ser95 100 105Trp Glu Lys Leu Ser Val Arg
Asn Arg Gly Phe Pro Leu Leu Thr110 115
120Val Tyr Leu Lys Val Phe Leu Ser Pro Thr Ser Leu Arg Glu Gly125
130 135Glu Cys Gln Trp Pro Gly Cys Ala Arg Val
Tyr Phe Ser Phe Phe140 145 150Asn Thr Ser
Phe Pro Ala Cys Ser Ser Leu Lys Pro Arg Glu Leu155 160
165Cys Phe Pro Glu Thr Arg Pro Ser Phe Arg Ile Arg Glu Asn
Arg170 175 180Pro Pro Gly Thr Phe His Gln
Phe Arg Leu Leu Pro Val Gln Phe185 190
195Leu Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu Leu Glu Gly Glu200
205 210Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser
Leu Glu Val Ser Thr215 220 225Arg Trp Ala
Leu Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val230 235
240Ala Val Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val
Met245 250 255Val Pro Phe Pro Val Thr Val
Tyr Asp Glu Asp Asp Ser Ala Pro260 265
270Thr Phe Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val Glu Phe275
280 285Lys Arg Lys Glu Asp Thr Val Val Ala Thr
Leu Arg Val Phe Asp290 295 300Ala Asp Val
Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr305 310
315Ser Thr Leu Leu Pro Gly Asp Thr Trp Ala Gln Gln Thr Phe
Arg320 325 330Val Glu His Trp Pro Asn Glu
Thr Ser Val Gln Ala Asn Gly Ser335 340
345Phe Val Arg Ala Thr Val His Asp Tyr Arg Leu Val Leu Asn Arg350
355 360Asn Leu Ser Ile Ser Glu Asn Arg Thr Met
Gln Leu Ala Val Leu365 370 375Val Asn Asp
Ser Asp Phe Gln Gly Pro Gly Ala Gly Val Leu Leu380 385
390Leu His Phe Asn Val Ser Val Leu Pro Val Ser Leu His Leu
Pro395 400 405Ser Thr Tyr Ser Leu Ser Val
Ser Arg Arg Ala Arg Arg Phe Ala410 415
420Gln Ile Gly Lys Val Cys Val Glu Asn Cys Gln Ala Phe Ser Gly425
430 435Ile Asn Val Gln Tyr Lys Leu His Ser Ser
Gly Ala Asn Cys Ser440 445 450Thr Leu Gly
Val Val Thr Ser Ala Glu Asp Thr Ser Gly Ile Leu455 460
465Phe Val Asn Asp Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala
Glu470 475 480Leu His Tyr Met Val Val Ala
Thr Asp Gln Gln Thr Ser Arg Gln485 490
495Ala Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala500
505 510Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala
Val Ser Lys Arg Arg515 520 525Leu Glu Cys
Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg530 535
540Cys Glu Trp Arg Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn
Phe545 550 555Ser Thr Cys Ser Pro Ser Thr
Lys Thr Cys Pro Asp Gly His Cys560 565
570Asp Val Val Glu Thr Gln Asp Ile Asn Ile Cys Pro Gln Asp Cys575
580 585Leu Arg Gly Ser Ile Val Gly Gly His Glu
Pro Gly Glu Pro Arg590 595 600Gly Ile Lys
Ala Gly Tyr Gly Thr Cys Asn Cys Phe Pro Glu Glu605 610
615Glu Lys Cys Phe Cys Glu Pro Glu Asp Ile Gln Asp Pro Leu
Cys620 625 630Asp Glu Leu Cys Arg Thr Val
Ile Ala Ala Ala Val Leu Phe Ser635 640
645Phe Ile Val Ser Val Leu Leu Ser Ala Phe Cys Ile His Cys Tyr650
655 660His Lys Phe Ala His Lys Pro Pro Ile Ser
Ser Ala Glu Met Thr665 670 675Phe Arg Arg
Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser Ser Ser680 685
690Gly Ala Arg Arg Pro Ser Leu Asp Ser Met Glu Asn Gln Val
Ser695 700 705Val Asp Ala Phe Lys Ile Leu
Glu Asp Pro Lys Trp Glu Phe Pro710 715
720Arg Lys Asn Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe725
730 735Gly Lys Val Val Lys Ala Thr Ala Phe His
Leu Lys Gly Arg Ala740 745 750Gly Tyr Thr
Thr Val Ala Val Lys Met Leu Lys Glu Asn Ala Ser755 760
765Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val Leu
Lys770 775 780Gln Val Asn His Pro His Val
Ile Lys Leu Tyr Gly Ala Cys Ser785 790
795Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr Gly800
805 810Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg
Lys Val Gly Pro Gly815 820 825Tyr Leu Gly
Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His830 835
840Pro Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe
Ala845 850 855Trp Gln Ile Ser Gln Gly Met
Gln Tyr Leu Ala Glu Met Lys Leu860 865
870Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly875
880 885Arg Lys Met Lys Ile Ser Asp Phe Gly Leu
Ser Arg Asp Val Tyr890 895 900Glu Glu Asp
Ser Tyr Val Lys Arg Ser Gln Gly Arg Ile Pro Val905 910
915Lys Trp Met Ala Ile Glu Ser Leu Phe Asp His Ile Tyr Thr
Thr920 925 930Gln Ser Asp Val Trp Ser Phe
Gly Val Leu Leu Trp Glu Ile Val935 940
945Thr Leu Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Glu Arg Leu950
955 960Phe Asn Leu Leu Lys Thr Gly His Arg Met
Glu Arg Pro Asp Asn965 970 975Cys Ser Glu
Glu Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gln980 985
990Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser Lys Asp
Leu995 1000 1005Glu Lys Met Met Val Lys Arg
Arg Asp Tyr Leu Asp Leu Ala Ala1010 1015
1020Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu Ser Glu1025
1030 1035Glu Glu Thr Pro Leu Val Asp Cys Asn Asn
Ala Pro Leu Pro Arg1040 1045 1050Ala Leu
Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser1055
1060 1065Asp Pro Asn Trp Pro Gly Glu Ser Pro Val Pro Leu
Thr Arg Ala1070 1075 1080Asp Gly Thr Asn
Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val1085 1090
1095Tyr Ala Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met
Asp1100 1105 1110Thr Phe Asp
Ser75790PRTHomo Sapien 75Met Arg Thr Tyr Arg Tyr Phe Leu Leu Leu Phe Trp
Val Gly Gln1 5 10 15Pro
Tyr Pro Thr Leu Ser Thr Pro Leu Ser Lys Arg Thr Ser Gly20
25 30Phe Pro Ala Lys Lys Arg Ala Leu Glu Leu Ser Gly
Asn Ser Lys35 40 45Asn Glu Leu Asn Arg
Ser Lys Arg Ser Trp Met Trp Asn Gln Phe50 55
60Phe Leu Leu Glu Glu Tyr Thr Gly Ser Asp Tyr Gln Tyr Val Gly65
70 75Lys Leu His Ser Asp Gln Asp Arg Gly Asp
Gly Ser Leu Lys Tyr80 85 90Ile Leu Ser
Gly Asp Gly Ala Gly Asp Leu Phe Ile Ile Asn Glu95 100
105Asn Thr Gly Asp Ile Gln Ala Thr Lys Arg Leu Asp Arg Glu
Glu110 115 120Lys Pro Val Tyr Ile Leu Arg
Ala Gln Ala Ile Asn Arg Arg Thr125 130
135Gly Arg Pro Val Glu Pro Glu Ser Glu Phe Ile Ile Lys Ile His140
145 150Asp Ile Asn Asp Asn Glu Pro Ile Phe Thr
Lys Glu Val Tyr Thr155 160 165Ala Thr Val
Pro Glu Met Ser Asp Val Gly Thr Phe Val Val Gln170 175
180Val Thr Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser
Ala185 190 195Lys Val Val Tyr Ser Ile Leu
Gln Gly Gln Pro Tyr Phe Ser Val200 205
210Glu Ser Glu Thr Gly Ile Ile Lys Thr Ala Leu Leu Asn Met Asp215
220 225Arg Glu Asn Arg Glu Gln Tyr Gln Val Val
Ile Gln Ala Lys Asp230 235 240Met Gly Gly
Gln Met Gly Gly Leu Ser Gly Thr Thr Thr Val Asn245 250
255Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro
Gln260 265 270Ser Thr Tyr Gln Phe Lys Thr
Pro Glu Ser Ser Pro Pro Gly Thr275 280
285Pro Ile Gly Arg Ile Lys Ala Ser Asp Ala Asp Val Gly Glu Asn290
295 300Ala Glu Ile Glu Tyr Ser Ile Thr Asp Gly
Glu Gly Leu Asp Met305 310 315Phe Asp Val
Ile Thr Asp Gln Glu Thr Gln Glu Gly Ile Ile Thr320 325
330Val Lys Lys Leu Leu Asp Phe Glu Lys Lys Lys Val Tyr Thr
Leu335 340 345Lys Val Glu Ala Ser Asn Pro
Tyr Val Glu Pro Arg Phe Leu Tyr350 355
360Leu Gly Pro Phe Lys Asp Ser Ala Thr Val Arg Ile Val Val Glu365
370 375Asp Val Asp Glu Pro Pro Val Phe Ser Lys
Leu Ala Tyr Ile Leu380 385 390Gln Ile Arg
Glu Asp Ala Gln Ile Asn Thr Thr Ile Gly Ser Val395 400
405Thr Ala Gln Asp Pro Asp Ala Ala Arg Asn Pro Val Lys Tyr
Ser410 415 420Val Asp Arg His Thr Asp Met
Asp Arg Ile Phe Asn Ile Asp Ser425 430
435Gly Asn Gly Ser Ile Phe Thr Ser Lys Leu Leu Asp Arg Glu Thr440
445 450Leu Leu Trp His Asn Ile Thr Val Ile Ala
Thr Glu Ile Asn Asn455 460 465Pro Lys Gln
Ser Ser Arg Val Pro Leu Tyr Ile Lys Val Leu Asp470 475
480Val Asn Asp Asn Ala Pro Glu Phe Ala Glu Phe Tyr Glu Thr
Phe485 490 495Val Cys Glu Lys Ala Lys Ala
Asp Gln Leu Ile Gln Thr Leu His500 505
510Ala Val Asp Lys Asp Asp Pro Tyr Ser Gly His Gln Phe Ser Phe515
520 525Ser Leu Ala Pro Glu Ala Ala Ser Gly Ser
Asn Phe Thr Ile Gln530 535 540Asp Asn Lys
Asp Asn Thr Ala Gly Ile Leu Thr Arg Lys Asn Gly545 550
555Tyr Asn Arg His Glu Met Ser Thr Tyr Leu Leu Pro Val Val
Ile560 565 570Ser Asp Asn Asp Tyr Pro Val
Gln Ser Ser Thr Gly Thr Val Thr575 580
585Val Arg Val Cys Ala Cys Asp His His Gly Asn Met Gln Ser Cys590
595 600His Ala Glu Ala Leu Ile His Pro Thr Gly
Leu Ser Thr Gly Ala605 610 615Leu Val Ala
Ile Leu Leu Cys Ile Val Ile Leu Leu Val Thr Val620 625
630Val Leu Phe Ala Ala Leu Arg Arg Gln Arg Lys Lys Glu Pro
Leu635 640 645Ile Ile Ser Lys Glu Asp Ile
Arg Asp Asn Ile Val Ser Tyr Asn650 655
660Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe Asp Ile Gly665
670 675Thr Leu Arg Asn Pro Glu Ala Ile Glu Asp
Asn Lys Leu Arg Arg680 685 690Asp Ile Val
Pro Glu Ala Leu Phe Leu Pro Arg Arg Thr Pro Thr695 700
705Ala Arg Asp Asn Thr Asp Val Arg Asp Phe Ile Asn Gln Arg
Leu710 715 720Lys Glu Asn Asp Thr Asp Pro
Thr Ala Pro Pro Tyr Asp Ser Leu725 730
735Ala Thr Tyr Ala Tyr Glu Gly Thr Gly Ser Val Ala Asp Ser Leu740
745 750Ser Ser Leu Glu Ser Val Thr Thr Asp Ala
Asp Gln Asp Tyr Asp755 760 765Tyr Leu Ser
Asp Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Met770 775
780Tyr Gly Gly Val Asp Ser Asp Lys Asp Ser785
79076794PRTHomo Sapien 76Met Leu Thr Arg Asn Cys Leu Ser Leu Leu Leu Trp
Val Leu Phe1 5 10 15Asp
Gly Gly Leu Leu Thr Pro Leu Gln Pro Gln Pro Gln Gln Thr20
25 30Leu Ala Thr Glu Pro Arg Glu Asn Val Ile His Leu
Pro Gly Gln35 40 45Arg Ser His Phe Gln
Arg Val Lys Arg Gly Trp Val Trp Asn Gln50 55
60Phe Phe Val Leu Glu Glu Tyr Val Gly Ser Glu Pro Gln Tyr Val65
70 75Gly Lys Leu His Ser Asp Leu Asp Lys Gly
Glu Gly Thr Val Lys80 85 90Tyr Thr Leu
Ser Gly Asp Gly Ala Gly Thr Val Phe Thr Ile Asp95 100
105Glu Thr Thr Gly Asp Ile His Ala Ile Arg Ser Leu Asp Arg
Glu110 115 120Glu Lys Pro Phe Tyr Thr Leu
Arg Ala Gln Ala Val Asp Ile Glu125 130
135Thr Arg Lys Pro Leu Glu Pro Glu Ser Glu Phe Ile Ile Lys Val140
145 150Gln Asp Ile Asn Asp Asn Glu Pro Lys Phe
Leu Asp Gly Pro Tyr155 160 165Val Ala Thr
Val Pro Glu Met Ser Pro Val Gly Ala Tyr Val Leu170 175
180Gln Val Lys Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly Asn
Ser185 190 195Ala Arg Val Val Tyr Ser Ile
Leu Gln Gly Gln Pro Tyr Phe Ser200 205
210Ile Asp Pro Lys Thr Gly Val Ile Arg Thr Ala Leu Pro Asn Met215
220 225Asp Arg Glu Val Lys Glu Gln Tyr Gln Val
Leu Ile Gln Ala Lys230 235 240Asp Met Gly
Gly Gln Leu Gly Gly Leu Ala Gly Thr Thr Ile Val245 250
255Asn Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe
Pro260 265 270Lys Ser Ile Phe His Leu Lys
Val Pro Glu Ser Ser Pro Ile Gly275 280
285Ser Ala Ile Gly Arg Ile Arg Ala Val Asp Pro Asp Phe Gly Gln290
295 300Asn Ala Glu Ile Glu Tyr Asn Ile Val Pro
Gly Asp Gly Gly Asn305 310 315Leu Phe Asp
Ile Val Thr Asp Glu Asp Thr Gln Glu Gly Val Ile320 325
330Lys Leu Lys Lys Pro Leu Asp Phe Glu Thr Lys Lys Ala Tyr
Thr335 340 345Phe Lys Val Glu Ala Ser Asn
Leu His Leu Asp His Arg Phe His350 355
360Ser Ala Gly Pro Phe Lys Asp Thr Ala Thr Val Lys Ile Ser Val365
370 375Leu Asp Val Asp Glu Pro Pro Val Phe Ser
Lys Pro Leu Tyr Thr380 385 390Met Glu Val
Tyr Glu Asp Thr Pro Val Gly Thr Ile Ile Gly Ala395 400
405Val Thr Ala Gln Asp Leu Asp Val Gly Ser Gly Ala Val Arg
Tyr410 415 420Phe Ile Asp Trp Lys Ser Asp
Gly Asp Ser Tyr Phe Thr Ile Asp425 430
435Gly Asn Glu Gly Thr Ile Ala Thr Asn Glu Leu Leu Asp Arg Glu440
445 450Ser Thr Ala Gln Tyr Asn Phe Ser Ile Ile
Ala Ser Lys Val Ser455 460 465Asn Pro Leu
Leu Thr Ser Lys Val Asn Ile Leu Ile Asn Val Leu470 475
480Asp Val Asn Glu Phe Pro Pro Glu Ile Ser Val Pro Tyr Glu
Thr485 490 495Ala Val Cys Glu Asn Ala Lys
Pro Gly Gln Ile Ile Gln Ile Val500 505
510Ser Ala Ala Asp Arg Asp Leu Ser Pro Ala Gly Gln Gln Phe Ser515
520 525Phe Arg Leu Ser Pro Glu Ala Ala Ile Lys
Pro Asn Phe Thr Val530 535 540Arg Asp Phe
Arg Asn Asn Thr Ala Gly Ile Glu Thr Arg Arg Asn545 550
555Gly Tyr Ser Arg Arg Gln Gln Glu Leu Tyr Phe Leu Pro Val
Val560 565 570Ile Glu Asp Ser Ser Tyr Pro
Val Gln Ser Ser Thr Asn Thr Met575 580
585Thr Ile Arg Val Cys Arg Cys Asp Ser Asp Gly Thr Ile Leu Ser590
595 600Cys Asn Val Glu Ala Ile Phe Leu Pro Val
Gly Leu Ser Thr Gly605 610 615Ala Leu Ile
Ala Ile Leu Leu Cys Ile Val Ile Leu Leu Ala Ile620 625
630Val Val Leu Tyr Val Ala Leu Arg Arg Gln Lys Lys Lys His
Thr635 640 645Leu Met Thr Ser Lys Glu Asp
Ile Arg Asp Asn Val Ile His Tyr650 655
660Asp Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe Asp Ile665
670 675Gly Ala Leu Arg Asn Pro Lys Val Ile Glu
Glu Asn Lys Ile Arg680 685 690Arg Asp Ile
Lys Pro Asp Ser Leu Cys Leu Pro Arg Gln Arg Pro695 700
705Pro Met Glu Asp Asn Thr Asp Ile Arg Asp Phe Ile His Gln
Arg710 715 720Leu Gln Glu Asn Asp Val Asp
Pro Thr Ala Pro Pro Ile Asp Ser725 730
735Leu Ala Thr Tyr Ala Tyr Glu Gly Ser Gly Ser Val Ala Glu Ser740
745 750Leu Ser Ser Ile Asp Ser Leu Thr Thr Glu
Ala Asp Gln Asp Tyr755 760 765Asp Tyr Leu
Thr Asp Trp Gly Pro Arg Phe Lys Val Leu Ala Asp770 775
780Met Phe Gly Glu Glu Glu Ser Tyr Asn Pro Asp Lys Val
Thr785 79077141PRTHomo Sapien 77Met Ala Arg Pro Leu Cys
Thr Leu Leu Leu Leu Met Ala Thr Leu1 5 10
15Ala Gly Ala Leu Ala Ser Ser Ser Lys Glu Glu Asn Arg Ile
Ile20 25 30Pro Gly Gly Ile Tyr Asp Ala
Asp Leu Asn Asp Glu Trp Val Gln35 40
45Arg Ala Leu His Phe Ala Ile Ser Glu Tyr Asn Lys Ala Thr Glu50
55 60Asp Glu Tyr Tyr Arg Arg Pro Leu Gln Val Leu
Arg Ala Arg Glu65 70 75Gln Thr Phe Gly
Gly Val Asn Tyr Phe Phe Asp Val Glu Val Gly80 85
90Arg Thr Ile Cys Thr Lys Ser Gln Pro Asn Leu Asp Thr Cys Ala95
100 105Phe His Glu Gln Pro Glu Leu Gln Lys
Lys Gln Leu Cys Ser Phe110 115 120Glu Ile
Tyr Glu Val Pro Trp Glu Asp Arg Met Ser Leu Val Asn125
130 135Ser Arg Cys Gln Glu Ala14078466PRTHomo Sapien
78Met Thr Thr Ser Pro Ile Leu Gln Leu Leu Leu Arg Leu Ser Leu1
5 10 15Cys Gly Leu Leu Leu Gln Arg
Ala Glu Thr Gly Ser Lys Gly Gln20 25
30Thr Ala Gly Glu Leu Tyr Gln Arg Trp Glu Arg Tyr Arg Arg Glu35
40 45Cys Gln Glu Thr Leu Ala Ala Ala Glu Pro Pro
Ser Gly Leu Ala50 55 60Cys Asn Gly Ser
Phe Asp Met Tyr Val Cys Trp Asp Tyr Ala Ala65 70
75Pro Asn Ala Thr Ala Arg Ala Ser Cys Pro Trp Tyr Leu Pro Trp80
85 90His His His Val Ala Ala Gly Phe Val
Leu Arg Gln Cys Gly Ser95 100 105Asp Gly
Gln Trp Gly Leu Trp Arg Asp His Thr Gln Cys Glu Asn110
115 120Pro Glu Lys Asn Glu Ala Phe Leu Asp Gln Arg Leu
Ile Leu Glu125 130 135Arg Leu Gln Val Met
Tyr Thr Val Gly Tyr Ser Leu Ser Leu Ala140 145
150Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser Leu Phe Arg Arg Leu155
160 165His Cys Thr Arg Asn Tyr Ile His Ile
Asn Leu Phe Thr Ser Phe170 175 180Met Leu
Arg Ala Ala Ala Ile Leu Ser Arg Asp Arg Leu Leu Pro185
190 195Arg Pro Gly Pro Tyr Leu Gly Asp Gln Ala Leu Ala
Leu Trp Asn200 205 210Gln Ala Leu Ala Ala
Cys Arg Thr Ala Gln Ile Val Thr Gln Tyr215 220
225Cys Val Gly Ala Asn Tyr Thr Trp Leu Leu Val Glu Gly Val Tyr230
235 240Leu His Ser Leu Leu Val Leu Val Gly
Gly Ser Glu Glu Gly His245 250 255Phe Arg
Tyr Tyr Leu Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe260
265 270Val Ile Pro Trp Val Ile Val Arg Tyr Leu Tyr Glu
Asn Thr Gln275 280 285Cys Trp Glu Arg Asn
Glu Val Lys Ala Ile Trp Trp Ile Ile Arg290 295
300Thr Pro Ile Leu Met Thr Ile Leu Ile Asn Phe Leu Ile Phe Ile305
310 315Arg Ile Leu Gly Ile Leu Leu Ser Lys
Leu Arg Thr Arg Gln Met320 325 330Arg Cys
Arg Asp Tyr Arg Leu Arg Leu Ala Arg Ser Thr Leu Thr335
340 345Leu Val Pro Leu Leu Gly Val His Glu Val Val Phe
Ala Pro Val350 355 360Thr Glu Glu Gln Ala
Arg Gly Ala Leu Arg Phe Ala Lys Leu Gly365 370
375Phe Glu Ile Phe Leu Ser Ser Phe Gln Gly Phe Leu Val Ser Val380
385 390Leu Tyr Cys Phe Ile Asn Lys Glu Val
Gln Ser Glu Ile Arg Arg395 400 405Gly Trp
His His Cys Arg Leu Arg Arg Ser Leu Gly Glu Glu Gln410
415 420Arg Gln Leu Pro Glu Arg Ala Phe Arg Ala Leu Pro
Ser Gly Ser425 430 435Gly Pro Gly Glu Val
Pro Thr Ser Arg Gly Leu Ser Ser Gly Thr440 445
450Leu Pro Gly Pro Gly Asn Glu Ala Ser Arg Glu Leu Glu Ser Tyr455
460 465Cys79506PRTHomo Sapien 79Met Leu Ser
Lys Val Leu Pro Val Leu Leu Gly Ile Leu Leu Ile1 5
10 15Leu Gln Ser Arg Val Glu Gly Pro Gln Thr Glu
Ser Lys Asn Glu20 25 30Ala Ser Ser Arg
Asp Val Val Tyr Gly Pro Gln Pro Gln Pro Leu35 40
45Glu Asn Gln Leu Leu Ser Glu Glu Thr Lys Ser Thr Glu Thr Glu50
55 60Thr Gly Ser Arg Val Gly Lys Leu Pro
Glu Ala Ser Arg Ile Leu65 70 75Asn Thr
Ile Leu Ser Asn Tyr Asp His Lys Leu Arg Pro Gly Ile80 85
90Gly Glu Lys Pro Thr Val Val Thr Val Glu Ile Ala Val
Asn Ser95 100 105Leu Gly Pro Leu Ser Ile
Leu Asp Met Glu Tyr Thr Ile Asp Ile110 115
120Ile Phe Ser Gln Thr Trp Tyr Asp Glu Arg Leu Cys Tyr Asn Asp125
130 135Thr Phe Glu Ser Leu Val Leu Asn Gly Asn
Val Val Ser Gln Leu140 145 150Trp Ile Pro
Asp Thr Phe Phe Arg Asn Ser Lys Arg Thr His Glu155 160
165His Glu Ile Thr Met Pro Asn Gln Met Val Arg Ile Tyr Lys
Asp170 175 180Gly Lys Val Leu Tyr Thr Ile
Arg Met Thr Ile Asp Ala Gly Cys185 190
195Ser Leu His Met Leu Arg Phe Pro Met Asp Ser His Ser Cys Pro200
205 210Leu Ser Phe Ser Ser Phe Ser Tyr Pro Glu
Asn Glu Met Ile Tyr215 220 225Lys Trp Glu
Asn Phe Lys Leu Glu Ile Asn Glu Lys Asn Ser Trp230 235
240Lys Leu Phe Gln Phe Asp Phe Thr Gly Val Ser Asn Lys Thr
Glu245 250 255Ile Ile Thr Thr Pro Val Gly
Asp Phe Met Val Met Thr Ile Phe260 265
270Phe Asn Val Ser Arg Arg Phe Gly Tyr Val Ala Phe Gln Asn Tyr275
280 285Val Pro Ser Ser Val Thr Thr Met Leu Ser
Trp Val Ser Phe Trp290 295 300Ile Lys Thr
Glu Ser Ala Pro Ala Arg Thr Ser Leu Gly Ile Thr305 310
315Ser Val Leu Thr Met Thr Thr Leu Gly Thr Phe Ser Arg Lys
Asn320 325 330Phe Pro Arg Val Ser Tyr Ile
Thr Ala Leu Asp Phe Tyr Ile Ala335 340
345Ile Cys Phe Val Phe Cys Phe Cys Ala Leu Leu Glu Phe Ala Val350
355 360Leu Asn Phe Leu Ile Tyr Asn Gln Thr Lys
Ala His Ala Ser Pro365 370 375Lys Leu Arg
His Pro Arg Ile Asn Ser Arg Ala His Ala Arg Thr380 385
390Arg Ala Arg Ser Arg Ala Cys Ala Arg Gln His Gln Glu Ala
Phe395 400 405Val Cys Gln Ile Val Thr Thr
Glu Gly Ser Asp Gly Glu Glu Arg410 415
420Pro Ser Cys Ser Ala Gln Gln Pro Pro Ser Pro Gly Ser Pro Glu425
430 435Gly Pro Arg Ser Leu Cys Ser Lys Leu Ala
Cys Cys Glu Trp Cys440 445 450Lys Arg Phe
Lys Lys Tyr Phe Cys Met Val Pro Asp Cys Glu Gly455 460
465Ser Thr Trp Gln Gln Gly Arg Leu Cys Ile His Val Tyr Arg
Leu470 475 480Asp Asn Tyr Ser Arg Val Val
Phe Pro Val Thr Phe Phe Phe Phe485 490
495Asn Val Leu Tyr Trp Leu Val Cys Leu Asn Leu500
505801212PRTHomo Sapien 80Met Glu Pro Arg Pro Thr Ala Pro Ser Ser Gly Ala
Pro Gly Leu1 5 10 15Ala
Gly Val Gly Glu Thr Pro Ser Ala Ala Ala Leu Ala Ala Ala20
25 30Arg Val Glu Leu Pro Gly Thr Ala Val Pro Ser Val
Pro Glu Asp35 40 45Ala Ala Pro Ala Ser
Arg Asp Gly Gly Gly Val Arg Asp Glu Gly50 55
60Pro Ala Ala Ala Gly Asp Gly Leu Gly Arg Pro Leu Gly Pro Thr65
70 75Pro Ser Gln Ser Arg Phe Gln Val Asp Leu
Val Ser Glu Asn Ala80 85 90Gly Arg Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala95 100
105Ala Ala Gly Ala Gly Ala Gly Ala Lys Gln Thr Pro Ala Asp
Gly110 115 120Glu Ala Ser Gly Glu Ser Glu
Pro Ala Lys Gly Ser Glu Glu Ala125 130
135Lys Gly Arg Phe Arg Val Asn Phe Val Asp Pro Ala Ala Ser Ser140
145 150Ser Ala Glu Asp Ser Leu Ser Asp Ala Ala
Gly Val Gly Val Asp155 160 165Gly Pro Asn
Val Ser Phe Gln Asn Gly Gly Asp Thr Val Leu Ser170 175
180Glu Gly Ser Ser Leu His Ser Gly Gly Gly Gly Gly Ser Gly
His185 190 195His Gln His Tyr Tyr Tyr Asp
Thr His Thr Asn Thr Tyr Tyr Leu200 205
210Arg Thr Phe Gly His Asn Thr Met Asp Ala Val Pro Arg Ile Asp215
220 225His Tyr Arg His Thr Ala Ala Gln Leu Gly
Glu Lys Leu Leu Arg230 235 240Pro Ser Leu
Ala Glu Leu His Asp Glu Leu Glu Lys Glu Pro Phe245 250
255Glu Asp Gly Phe Ala Asn Gly Glu Glu Ser Thr Pro Thr Arg
Asp260 265 270Ala Val Val Thr Tyr Thr Ala
Glu Ser Lys Gly Val Val Lys Phe275 280
285Gly Trp Ile Lys Gly Val Leu Val Arg Cys Met Leu Asn Ile Trp290
295 300Gly Val Met Leu Phe Ile Arg Leu Ser Trp
Ile Val Gly Gln Ala305 310 315Gly Ile Gly
Leu Ser Val Leu Val Ile Met Met Ala Thr Val Val320 325
330Thr Thr Ile Thr Gly Leu Ser Thr Ser Ala Ile Ala Thr Asn
Gly335 340 345Phe Val Arg Gly Gly Gly Ala
Tyr Tyr Leu Ile Ser Arg Ser Leu350 355
360Gly Pro Glu Phe Gly Gly Ala Ile Gly Leu Ile Phe Ala Phe Ala365
370 375Asn Ala Val Ala Val Ala Met Tyr Val Val
Gly Phe Ala Glu Thr380 385 390Val Val Glu
Leu Leu Lys Glu His Ser Ile Leu Met Ile Asp Glu395 400
405Ile Asn Asp Ile Arg Ile Ile Gly Ala Ile Thr Val Val Ile
Leu410 415 420Leu Gly Ile Ser Val Ala Gly
Met Glu Trp Glu Ala Lys Ala Gln425 430
435Ile Val Leu Leu Val Ile Leu Leu Leu Ala Ile Gly Asp Phe Val440
445 450Ile Gly Thr Phe Ile Pro Leu Glu Ser Lys
Lys Pro Lys Gly Phe455 460 465Phe Gly Tyr
Lys Ser Glu Ile Phe Asn Glu Asn Phe Gly Pro Asp470 475
480Phe Arg Glu Glu Glu Thr Phe Phe Ser Val Phe Ala Ile Phe
Phe485 490 495Pro Ala Ala Thr Gly Ile Leu
Ala Gly Ala Asn Ile Ser Gly Asp500 505
510Leu Ala Asp Pro Gln Ser Ala Ile Pro Lys Gly Thr Leu Leu Ala515
520 525Ile Leu Ile Thr Thr Leu Val Tyr Val Gly
Ile Ala Val Ser Val530 535 540Gly Ser Cys
Val Val Arg Asp Ala Thr Gly Asn Val Asn Asp Thr545 550
555Ile Val Thr Glu Leu Thr Asn Cys Thr Ser Ala Ala Cys Lys
Leu560 565 570Asn Phe Asp Phe Ser Ser Cys
Glu Ser Ser Pro Cys Ser Tyr Gly575 580
585Leu Met Asn Asn Phe Gln Val Met Ser Met Val Ser Gly Phe Thr590
595 600Pro Leu Ile Ser Ala Gly Ile Phe Ser Ala
Thr Leu Ser Ser Ala605 610 615Leu Ala Ser
Leu Val Ser Ala Pro Lys Ile Phe Gln Ala Leu Cys620 625
630Lys Asp Asn Ile Tyr Pro Ala Phe Gln Met Phe Ala Lys Gly
Tyr635 640 645Gly Lys Asn Asn Glu Pro Leu
Arg Gly Tyr Ile Leu Thr Phe Leu650 655
660Ile Ala Leu Gly Phe Ile Leu Ile Ala Glu Leu Asn Val Ile Ala665
670 675Pro Ile Ile Ser Asn Phe Phe Leu Ala Ser
Tyr Ala Leu Ile Asn680 685 690Phe Ser Val
Phe His Ala Ser Leu Ala Lys Ser Pro Gly Trp Arg695 700
705Pro Ala Phe Lys Tyr Tyr Asn Met Trp Ile Ser Leu Leu Gly
Ala710 715 720Ile Leu Cys Cys Ile Val Met
Phe Val Ile Asn Trp Trp Ala Ala725 730
735Leu Leu Thr Tyr Val Ile Val Leu Gly Leu Tyr Ile Tyr Val Thr740
745 750Tyr Lys Lys Pro Asp Val Asn Trp Gly Ser
Ser Thr Gln Ala Leu755 760 765Thr Tyr Leu
Asn Ala Leu Gln His Ser Ile Arg Leu Ser Gly Val770 775
780Glu Asp His Val Lys Asn Phe Arg Pro Gln Cys Leu Val Met
Thr785 790 795Gly Ala Pro Asn Ser Arg Pro
Ala Leu Leu His Leu Val His Asp800 805
810Phe Thr Lys Asn Val Gly Leu Met Ile Cys Gly His Val His Met815
820 825Gly Pro Arg Arg Gln Ala Met Lys Glu Met
Ser Ile Asp Gln Ala830 835 840Lys Tyr Gln
Arg Trp Leu Ile Lys Asn Lys Met Lys Ala Phe Tyr845 850
855Ala Pro Val His Ala Asp Asp Leu Arg Glu Gly Ala Gln Tyr
Leu860 865 870Met Gln Ala Ala Gly Leu Gly
Arg Met Lys Pro Asn Thr Leu Val875 880
885Leu Gly Phe Lys Lys Asp Trp Leu Gln Ala Asp Met Arg Asp Val890
895 900Asp Met Tyr Ile Asn Leu Phe His Asp Ala
Phe Asp Ile Gln Tyr905 910 915Gly Val Val
Val Ile Arg Leu Lys Glu Gly Leu Asp Ile Ser His920 925
930Leu Gln Gly Gln Glu Glu Leu Leu Ser Ser Gln Glu Lys Ser
Pro935 940 945Gly Thr Lys Asp Val Val Val
Ser Val Glu Tyr Ser Lys Lys Ser950 955
960Asp Leu Asp Thr Ser Lys Pro Leu Ser Glu Lys Pro Ile Thr His965
970 975Lys Val Glu Glu Glu Asp Gly Lys Thr Ala
Thr Gln Pro Leu Leu980 985 990Lys Lys Glu
Ser Lys Gly Pro Ile Val Pro Leu Asn Val Ala Asp995 1000
1005Gln Lys Leu Leu Glu Ala Ser Thr Gln Phe Gln Lys Lys Gln
Gly1010 1015 1020Lys Asn Thr Ile Asp Val
Trp Trp Leu Phe Asp Asp Gly Gly Leu1025 1030
1035Thr Leu Leu Ile Pro Tyr Leu Leu Thr Thr Lys Lys Lys Trp Lys1040
1045 1050Asp Cys Lys Ile Arg Val Phe Ile Gly Gly
Lys Ile Asn Arg Ile1055 1060 1065Asp His
Asp Arg Arg Ala Met Ala Thr Leu Leu Ser Lys Phe Arg1070
1075 1080Ile Asp Phe Ser Asp Ile Met Val Leu Gly Asp Ile
Asn Thr Lys1085 1090 1095Pro Lys Lys Glu
Asn Ile Ile Ala Phe Glu Glu Ile Ile Glu Pro1100 1105
1110Tyr Arg Leu His Glu Asp Asp Lys Glu Gln Asp Ile Ala Asp
Lys1115 1120 1125Met Lys Glu Asp Glu Pro
Trp Arg Ile Thr Asp Asn Glu Leu Glu1130 1135
1140Leu Tyr Lys Thr Lys Thr Tyr Arg Gln Ile Arg Leu Asn Glu Leu1145
1150 1155Leu Lys Glu His Ser Ser Thr Ala Asn Ile
Ile Val Met Ser Leu1160 1165 1170Pro Val
Ala Arg Lys Gly Ala Val Ser Ser Ala Leu Tyr Met Ala1175
1180 1185Trp Leu Glu Ala Leu Ser Lys Asp Leu Pro Pro Ile
Leu Leu Val1190 1195 1200Arg Gly Asn His
Gln Ser Val Leu Thr Phe Tyr Ser1205 121081674PRTHomo
Sapien 81Met Ala Thr Ala Val Ser Arg Pro Cys Ala Gly Arg Ser Arg Asp1
5 10 15Ile Leu Trp Arg Val
Leu Gly Trp Arg Ile Val Ala Ser Ile Val20 25
30Trp Ser Val Leu Phe Leu Pro Ile Cys Thr Thr Val Phe Ile Ile35
40 45Phe Ser Arg Ile Asp Leu Phe His Pro Ile
Gln Trp Leu Ser Asp50 55 60Ser Phe Ser
Asp Leu Tyr Ser Ser Tyr Val Ile Phe Tyr Phe Leu65 70
75Leu Leu Ser Val Val Ile Ile Ile Ile Ser Ile Phe Asn Val
Glu80 85 90Phe Tyr Ala Val Val Pro Ser
Ile Pro Cys Ser Arg Leu Ala Leu95 100
105Ile Gly Lys Ile Ile His Pro Gln Gln Leu Met His Ser Phe Ile110
115 120His Ala Ala Met Gly Met Val Met Ala Trp
Cys Ala Ala Val Ile125 130 135Thr Gln Gly
Gln Tyr Ser Phe Leu Val Val Pro Cys Thr Gly Thr140 145
150Asn Ser Phe Gly Ser Pro Ala Ala Gln Thr Cys Leu Asn Glu
Tyr155 160 165His Leu Phe Phe Leu Leu Thr
Gly Ala Phe Met Gly Tyr Ser Tyr170 175
180Ser Leu Leu Tyr Phe Val Asn Asn Met Asn Tyr Leu Pro Phe Pro185
190 195Ile Ile Gln Gln Tyr Lys Phe Leu Arg Phe
Arg Arg Ser Leu Leu200 205 210Leu Leu Val
Lys His Ser Cys Val Glu Ser Leu Phe Leu Val Arg215 220
225Asn Phe Cys Ile Leu Tyr Tyr Phe Leu Gly Tyr Ile Pro Lys
Ala230 235 240Trp Ile Ser Thr Ala Met Asn
Leu His Ile Asp Glu Gln Val His245 250
255Arg Pro Leu Asp Thr Val Ser Gly Leu Leu Asn Leu Ser Leu Leu260
265 270Tyr His Val Trp Leu Cys Gly Val Phe Leu
Leu Thr Thr Trp Tyr275 280 285Val Ser Trp
Ile Leu Phe Lys Ile Tyr Ala Thr Glu Ala His Val290 295
300Phe Pro Val Gln Pro Pro Phe Ala Glu Gly Ser Asp Glu Cys
Leu305 310 315Pro Lys Val Leu Asn Ser Asn
Pro Pro Pro Ile Ile Lys Tyr Leu320 325
330Ala Leu Gln Asp Leu Met Leu Leu Ser Gln Tyr Ser Pro Ser Arg335
340 345Arg Gln Glu Val Phe Ser Leu Ser Gln Pro
Gly Gly His Pro His350 355 360Asn Trp Thr
Ala Ile Ser Arg Glu Cys Leu Asn Leu Leu Asn Gly365 370
375Met Thr Gln Lys Leu Ile Leu Tyr Gln Glu Ala Ala Ala Thr
Asn380 385 390Gly Arg Val Ser Ser Ser Tyr
Pro Val Glu Pro Lys Lys Leu Asn395 400
405Ser Pro Glu Glu Thr Ala Phe Gln Thr Pro Lys Ser Ser Gln Met410
415 420Pro Arg Pro Ser Val Pro Pro Leu Val Lys
Thr Ser Leu Phe Ser425 430 435Ser Lys Leu
Ser Thr Pro Asp Val Val Ser Pro Phe Gly Thr Pro440 445
450Phe Gly Ser Ser Val Met Asn Arg Met Ala Gly Ile Phe Asp
Val455 460 465Asn Thr Cys Tyr Gly Ser Pro
Gln Ser Pro Gln Leu Ile Arg Arg470 475
480Gly Pro Arg Leu Trp Thr Ser Ala Ser Asp Gln Gln Met Thr Glu485
490 495Phe Ser Asn Pro Ser Pro Ser Thr Ser Ile
Ser Ala Glu Gly Lys500 505 510Thr Met Arg
Gln Pro Ser Val Ile Tyr Ser Trp Ile Gln Asn Lys515 520
525Arg Glu Gln Ile Lys Asn Phe Leu Ser Lys Arg Val Leu Ile
Met530 535 540Tyr Phe Phe Ser Lys His Pro
Glu Ala Ser Ile Gln Ala Val Phe545 550
555Ser Asp Ala Gln Met His Ile Trp Ala Leu Glu Gly Leu Ser His560
565 570Leu Val Ala Ala Ser Phe Thr Glu Asp Arg
Phe Gly Val Val Gln575 580 585Thr Thr Leu
Pro Ala Ile Leu Asn Thr Leu Leu Thr Leu Gln Glu590 595
600Ala Val Asp Lys Tyr Phe Lys Leu Pro His Ala Ser Ser Lys
Pro605 610 615Pro Arg Ile Ser Gly Ser Leu
Val Asp Thr Ser Tyr Lys Thr Leu620 625
630Arg Phe Ala Phe Arg Ala Ser Leu Lys Thr Ala Ile Tyr Arg Ile635
640 645Thr Thr Thr Phe Gly Glu His Leu Asn Ala
Val Gln Ala Ser Ala650 655 660Glu His Gln
Lys Arg Leu Gln Gln Phe Leu Glu Phe Lys Glu665
670821321PRTHomo Sapien 82Met Gly Ala Pro Phe Val Trp Ala Leu Gly Leu Leu
Met Leu Gln1 5 10 15Met
Leu Leu Phe Val Ala Gly Glu Gln Gly Thr Gln Asp Ile Thr20
25 30Asp Ala Ser Glu Arg Gly Leu His Met Gln Lys Leu
Gly Ser Gly35 40 45Ser Val Gln Ala Ala
Leu Ala Glu Leu Val Ala Leu Pro Cys Leu50 55
60Phe Thr Leu Gln Pro Arg Pro Ser Ala Ala Arg Asp Ala Pro Arg65
70 75Ile Lys Trp Thr Lys Val Arg Thr Ala Ser
Gly Gln Arg Gln Asp80 85 90Leu Pro Ile
Leu Val Ala Lys Asp Asn Val Val Arg Val Ala Lys95 100
105Ser Trp Gln Gly Arg Val Ser Leu Pro Ser Tyr Pro Arg Arg
Arg110 115 120Ala Asn Ala Thr Leu Leu Leu
Gly Pro Leu Arg Ala Ser Asp Ser125 130
135Gly Leu Tyr Arg Cys Gln Val Val Arg Gly Ile Glu Asp Glu Gln140
145 150Asp Leu Val Pro Leu Glu Val Thr Gly Val
Val Phe His Tyr Arg155 160 165Ser Ala Arg
Asp Arg Tyr Ala Leu Thr Phe Ala Glu Ala Gln Glu170 175
180Ala Cys Arg Leu Ser Ser Ala Ile Ile Ala Ala Pro Arg His
Leu185 190 195Gln Ala Ala Phe Glu Asp Gly
Phe Asp Asn Cys Asp Ala Gly Trp200 205
210Leu Ser Asp Arg Thr Val Arg Tyr Pro Ile Thr Gln Ser Arg Pro215
220 225Gly Cys Tyr Gly Asp Arg Ser Ser Leu Pro
Gly Val Arg Ser Tyr230 235 240Gly Arg Arg
Asn Pro Gln Glu Leu Tyr Asp Val Tyr Cys Phe Ala245 250
255Arg Glu Leu Gly Gly Glu Val Phe Tyr Val Gly Pro Ala Arg
Arg260 265 270Leu Thr Leu Ala Gly Ala Arg
Ala Gln Cys Arg Arg Gln Gly Ala275 280
285Ala Leu Ala Ser Val Gly Gln Leu His Leu Ala Trp His Glu Gly290
295 300Leu Asp Gln Cys Asp Pro Gly Trp Leu Ala
Asp Gly Ser Val Arg305 310 315Tyr Pro Ile
Gln Thr Pro Arg Arg Arg Cys Gly Gly Pro Ala Pro320 325
330Gly Val Arg Thr Val Tyr Arg Phe Ala Asn Arg Thr Gly Phe
Pro335 340 345Ser Pro Ala Glu Arg Phe Asp
Ala Tyr Cys Phe Arg Ala His His350 355
360Pro Thr Ser Gln His Gly Asp Leu Glu Thr Pro Ser Ser Gly Asp365
370 375Glu Gly Glu Ile Leu Ser Ala Glu Gly Pro
Pro Val Arg Glu Leu380 385 390Glu Pro Thr
Leu Glu Glu Glu Glu Val Val Thr Pro Asp Phe Gln395 400
405Glu Pro Leu Val Ser Ser Gly Glu Glu Glu Thr Leu Ile Leu
Glu410 415 420Glu Lys Gln Glu Ser Gln Gln
Thr Leu Ser Pro Thr Pro Gly Asp425 430
435Pro Met Leu Ala Ser Trp Pro Thr Gly Glu Val Trp Leu Ser Thr440
445 450Val Ala Pro Ser Pro Ser Asp Met Gly Ala
Gly Thr Ala Ala Ser455 460 465Ser His Thr
Glu Val Ala Pro Thr Asp Pro Met Pro Arg Arg Arg470 475
480Gly Arg Phe Lys Gly Leu Asn Gly Arg Tyr Phe Gln Gln Gln
Glu485 490 495Pro Glu Pro Gly Leu Gln Gly
Gly Met Glu Ala Ser Ala Gln Pro500 505
510Pro Thr Ser Glu Ala Ala Val Asn Gln Met Glu Pro Pro Leu Ala515
520 525Met Ala Val Thr Glu Met Leu Gly Ser Gly
Gln Ser Arg Ser Pro530 535 540Trp Ala Asp
Leu Thr Asn Glu Val Asp Met Pro Gly Ala Gly Ser545 550
555Ala Gly Gly Lys Ser Ser Pro Glu Pro Trp Leu Trp Pro Pro
Thr560 565 570Met Val Pro Pro Ser Ile Ser
Gly His Ser Arg Ala Pro Val Leu575 580
585Glu Leu Glu Lys Ala Glu Gly Pro Ser Ala Arg Pro Ala Thr Pro590
595 600Asp Leu Phe Trp Ser Pro Leu Glu Ala Thr
Val Ser Ala Pro Ser605 610 615Pro Ala Pro
Trp Glu Ala Phe Pro Val Ala Thr Ser Pro Asp Leu620 625
630Pro Met Met Ala Met Leu Arg Gly Pro Lys Glu Trp Met Leu
Pro635 640 645His Pro Thr Pro Ile Ser Thr
Glu Ala Asn Arg Val Glu Ala His650 655
660Gly Glu Ala Thr Ala Thr Ala Pro Pro Ser Pro Ala Ala Glu Thr665
670 675Lys Val Tyr Ser Leu Pro Leu Ser Leu Thr
Pro Thr Gly Gln Gly680 685 690Gly Glu Ala
Met Pro Thr Thr Pro Glu Ser Pro Arg Ala Asp Phe695 700
705Arg Glu Thr Gly Glu Thr Ser Pro Ala Gln Val Asn Lys Ala
Glu710 715 720His Ser Ser Ser Ser Pro Trp
Pro Ser Val Asn Arg Asn Val Ala725 730
735Val Gly Phe Val Pro Thr Glu Thr Ala Thr Glu Pro Thr Gly Leu740
745 750Arg Gly Ile Pro Gly Ser Glu Ser Gly Val
Phe Asp Thr Ala Glu755 760 765Ser Pro Thr
Ser Gly Leu Gln Ala Thr Val Asp Glu Val Gln Asp770 775
780Pro Trp Pro Ser Val Tyr Ser Lys Gly Leu Asp Ala Ser Ser
Pro785 790 795Ser Ala Pro Leu Gly Ser Pro
Gly Val Phe Leu Val Pro Lys Val800 805
810Thr Pro Asn Leu Glu Pro Trp Val Ala Thr Asp Glu Gly Pro Thr815
820 825Val Asn Pro Met Asp Ser Thr Val Thr Pro
Ala Pro Ser Asp Ala830 835 840Ser Gly Ile
Trp Glu Pro Gly Ser Gln Val Phe Glu Glu Ala Glu845 850
855Ser Thr Thr Leu Ser Pro Gln Val Ala Leu Asp Thr Ser Ile
Val860 865 870Thr Pro Leu Thr Thr Leu Glu
Gln Gly Asp Lys Val Gly Val Pro875 880
885Ala Met Ser Thr Leu Gly Ser Ser Ser Ser Gln Pro His Pro Glu890
895 900Pro Glu Asp Gln Val Glu Thr Gln Gly Thr
Ser Gly Ala Ser Val905 910 915Pro Pro His
Gln Ser Ser Pro Leu Gly Lys Pro Ala Val Pro Pro920 925
930Gly Thr Pro Thr Ala Ala Ser Val Gly Glu Ser Ala Ser Val
Ser935 940 945Ser Gly Glu Pro Thr Val Pro
Trp Asp Pro Ser Ser Thr Leu Leu950 955
960Pro Val Thr Leu Gly Ile Glu Asp Phe Glu Leu Glu Val Leu Ala965
970 975Gly Ser Pro Gly Val Glu Ser Phe Trp Glu
Glu Val Ala Ser Gly980 985 990Glu Glu Pro
Ala Leu Pro Gly Thr Pro Met Asn Ala Gly Ala Glu995 1000
1005Glu Val His Ser Asp Pro Cys Glu Asn Asn Pro Cys Leu His
Gly1010 1015 1020Gly Thr Cys Asn Ala Asn
Gly Thr Met Tyr Gly Cys Ser Cys Asp1025 1030
1035Gln Gly Phe Ala Gly Glu Asn Cys Glu Ile Asp Ile Asp Asp Cys1040
1045 1050Leu Cys Ser Pro Cys Glu Asn Gly Gly Thr
Cys Ile Asp Glu Val1055 1060 1065Asn Gly
Phe Val Cys Leu Cys Leu Pro Ser Tyr Gly Gly Ser Phe1070
1075 1080Cys Glu Lys Asp Thr Glu Gly Cys Asp Arg Gly Trp
His Lys Phe1085 1090 1095Gln Gly His Cys
Tyr Arg Tyr Phe Ala His Arg Arg Ala Trp Glu1100 1105
1110Asp Ala Glu Lys Asp Cys Arg Arg Arg Ser Gly His Leu Thr
Ser1115 1120 1125Val His Ser Pro Glu Glu
His Ser Phe Ile Asn Ser Phe Gly His1130 1135
1140Glu Asn Thr Trp Ile Gly Leu Asn Asp Arg Ile Val Glu Arg Asp1145
1150 1155Phe Gln Trp Thr Asp Asn Thr Gly Leu Gln
Phe Glu Asn Trp Arg1160 1165 1170Glu Asn
Gln Pro Asp Asn Phe Phe Ala Gly Gly Glu Asp Cys Val1175
1180 1185Val Met Val Ala His Glu Ser Gly Arg Trp Asn Asp
Val Pro Cys1190 1195 1200Asn Tyr Asn Leu
Pro Tyr Val Cys Lys Lys Gly Thr Val Leu Cys1205 1210
1215Gly Pro Pro Pro Ala Val Glu Asn Ala Ser Leu Ile Gly Ala
Arg1220 1225 1230Lys Ala Lys Asn Asn Val
His Ala Thr Val Arg Tyr Gln Cys Asn1235 1240
1245Glu Gly Phe Ala Gln His His Val Val Thr Ile Arg Cys Arg Ser1250
1255 1260Asn Gly Lys Trp Asp Arg Pro Gln Ile Val
Cys Thr Lys Pro Arg1265 1270 1275Arg Ser
His Arg Met Arg Gly His His His His His Gln His His1280
1285 1290His Gln His His His His Lys Ser Arg Lys Glu Arg
Arg Lys His1295 1300 1305Lys Lys His Pro
Thr Glu Asp Trp Glu Lys Asp Glu Gly Asn Phe1310 1315
1320Cys83696PRTHomo Sapien 83Met Lys Phe Ala Glu His Leu Ser Ala
His Ile Thr Pro Glu Trp1 5 10
15Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met Leu Tyr20
25 30Ser Ala Gln Asp Gln Ala Pro Ser Val Glu
Val Thr Asp Glu Asp35 40 45Thr Val Lys
Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln50 55
60Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr Phe Tyr Ser
Glu65 70 75Lys Leu Ala Glu Ala Gln Arg
Arg Phe Ala Thr Leu Gln Asn Glu80 85
90Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val Thr95
100 105Thr Leu Arg Gln Arg Arg Lys Pro Val Phe His
Leu Ser His Glu110 115 120Glu Arg Val Gln
His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe125 130
135Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn Tyr Gln
Asn140 145 150Leu Asn Phe Thr Gly Phe Arg
Lys Ile Leu Lys Lys His Asp Lys155 160
165Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val170
175 180Glu Val Ala Pro Phe Tyr Thr Cys Lys Lys
Ile Asn Gln Leu Ile185 190 195Ser Glu Thr
Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp200 205
210Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro Leu Gly
Ala215 220 225Ala Gln Pro Ala Pro Ala Trp
Thr Thr Phe Arg Val Gly Leu Phe230 235
240Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala245
250 255Val Phe Lys Leu Glu Thr Asp Arg Ser Ile
Trp Pro Leu Ile Arg260 265 270Ile Tyr Arg
Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu275 280
285Gly Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val Asn His
Val290 295 300Leu Ile Phe Glu Leu Asn Pro
Arg Ser Asn Leu Ser His Gln His305 310
315Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser320
325 330Leu Leu Ala Cys Phe Phe Ala Pro Ile Ser
Val Ile Pro Thr Tyr335 340 345Val Tyr Pro
Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu Ile350 355
360Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe Trp Leu
Leu365 370 375Lys Leu Leu Phe Arg Val Phe
Thr Ala Pro Phe His Lys Val Gly380 385
390Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val395
400 405Ile Leu Met Asp Leu Glu Tyr Met Ile Cys
Phe Tyr Ser Leu Glu410 415 420Leu Lys Trp
Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu425 430
435Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val Arg Ala
Ile440 445 450Val Gln Cys Ile Pro Ala Trp
Leu Arg Phe Ile Gln Cys Leu Arg455 460
465Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn Ala470
475 480Gly Lys Tyr Ser Thr Thr Phe Phe Met Val
Ala Phe Ala Ala Leu485 490 495Tyr Ser Thr
His Lys Glu Arg Gly His Ser Asp Thr Met Val Phe500 505
510Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr
Thr515 520 525Leu Ile Trp Asp Leu Lys Met
Asp Trp Gly Leu Phe Asp Lys Asn530 535
540Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln545
550 555Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu
Asp Val Ile Leu Arg560 565 570Phe Ala Trp
Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu575 580
585Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala Pro Leu
Glu590 595 600Val Phe Arg Arg Phe Val Trp
Asn Phe Phe Arg Leu Glu Asn Glu605 610
615His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp Ile Ser620
625 630Val Ala Pro Leu Asn Ala Asp Asp Gln Thr
Leu Leu Glu Gln Met635 640 645Met Asp Gln
Asp Asp Gly Val Arg Asn Arg Gln Lys Asn Arg Ser650 655
660Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg Arg Pro Arg Leu
Ala665 670 675Ser Gln Ser Lys Ala Arg Asp
Thr Lys Val Leu Ile Glu Asp Thr680 685
690Asp Asp Glu Ala Asn Thr69584696PRTHomo Sapien 84Met Lys Phe Ala Glu
His Leu Ser Ala His Ile Thr Pro Glu Trp1 5
10 15Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met
Leu Tyr20 25 30Ser Ala Gln Asp Gln Ala
Pro Ser Val Glu Val Thr Asp Glu Asp35 40
45Thr Val Lys Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln50
55 60Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr
Phe Tyr Ser Glu65 70 75Lys Leu Ala Glu
Ala Gln Arg Arg Phe Ala Thr Leu Gln Asn Glu80 85
90Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val Thr95
100 105Thr Leu Arg Gln Arg Arg Lys Pro Val
Phe His Leu Ser His Glu110 115 120Glu Arg
Val Gln His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe125
130 135Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn
Tyr Gln Asn140 145 150Leu Asn Phe Thr Gly
Phe Arg Lys Ile Leu Lys Lys His Asp Lys155 160
165Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val170
175 180Glu Val Ala Pro Phe Tyr Thr Cys Lys
Lys Ile Asn Gln Leu Ile185 190 195Ser Glu
Thr Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp200
205 210Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro
Leu Gly Ala215 220 225Ala Gln Pro Ala Pro
Ala Trp Thr Thr Phe Arg Val Gly Leu Phe230 235
240Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala245
250 255Val Phe Lys Leu Glu Thr Asp Arg Ser
Ile Trp Pro Leu Ile Arg260 265 270Ile Tyr
Arg Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu275
280 285Gly Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val
Asn His Val290 295 300Leu Ile Phe Glu Leu
Asn Pro Arg Ser Asn Leu Ser His Gln His305 310
315Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser320
325 330Leu Leu Ala Cys Phe Phe Ala Pro Ile
Ser Val Ile Pro Thr Tyr335 340 345Val Tyr
Pro Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu Ile350
355 360Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe
Trp Leu Leu365 370 375Lys Leu Leu Phe Arg
Val Phe Thr Ala Pro Phe His Lys Val Gly380 385
390Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val395
400 405Ile Leu Met Asp Leu Glu Tyr Met Ile
Cys Phe Tyr Ser Leu Glu410 415 420Leu Lys
Trp Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu425
430 435Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val
Arg Ala Ile440 445 450Val Gln Cys Ile Pro
Ala Trp Leu Arg Phe Ile Gln Cys Leu Arg455 460
465Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn Ala470
475 480Gly Lys Tyr Ser Thr Thr Phe Phe Met
Val Thr Phe Ala Ala Leu485 490 495Tyr Ser
Thr His Lys Glu Arg Gly His Ser Asp Thr Met Val Phe500
505 510Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser
Cys Tyr Thr515 520 525Leu Ile Trp Asp Leu
Lys Met Asp Trp Gly Leu Phe Asp Lys Asn530 535
540Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln545
550 555Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile
Glu Asp Val Ile Leu Arg560 565 570Phe Ala
Trp Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu575
580 585Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala
Pro Leu Glu590 595 600Val Phe Arg Arg Phe
Val Trp Asn Phe Phe Arg Leu Glu Asn Glu605 610
615His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp Ile Ser620
625 630Val Ala Pro Leu Asn Ala Asp Asp Gln
Thr Leu Leu Glu Gln Met635 640 645Met Asp
Gln Asp Asp Gly Val Arg Asn Arg Gln Lys Asn Arg Ser650
655 660Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg Arg Pro
Arg Leu Ala665 670 675Ser Gln Ser Lys Ala
Arg Asp Thr Lys Val Leu Ile Glu Asp Thr680 685
690Asp Asp Glu Ala Asn Thr69585635PRTHomo Sapien 85Met Ser Val Gly
Val Ser Thr Ser Ala Pro Leu Ser Pro Thr Ser1 5
10 15Gly Thr Ser Val Gly Met Ser Thr Phe Ser Ile Met
Asp Tyr Val20 25 30Val Phe Val Leu Leu
Leu Val Leu Ser Leu Ala Ile Gly Leu Tyr35 40
45His Ala Cys Arg Gly Trp Gly Arg His Thr Val Gly Glu Leu Leu50
55 60Met Ala Asp Arg Lys Met Gly Cys Leu Pro
Val Ala Leu Ser Leu65 70 75Leu Ala Thr
Phe Gln Ser Ala Val Ala Ile Leu Gly Val Pro Ser80 85
90Glu Ile Tyr Arg Phe Gly Thr Gln Tyr Trp Phe Leu Gly Cys
Cys95 100 105Tyr Phe Leu Gly Leu Leu Ile
Pro Ala His Ile Phe Ile Pro Val110 115
120Phe Tyr Arg Leu His Leu Thr Ser Ala Tyr Glu Tyr Leu Glu Leu125
130 135Arg Phe Asn Lys Thr Val Arg Val Cys Gly
Thr Val Thr Phe Ile140 145 150Phe Gln Met
Val Ile Tyr Met Gly Val Val Leu Tyr Ala Pro Ser155 160
165Leu Ala Leu Asn Ala Val Thr Gly Phe Asp Leu Trp Leu Ser
Val170 175 180Leu Ala Leu Gly Ile Val Cys
Thr Val Tyr Thr Ala Leu Gly Gly185 190
195Leu Lys Ala Val Ile Trp Thr Asp Val Phe Gln Thr Leu Val Met200
205 210Phe Leu Gly Gln Leu Ala Val Ile Ile Val
Gly Ser Ala Lys Val215 220 225Gly Gly Leu
Gly Arg Val Trp Ala Val Ala Ser Gln His Gly Arg230 235
240Ile Ser Gly Phe Glu Leu Asp Pro Asp Pro Phe Val Arg His
Thr245 250 255Phe Trp Thr Leu Ala Phe Gly
Gly Val Phe Met Met Leu Ser Leu260 265
270Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Leu Ser Ser Arg275
280 285Thr Glu Lys Ala Ala Val Leu Ser Cys Tyr
Ala Val Phe Pro Phe290 295 300Gln Gln Val
Ser Leu Cys Val Gly Cys Leu Ile Gly Leu Val Met305 310
315Phe Ala Tyr Tyr Gln Glu Tyr Pro Met Ser Ile Gln Gln Ala
Gln320 325 330Ala Ala Pro Asp Gln Phe Val
Leu Tyr Phe Val Met Asp Leu Leu335 340
345Lys Gly Leu Pro Gly Leu Pro Gly Leu Phe Ile Ala Cys Leu Phe350
355 360Ser Gly Ser Leu Ser Thr Ile Ser Ser Ala
Phe Asn Ser Leu Ala365 370 375Thr Val Thr
Met Glu Asp Leu Ile Arg Pro Trp Phe Pro Glu Phe380 385
390Ser Glu Ala Arg Ala Ile Met Leu Ser Arg Gly Leu Ala Phe
Gly395 400 405Tyr Gly Leu Leu Cys Leu Gly
Met Ala Tyr Ile Ser Ser Gln Met410 415
420Gly Pro Val Leu Gln Ala Ala Ile Ser Ile Phe Gly Met Val Gly425
430 435Gly Pro Leu Leu Gly Leu Phe Cys Leu Gly
Met Phe Phe Pro Cys440 445 450Ala Asn Pro
Pro Gly Ala Val Val Gly Leu Leu Ala Gly Leu Val455 460
465Met Ala Phe Trp Ile Gly Ile Gly Ser Ile Val Thr Ser Met
Gly470 475 480Phe Ser Met Pro Pro Ser Pro
Ser Asn Gly Ser Ser Phe Ser Leu485 490
495Pro Thr Asn Leu Thr Val Ala Thr Val Thr Thr Leu Met Pro Leu500
505 510Thr Thr Phe Ser Lys Pro Thr Gly Leu Gln
Arg Phe Tyr Ser Leu515 520 525Ser Tyr Leu
Trp Tyr Ser Ala His Asn Ser Thr Thr Val Ile Val530 535
540Val Gly Leu Ile Val Ser Leu Leu Thr Gly Arg Met Arg Gly
Arg545 550 555Ser Leu Asn Pro Ala Thr Ile
Tyr Pro Val Leu Pro Lys Leu Leu560 565
570Ser Leu Leu Pro Leu Ser Cys Gln Lys Arg Leu His Cys Arg Ser575
580 585Tyr Gly Gln Asp His Leu Asp Thr Gly Leu
Phe Pro Glu Lys Pro590 595 600Arg Asn Gly
Val Leu Gly Asp Ser Arg Asp Lys Glu Ala Met Ala605 610
615Leu Asp Gly Thr Ala Tyr Gln Gly Ser Ser Ser Thr Cys Ile
Leu620 625 630Gln Glu Thr Ser
Leu63586351PRTHomo Sapien 86Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala
Glu Ala Glu Ala1 5 10
15Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu20
25 30Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser
Met Val Phe Leu35 40 45Asn Lys Tyr Leu
Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro50 55
60Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys65
70 75Lys Gly Leu Ser Ala Leu Ala Ala Cys
Cys Pro Gly Ala Val Asp80 85 90Phe Pro
Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser Val Leu95 100
105Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe Asn
Asn Leu110 115 120Cys Leu Lys Tyr Val Gly
Val Ala Phe Tyr Asn Val Gly Arg Ser125 130
135Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu Leu Leu Lys140
145 150Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr
Cys Gly Ile Ile Ile155 160 165Gly Gly Phe
Trp Leu Gly Val Asp Gln Glu Gly Ala Glu Gly Thr170 175
180Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu Ala Ser Leu
Cys185 190 195Val Ser Leu Asn Ala Ile Tyr
Thr Thr Lys Val Leu Pro Ala Val200 205
210Asp Gly Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala215
220 225Cys Ile Leu Phe Leu Pro Leu Leu Leu Leu
Leu Gly Glu Leu Gln230 235 240Ala Leu Arg
Asp Leu Ala Gln Leu Gly Ser Ala His Phe Trp Gly245 250
255Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr
Val260 265 270Thr Gly Leu Gln Ile Lys Phe
Thr Ser Pro Leu Thr His Asn Val275 280
285Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu290
295 300Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp
Trp Thr Ser Asn Met305 310 315Met Val Leu
Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp320 325
330Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser
Glu335 340 345Lys Ser Ala Met Gly
Val35087351PRTHomo Sapien 87Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala
Glu Ala Glu Ala1 5 10
15Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu20
25 30Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser
Met Val Phe Leu35 40 45Asn Lys Tyr Leu
Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro50 55
60Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys65
70 75Lys Gly Leu Ser Ala Leu Ala Ala Cys
Cys Pro Gly Ala Val Asp80 85 90Phe Pro
Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser Val Leu95 100
105Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe Asn
Asn Leu110 115 120Cys Leu Lys Tyr Val Gly
Val Ala Phe Tyr Asn Val Gly Arg Ser125 130
135Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu Leu Leu Lys140
145 150Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr
Cys Gly Ile Ile Ile155 160 165Gly Gly Phe
Trp Leu Gly Val Asp Gln Glu Gly Ala Glu Gly Thr170 175
180Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu Ala Ser Leu
Cys185 190 195Val Ser Leu Asn Ala Ile Tyr
Thr Thr Lys Val Leu Pro Ala Val200 205
210Asp Gly Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala215
220 225Cys Ile Leu Phe Leu Pro Leu Leu Leu Leu
Leu Gly Glu Leu Gln230 235 240Ala Leu Arg
Asp Phe Ala Gln Leu Gly Ser Ala His Phe Trp Gly245 250
255Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr
Val260 265 270Thr Gly Leu Gln Ile Lys Phe
Thr Ser Pro Leu Thr His Asn Val275 280
285Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu290
295 300Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp
Trp Thr Ser Asn Met305 310 315Met Val Leu
Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp320 325
330Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser
Glu335 340 345Lys Ser Ala Met Gly
Val35088208PRTHomo Sapien 88Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val
Val Leu Cys Ala1 5 10
15Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu20
25 30Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe
Val His Ile Ile35 40 45Ile Val Ile Leu
Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg50 55
60Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp65
70 75Asn Val Phe Ile Ile Cys Phe Tyr Leu
Glu Val Gly Gly Leu Leu80 85 90Gln Asp
Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser95 100
105Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu
Val Pro110 115 120Ala Val Gly Leu Gly Ala
Pro His Gly Gln Ala Leu Val Ser Gly125 130
135Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser140
145 150Gly Leu Gln Ile Leu Ile Ala Leu Leu Gly
Phe Val Cys Gly Cys155 160 165Gln Val Val
Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe170 175
180Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu
Lys185 190 195Pro Ser Ser Leu Leu Ser Lys
Gln Val Tyr Leu Pro Ala200 20589208PRTHomo Sapien 89Met
Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala1 5
10 15Phe Gln Leu Val Ala Ala Leu Glu Arg
Gln Val Phe Asp Phe Leu20 25 30Gly Tyr
Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile35 40
45Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg
Leu Arg50 55 60Tyr Val Met Val Tyr Thr
Leu Trp Ala Ala Val Trp Val Thr Trp65 70
75Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu80
85 90Lys Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser
Arg His Arg Ser95 100 105Trp Trp Arg Glu
Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro110 115
120Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser
Gly125 130 135Ala Gly Cys Ala Leu Glu Pro
Ser Tyr Val Glu Ala Leu His Ser140 145
150Cys Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys155
160 165Gln Val Val Ser Val Phe Thr Glu Glu Glu
Asp Ser Phe Asp Phe170 175 180Ile Gly Gly
Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys185 190
195Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala200
20590181PRTHomo Sapien 90Met Gly Ser Cys Ser Gly Arg Cys Ala
Leu Val Val Leu Cys Ala1 5 10
15Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu20
25 30Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn
Phe Val His Ile Ile35 40 45Ile Val Ile
Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg50 55
60Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr
Trp65 70 75Asn Val Phe Ile Ile Cys Phe
Tyr Leu Glu Val Gly Gly Leu Leu80 85
90Lys Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser95
100 105Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His
Glu Glu Val Pro110 115 120Ala Val Gly Leu
Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly125 130
135Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His
Ser140 145 150Cys Leu Gln Ile Leu Ile Ala
Leu Leu Gly Phe Val Cys Gly Cys155 160
165Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu Arg170
175 180Lys91181PRTHomo Sapien 91Met Gly Ser Cys
Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala1 5
10 15Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe
Asp Phe Leu20 25 30Gly Tyr Gln Trp Ala
Pro Ile Leu Ala Asn Phe Val His Ile Ile35 40
45Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg50
55 60Tyr Val Met Val Tyr Thr Leu Trp Ala Ala
Val Trp Val Thr Trp65 70 75Asn Val Phe
Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu80 85
90Gln Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg
Ser95 100 105Trp Trp Arg Glu Arg Trp Pro
Gly Cys Leu His Glu Glu Val Pro110 115
120Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly125
130 135Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val
Glu Ala Leu His Ser140 145 150Gly Leu Gln
Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys155 160
165Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu
Arg170 175 180Lys92382PRTHomo Sapien
92Met Ala Val Leu Phe Leu Leu Leu Phe Leu Cys Gly Thr Pro Gln1
5 10 15Ala Ala Asp Asn Met Gln Ala
Ile Tyr Val Ala Leu Gly Glu Ala20 25
30Val Glu Leu Pro Cys Pro Ser Pro Pro Thr Leu His Gly Asp Glu35
40 45His Leu Ser Trp Phe Cys Ser Pro Ala Ala Gly
Ser Phe Thr Thr50 55 60Leu Val Ala Gln
Val Gln Val Gly Arg Pro Ala Pro Asp Pro Gly65 70
75Lys Pro Gly Arg Glu Ser Arg Leu Arg Leu Leu Gly Asn Tyr Ser80
85 90Leu Trp Leu Glu Gly Ser Lys Glu Glu
Asp Ala Gly Arg Tyr Trp95 100 105Cys Ala
Val Leu Gly Gln His His Asn Tyr Gln Asn Trp Arg Val110
115 120Tyr Asp Val Leu Val Leu Lys Gly Ser Gln Leu Ser
Ala Arg Ala125 130 135Ala Asp Gly Ser Pro
Cys Asn Val Leu Leu Cys Ser Val Val Pro140 145
150Ser Arg Arg Met Asp Ser Val Thr Trp Gln Glu Gly Lys Gly Pro155
160 165Val Arg Gly Arg Val Gln Ser Phe Trp
Gly Ser Glu Ala Ala Leu170 175 180Leu Leu
Val Cys Pro Gly Glu Gly Leu Ser Glu Pro Arg Ser Arg185
190 195Arg Pro Arg Ile Ile Arg Cys Leu Met Thr His Asn
Lys Gly Val200 205 210Ser Phe Ser Leu Ala
Ala Ser Ile Asp Ala Ser Pro Ala Leu Cys215 220
225Ala Pro Ser Thr Gly Trp Asp Met Pro Trp Ile Leu Met Leu Leu230
235 240Leu Thr Met Gly Gln Gly Val Val Ile
Leu Ala Leu Ser Ile Val245 250 255Leu Trp
Arg Gln Arg Val Arg Gly Ala Pro Gly Arg Gly Asn Arg260
265 270Met Arg Cys Tyr Asn Cys Gly Gly Ser Pro Ser Ser
Ser Cys Lys275 280 285Glu Ala Val Thr Thr
Cys Gly Glu Gly Arg Pro Gln Pro Gly Leu290 295
300Glu Gln Ile Lys Leu Pro Gly Asn Pro Pro Val Thr Leu Ile His305
310 315Gln His Pro Ala Cys Val Ala Ala His
His Cys Asn Gln Val Glu320 325 330Thr Glu
Ser Val Gly Asp Val Thr Tyr Pro Ala His Arg Asp Cys335
340 345Tyr Leu Gly Asp Leu Cys Asn Ser Ala Val Ala Ser
His Val Ala350 355 360Pro Ala Gly Ile Leu
Ala Ala Ala Ala Thr Ala Leu Thr Cys Leu365 370
375Leu Pro Gly Leu Trp Ser Gly38093783PRTHomo Sapien 93Met Ser Gly
Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp1 5
10 15Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly
Arg Thr Gly Leu20 25 30Val Leu Ala Ala
Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys35 40
45Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys50
55 60Leu Asn Leu Thr Leu Glu Gly Val Phe
Ala Gly Val Ala Glu Ile65 70 75Thr Pro
Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu80 85
90Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe
Ile Ser95 100 105Ile Val Lys Leu Glu Ser
Pro Arg Arg Ala Pro Arg Pro Cys Leu110 115
120Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser125
130 135Ala Val Leu Phe Asp Ile Thr Glu Asp Arg
Ala Ala Ala Glu Gln140 145 150Leu Gln Gln
Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp155 160
165Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn
Gln170 175 180Lys Ala His Val Arg Ile Glu
Leu Lys Glu Pro Pro Ala Trp Pro185 190
195Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe200
205 210Val Ile Ile Leu Ala Ser Val Leu Arg Ile
Arg Cys Arg Pro Arg215 220 225His Ser Arg
Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile230 235
240Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln
Ala245 250 255Arg Gly Glu Trp Pro Asp Ser
Gly Ser Ser Cys Ser Ser Ala Pro260 265
270Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu275
280 285Arg Val Ile Ser Cys Leu His Glu Phe His
Arg Asn Cys Val Asp290 295 300Pro Trp Leu
His Gln His Arg Thr Cys Pro Leu Cys Val Phe Asn305 310
315Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser
Arg320 325 330Ser Tyr Gln Glu Pro Gly Arg
Arg Leu His Leu Ile Arg Gln His335 340
345Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly350
355 360Pro Ser Arg Ser Ala Val Ala Arg Pro Pro
Arg Pro Gly Pro Phe365 370 375Leu Pro Ser
Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe380 385
390Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg
Leu395 400 405Ala Gly Ala Gln His Pro Tyr
Ala Gln Gly Trp Gly Met Ser His410 415
420Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu425
430 435Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly
Ser Gly Glu Ser Tyr440 445 450Cys Thr Glu
Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp455 460
465Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val
Val470 475 480Asn Cys Thr Asp Ile Ser Leu
Gln Gly Val His Gly Ser Ser Ser485 490
495Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr500
505 510Cys Ser Pro Lys Gly Asp Pro Gln Arg Val
Asp Met Gln Pro Ser515 520 525Val Thr Ser
Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly530 535
540Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg
His545 550 555His His Tyr Lys Lys Arg Phe
Gln Trp His Gly Arg Lys Pro Gly560 565
570Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr575
580 585Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp
Gln Gln Val Thr Gly590 595 600Ser Asn Ser
Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys605 610
615Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala
Ser620 625 630Ser Ile Cys Pro Ser Thr Ser
Ser Leu Phe Asn Leu Gln Lys Ser635 640
645Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro650
655 660Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln
Asp Ala Thr Val His665 670 675Pro Ala Cys
Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr680 685
690Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro
Gly695 700 705Leu Asp Lys Arg Leu Leu Pro
Glu Thr Pro Gly Pro Cys Tyr Ser710 715
720Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu725
730 735Glu Pro His Pro Pro Gly Glu Gly Pro Ser
Glu Trp Ser Ser Asp740 745 750Thr Ala Glu
Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu755 760
765Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys
Glu770 775 780Gln Ala Val94510PRTHomo
Sapien 94Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp1
5 10 15Leu Leu Leu Leu Leu
Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro20 25
30Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly35
40 45Gln Asp Ala Lys Leu Pro Cys Phe Tyr Arg
Gly Asp Ser Gly Glu50 55 60Gln Val Gly
Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly65 70
75Ala Gln Glu Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His
Val80 85 90Ser Pro Ala Tyr Glu Gly Arg
Val Glu Gln Pro Pro Pro Pro Arg95 100
105Asn Pro Leu Asp Gly Ser Val Leu Leu Arg Asn Ala Val Gln Ala110
115 120Asp Glu Gly Glu Tyr Glu Cys Arg Val Ser
Thr Phe Pro Ala Gly125 130 135Ser Phe Gln
Ala Arg Leu Arg Leu Arg Val Leu Val Pro Pro Leu140 145
150Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu Glu Gly Gln Gly
Leu155 160 165Thr Leu Ala Ala Ser Cys Thr
Ala Glu Gly Ser Pro Ala Pro Ser170 175
180Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser Ser Arg Ser185
190 195Phe Lys His Ser Arg Ser Ala Ala Val Thr
Ser Glu Phe His Leu200 205 210Val Pro Ser
Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val Val215 220
225Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile
Leu230 235 240His Val Ser Phe Leu Ala Glu
Ala Ser Val Arg Gly Leu Glu Asp245 250
255Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys260
265 270Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr
Asn Trp Thr Arg Leu275 280 285Asp Gly Pro
Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu290 295
300Gly Phe Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val
Cys305 310 315His Val Ser Asn Glu Phe Ser
Ser Arg Asp Ser Gln Val Thr Val320 325
330Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu335
340 345Val Ser Ala Ser Val Val Val Val Gly Val
Ile Ala Ala Leu Leu350 355 360Phe Cys Leu
Leu Val Val Val Val Val Leu Met Ser Arg Tyr His365 370
375Arg Arg Lys Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu
Leu380 385 390Thr Leu Thr Arg Glu Asn Ser
Ile Arg Arg Leu His Ser His His395 400
405Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val Gly Leu Arg Ala410
415 420Glu Gly His Pro Asp Ser Leu Lys Asp Asn
Ser Ser Cys Ser Val425 430 435Met Ser Glu
Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu Thr Thr440 445
450Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly
Ser455 460 465Gly Arg Ala Glu Glu Glu Glu
Asp Gln Asp Glu Gly Ile Lys Gln470 475
480Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys485
490 495Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly
Arg Gly His Leu Val500 505
51095523PRTHomo Sapien 95Met Thr Gln Asn Lys Leu Lys Leu Cys Ser Lys Ala
Asn Val Tyr1 5 10 15Thr
Glu Val Pro Asp Gly Gly Trp Gly Trp Ala Val Ala Val Ser20
25 30Phe Phe Phe Val Glu Val Phe Thr Tyr Gly Ile Ile
Lys Thr Phe35 40 45Gly Val Phe Phe Asn
Asp Leu Met Asp Ser Phe Asn Glu Ser Asn50 55
60Ser Arg Ile Ser Trp Ile Ile Ser Ile Cys Val Phe Val Leu Thr65
70 75Phe Ser Ala Pro Leu Ala Thr Val Leu Ser
Asn Arg Phe Gly His80 85 90Arg Leu Val
Val Met Leu Gly Gly Leu Leu Val Ser Thr Gly Met95 100
105Val Ala Ala Ser Phe Ser Gln Glu Val Ser His Met Tyr Val
Ala110 115 120Ile Gly Ile Ile Ser Gly Leu
Gly Tyr Cys Phe Ser Phe Leu Pro125 130
135Thr Val Thr Ile Leu Ser Gln Tyr Phe Gly Lys Arg Arg Ser Ile140
145 150Val Thr Ala Val Ala Ser Thr Gly Glu Cys
Phe Ala Val Phe Ala155 160 165Phe Ala Pro
Ala Ile Met Ala Leu Lys Glu Arg Ile Gly Trp Arg170 175
180Tyr Ser Leu Leu Phe Val Gly Leu Leu Gln Leu Asn Ile Val
Ile185 190 195Phe Gly Ala Leu Leu Arg Pro
Ile Ile Ile Arg Gly Pro Ala Ser200 205
210Pro Lys Ile Val Ile Gln Glu Asn Arg Lys Glu Ala Gln Tyr Met215
220 225Leu Glu Asn Glu Lys Thr Arg Thr Ser Ile
Asp Ser Ile Asp Ser230 235 240Gly Val Glu
Leu Thr Thr Ser Pro Lys Asn Val Pro Thr His Thr245 250
255Asn Leu Glu Leu Glu Pro Lys Ala Asp Met Gln Gln Val Leu
Val260 265 270Lys Thr Ser Pro Arg Pro Ser
Glu Lys Lys Ala Pro Leu Leu Asp275 280
285Phe Ser Ile Leu Lys Glu Lys Ser Phe Ile Cys Tyr Ala Leu Phe290
295 300Gly Leu Phe Ala Thr Leu Gly Phe Phe Ala
Pro Ser Leu Tyr Ile305 310 315Ile Pro Leu
Gly Ile Ser Leu Gly Ile Asp Gln Asp Arg Ala Ala320 325
330Phe Leu Leu Ser Thr Met Ala Ile Ala Glu Val Phe Gly Arg
Ile335 340 345Gly Ala Gly Phe Val Leu Asn
Arg Glu Pro Ile Arg Lys Ile Tyr350 355
360Ile Glu Leu Ile Cys Val Ile Leu Leu Thr Val Ser Leu Phe Ala365
370 375Phe Thr Phe Ala Thr Glu Phe Trp Gly Leu
Met Ser Cys Ser Ile380 385 390Phe Phe Gly
Phe Met Val Gly Thr Ile Gly Gly Leu Thr Phe His395 400
405Cys Leu Leu Lys Met Met Ser Trp Ala Leu Gln Lys Met Ser
Ser410 415 420Ala Ala Gly Val Tyr Ile Phe
Ile Gln Ser Ile Ala Gly Leu Ala425 430
435Gly Pro Pro Leu Ala Gly Leu Leu Val Asp Gln Ser Lys Ile Tyr440
445 450Ser Arg Ala Phe Tyr Ser Cys Ala Ala Gly
Met Ala Leu Ala Ala455 460 465Val Cys Leu
Ala Leu Val Arg Pro Cys Lys Met Gly Leu Cys Gln470 475
480Arg His His Ser Gly Glu Thr Lys Val Val Ser His Arg Gly
Lys485 490 495Thr Leu Gln Asp Ile Pro Glu
Asp Phe Leu Glu Met Asp Leu Ala500 505
510Lys Asn Glu His Arg Val His Val Gln Met Glu Pro Val515
52096124PRTHomo Sapien 96Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro
Val Ser Gly1 5 10 15Gln
Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro20
25 30Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu
Thr Cys Lys35 40 45Gly Phe Arg Phe Tyr
Ser Pro Gln Lys Thr Lys Trp Tyr His Arg50 55
60Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu65
70 75Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys
Gln Ala Gln Gly Ser80 85 90Pro Leu Ser
Ser Pro Val His Leu Asp Phe Ser Ser Glu Met Gly95 100
105Phe Pro His Ala Ala Gln Ala Asn Val Glu Leu Leu Gly Ser
Ser110 115 120Asp Leu Leu Thr97977PRTHomo
Sapien 97Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly1
5 10 15Gln Phe Ala Arg Thr
Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro20 25
30Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys Lys35
40 45Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr
Lys Trp Tyr His Arg50 55 60Tyr Leu Gly
Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu65 70
75Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln Gly
Ser80 85 90Pro Leu Ser Ser Pro Val His
Leu Asp Phe Ser Ser Ala Ser Leu95 100
105Ile Leu Gln Ala Pro Leu Ser Val Phe Glu Gly Asp Ser Val Val110
115 120Leu Arg Cys Arg Ala Lys Ala Glu Val Thr
Leu Asn Asn Thr Ile125 130 135Tyr Lys Asn
Asp Asn Val Leu Ala Phe Leu Asn Lys Arg Thr Asp140 145
150Phe His Ile Pro His Ala Cys Leu Lys Asp Asn Gly Ala Tyr
Arg155 160 165Cys Thr Gly Tyr Lys Glu Ser
Cys Cys Pro Val Ser Ser Asn Thr170 175
180Val Lys Ile Gln Val Gln Glu Pro Phe Thr Arg Pro Val Leu Arg185
190 195Ala Ser Ser Phe Gln Pro Ile Ser Gly Asn
Pro Val Thr Leu Thr200 205 210Cys Glu Thr
Gln Leu Ser Leu Glu Arg Ser Asp Val Pro Leu Arg215 220
225Phe Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu Gly Trp
Ser230 235 240Leu Ser Pro Asn Phe Gln Ile
Thr Ala Met Trp Ser Lys Asp Ser245 250
255Gly Phe Tyr Trp Cys Lys Ala Ala Thr Met Pro His Ser Val Ile260
265 270Ser Asp Ser Pro Arg Ser Trp Ile Gln Val
Gln Ile Pro Ala Ser275 280 285His Pro Val
Leu Thr Leu Ser Pro Glu Lys Ala Leu Asn Phe Glu290 295
300Gly Thr Lys Val Thr Leu His Cys Glu Thr Gln Glu Asp Ser
Leu305 310 315Arg Thr Leu Tyr Arg Phe Tyr
His Glu Gly Val Pro Leu Arg His320 325
330Lys Ser Val Arg Cys Glu Arg Gly Ala Ser Ile Ser Phe Ser Leu335
340 345Thr Thr Glu Asn Ser Gly Asn Tyr Tyr Cys
Thr Ala Asp Asn Gly350 355 360Leu Gly Ala
Lys Pro Ser Lys Ala Val Ser Leu Ser Val Thr Val365 370
375Pro Val Ser His Pro Val Leu Asn Leu Ser Ser Pro Glu Asp
Leu380 385 390Ile Phe Glu Gly Ala Lys Val
Thr Leu His Cys Glu Ala Gln Arg395 400
405Gly Ser Leu Pro Ile Leu Tyr Gln Phe His His Glu Asp Ala Ala410
415 420Leu Glu Arg Arg Ser Ala Asn Ser Ala Gly
Gly Val Ala Ile Ser425 430 435Phe Ser Leu
Thr Ala Glu His Ser Gly Asn Tyr Tyr Cys Thr Ala440 445
450Asp Asn Gly Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu
Ser455 460 465Ile Thr Val Pro Val Ser His
Pro Val Leu Thr Leu Ser Ser Ala470 475
480Glu Ala Leu Thr Phe Glu Gly Ala Thr Val Thr Leu His Cys Glu485
490 495Val Gln Arg Gly Ser Pro Gln Ile Leu Tyr
Gln Phe Tyr His Glu500 505 510Asp Met Pro
Leu Trp Ser Ser Ser Thr Pro Ser Val Gly Arg Val515 520
525Ser Phe Ser Phe Ser Leu Thr Glu Gly His Ser Gly Asn Tyr
Tyr530 535 540Cys Thr Ala Asp Asn Gly Phe
Gly Pro Gln Arg Ser Glu Val Val545 550
555Ser Leu Phe Val Thr Val Pro Val Ser Arg Pro Ile Leu Thr Leu560
565 570Arg Val Pro Arg Ala Gln Ala Val Val Gly
Asp Leu Leu Glu Leu575 580 585His Cys Glu
Ala Pro Arg Gly Ser Pro Pro Ile Leu Tyr Trp Phe590 595
600Tyr His Glu Asp Val Thr Leu Gly Ser Ser Ser Ala Pro Ser
Gly605 610 615Gly Glu Ala Ser Phe Asn Leu
Ser Leu Thr Ala Glu His Ser Gly620 625
630Asn Tyr Ser Cys Glu Ala Asn Asn Gly Leu Val Ala Gln His Ser635
640 645Asp Thr Ile Ser Leu Ser Val Ile Val Pro
Val Ser Arg Pro Ile650 655 660Leu Thr Phe
Arg Ala Pro Arg Ala Gln Ala Val Val Gly Asp Leu665 670
675Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Ser Pro Ile
Leu680 685 690Tyr Trp Phe Tyr His Glu Asp
Val Thr Leu Gly Lys Ile Ser Ala695 700
705Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr Glu710
715 720His Ser Gly Ile Tyr Ser Cys Glu Ala Asp
Asn Gly Pro Glu Ala725 730 735Gln Arg Ser
Glu Met Val Thr Leu Lys Val Ala Val Pro Val Ser740 745
750Arg Pro Val Leu Thr Leu Arg Ala Pro Gly Thr His Ala Ala
Val755 760 765Gly Asp Leu Leu Glu Leu His
Cys Glu Ala Leu Arg Gly Ser Pro770 775
780Leu Ile Leu Tyr Arg Phe Phe His Glu Asp Val Thr Leu Gly Asn785
790 795Arg Ser Ser Pro Ser Gly Gly Ala Ser Leu
Asn Leu Ser Leu Thr800 805 810Ala Glu His
Ser Gly Asn Tyr Ser Cys Glu Ala Asp Asn Gly Leu815 820
825Gly Ala Gln Arg Ser Glu Thr Val Thr Leu Tyr Ile Thr Gly
Leu830 835 840Thr Ala Asn Arg Ser Gly Pro
Phe Ala Thr Gly Val Ala Gly Gly845 850
855Leu Leu Ser Ile Ala Gly Leu Ala Ala Gly Ala Leu Leu Leu Tyr860
865 870Cys Trp Leu Ser Arg Lys Ala Gly Arg Lys
Pro Ala Ser Asp Pro875 880 885Ala Arg Ser
Pro Pro Asp Ser Asp Ser Gln Glu Pro Thr Tyr His890 895
900Asn Val Pro Ala Trp Glu Glu Leu Gln Pro Val Tyr Thr Asn
Ala905 910 915Asn Pro Arg Gly Glu Asn Val
Val Tyr Ser Glu Val Arg Ile Ile920 925
930Gln Glu Lys Lys Lys His Ala Val Ala Ser Asp Pro Arg His Leu935
940 945Arg Asn Lys Gly Ser Pro Ile Ile Tyr Ser
Glu Val Lys Val Ala950 955 960Ser Thr Pro
Val Ser Gly Ser Leu Phe Leu Ala Ser Ser Ala Pro965 970
975His Arg98146PRTHomo Sapien 98Met Leu Leu Trp Cys Pro Pro
Gln Cys Ala Cys Ser Leu Gly Val1 5 10
15Phe Pro Ser Ala Pro Ser Pro Val Trp Gly Thr Arg Arg Ser
Cys20 25 30Glu Pro Ala Thr Arg Val Pro
Glu Val Trp Ile Leu Ser Pro Leu35 40
45Leu Arg His Gly Gly His Thr Gln Thr Gln Asn His Thr Ala Ser50
55 60Pro Arg Ser Pro Val Met Glu Ser Pro Lys Lys
Lys Asn Gln Gln65 70 75Leu Lys Val Gly
Ile Leu His Leu Gly Ser Arg Gln Lys Lys Ile80 85
90Arg Ile Gln Leu Arg Ser Gln Cys Ala Thr Trp Lys Val Ile Cys95
100 105Lys Ser Cys Ile Ser Gln Thr Pro Gly
Ile Asn Leu Asp Leu Gly110 115 120Ser Gly
Val Lys Val Lys Ile Ile Pro Lys Glu Glu His Cys Lys125
130 135Met Pro Glu Ala Gly Glu Glu Gln Pro Gln Val140
14599235PRTHomo Sapien 99Met Arg Glu Leu Ala Ile Glu Ile Gly
Val Arg Ala Leu Leu Phe1 5 10
15Gly Val Phe Val Phe Thr Glu Phe Leu Asp Pro Phe Gln Arg Val20
25 30Ile Gln Pro Glu Glu Ile Trp Leu Tyr Lys
Asn Pro Leu Val Gln35 40 45Ser Asp Asn
Ile Pro Thr Arg Leu Met Phe Ala Ile Ser Phe Leu50 55
60Thr Pro Leu Ala Val Ile Cys Val Val Lys Ile Ile Arg Arg
Thr65 70 75Asp Lys Thr Glu Ile Lys Glu
Ala Phe Leu Ala Val Ser Leu Ala80 85
90Leu Ala Leu Asn Gly Val Cys Thr Asn Thr Ile Lys Leu Ile Val95
100 105Gly Arg Pro Arg Ala Asp Phe Phe Tyr Arg Cys
Phe Pro Asp Gly110 115 120Val Met Asn Ser
Glu Met His Cys Thr Gly Asp Pro Asp Leu Val125 130
135Ser Glu Gly Arg Lys Ser Phe Pro Ser Ile His Ser Ser Phe
Ala140 145 150Phe Ser Gly Leu Gly Phe Thr
Thr Phe Tyr Leu Ala Gly Lys Leu155 160
165His Cys Phe Thr Glu Ser Gly Arg Gly Lys Ser Trp Arg Leu Cys170
175 180Ala Ala Ile Leu Pro Leu Tyr Cys Ala Met
Met Ile Ala Leu Ser185 190 195Arg Met Cys
Asp Tyr Lys His His Trp Gln Asp Ser Phe Val Gly200 205
210Gly Val Ile Ala Leu Ile Phe Ala Tyr Ile Cys Tyr Arg Gln
His215 220 225Tyr Pro Pro Leu Gly Gln His
Ser Leu Pro230 235100252PRTHomo Sapien 100Met Ala Glu Leu
Glu Phe Val Gln Ile Ile Ile Ile Val Val Val1 5
10 15Met Met Val Met Val Val Val Ile Thr Cys Leu Leu
Ser His Tyr20 25 30Lys Leu Ser Ala Arg
Ser Phe Ile Ser Arg His Ser Gln Gly Arg35 40
45Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser50
55 60Glu Ser Thr Val Ser Gly Asn Gly Ile Pro
Glu Pro Gln Val Tyr65 70 75Ala Pro Pro
Arg Pro Thr Asp Arg Leu Ala Val Pro Pro Phe Ala80 85
90Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr
Leu95 100 105Gln His Glu Ile Asp Leu Pro
Pro Thr Ile Ser Leu Ser Asp Gly110 115
120Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg125
130 135Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg
Glu Ser Val Arg Ala140 145 150Pro Pro Asn
Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala155 160
165Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile
Ser170 175 180Ala Thr Cys Tyr Gly Ser Gly
Gly Arg Met Glu Gly Pro Pro Pro185 190
195Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln200
205 210His Gln Gln Ser Ser Gly Pro Pro Ser Leu
Leu Glu Gly Thr Arg215 220 225Leu His His
Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp230 235
240Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu245
250101252PRTHomo Sapien 101Met Ala Glu Leu Glu Phe Val Gln Ile
Ile Ile Ile Val Val Val1 5 10
15Met Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr20
25 30Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg
His Ser Gln Gly Arg35 40 45Arg Arg Glu
Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser50 55
60Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu Pro Gln Val
Tyr65 70 75Ala Pro Pro Arg Pro Thr Asp
Arg Leu Ala Val Pro Pro Phe Ala80 85
90Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr Leu95
100 105Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser
Leu Ser Asp Gly110 115 120Glu Glu Pro Pro
Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg125 130
135Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg
Ala140 145 150Pro Pro Asn Arg Thr Ile Phe
Asp Ser Asp Leu Met Asp Ser Ala155 160
165Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser170
175 180Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met
Glu Gly Pro Pro Pro185 190 195Thr Tyr Ser
Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln200 205
210His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr
Arg215 220 225Leu His His Thr His Ile Ala
Pro Leu Glu Ser Ala Ala Ile Trp230 235
240Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu245
250102465PRTHomo Sapien 102Met Gly Gly Ala Val Val Asp Glu Gly Pro Thr
Gly Val Lys Ala1 5 10
15Pro Asp Gly Gly Trp Gly Trp Ala Val Leu Phe Gly Cys Phe Val20
25 30Ile Thr Gly Phe Ser Tyr Ala Phe Pro Lys Ala
Val Ser Val Phe35 40 45Phe Lys Glu Leu
Ile Gln Glu Phe Gly Ile Gly Tyr Ser Asp Thr50 55
60Ala Trp Ile Ser Ser Ile Leu Leu Ala Met Leu Tyr Gly Thr Gly65
70 75Pro Leu Cys Ser Val Cys Val Asn Arg
Phe Gly Cys Arg Pro Val80 85 90Met Leu
Val Gly Gly Leu Phe Ala Ser Leu Gly Met Val Ala Ala95 100
105Ser Phe Cys Arg Ser Ile Ile Gln Val Tyr Leu Thr Thr
Gly Val110 115 120Ile Thr Gly Leu Gly Leu
Ala Leu Asn Phe Gln Pro Ser Leu Ile125 130
135Met Leu Asn Arg Tyr Phe Ser Lys Arg Arg Pro Met Ala Asn Gly140
145 150Leu Ala Ala Ala Gly Ser Pro Val Phe Leu
Cys Ala Leu Ser Pro155 160 165Leu Gly Gln
Leu Leu Gln Asp Arg Tyr Gly Trp Arg Gly Gly Phe170 175
180Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Cys Ala
Ala185 190 195Leu Met Arg Pro Leu Val Val
Thr Ala Gln Pro Gly Ser Gly Pro200 205
210Pro Arg Pro Ser Arg Arg Leu Leu Asp Leu Ser Val Phe Arg Asp215
220 225Arg Gly Phe Val Leu Tyr Ala Val Ala Ala
Ser Val Met Val Leu230 235 240Gly Leu Phe
Val Pro Pro Val Phe Val Val Ser Tyr Ala Lys Asp245 250
255Leu Gly Val Pro Asp Thr Lys Ala Ala Phe Leu Leu Thr Ile
Leu260 265 270Gly Phe Ile Asp Ile Phe Ala
Arg Pro Ala Ala Gly Phe Val Ala275 280
285Gly Leu Gly Lys Val Arg Pro Tyr Ser Val Tyr Leu Phe Ser Phe290
295 300Ser Met Phe Phe Asn Gly Leu Ala Asp Leu
Ala Gly Ser Thr Ala305 310 315Gly Asp Tyr
Gly Gly Leu Val Val Phe Cys Ile Phe Phe Gly Ile320 325
330Ser Tyr Gly Met Val Gly Ala Leu Gln Phe Glu Val Leu Met
Ala335 340 345Ile Val Gly Thr His Lys Phe
Ser Ser Ala Ile Gly Leu Val Leu350 355
360Leu Met Glu Ala Val Ala Val Leu Val Gly Pro Pro Ser Gly Gly365
370 375Lys Leu Leu Asp Ala Thr His Val Tyr Met
Tyr Val Phe Ile Leu380 385 390Ala Gly Ala
Glu Val Leu Thr Ser Ser Leu Ile Leu Leu Leu Gly395 400
405Asn Phe Phe Cys Ile Arg Lys Lys Pro Lys Glu Pro Gln Pro
Glu410 415 420Val Ala Ala Ala Glu Glu Glu
Lys Leu His Lys Pro Pro Ala Asp425 430
435Ser Gly Val Asp Leu Arg Glu Val Glu His Phe Leu Lys Ala Glu440
445 450Pro Glu Lys Asn Gly Glu Val Val His Thr
Pro Glu Thr Ser Val455 460
465103445PRTHomo Sapien 103Met Ala Ala Pro Thr Pro Ala Arg Pro Val Leu
Thr His Leu Leu1 5 10
15Val Ala Leu Phe Gly Met Gly Ser Trp Ala Ala Val Asn Gly Ile20
25 30Trp Val Glu Leu Pro Val Val Val Lys Glu Leu
Pro Glu Gly Trp35 40 45Ser Leu Pro Ser
Tyr Val Ser Val Leu Val Ala Leu Gly Asn Leu50 55
60Gly Leu Leu Val Val Thr Leu Trp Arg Arg Leu Ala Pro Gly Lys65
70 75Asp Glu Gln Val Pro Ile Arg Val Val
Gln Val Leu Gly Met Val80 85 90Gly Thr
Ala Leu Leu Ala Ser Leu Trp His His Val Ala Pro Val95 100
105Ala Gly Gln Leu His Ser Val Ala Phe Leu Ala Leu Ala
Phe Val110 115 120Leu Ala Leu Ala Cys Cys
Ala Ser Asn Val Thr Phe Leu Pro Phe125 130
135Leu Ser His Leu Pro Pro Arg Phe Leu Arg Ser Phe Phe Leu Gly140
145 150Gln Gly Leu Ser Ala Leu Leu Pro Cys Val
Leu Ala Leu Val Gln155 160 165Gly Val Gly
Arg Leu Glu Cys Pro Pro Ala Pro Ile Asn Gly Thr170 175
180Pro Gly Pro Pro Leu Asp Phe Leu Glu Arg Phe Pro Ala Ser
Thr185 190 195Phe Phe Trp Ala Leu Thr Ala
Leu Leu Val Ala Ser Ala Ala Ala200 205
210Phe Gln Gly Leu Leu Leu Leu Leu Pro Pro Pro Pro Ser Val Pro215
220 225Thr Gly Glu Leu Gly Ser Gly Leu Gln Val
Gly Ala Pro Gly Ala230 235 240Glu Glu Glu
Val Glu Glu Ser Ser Pro Leu Gln Glu Pro Pro Ser245 250
255Gln Ala Ala Gly Thr Thr Pro Gly Pro Asp Pro Lys Ala Tyr
Gln260 265 270Leu Leu Ser Ala Arg Ser Ala
Cys Leu Leu Gly Leu Leu Ala Ala275 280
285Thr Asn Ala Leu Thr Asn Gly Val Leu Pro Ala Val Gln Ser Phe290
295 300Ser Cys Leu Pro Tyr Gly Arg Leu Ala Tyr
His Leu Ala Val Val305 310 315Leu Gly Ser
Ala Ala Asn Pro Leu Ala Cys Phe Leu Ala Met Gly320 325
330Val Leu Cys Arg Ser Leu Ala Gly Leu Gly Gly Leu Ser Leu
Leu335 340 345Gly Val Phe Cys Gly Gly Tyr
Leu Met Ala Leu Ala Val Leu Ser350 355
360Pro Cys Pro Pro Leu Val Gly Thr Ser Ala Gly Val Val Leu Val365
370 375Val Leu Ser Trp Val Leu Cys Leu Gly Val
Phe Ser Tyr Val Lys380 385 390Val Ala Ala
Ser Ser Leu Leu His Gly Gly Gly Arg Pro Ala Leu395 400
405Leu Ala Ala Gly Val Ala Ile Gln Val Gly Ser Leu Leu Gly
Ala410 415 420Val Ala Met Phe Pro Pro Thr
Ser Ile Tyr His Val Phe His Ser425 430
435Arg Lys Asp Cys Ala Asp Pro Cys Asp Ser440
445104398PRTHomo Sapien 104Met His Thr Val Ala Thr Ser Gly Pro Asn Ala
Ser Trp Gly Ala1 5 10
15Pro Ala Asn Ala Ser Gly Cys Pro Gly Cys Gly Ala Asn Ala Ser20
25 30Asp Gly Pro Val Pro Ser Pro Arg Ala Val Asp
Ala Trp Leu Val35 40 45Pro Leu Phe Phe
Ala Ala Leu Met Leu Leu Gly Leu Val Gly Asn50 55
60Ser Leu Val Ile Tyr Val Ile Cys Arg His Lys Pro Met Arg Thr65
70 75Val Thr Asn Phe Tyr Ile Ala Asn Leu
Ala Ala Thr Asp Val Thr80 85 90Phe Leu
Leu Cys Cys Val Pro Phe Thr Ala Leu Leu Tyr Pro Leu95 100
105Pro Gly Trp Val Leu Gly Asp Phe Met Cys Lys Phe Val
Asn Tyr110 115 120Ile Gln Gln Val Ser Val
Gln Ala Thr Cys Ala Thr Leu Thr Ala125 130
135Met Ser Val Asp Arg Trp Tyr Val Thr Val Phe Pro Leu Arg Ala140
145 150Leu His Arg Arg Thr Pro Arg Leu Ala Leu
Ala Val Ser Leu Ser155 160 165Ile Trp Val
Gly Ser Ala Ala Val Ser Ala Pro Val Leu Ala Leu170 175
180His Arg Leu Ser Pro Gly Pro Arg Ala Tyr Cys Ser Glu Ala
Phe185 190 195Pro Ser Arg Ala Leu Glu Arg
Ala Phe Ala Leu Tyr Asn Leu Leu200 205
210Ala Leu Tyr Leu Leu Pro Leu Leu Ala Thr Cys Ala Cys Tyr Ala215
220 225Ala Met Leu Arg His Leu Gly Arg Val Ala
Val Arg Pro Ala Pro230 235 240Ala Asp Ser
Ala Leu Gln Gly Gln Val Leu Ala Glu Arg Ala Gly245 250
255Ala Val Arg Ala Lys Val Ser Arg Leu Val Ala Ala Val Val
Leu260 265 270Leu Phe Ala Ala Cys Trp Gly
Pro Ile Gln Leu Phe Leu Val Leu275 280
285Gln Ala Leu Gly Pro Ala Gly Ser Trp His Pro Arg Ser Tyr Ala290
295 300Ala Tyr Ala Leu Lys Thr Trp Ala His Cys
Met Ser Tyr Ser Asn305 310 315Ser Ala Leu
Asn Pro Leu Leu Tyr Ala Phe Leu Gly Ser His Phe320 325
330Arg Gln Ala Phe Arg Arg Val Cys Pro Cys Ala Pro Arg Arg
Pro335 340 345Arg Arg Pro Arg Arg Pro Gly
Pro Ser Asp Pro Ala Ala Pro His350 355
360Ala Glu Leu His Arg Leu Gly Ser His Pro Ala Pro Ala Arg Ala365
370 375Gln Lys Pro Gly Ser Ser Gly Leu Ala Ala
Arg Gly Leu Cys Val380 385 390Leu Gly Glu
Asp Asn Ala Pro Leu395105359PRTHomo Sapien 105Met Ser Met Asn Asn Ser Lys
Gln Leu Val Ser Pro Ala Ala Ala1 5 10
15Leu Leu Ser Asn Thr Thr Cys Gln Thr Glu Asn Arg Leu Ser
Val20 25 30Phe Phe Ser Val Ile Phe Met
Thr Val Gly Ile Leu Ser Asn Ser35 40
45Leu Ala Ile Ala Ile Leu Met Lys Ala Tyr Gln Arg Phe Arg Gln50
55 60Lys Ser Lys Ala Ser Phe Leu Leu Leu Ala Ser
Gly Leu Val Ile65 70 75Thr Asp Phe Phe
Gly His Leu Ile Asn Gly Ala Ile Ala Val Phe80 85
90Val Tyr Ala Ser Asp Lys Glu Trp Ile Arg Phe Asp Gln Ser Asn95
100 105Val Leu Cys Ser Ile Phe Gly Ile Cys
Met Val Phe Ser Gly Leu110 115 120Cys Pro
Leu Leu Leu Gly Ser Val Met Ala Ile Glu Arg Cys Ile125
130 135Gly Val Thr Lys Pro Ile Phe His Ser Thr Lys Ile
Thr Ser Lys140 145 150His Val Lys Met Met
Leu Ser Gly Val Cys Leu Phe Ala Val Phe155 160
165Ile Ala Leu Leu Pro Ile Leu Gly His Arg Asp Tyr Lys Ile Gln170
175 180Ala Ser Arg Thr Trp Cys Phe Tyr Asn
Thr Glu Asp Ile Lys Asp185 190 195Trp Glu
Asp Arg Phe Tyr Leu Leu Leu Phe Ser Phe Leu Gly Leu200
205 210Leu Ala Leu Gly Val Ser Leu Leu Cys Asn Ala Ile
Thr Gly Ile215 220 225Thr Leu Leu Arg Val
Lys Phe Lys Ser Gln Gln His Arg Gln Gly230 235
240Arg Ser His His Leu Glu Met Val Ile Gln Leu Leu Ala Ile Met245
250 255Cys Val Ser Cys Ile Cys Trp Ser Pro
Phe Leu Val Thr Met Ala260 265 270Asn Ile
Gly Ile Asn Gly Asn His Ser Leu Glu Thr Cys Glu Thr275
280 285Thr Leu Phe Ala Leu Arg Met Ala Thr Trp Asn Gln
Ile Leu Asp290 295 300Pro Trp Val Tyr Ile
Leu Leu Arg Lys Ala Val Leu Lys Asn Leu305 310
315Tyr Lys Leu Ala Ser Gln Cys Cys Gly Val His Val Ile Ser Leu320
325 330His Ile Trp Glu Leu Ser Ser Ile Lys
Asn Ser Leu Lys Val Ala335 340 345Ala Ile
Ser Glu Ser Pro Val Ala Glu Lys Ser Ala Ser Thr350
355106819PRTHomo Sapien 106Met Ser Arg Met Ser Arg His Pro Asp Lys Asp
Leu Ala Gln Gly1 5 10
15Pro Phe Asn Thr Cys Cys Gly Cys Thr Leu Met Ala Ser Pro Ala20
25 30Asn Leu Pro Pro Asn Thr Gln Ala Ala Ala Glu
Arg Ala Leu Ser35 40 45Gln Ser Arg Trp
Lys Arg Val Gln Val Pro Ala Pro Ala Ser Leu50 55
60Ser Pro Phe Pro Leu Ala Met Ala Ser Val Ala Phe Trp Ile Ser65
70 75Ile Leu Ile Gly Cys Glu Glu Gln Thr
Leu Cys Arg Gly Trp Arg80 85 90Ser Pro
Val Gly Asp Gly Cys Ala His Val Pro Pro Gln Glu Arg95 100
105Ala Thr Ala Glu Ala Asp Pro Pro Gly Arg Cys Ser Thr
Ser Thr110 115 120Ala Ser Ser Thr Ile Cys
Gly Leu Trp His Leu Ser Pro Arg Leu125 130
135Gln Leu Leu Pro Pro Leu His Ser Arg Gln Gly Glu Glu Ser Gly140
145 150Lys Thr Glu Lys Val Leu Leu Trp Gly Arg
Glu Gly Leu His Val155 160 165Trp Lys Pro
Gly Val Leu Gln Pro Asp Val His Gly Thr Ser Asn170 175
180Leu Gly Asn Cys Ser Phe Leu His Gly Leu Val Thr Ala Pro
Ser185 190 195Cys Pro Arg Arg Ala Gly Ala
Glu Leu Leu Asn Ser Leu Gly Ser200 205
210Gln Phe Ala Ile Ser Leu Phe Glu Val Gln Ser Gly Thr Glu Pro215
220 225Ser Ile Thr Gly Val Ala Thr Ser Gly Gln
Cys Arg Ala Met Pro230 235 240Leu Lys His
Tyr Leu Leu Leu Leu Val Gly Cys Gln Ala Trp Gly245 250
255Ala Gly Leu Ala Tyr His Gly Cys Pro Ser Glu Cys Thr Cys
Ser260 265 270Arg Ala Ser Gln Val Glu Cys
Thr Gly Ala Arg Ile Val Ala Val275 280
285Pro Thr Pro Leu Pro Trp Asn Ala Met Ser Leu Gln Ile Leu Asn290
295 300Thr His Ile Thr Glu Leu Asn Glu Ser Pro
Phe Leu Asn Ile Ser305 310 315Ala Leu Ile
Ala Leu Arg Ile Glu Lys Asn Glu Leu Ser Arg Ile320 325
330Thr Pro Gly Ala Phe Arg Asn Leu Gly Ser Leu Arg Tyr Leu
Ser335 340 345Leu Ala Asn Asn Lys Leu Gln
Val Leu Pro Ile Gly Leu Phe Gln350 355
360Gly Leu Asp Ser Leu Glu Ser Leu Leu Leu Ser Ser Asn Gln Leu365
370 375Leu Gln Ile Gln Pro Ala His Phe Ser Gln
Cys Ser Asn Leu Lys380 385 390Glu Leu Gln
Leu His Gly Asn His Leu Glu Tyr Ile Pro Asp Gly395 400
405Ala Phe Asp His Leu Val Gly Leu Thr Lys Leu Asn Leu Gly
Lys410 415 420Asn Ser Leu Thr His Ile Ser
Pro Arg Val Phe Gln His Leu Gly425 430
435Asn Leu Gln Val Leu Arg Leu Tyr Glu Asn Arg Leu Thr Asp Ile440
445 450Pro Met Gly Thr Phe Asp Gly Leu Val Asn
Leu Gln Glu Leu Ala455 460 465Leu Gln Gln
Asn Gln Ile Gly Leu Leu Ser Pro Gly Leu Phe His470 475
480Asn Asn His Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His
Ile485 490 495Ser Gln Leu Pro Pro Ser Ile
Phe Met Gln Leu Pro Gln Leu Asn500 505
510Arg Leu Thr Leu Phe Gly Asn Ser Leu Lys Glu Leu Ser Leu Gly515
520 525Ile Phe Gly Pro Met Pro Asn Leu Arg Glu
Leu Trp Leu Tyr Asp530 535 540Asn His Ile
Ser Ser Leu Pro Asp Asn Val Phe Ser Asn Leu Arg545 550
555Gln Leu Gln Val Leu Ile Leu Ser Arg Asn Gln Ile Ser Phe
Ile560 565 570Ser Pro Gly Ala Phe Asn Gly
Leu Thr Glu Leu Arg Glu Leu Ser575 580
585Leu His Thr Asn Ala Leu Gln Asp Leu Asp Gly Asn Val Phe Arg590
595 600Met Leu Ala Asn Leu Gln Asn Ile Ser Leu
Gln Asn Asn Arg Leu605 610 615Arg Gln Leu
Pro Gly Asn Ile Phe Ala Asn Val Asn Gly Leu Met620 625
630Ala Ile Gln Leu Gln Asn Asn Gln Leu Glu Asn Leu Pro Leu
Gly635 640 645Ile Phe Asp His Leu Gly Lys
Leu Cys Glu Leu Arg Leu Tyr Asp650 655
660Asn Pro Trp Arg Cys Asp Ser Asp Ile Leu Pro Leu Arg Asn Trp665
670 675Leu Leu Leu Asn Gln Pro Arg Leu Gly Thr
Asp Thr Val Pro Val680 685 690Cys Phe Ser
Pro Ala Asn Val Arg Gly Gln Ser Leu Ile Ile Ile695 700
705Asn Val Asn Val Ala Val Pro Ser Val His Val Pro Glu Val
Pro710 715 720Ser Tyr Pro Glu Thr Pro Trp
Tyr Pro Asp Thr Pro Ser Tyr Pro725 730
735Asp Thr Thr Ser Val Ser Ser Thr Thr Glu Leu Thr Ser Pro Val740
745 750Glu Asp Tyr Thr Asp Leu Thr Thr Ile Gln
Val Thr Asp Asp Arg755 760 765Ser Val Trp
Gly Met Thr His Ala His Ser Gly Leu Ala Ile Ala770 775
780Ala Ile Val Ile Gly Ile Val Ala Leu Ala Cys Ser Leu Ala
Ala785 790 795Cys Val Gly Cys Cys Cys Cys
Lys Lys Arg Ser Gln Ala Val Leu800 805
810Met Gln Met Lys Ala Pro Asn Glu Cys8151073014PRTHomo Sapien 107Met Ala
Pro Pro Pro Pro Pro Val Leu Pro Val Leu Leu Leu Leu1 5
10 15Ala Ala Ala Ala Ala Leu Pro Ala Met Gly
Leu Arg Ala Ala Ala20 25 30Trp Glu Pro
Arg Val Pro Gly Gly Thr Arg Ala Phe Ala Leu Arg35 40
45Pro Gly Cys Thr Tyr Ala Val Gly Ala Ala Cys Thr Pro Arg
Ala50 55 60Pro Arg Glu Leu Leu Asp Val
Gly Arg Asp Gly Arg Leu Ala Gly65 70
75Arg Arg Arg Val Ser Gly Ala Gly Arg Pro Leu Pro Leu Gln Val80
85 90Arg Leu Val Ala Arg Ser Ala Pro Thr Ala Leu
Ser Arg Arg Leu95 100 105Arg Ala Arg Thr
His Leu Pro Gly Cys Gly Ala Arg Ala Arg Leu110 115
120Cys Gly Thr Gly Ala Arg Leu Cys Gly Ala Leu Cys Phe Pro
Val125 130 135Pro Gly Gly Cys Ala Ala Ala
Gln His Ser Ala Leu Ala Ala Pro140 145
150Thr Thr Leu Pro Ala Cys Arg Cys Pro Pro Arg Pro Arg Pro Arg155
160 165Cys Pro Gly Arg Pro Ile Cys Leu Pro Pro
Gly Gly Ser Val Arg170 175 180Leu Arg Leu
Leu Cys Ala Leu Arg Arg Ala Ala Gly Ala Val Arg185 190
195Val Gly Leu Ala Leu Glu Ala Ala Thr Ala Gly Thr Pro Ser
Ala200 205 210Ser Pro Ser Pro Ser Pro Pro
Leu Pro Pro Asn Leu Pro Glu Ala215 220
225Arg Ala Gly Pro Ala Arg Arg Ala Arg Arg Gly Thr Ser Gly Arg230
235 240Gly Ser Leu Lys Phe Pro Met Pro Asn Tyr
Gln Val Ala Leu Phe245 250 255Glu Asn Glu
Pro Ala Gly Thr Leu Ile Leu Gln Leu His Ala His260 265
270Tyr Thr Ile Glu Gly Glu Glu Glu Arg Val Ser Tyr Tyr Met
Glu275 280 285Gly Leu Phe Asp Glu Arg Ser
Arg Gly Tyr Phe Arg Ile Asp Ser290 295
300Ala Thr Gly Ala Val Ser Thr Asp Ser Val Leu Asp Arg Glu Thr305
310 315Lys Glu Thr His Val Leu Arg Val Lys Ala
Val Asp Tyr Ser Thr320 325 330Pro Pro Arg
Ser Ala Thr Thr Tyr Ile Thr Val Leu Val Lys Asp335 340
345Thr Asn Asp His Ser Pro Val Phe Glu Gln Ser Glu Tyr Arg
Glu350 355 360Arg Val Arg Glu Asn Leu Glu
Val Gly Tyr Glu Val Leu Thr Ile365 370
375Arg Ala Ser Asp Arg Asp Ser Pro Ile Asn Ala Asn Leu Arg Tyr380
385 390Arg Val Leu Gly Gly Ala Trp Asp Val Phe
Gln Leu Asn Glu Ser395 400 405Ser Gly Val
Val Ser Thr Arg Ala Val Leu Asp Arg Glu Glu Ala410 415
420Ala Glu Tyr Gln Leu Leu Val Glu Ala Asn Asp Gln Gly Arg
Asn425 430 435Pro Gly Pro Leu Ser Ala Thr
Ala Thr Val Tyr Ile Glu Val Glu440 445
450Asp Glu Asn Asp Asn Tyr Pro Gln Phe Ser Glu Gln Asn Tyr Val455
460 465Val Gln Val Pro Glu Asp Val Gly Leu Asn
Thr Ala Val Leu Arg470 475 480Val Gln Ala
Thr Asp Arg Asp Gln Gly Gln Asn Ala Ala Ile His485 490
495Tyr Ser Ile Leu Ser Gly Asn Val Ala Gly Gln Phe Tyr Leu
His500 505 510Ser Leu Ser Gly Ile Leu Asp
Val Ile Asn Pro Leu Asp Phe Glu515 520
525Asp Val Gln Lys Tyr Ser Leu Ser Ile Lys Ala Gln Asp Gly Gly530
535 540Arg Pro Pro Leu Ile Asn Ser Ser Gly Val
Val Ser Val Gln Val545 550 555Leu Asp Val
Asn Asp Asn Glu Pro Ile Phe Val Ser Ser Pro Phe560 565
570Gln Ala Thr Val Leu Glu Asn Val Pro Leu Gly Tyr Pro Val
Val575 580 585His Ile Gln Ala Val Asp Ala
Asp Ser Gly Glu Asn Ala Arg Leu590 595
600His Tyr Arg Leu Val Asp Thr Ala Ser Thr Phe Leu Gly Gly Gly605
610 615Ser Ala Gly Pro Lys Asn Pro Ala Pro Thr
Pro Asp Phe Pro Phe620 625 630Gln Ile His
Asn Ser Ser Gly Trp Ile Thr Val Cys Ala Glu Leu635 640
645Asp Arg Glu Glu Val Glu His Tyr Ser Phe Gly Val Glu Ala
Val650 655 660Asp His Gly Ser Pro Pro Met
Ser Ser Ser Thr Ser Val Ser Ile665 670
675Thr Val Leu Asp Val Asn Asp Asn Asp Pro Val Phe Thr Gln Pro680
685 690Thr Tyr Glu Leu Arg Leu Asn Glu Asp Ala
Ala Val Gly Ser Ser695 700 705Val Leu Thr
Leu Gln Ala Arg Asp Arg Asp Ala Asn Ser Val Ile710 715
720Thr Tyr Gln Leu Thr Gly Gly Asn Thr Arg Asn Arg Phe Ala
Leu725 730 735Ser Ser Gln Arg Gly Gly Gly
Leu Ile Thr Leu Ala Leu Pro Leu740 745
750Asp Tyr Lys Gln Glu Gln Gln Tyr Val Leu Ala Val Thr Ala Ser755
760 765Asp Gly Thr Arg Ser His Thr Ala His Val
Leu Ile Asn Val Thr770 775 780Asp Ala Asn
Thr His Arg Pro Val Phe Gln Ser Ser His Tyr Thr785 790
795Val Ser Val Ser Glu Asp Arg Pro Val Gly Thr Ser Ile Ala
Thr800 805 810Leu Ser Ala Asn Asp Glu Asp
Thr Gly Glu Asn Ala Arg Ile Thr815 820
825Tyr Val Ile Gln Asp Pro Val Pro Gln Phe Arg Ile Asp Pro Asp830
835 840Ser Gly Thr Met Tyr Thr Met Met Glu Leu
Asp Tyr Glu Asn Gln845 850 855Val Ala Tyr
Thr Leu Thr Ile Met Ala Gln Asp Asn Gly Ile Pro860 865
870Gln Lys Ser Asp Thr Thr Thr Leu Glu Ile Leu Ile Leu Asp
Ala875 880 885Asn Asp Asn Ala Pro Gln Phe
Leu Trp Asp Phe Tyr Gln Gly Ser890 895
900Ile Phe Glu Asp Ala Pro Pro Ser Thr Ser Ile Leu Gln Val Ser905
910 915Ala Thr Asp Arg Asp Ser Gly Pro Asn Gly
Arg Leu Leu Tyr Thr920 925 930Phe Gln Gly
Gly Asp Asp Gly Asp Gly Asp Phe Tyr Ile Glu Pro935 940
945Thr Ser Gly Val Ile Arg Thr Gln Arg Arg Leu Asp Arg Glu
Asn950 955 960Val Ala Val Tyr Asn Leu Trp
Ala Leu Ala Val Asp Arg Gly Ser965 970
975Pro Thr Pro Leu Ser Ala Ser Val Glu Ile Gln Val Thr Ile Leu980
985 990Asp Ile Asn Asp Asn Ala Pro Met Phe Glu
Lys Asp Glu Leu Glu995 1000 1005Leu Phe Val
Glu Glu Asn Asn Pro Val Gly Ser Val Val Ala Lys1010 1015
1020Ile Arg Ala Asn Asp Pro Asp Glu Gly Pro Asn Ala Gln Ile
Met1025 1030 1035Tyr Gln Ile Val Glu Gly
Asp Met Arg His Phe Phe Gln Leu Asp1040 1045
1050Leu Leu Asn Gly Asp Leu Arg Ala Met Val Glu Leu Asp Phe Glu1055
1060 1065Val Arg Arg Glu Tyr Val Leu Val Val Gln
Ala Thr Ser Ala Pro1070 1075 1080Leu Val
Ser Arg Ala Thr Val His Ile Leu Leu Val Asp Gln Asn1085
1090 1095Asp Asn Pro Pro Val Leu Pro Asp Phe Gln Ile Leu
Phe Asn Asn1100 1105 1110Tyr Val Thr Asn
Lys Ser Asn Ser Phe Pro Thr Gly Val Ile Gly1115 1120
1125Cys Ile Pro Ala His Asp Pro Asp Val Ser Asp Ser Leu Asn
Tyr1130 1135 1140Thr Phe Val Gln Gly Asn
Glu Leu Arg Leu Leu Leu Leu Asp Pro1145 1150
1155Ala Thr Gly Glu Leu Gln Leu Ser Arg Asp Leu Asp Asn Asn Arg1160
1165 1170Pro Leu Glu Ala Leu Met Glu Val Ser Val
Ser Asp Gly Ile His1175 1180 1185Ser Val
Thr Ala Phe Cys Thr Leu Arg Val Thr Ile Ile Thr Asp1190
1195 1200Asp Met Leu Thr Asn Ser Ile Thr Val Arg Leu Glu
Asn Met Ser1205 1210 1215Gln Glu Lys Phe
Leu Ser Pro Leu Leu Ala Leu Phe Val Glu Gly1220 1225
1230Val Ala Ala Val Leu Ser Thr Thr Lys Asp Asp Val Phe Val
Phe1235 1240 1245Asn Val Gln Asn Asp Thr
Asp Val Ser Ser Asn Ile Leu Asn Val1250 1255
1260Thr Phe Ser Ala Leu Leu Pro Gly Gly Val Arg Gly Gln Phe Phe1265
1270 1275Pro Ser Glu Asp Leu Gln Glu Gln Ile Tyr
Leu Asn Arg Thr Leu1280 1285 1290Leu Thr
Thr Ile Ser Thr Gln Arg Val Leu Pro Phe Asp Asp Asn1295
1300 1305Ile Cys Leu Arg Glu Pro Cys Glu Asn Tyr Met Lys
Cys Val Ser1310 1315 1320Val Leu Arg Phe
Asp Ser Ser Ala Pro Phe Leu Ser Ser Thr Thr1325 1330
1335Val Leu Phe Arg Pro Ile His Pro Ile Asn Gly Leu Arg Cys
Arg1340 1345 1350Cys Pro Pro Gly Phe Thr
Gly Asp Tyr Cys Glu Thr Glu Ile Asp1355 1360
1365Leu Cys Tyr Ser Asp Pro Cys Gly Ala Asn Gly Arg Cys Arg Ser1370
1375 1380Arg Glu Gly Gly Tyr Thr Cys Glu Cys Phe
Glu Asp Phe Thr Gly1385 1390 1395Glu His
Cys Glu Val Asp Ala Arg Ser Gly Arg Cys Ala Asn Gly1400
1405 1410Val Cys Lys Asn Gly Gly Thr Cys Val Asn Leu Leu
Ile Gly Gly1415 1420 1425Phe His Cys Val
Cys Pro Pro Gly Glu Tyr Glu Arg Pro Tyr Cys1430 1435
1440Glu Val Thr Thr Arg Ser Phe Pro Pro Gln Ser Phe Val Thr
Phe1445 1450 1455Arg Gly Leu Arg Gln Arg
Phe His Phe Thr Ile Ser Leu Thr Phe1460 1465
1470Ala Thr Gln Glu Arg Asn Gly Leu Leu Leu Tyr Asn Gly Arg Phe1475
1480 1485Asn Glu Lys His Asp Phe Ile Ala Leu Glu
Ile Val Asp Glu Gln1490 1495 1500Val Gln
Leu Thr Phe Ser Ala Gly Glu Thr Thr Thr Thr Val Ala1505
1510 1515Pro Lys Val Pro Ser Gly Val Ser Asp Gly Arg Trp
His Ser Val1520 1525 1530Gln Val Gln Tyr
Tyr Asn Lys Pro Asn Ile Gly His Leu Gly Leu1535 1540
1545Pro His Gly Pro Ser Gly Glu Lys Met Ala Val Val Thr Val
Asp1550 1555 1560Asp Cys Asp Thr Thr Met
Ala Val Arg Phe Gly Lys Asp Ile Gly1565 1570
1575Asn Tyr Ser Cys Ala Ala Gln Gly Thr Gln Thr Gly Ser Lys Lys1580
1585 1590Ser Leu Asp Leu Thr Gly Pro Leu Leu Leu
Gly Gly Val Pro Asn1595 1600 1605Leu Pro
Glu Asp Phe Pro Val His Asn Arg Gln Phe Val Gly Cys1610
1615 1620Met Arg Asn Leu Ser Val Asp Gly Lys Asn Val Asp
Met Ala Gly1625 1630 1635Phe Ile Ala Asn
Asn Gly Thr Arg Glu Gly Cys Ala Ala Arg Arg1640 1645
1650Asn Phe Cys Asp Gly Arg Arg Cys Gln Asn Gly Gly Thr Cys
Val1655 1660 1665Asn Arg Trp Asn Met Tyr
Leu Cys Glu Cys Pro Leu Arg Phe Gly1670 1675
1680Gly Lys Asn Cys Glu Gln Ala Met Pro His Pro Gln Leu Phe Ser1685
1690 1695Gly Glu Ser Val Val Ser Trp Ser Asp Leu
Asn Ile Ile Ile Ser1700 1705 1710Val Pro
Trp Tyr Leu Gly Leu Met Phe Arg Thr Arg Lys Glu Asp1715
1720 1725Ser Val Leu Met Glu Ala Thr Ser Gly Gly Pro Thr
Ser Phe Arg1730 1735 1740Leu Gln Ile Leu
Asn Asn Tyr Leu Gln Phe Glu Val Ser His Gly1745 1750
1755Pro Ser Asp Val Glu Ser Val Met Leu Ser Gly Leu Arg Val
Thr1760 1765 1770Asp Gly Glu Trp His His
Leu Leu Ile Glu Leu Lys Asn Val Lys1775 1780
1785Glu Asp Ser Glu Met Lys His Leu Val Thr Met Thr Leu Asp Tyr1790
1795 1800Gly Met Asp Gln Asn Lys Ala Asp Ile Gly
Gly Met Leu Pro Gly1805 1810 1815Leu Thr
Val Arg Ser Val Val Val Gly Gly Ala Ser Glu Asp Lys1820
1825 1830Val Ser Val Arg Arg Gly Phe Arg Gly Cys Met Gln
Gly Val Arg1835 1840 1845Met Gly Gly Thr
Pro Thr Asn Val Ala Thr Leu Asn Met Asn Asn1850 1855
1860Ala Leu Lys Val Arg Val Lys Asp Gly Cys Asp Val Asp Asp
Pro1865 1870 1875Cys Thr Ser Ser Pro Cys
Pro Pro Asn Ser Arg Cys His Asp Ala1880 1885
1890Trp Glu Asp Tyr Ser Cys Val Cys Asp Lys Gly Tyr Leu Gly Ile1895
1900 1905Asn Cys Val Asp Ala Cys His Leu Asn Pro
Cys Glu Asn Met Gly1910 1915 1920Ala Cys
Val Arg Ser Pro Gly Ser Pro Gln Gly Tyr Val Cys Glu1925
1930 1935Cys Gly Pro Ser His Tyr Gly Pro Tyr Cys Glu Asn
Lys Leu Asp1940 1945 1950Leu Pro Cys Pro
Arg Gly Trp Trp Gly Asn Pro Val Cys Gly Pro1955 1960
1965Cys His Cys Ala Val Ser Lys Gly Phe Asp Pro Asp Cys Asn
Lys1970 1975 1980Thr Asn Gly Gln Cys Gln
Cys Lys Glu Asn Tyr Tyr Lys Leu Leu1985 1990
1995Ala Gln Asp Thr Cys Leu Pro Cys Asp Cys Phe Pro His Gly Ser2000
2005 2010His Ser Arg Thr Cys Asp Met Ala Thr Gly
Gln Cys Ala Cys Lys2015 2020 2025Pro Gly
Val Ile Gly Arg Gln Cys Asn Arg Cys Asp Asn Pro Phe2030
2035 2040Ala Glu Val Thr Thr Leu Gly Cys Glu Val Ile Tyr
Asn Gly Cys2045 2050 2055Pro Lys Ala Phe
Glu Ala Gly Ile Trp Trp Pro Gln Thr Lys Phe2060 2065
2070Gly Gln Pro Ala Ala Val Pro Cys Pro Lys Gly Ser Val Gly
Asn2075 2080 2085Ala Val Arg His Cys Ser
Gly Glu Lys Gly Trp Leu Pro Pro Glu2090 2095
2100Leu Phe Asn Cys Thr Thr Ile Ser Phe Val Asp Leu Arg Ala Met2105
2110 2115Asn Glu Lys Leu Ser Arg Asn Glu Thr Gln
Val Asp Gly Ala Arg2120 2125 2130Ala Leu
Gln Leu Val Arg Ala Leu Arg Ser Ala Thr Gln His Thr2135
2140 2145Gly Thr Leu Phe Gly Asn Asp Val Arg Thr Ala Tyr
Gln Leu Leu2150 2155 2160Gly His Val Leu
Gln His Glu Ser Trp Gln Gln Gly Phe Asp Leu2165 2170
2175Ala Ala Thr Gln Asp Ala Asp Phe His Glu Asp Val Ile His
Ser2180 2185 2190Gly Ser Ala Leu Leu Ala
Pro Ala Thr Arg Ala Ala Trp Glu Gln2195 2200
2205Ile Gln Arg Ser Glu Gly Gly Thr Ala Gln Leu Leu Arg Arg Leu2210
2215 2220Glu Gly Tyr Phe Ser Asn Val Ala Arg Asn
Val Arg Arg Thr Tyr2225 2230 2235Leu Arg
Pro Phe Val Ile Val Thr Ala Asn Met Ile Leu Ala Val2240
2245 2250Asp Ile Phe Asp Lys Phe Asn Phe Thr Gly Ala Arg
Val Pro Arg2255 2260 2265Phe Asp Thr Ile
His Glu Glu Phe Pro Arg Glu Leu Glu Ser Ser2270 2275
2280Val Ser Phe Pro Ala Asp Phe Phe Arg Pro Pro Glu Glu Lys
Glu2285 2290 2295Gly Pro Leu Leu Arg Pro
Ala Gly Arg Arg Thr Thr Pro Gln Thr2300 2305
2310Thr Arg Pro Gly Pro Gly Thr Glu Arg Glu Ala Pro Ile Ser Arg2315
2320 2325Arg Arg Arg His Pro Asp Asp Ala Gly Gln
Phe Ala Val Ala Leu2330 2335 2340Val Ile
Ile Tyr Arg Thr Leu Gly Gln Leu Leu Pro Glu Arg Tyr2345
2350 2355Asp Pro Asp Arg Arg Ser Leu Arg Leu Pro His Arg
Pro Ile Ile2360 2365 2370Asn Thr Pro Met
Val Ser Thr Leu Val Tyr Ser Glu Gly Ala Pro2375 2380
2385Leu Pro Arg Pro Leu Glu Arg Pro Val Leu Val Glu Phe Ala
Leu2390 2395 2400Leu Glu Val Glu Glu Arg
Thr Lys Pro Val Cys Val Phe Trp Asn2405 2410
2415His Ser Leu Ala Val Gly Gly Thr Gly Gly Trp Ser Ala Arg Gly2420
2425 2430Cys Glu Leu Leu Ser Arg Asn Arg Thr His
Val Ala Cys Gln Cys2435 2440 2445Ser His
Thr Ala Ser Phe Ala Val Leu Met Asp Ile Ser Arg Arg2450
2455 2460Glu Asn Gly Glu Val Leu Pro Leu Lys Ile Val Thr
Tyr Ala Ala2465 2470 2475Val Ser Leu Ser
Leu Ala Ala Leu Leu Val Ala Phe Val Leu Leu2480 2485
2490Ser Leu Val Arg Met Leu Arg Ser Asn Leu His Ser Ile His
Lys2495 2500 2505His Leu Ala Val Ala Leu
Phe Leu Ser Gln Leu Val Phe Val Ile2510 2515
2520Gly Ile Asn Gln Thr Glu Asn Pro Phe Leu Cys Thr Val Val Ala2525
2530 2535Ile Leu Leu His Tyr Ile Tyr Met Ser Thr
Phe Ala Trp Thr Leu2540 2545 2550Val Glu
Ser Leu His Val Tyr Arg Met Leu Thr Glu Val Arg Asn2555
2560 2565Ile Asp Thr Gly Pro Met Arg Phe Tyr Tyr Val Val
Gly Trp Gly2570 2575 2580Ile Pro Ala Ile
Val Thr Gly Leu Ala Val Gly Leu Asp Pro Gln2585 2590
2595Gly Tyr Gly Asn Pro Asp Phe Cys Trp Leu Ser Leu Gln Asp
Thr2600 2605 2610Leu Ile Trp Ser Phe Ala
Gly Pro Ile Gly Ala Val Ile Ile Ile2615 2620
2625Asn Thr Val Thr Ser Val Leu Ser Ala Lys Val Ser Cys Gln Arg2630
2635 2640Lys His His Tyr Tyr Gly Lys Lys Gly Ile
Val Ser Leu Leu Arg2645 2650 2655Thr Ala
Phe Leu Leu Leu Leu Leu Ile Ser Ala Thr Trp Leu Leu2660
2665 2670Gly Leu Leu Ala Val Asn Arg Asp Ala Leu Ser Phe
His Tyr Leu2675 2680 2685Phe Ala Ile Phe
Ser Gly Leu Gln Gly Pro Phe Val Leu Leu Phe2690 2695
2700His Cys Val Leu Asn Gln Glu Val Arg Lys His Leu Lys Gly
Val2705 2710 2715Leu Gly Gly Arg Lys Leu
His Leu Glu Asp Ser Ala Thr Thr Arg2720 2725
2730Ala Thr Leu Leu Thr Arg Ser Leu Asn Cys Asn Thr Thr Phe Gly2735
2740 2745Asp Gly Pro Asp Met Leu Arg Thr Asp Leu
Gly Glu Ser Thr Ala2750 2755 2760Ser Leu
Asp Ser Ile Val Arg Asp Glu Gly Ile Gln Lys Leu Gly2765
2770 2775Val Ser Ser Gly Leu Val Arg Gly Ser His Gly Glu
Pro Asp Ala2780 2785 2790Ser Leu Met Pro
Arg Ser Cys Lys Asp Pro Pro Gly His Asp Ser2795 2800
2805Asp Ser Asp Ser Glu Leu Ser Leu Asp Glu Gln Ser Ser Ser
Tyr2810 2815 2820Ala Ser Ser His Ser Ser
Asp Ser Glu Asp Asp Gly Val Gly Ala2825 2830
2835Glu Glu Lys Trp Asp Pro Ala Arg Gly Ala Val His Ser Thr Pro2840
2845 2850Lys Gly Asp Ala Val Ala Asn His Val Pro
Ala Gly Trp Pro Asp2855 2860 2865Gln Ser
Leu Ala Glu Ser Asp Ser Glu Asp Pro Ser Gly Lys Pro2870
2875 2880Arg Leu Lys Val Glu Thr Lys Val Ser Val Glu Leu
His Arg Glu2885 2890 2895Glu Gln Gly Ser
His Arg Gly Glu Tyr Pro Pro Asp Gln Glu Ser2900 2905
2910Gly Gly Ala Ala Arg Leu Ala Ser Ser Gln Pro Pro Glu Gln
Arg2915 2920 2925Lys Gly Ile Leu Lys Asn
Lys Val Thr Tyr Pro Pro Pro Leu Thr2930 2935
2940Leu Thr Glu Gln Thr Leu Lys Gly Arg Leu Arg Glu Lys Leu Ala2945
2950 2955Asp Cys Glu Gln Ser Pro Thr Ser Ser Arg
Thr Ser Ser Leu Gly2960 2965 2970Ser Gly
Gly Pro Asp Cys Ala Ile Thr Val Lys Ser Pro Gly Arg2975
2980 2985Glu Pro Gly Arg Asp His Leu Asn Gly Val Ala Met
Asn Val Arg2990 2995 3000Thr Gly Ser Ala
Gln Ala Asp Gly Ser Asp Ser Glu Lys Pro3005
3010108181PRTHomo Sapien 108Met Val Asp Val Lys Cys Leu Ser Asp Cys Lys
Leu Gln Asn Gln1 5 10
15Leu Glu Lys Leu Gly Phe Ser Pro Gly Pro Ile Leu Pro Ser Thr20
25 30Arg Lys Leu Tyr Glu Lys Lys Leu Val Gln Leu
Leu Val Ser Pro35 40 45Pro Cys Ala Pro
Pro Val Met Asn Gly Pro Arg Glu Leu Asp Gly50 55
60Ala Gln Asp Ser Asp Asp Ser Glu Glu Leu Asn Ile Ile Leu Gln65
70 75Gly Asn Ile Ile Leu Ser Thr Glu Lys
Ser Lys Lys Leu Lys Lys80 85 90Trp Pro
Glu Ala Ser Thr Thr Lys Arg Lys Ala Val Asp Thr Tyr95 100
105Cys Leu Asp Tyr Lys Pro Ser Lys Gly Arg Arg Trp Ala
Ala Arg110 115 120Ala Pro Ser Thr Arg Ile
Thr Tyr Gly Thr Ile Thr Lys Glu Arg125 130
135Asp Tyr Cys Ala Glu Asp Gln Thr Ile Glu Ser Trp Arg Glu Glu140
145 150Gly Phe Pro Val Gly Leu Lys Leu Ala Val
Leu Gly Ile Phe Ile155 160 165Ile Val Val
Phe Val Tyr Leu Thr Val Glu Asn Lys Ser Leu Phe170 175
180Gly109620PRTHomo Sapien 109Met Ser Lys Ser Lys Cys Ser
Val Gly Leu Met Ser Ser Val Val1 5 10
15Ala Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys Glu Val
Glu20 25 30Leu Ile Leu Val Lys Glu Gln
Asn Gly Val Gln Leu Thr Ser Ser35 40
45Thr Leu Thr Asn Pro Arg Gln Ser Pro Val Glu Ala Gln Asp Arg50
55 60Glu Thr Trp Gly Lys Lys Ile Asp Phe Leu Leu
Ser Val Ile Gly65 70 75Phe Ala Val Asp
Leu Ala Asn Val Trp Arg Phe Pro Tyr Leu Cys80 85
90Tyr Lys Asn Gly Gly Gly Ala Phe Leu Val Pro Tyr Leu Leu Phe95
100 105Met Val Ile Ala Gly Met Pro Leu Phe
Tyr Met Glu Leu Ala Leu110 115 120Gly Gln
Phe Asn Arg Glu Gly Ala Ala Gly Val Trp Lys Ile Cys125
130 135Pro Ile Leu Lys Gly Val Gly Phe Thr Val Ile Leu
Ile Ser Leu140 145 150Tyr Val Gly Phe Phe
Tyr Asn Val Ile Ile Ala Trp Ala Leu His155 160
165Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro Trp Ile His Cys170
175 180Asn Asn Ser Trp Asn Ser Pro Asn Cys
Ser Asp Ala His Pro Gly185 190 195Asp Ser
Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe Gly Thr200
205 210Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu
His Leu His215 220 225Gln Ser His Gly Ile
Asp Asp Leu Gly Pro Pro Arg Trp Gln Leu230 235
240Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser Leu245
250 255Trp Lys Gly Val Lys Thr Ser Gly Lys
Val Val Trp Ile Thr Ala260 265 270Thr Met
Pro Tyr Val Val Leu Thr Ala Leu Leu Leu Arg Gly Val275
280 285Thr Leu Pro Gly Ala Ile Asp Gly Ile Arg Ala Tyr
Leu Ser Val290 295 300Asp Phe Tyr Arg Leu
Cys Glu Ala Ser Val Trp Ile Asp Ala Ala305 310
315Thr Gln Val Cys Phe Ser Leu Gly Val Gly Phe Gly Val Leu Ile320
325 330Ala Phe Ser Ser Tyr Asn Lys Phe Thr
Asn Asn Cys Tyr Arg Asp335 340 345Ala Ile
Val Thr Thr Ser Ile Asn Ser Leu Thr Ser Phe Ser Ser350
355 360Gly Phe Val Val Phe Ser Phe Leu Gly Tyr Met Ala
Gln Lys His365 370 375Ser Val Pro Ile Gly
Asp Val Ala Lys Asp Gly Pro Gly Leu Ile380 385
390Phe Ile Ile Tyr Pro Glu Ala Ile Ala Thr Leu Pro Leu Ser Ser395
400 405Ala Trp Ala Val Val Phe Phe Ile Met
Leu Leu Thr Leu Gly Ile410 415 420Asp Ser
Ala Met Gly Gly Met Glu Ser Val Ile Thr Gly Leu Ile425
430 435Asp Glu Phe Gln Leu Leu His Arg His Arg Glu Leu
Phe Thr Leu440 445 450Phe Ile Val Leu Ala
Thr Phe Leu Leu Ser Leu Phe Cys Val Thr455 460
465Asn Gly Gly Ile Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala470
475 480Gly Thr Ser Ile Leu Phe Gly Val Leu
Ile Glu Ala Ile Gly Val485 490 495Ala Trp
Phe Tyr Gly Val Gly Gln Phe Ser Asp Asp Ile Gln Gln500
505 510Met Thr Gly Gln Arg Pro Ser Leu Tyr Trp Arg Leu
Cys Trp Lys515 520 525Leu Val Ser Pro Cys
Phe Leu Leu Phe Val Val Val Val Ser Ile530 535
540Val Thr Phe Arg Pro Pro His Tyr Gly Ala Tyr Ile Phe Pro Asp545
550 555Trp Ala Asn Ala Leu Gly Trp Val Ile
Ala Thr Ser Ser Met Ala560 565 570Met Val
Pro Ile Tyr Ala Ala Tyr Lys Phe Cys Ser Leu Pro Gly575
580 585Ser Phe Arg Glu Lys Leu Ala Tyr Ala Ile Ala Pro
Glu Lys Asp590 595 600Arg Glu Leu Val Asp
Arg Gly Glu Val Arg Gln Phe Thr Leu Arg605 610
615His Trp Leu Lys Val620110442PRTHomo Sapien 110Met Gly Leu Ala Met
Glu His Gly Gly Ser Tyr Ala Arg Ala Gly1 5
10 15Gly Ser Ser Arg Gly Cys Trp Tyr Tyr Leu Arg Tyr Phe
Phe Leu20 25 30Phe Val Ser Leu Ile Gln
Phe Leu Ile Ile Leu Gly Leu Val Leu35 40
45Phe Met Val Tyr Gly Asn Val His Val Ser Thr Glu Ser Asn Leu50
55 60Gln Ala Thr Glu Arg Arg Ala Glu Gly Leu Tyr
Ser Gln Leu Leu65 70 75Gly Leu Thr Ala
Ser Gln Ser Asn Leu Thr Lys Glu Leu Asn Phe80 85
90Thr Thr Arg Ala Lys Asp Ala Ile Met Gln Met Trp Leu Asn Ala95
100 105Arg Arg Asp Leu Asp Arg Ile Asn Ala
Ser Phe Arg Gln Cys Gln110 115 120Gly Asp
Arg Val Ile Tyr Thr Asn Asn Gln Arg Tyr Met Ala Ala125
130 135Ile Ile Leu Ser Glu Lys Gln Cys Arg Asp Gln Phe
Lys Asp Met140 145 150Asn Lys Ser Cys Asp
Ala Leu Leu Phe Met Leu Asn Gln Lys Val155 160
165Lys Thr Leu Glu Val Glu Ile Ala Lys Glu Lys Thr Ile Cys Thr170
175 180Lys Asp Lys Glu Ser Val Leu Leu Asn
Lys Arg Val Ala Glu Glu185 190 195Gln Leu
Val Glu Cys Val Lys Thr Arg Glu Leu Gln His Gln Glu200
205 210Arg Gln Leu Ala Lys Glu Gln Leu Gln Lys Val Gln
Ala Leu Cys215 220 225Leu Pro Leu Asp Lys
Asp Lys Phe Glu Met Asp Leu Arg Asn Leu230 235
240Trp Arg Asp Ser Ile Ile Pro Arg Ser Leu Asp Asn Leu Gly Tyr245
250 255Asn Leu Tyr His Pro Leu Gly Ser Glu
Leu Ala Ser Ile Arg Arg260 265 270Ala Cys
Asp His Met Pro Ser Leu Met Ser Ser Lys Val Glu Glu275
280 285Leu Ala Arg Ser Leu Arg Ala Asp Ile Glu Arg Val
Ala Arg Glu290 295 300Asn Ser Asp Leu Gln
Arg Gln Lys Leu Glu Ala Gln Gln Gly Leu305 310
315Arg Ala Ser Gln Glu Ala Lys Gln Lys Val Glu Lys Glu Ala Gln320
325 330Ala Arg Glu Ala Lys Leu Gln Ala Glu
Cys Ser Arg Gln Thr Gln335 340 345Leu Ala
Leu Glu Glu Lys Ala Val Leu Arg Lys Glu Arg Asp Asn350
355 360Leu Ala Lys Glu Leu Glu Glu Lys Lys Arg Glu Ala
Glu Gln Leu365 370 375Arg Met Glu Leu Ala
Ile Arg Asn Ser Ala Leu Asp Thr Cys Ile380 385
390Lys Thr Lys Ser Gln Pro Met Met Pro Val Ser Arg Pro Met Gly395
400 405Pro Val Pro Asn Pro Gln Pro Ile Asp
Pro Ala Ser Leu Glu Glu410 415 420Phe Lys
Arg Lys Ile Leu Glu Ser Gln Arg Pro Pro Ala Gly Ile425
430 435Pro Val Ala Pro Ser Ser Gly440111170PRTHomo Sapien
111Met Met Ala Gly Met Lys Ile Gln Leu Val Cys Met Leu Leu Leu1
5 10 15Ala Phe Ser Ser Trp Ser Leu
Cys Ser Asp Ser Glu Glu Glu Met20 25
30Lys Ala Leu Glu Ala Asp Phe Leu Thr Asn Met His Thr Ser Lys35
40 45Ile Ser Lys Ala His Val Pro Ser Trp Lys Met
Thr Leu Leu Asn50 55 60Val Cys Ser Leu
Val Asn Asn Leu Asn Ser Pro Ala Glu Glu Thr65 70
75Gly Glu Val His Glu Glu Glu Leu Val Ala Arg Arg Lys Leu Pro80
85 90Thr Ala Leu Asp Gly Phe Ser Leu Glu
Ala Met Leu Thr Ile Tyr95 100 105Gln Leu
His Lys Ile Cys His Ser Arg Ala Phe Gln His Trp Glu110
115 120Leu Ile Gln Glu Asp Ile Leu Asp Thr Gly Asn Asp
Lys Asn Gly125 130 135Lys Glu Glu Val Ile
Lys Arg Lys Ile Pro Tyr Ile Leu Lys Arg140 145
150Gln Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu Lys Arg155
160 165Asp Ser Tyr Tyr Tyr170112502PRTHomo
Sapien 112Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys Lys1
5 10 15Glu Asp Gly Glu Ser
Thr Ala Pro Thr Pro Arg Pro Lys Val Leu20 25
30Arg Cys Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn35
40 45Ile Cys Ser Thr Asp Gly Tyr Cys Phe Thr
Met Ile Glu Glu Asp50 55 60Asp Ser Gly
Leu Pro Val Val Thr Ser Gly Cys Leu Gly Leu Glu65 70
75Gly Ser Asp Phe Gln Cys Arg Asp Thr Pro Ile Pro His Gln
Arg80 85 90Arg Ser Ile Glu Cys Cys Thr
Glu Arg Asn Glu Cys Asn Lys Asp95 100
105Leu His Pro Thr Leu Pro Pro Leu Lys Asn Arg Asp Phe Val Asp110
115 120Gly Pro Ile His His Arg Ala Leu Leu Ile
Ser Val Thr Val Cys125 130 135Ser Leu Leu
Leu Val Leu Ile Ile Leu Phe Cys Tyr Phe Arg Tyr140 145
150Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser Ile Gly Leu Glu
Gln155 160 165Asp Glu Thr Tyr Ile Pro Pro
Gly Glu Ser Leu Arg Asp Leu Ile170 175
180Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu185
190 195Val Gln Arg Thr Ile Ala Lys Gln Ile Gln
Met Val Lys Gln Ile200 205 210Gly Lys Gly
Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly215 220
225Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala
Ser230 235 240Trp Phe Arg Glu Thr Glu Ile
Tyr Gln Thr Val Leu Met Arg His245 250
255Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly260
265 270Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp
Tyr His Glu Asn Gly275 280 285Ser Leu Tyr
Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala Lys Ser290 295
300Met Leu Lys Leu Ala Tyr Ser Ser Val Ser Gly Leu Cys His
Leu305 310 315His Thr Glu Ile Phe Ser Thr
Gln Gly Lys Pro Ala Ile Ala His320 325
330Arg Asp Leu Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr335
340 345Cys Cys Ile Ala Asp Leu Gly Leu Ala Val
Lys Phe Ile Ser Asp350 355 360Thr Asn Glu
Val Asp Ile Pro Pro Asn Thr Arg Val Gly Thr Lys365 370
375Arg Tyr Met Pro Pro Glu Val Leu Asp Glu Ser Leu Asn Arg
Asn380 385 390His Phe Gln Ser Tyr Ile Met
Ala Asp Met Tyr Ser Phe Gly Leu395 400
405Ile Leu Trp Glu Val Ala Arg Arg Cys Val Ser Gly Gly Ile Val410
415 420Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu
Val Pro Ser Asp Pro425 430 435Ser Tyr Glu
Asp Met Arg Glu Ile Val Cys Ile Lys Lys Leu Arg440 445
450Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg
Gln455 460 465Met Gly Lys Leu Met Thr Glu
Cys Trp Ala His Asn Pro Ala Ser470 475
480Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser485
490 495Glu Ser Gln Asp Ile Lys
Leu5001132403DNAHomo Sapien 113ttgaagtgca ttgctgcagc tggtagcatg
agtggtggcc accacctgca 50gctggctgcc ctctggccct ggctgctgat
ggctaccctg caggcaggct 100ttggacgcac aggactggta ctggcagcag
cggtggagtc tgaaagatca 150gcagaacaga aagctgttat cagagtgatc
cccttgaaaa tggaccccac 200aggaaaactg aatctcactt tggaaggtgt
gtttgctggt gttgctgaaa 250taactccagc agaaggaaaa ttaatgcagt
cccacccgct gtacctgtgc 300aatgccagtg atgacgacaa tctggagcct
ggattcatca gcatcgtcaa 350gctggagagt cctcgacggg ccccccaccc
ctgcctgtca ctggctagca 400aggctcggat ggcgggtgag cgaggagcca
gtgctgtcct ctttgacatc 450actgaggatc gagctgctgc tgagcagctg
cagcagccgc tggggctgac 500ctggccagtg gtgttgatct ggggtaatga
cgctgagaag ctgatggagt 550ttgtgtacaa gaaccaaaag gcccatgtga
ggattgagct gaaggagccc 600ccggcctggc cagattatga tgtgtggatc
ctaatgacag tggtgggcac 650catctttgtg atcatcctgg cttcggtgct
gcgcatccgg tgccgccccc 700gccacagcag gccggatccg cttcagcaga
gaacagcctg ggccatcagc 750cagctggcca ccaggaggta ccaggccagc
tgcaggcagg cccggggtga 800gtggccagac tcagggagca gctgcagctc
agcccctgtg tgtgccatct 850gtctggagga gttctctgag gggcaggagc
tacgggtcat ttcctgcctc 900catgagttcc atcgtaactg tgtggacccc
tggttacatc agcatcggac 950ttgccccctc tgcatgttca acatcacaga
gggagattca ttttcccagt 1000ccctgggacc ctctcgatct taccaagaac
caggtcgaag actccacctc 1050attcgccagc atcccggcca tgcccactac
cacctccctg ctgcctacct 1100gttgggccct tcccggagtg cagtggctcg
gcccccacga cctggtccct 1150tcctgccatc ccaggagcca ggcatgggcc
ctcggcatca ccgcttcccc 1200agagctacac atccccgggc tccaggagag
cagcagcgcc tggcaggagc 1250ccagcacccc tatgcacaag gctggggact
gagccacctc caatccacct 1300cacagcaccc tgctgcttgc ccagtgcccc
tacgccgggc caggccccct 1350gacagcagtg gatctggaga aagctattgc
acagaacgca gtgggtacct 1400ggcagatggg ccagccagtg actccagctc
agggccctgt catggctctt 1450ccagtgactc tgtggtcaac tgcacggaca
tcagcctaca gggggtccat 1500ggcagcagtt ctactttctg cagctcccta
agcagtgact ttgaccccct 1550agtgtactgc agccctaaag gggatcccca
gcgagtggac atgcagccta 1600gtgtgacctc tcggcctcgt tccttggact
cggtggtgcc cacaggggaa 1650acccaggttt ccagccatgt ccactaccac
cgccaccggc accaccacta 1700caaaaagcgg ttccagtggc atggcaggaa
gcctggccca gaaaccggag 1750tcccccagtc caggcctcct attcctcgga
cacagcccca gccagagcca 1800ccttctcctg atcagcaagt caccagatcc
aactcagcag ccccttcggg 1850gcggctctct aacccacagt gccccagggc
cctccctgag ccagcccctg 1900gcccagttga cgcctccagc atctgcccca
gtaccagcag tctgttcaac 1950ttgcaaaaat ccagcctctc tgcccgacac
ccacagagga aaaggcgggg 2000gggtccctcc gagcccaccc ctggctctcg
gccccaggat gcaactgtgc 2050acccagcttg ccagattttt ccccattaca
cccccagtgt ggcatatcct 2100tggtccccag aggcacaccc cttgatctgt
ggacctccag gcctggacaa 2150gaggctgcta ccagaaaccc caggcccctg
ttactcaaat tcacagccag 2200tgtggttgtg cctgactcct cgccagcccc
tggaaccaca tccacctggg 2250gaggggcctt ctgaatggag ttctgacacc
gcagagggca ggccatgccc 2300ttatccgcac tgccaggtgc tgtcggccca
gcctggctca gaggaggaac 2350tcgaggagct gtgtgaacag gctgtgtgag
atgttcaggc ctagctccaa 2400cca 2403114783PRTHomo Sapien
114Met Ser Gly Gly His His Leu Gln Leu Ala Ala Leu Trp Pro Trp1
5 10 15Leu Leu Met Ala Thr Leu Gln
Ala Gly Phe Gly Arg Thr Gly Leu20 25
30Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys35
40 45Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp
Pro Thr Gly Lys50 55 60Leu Asn Leu Thr
Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile65 70
75Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu80
85 90Cys Asn Ala Ser Asp Asp Asp Asn Leu
Glu Pro Gly Phe Ile Ser95 100 105Ile Val
Lys Leu Glu Ser Pro Arg Arg Ala Pro His Pro Cys Leu110
115 120Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg
Gly Ala Ser125 130 135Ala Val Leu Phe Asp
Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln140 145
150Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp155
160 165Gly Asn Asp Ala Glu Lys Leu Met Glu
Phe Val Tyr Lys Asn Gln170 175 180Lys Ala
His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro185
190 195Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly
Thr Ile Phe200 205 210Val Ile Ile Leu Ala
Ser Val Leu Arg Ile Arg Cys Arg Pro Arg215 220
225His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile230
235 240Ser Gln Leu Ala Thr Arg Arg Tyr Gln
Ala Ser Cys Arg Gln Ala245 250 255Arg Gly
Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro260
265 270Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly
Gln Glu Leu275 280 285Arg Val Ile Ser Cys
Leu His Glu Phe His Arg Asn Cys Val Asp290 295
300Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn305
310 315Ile Thr Glu Gly Asp Ser Phe Ser Gln
Ser Leu Gly Pro Ser Arg320 325 330Ser Tyr
Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His335
340 345Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr
Leu Leu Gly350 355 360Pro Ser Arg Ser Ala
Val Ala Arg Pro Pro Arg Pro Gly Pro Phe365 370
375Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe380
385 390Pro Arg Ala Thr His Pro Arg Ala Pro
Gly Glu Gln Gln Arg Leu395 400 405Ala Gly
Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His410
415 420Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro
Val Pro Leu425 430 435Arg Arg Ala Arg Pro
Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr440 445
450Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp455
460 465Ser Ser Ser Gly Pro Cys His Gly Ser
Ser Ser Asp Ser Val Val470 475 480Asn Cys
Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser485
490 495Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro
Leu Val Tyr500 505 510Cys Ser Pro Lys Gly
Asp Pro Gln Arg Val Asp Met Gln Pro Ser515 520
525Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly530
535 540Glu Thr Gln Val Ser Ser His Val His
Tyr His Arg His Arg His545 550 555His His
Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly560
565 570Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile
Pro Arg Thr575 580 585Gln Pro Gln Pro Glu
Pro Pro Ser Pro Asp Gln Gln Val Thr Arg590 595
600Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys605
610 615Pro Arg Ala Leu Pro Glu Pro Ala Pro
Gly Pro Val Asp Ala Ser620 625 630Ser Ile
Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser635
640 645Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg
Gly Gly Pro650 655 660Ser Glu Pro Thr Pro
Gly Ser Arg Pro Gln Asp Ala Thr Val His665 670
675Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr680
685 690Pro Trp Ser Pro Glu Ala His Pro Leu
Ile Cys Gly Pro Pro Gly695 700 705Leu Asp
Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser710
715 720Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg
Gln Pro Leu725 730 735Glu Pro His Pro Pro
Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp740 745
750Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu755
760 765Ser Ala Gln Pro Gly Ser Glu Glu Glu
Leu Glu Glu Leu Cys Glu770 775 780Gln Ala
Val1152407DNAHomo Sapien 115ccctttgaag tgcattgctg cagctggtag catgagtggt
ggccaccagc 50tgcagctggc tgccctctgg ccctggctgc tgatggctac
cctgcaggca 100ggctttggac gcacaggact ggtactggca gcagcggtgg
agtctgaaag 150atcagcagaa cagaaagctg ttatcagagt gatccccttg
aaaatggacc 200ccacaggaaa actgaatctc actttggaag gtgtgtttgc
tggtgttgct 250gaaataactc cagcagaagg aaaattaatg cagtcccacc
cgctgtacct 300gtgcaatgcc agtgatgacg acaatctgga gcctggattc
atcagcatcg 350tcaagctgga gagtcctcga cgggcccccc gcccctgcct
gtcactggct 400agcaaggctc ggatggcggg tgagcgagga gccagtgctg
tcctctttga 450catcactgag gatcgagctg ctgctgagca gctgcagcag
ccgctggggc 500tgacctggcc agtggtgttg atctggggta atgacgctga
gaagctgatg 550gagtttgtgt acaagaacca aaaggcccat gtgaggattg
agctgaagga 600gcccccggcc tggccagatt atgatgtgtg gatcctaatg
acagtggtgg 650gcaccatctt tgtgatcatc ctggcttcgg tgctgcgcat
ccagtgccgc 700ccccgccaca gcaggccgga tccgcttcag cagagaacag
cctgggccat 750cagccagctg gccaccagga ggtaccaggc cagctgcagg
caggcccggg 800gtgagtggcc agactcaggg agcagctgca gctcagcccc
tgtgtgtgcc 850atctgtctgg aggagttctc tgaggggcag gagctacggg
tcatttcctg 900cctccatgag ttccatcgta actgtgtgga cccctggtta
catcagcatc 950ggacttgccc cctctgcatg ttcaacatca cagagggaga
ttcattttcc 1000cagtccctgg gaccctctcg atcttaccaa gaaccaggtc
gaagactcca 1050cctcattcgc cagcatcccg gccatgccca ctaccacctc
cctgctgcct 1100acctgttggg cccttcccgg agtgcagtgg ctcggccccc
acgacctggt 1150cccttcctgc catcccagga gccaggcatg ggccctcggc
atcaccgctt 1200ccccagagct gcacatcccc gggctccagg agagcagcag
cgcctggcag 1250gagcccagca cccctatgca caaggctggg gactgagcca
cctccaatcc 1300acctcacagc accctgctgc ttgcccagtg cccctacgcc
gggccaggcc 1350ccctgacagc agtggatctg gagaaagcta ttgcacagaa
cgcagtgggt 1400acctggcaga tgggccagcc agtgactcca gctcagggcc
ctgtcatggc 1450tcttccagtg actctgtggt caactgcacg gacatcagcc
tacagggggt 1500ccatggcagc agttctactt tctgcagctc cctaagcagt
gactttgacc 1550ccctagtgta ctgcagccct aaaggggatc cccagcgagt
ggacatgcag 1600cctagtgtga cctctcggcc tcgttccttg gactcggtgg
tgcccacagg 1650ggaaacccag gtttccagcc atgtccacta ccaccgccac
cggcaccacc 1700actacaaaaa gcggttccag tggcatggca ggaagcctgg
cccagaaacc 1750ggagtccccc agtccaggcc tcctattcct cggacacagc
cccagccaga 1800gccaccttct cctgatcagc aagtcaccag atccaactca
gcagcccctt 1850cggggcggct ctctaaccca cagtgcccca gggccctccc
tgagccagcc 1900cctggcccag ttgacgcctc cagcatctgc cccagtacca
gcagtctgtt 1950caacttgcaa aaatccagcc tctctgcccg acacccacag
aggaaaaggc 2000gggggggtcc ctccgagccc acccctggct ctcggcccca
ggatgcaact 2050gtgcacccag cttgccagat ttttccccat tacaccccca
gtgtggcata 2100tccttggtcc ccagaggcac accccttgat ctgtggacct
ccaggcctgg 2150acaagaggct gctaccagaa accccaggcc cctgttactc
aaattcacag 2200ccagtgtggt tgtgcctgac tcctcgccag cccctggaac
cacatccacc 2250tggggagggg ccttctgaat ggagttctga caccgcagag
ggcaggccat 2300gcccttgtcc gcactgccag gtgctgtcgg cccagcctgg
ctcagaggag 2350gaactcgagg agctgtgtga acaggctgtg tgagatgttc
aggcctagct 2400ccaacca
2407116783PRTHomo Sapien 116Met Ser Gly Gly His Gln
Leu Gln Leu Ala Ala Leu Trp Pro Trp1 5 10
15Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly
Leu20 25 30Val Leu Ala Ala Ala Val Glu
Ser Glu Arg Ser Ala Glu Gln Lys35 40
45Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys50
55 60Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly
Val Ala Glu Ile65 70 75Thr Pro Ala Glu
Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu80 85
90Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser95
100 105Ile Val Lys Leu Glu Ser Pro Arg Arg
Ala Pro Arg Pro Cys Leu110 115 120Ser Leu
Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser125
130 135Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala
Ala Glu Gln140 145 150Leu Gln Gln Pro Leu
Gly Leu Thr Trp Pro Val Val Leu Ile Trp155 160
165Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln170
175 180Lys Ala His Val Arg Ile Glu Leu Lys
Glu Pro Pro Ala Trp Pro185 190 195Asp Tyr
Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe200
205 210Val Ile Ile Leu Ala Ser Val Leu Arg Ile Gln Cys
Arg Pro Arg215 220 225His Ser Arg Pro Asp
Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile230 235
240Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala245
250 255Arg Gly Glu Trp Pro Asp Ser Gly Ser
Ser Cys Ser Ser Ala Pro260 265 270Val Cys
Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu275
280 285Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn
Cys Val Asp290 295 300Pro Trp Leu His Gln
His Arg Thr Cys Pro Leu Cys Met Phe Asn305 310
315Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg320
325 330Ser Tyr Gln Glu Pro Gly Arg Arg Leu
His Leu Ile Arg Gln His335 340 345Pro Gly
His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly350
355 360Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro
Gly Pro Phe365 370 375Leu Pro Ser Gln Glu
Pro Gly Met Gly Pro Arg His His Arg Phe380 385
390Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu395
400 405Ala Gly Ala Gln His Pro Tyr Ala Gln
Gly Trp Gly Leu Ser His410 415 420Leu Gln
Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu425
430 435Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly
Glu Ser Tyr440 445 450Cys Thr Glu Arg Ser
Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp455 460
465Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val470
475 480Asn Cys Thr Asp Ile Ser Leu Gln Gly
Val His Gly Ser Ser Ser485 490 495Thr Phe
Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr500
505 510Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met
Gln Pro Ser515 520 525Val Thr Ser Arg Pro
Arg Ser Leu Asp Ser Val Val Pro Thr Gly530 535
540Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His545
550 555His His Tyr Lys Lys Arg Phe Gln Trp
His Gly Arg Lys Pro Gly560 565 570Pro Glu
Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr575
580 585Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln
Val Thr Arg590 595 600Ser Asn Ser Ala Ala
Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys605 610
615Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser620
625 630Ser Ile Cys Pro Ser Thr Ser Ser Leu
Phe Asn Leu Gln Lys Ser635 640 645Ser Leu
Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro650
655 660Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala
Thr Val His665 670 675Pro Ala Cys Gln Ile
Phe Pro His Tyr Thr Pro Ser Val Ala Tyr680 685
690Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly695
700 705Leu Asp Lys Arg Leu Leu Pro Glu Thr
Pro Gly Pro Cys Tyr Ser710 715 720Asn Ser
Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu725
730 735Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp
Ser Ser Asp740 745 750Thr Ala Glu Gly Arg
Pro Cys Pro Cys Pro His Cys Gln Val Leu755 760
765Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu770
775 780Gln Ala Val1172403DNAHomo Sapien
117ttgaagtgca ttgctgcagc tggtagcatg agtggtggcc accacctgca
50gctggctgcc ctctggccct ggctgctgat ggctaccctg caggcaggct
100ttggacgcac aggactggta ctggcagcag cggtggagtc tgaaagatca
150gcagaacaga aagctgttat cagagtgatc cccttgaaaa tggaccccac
200aggaaaactg aatctcactt tggaaggtgt gtttgctggt gttgctgaaa
250taactccagc agaaggaaaa ttaatgcagt cccacccgct gtacctgtgc
300aatgccagtg atgacgacaa tctggagcct ggattcatca gcatcgtcaa
350gctggagagt cctcgacggg ccccccaccc ctgcctgtca ctggctagca
400aggctcggat ggcgggtgag cgaggagcca gtgctgtcct ctttgacatc
450actgaggatc gagctgctgc tgagcagctg cagcagccgc tggggctgac
500ctggccagtg gtgttgatct ggggtaatga cgctgagaag ctgatggagt
550ttgtgtacaa gaaccaaaag gcccatgtga ggattgagct gaaggagccc
600ccggcctggc cagattatga tgtgtggatc ctaatgacag tggtgggcac
650catctttgtg atcatcctgg cttcggtgct gcgcatccgg tgccgccccc
700gccacagcag gccggatccg cttcagcaga gaacagcctg ggccatcagc
750cagctggcca ccaggaggta ccaggccagc tgcaggcagg cccggggtga
800gtggccagac tcagggagca gctgcagctc agcccctgtg tgtgccatct
850gtctggagga gttctctgag gggcaggagc tacgggtcat ttcctgcctc
900catgagttcc atcgtaactg tgtggacccc tggttacatc agcatcggac
950ttgccccctc tgcatgttca acatcacaga gggagattca ttttcccagt
1000ccctgggacc ctctcgatct taccaagaac caggtcgaag actccacctc
1050attcgccagc atcccggcca tgcccactac cacctccctg ctgcctacct
1100gttgggccct tcccggagtg cagtggctcg gcccccacga cctggtccct
1150tcctgccatc ccaggagcca ggcatgggcc ctcggcatca ccgcttcccc
1200agagctgcac atccccgggc tccaggagag cagcagcgcc tggcaggagc
1250ccagcacccc tatgcacaag gctggggaat gagccacctc caatccacct
1300cacagcaccc tgctgcttgc ccagtgcccc tacgccgggc caggccccct
1350gacagcagtg gatctggaga aagctattgc acagaacgca gtgggtacct
1400ggcagatggg ccagccagtg actccagctc agggccctgt catggctctt
1450ccagtgactc tgtggtcaac tgcacggaca tcagcctaca gggggtccat
1500ggcagcagtt ctactttctg cagctcccta agcagtgact ttgaccccct
1550agtgtactgc agccctaaag gggatcccca gcgagtggac atgcagccta
1600gtgtgacctc tcggcctcgt tccttggact cggtggtgcc cacaggggaa
1650acccaggttt ccagccatgt ccactaccac cgccaccggc accaccacta
1700caaaaagcgg ttccagtggc atggcaggaa gcctggccca gaaaccggag
1750tcccccagtc caggcctcct attcctcgga cacagcccca gccagagcca
1800ccttctcctg atcagcaagt caccagatcc aactcagcag ccccttcggg
1850gcggctctct aacccacagt gccccagggc cctccctgag ccagcccctg
1900gcccagttga cgcctccagc atctgcccca gtaccagcag tctgttcaac
1950ttgcaaaaat ccagcctctc tgcccgacac ccacagagga aaaggcgggg
2000gggtccctcc gagcccaccc ctggctctcg gccccaggat gcaactgtgc
2050acccagcttg ccagattttt ccccattaca cccccagtgt ggcatatcct
2100tggtccccag aggcacaccc cttgatctgt ggacctccag gcctggacaa
2150gaggctgcta ccagaaaccc caggcccctg ttactcaaat tcacagccag
2200tgtggttgtg cctgactcct cgccagcccc tggaaccaca tccacctggg
2250gaggggcctt ctgaatggag ttctgacacc gcagagggca ggccatgccc
2300ttatccgcac tgccaggtgc tgtcggccca gcctggctca gaggaggaac
2350tcgaggagct gtgtgaacag gctgtgtgag atgttcaggc ctagctccaa
2400cca 2403118783PRTHomo Sapien 118Met Ser Gly Gly His His Leu Gln Leu
Ala Ala Leu Trp Pro Trp1 5 10
15Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu20
25 30Val Leu Ala Ala Ala Val Glu Ser Glu Arg
Ser Ala Glu Gln Lys35 40 45Ala Val Ile
Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys50 55
60Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu
Ile65 70 75Thr Pro Ala Glu Gly Lys Leu
Met Gln Ser His Pro Leu Tyr Leu80 85
90Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser95
100 105Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro
His Pro Cys Leu110 115 120Ser Leu Ala Ser
Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser125 130
135Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu
Gln140 145 150Leu Gln Gln Pro Leu Gly Leu
Thr Trp Pro Val Val Leu Ile Trp155 160
165Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln170
175 180Lys Ala His Val Arg Ile Glu Leu Lys Glu
Pro Pro Ala Trp Pro185 190 195Asp Tyr Asp
Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe200 205
210Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro
Arg215 220 225His Ser Arg Pro Asp Pro Leu
Gln Gln Arg Thr Ala Trp Ala Ile230 235
240Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala245
250 255Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser
Cys Ser Ser Ala Pro260 265 270Val Cys Ala
Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu275 280
285Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val
Asp290 295 300Pro Trp Leu His Gln His Arg
Thr Cys Pro Leu Cys Met Phe Asn305 310
315Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg320
325 330Ser Tyr Gln Glu Pro Gly Arg Arg Leu His
Leu Ile Arg Gln His335 340 345Pro Gly His
Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly350 355
360Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro
Phe365 370 375Leu Pro Ser Gln Glu Pro Gly
Met Gly Pro Arg His His Arg Phe380 385
390Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu395
400 405Ala Gly Ala Gln His Pro Tyr Ala Gln Gly
Trp Gly Met Ser His410 415 420Leu Gln Ser
Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu425 430
435Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser
Tyr440 445 450Cys Thr Glu Arg Ser Gly Tyr
Leu Ala Asp Gly Pro Ala Ser Asp455 460
465Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val470
475 480Asn Cys Thr Asp Ile Ser Leu Gln Gly Val
His Gly Ser Ser Ser485 490 495Thr Phe Cys
Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr500 505
510Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro
Ser515 520 525Val Thr Ser Arg Pro Arg Ser
Leu Asp Ser Val Val Pro Thr Gly530 535
540Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His545
550 555His His Tyr Lys Lys Arg Phe Gln Trp His
Gly Arg Lys Pro Gly560 565 570Pro Glu Thr
Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr575 580
585Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr
Arg590 595 600Ser Asn Ser Ala Ala Pro Ser
Gly Arg Leu Ser Asn Pro Gln Cys605 610
615Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser620
625 630Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe
Asn Leu Gln Lys Ser635 640 645Ser Leu Ser
Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro650 655
660Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val
His665 670 675Pro Ala Cys Gln Ile Phe Pro
His Tyr Thr Pro Ser Val Ala Tyr680 685
690Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly695
700 705Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro
Gly Pro Cys Tyr Ser710 715 720Asn Ser Gln
Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu725 730
735Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser
Asp740 745 750Thr Ala Glu Gly Arg Pro Cys
Pro Tyr Pro His Cys Gln Val Leu755 760
765Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu770
775 780Gln Ala Val1194839DNAHomo Sapien
119ggaaagctag cggcagaggc tcagccccgg cggcagcgcg cgccccgctg
50ccagcccatt ttccggacgc cacccgcggg cactgccgac gcccccgggg
100ctgccgaggg gaggccgggg gggcgcagcg gagcgcggtc ccgcgcactg
150agccccgcgg cgccccggga acttggcggc gacccgagcc cggcgagccg
200gggcgcgcct cccccgccgc gcgcctcctg catgcggggc cccagctccg
250ggcgccggcc ggagcccccc ccggccgccc ccgagccccc cgcgccccgc
300gccgcgccgc cgcgccgtcc atgcaccgct tgatgggggt caacagcacc
350gccgccgccg ccgccgggca gcccaatgtc tcctgcacgt gcaactgcaa
400acgctctttg ttccagagca tggagatcac ggagctggag tttgttcaga
450tcatcatcat cgtggtggtg atgatggtga tggtggtggt gatcacgtgc
500ctgctgagcc actacaagct gtctgcacgg tccttcatca gccggcacag
550ccaggggcgg aggagagaag atgccctgtc ctcagaagga tgcctgtggc
600cctcggagag cacagtgtca ggcaacggaa tcccagagcc gcaggtctac
650gccccgcctc ggcccaccga ccgcctggcc gtgccgccct tcgcccagcg
700ggagcgcttc caccgcttcc agcccaccta tccgtacctg cagcacgaga
750tcgacctgcc acccaccatc tcgctgtcag acggggagga gcccccaccc
800taccagggcc cctgcaccct ccagcttcgg gaccccgagc agcagctgga
850actgaaccgg gagtcggtgc gcgcaccccc aaacagaacc atcttcgaca
900gtgacctgat ggatagtgcc aggctgggcg gcccctgccc ccccagcagt
950aactcgggca tcagcgccac gtgctacggc agcggcgggc gcatggaggg
1000gccgccgccc acctacagcg aggtcatcgg ccactacccg gggtcctcct
1050tccagcacca gcagagcagt gggccgccct ccttgctgga ggggacccgg
1100ctccaccaca cacacatcgc gcccctagag agcgcagcca tctggagcaa
1150agagaaggat aaacagaaag gacaccctct ctagggtccc caggggggcc
1200gggctggggc tgcgtaggtg aaaaggcaga acactccgcg cttcttagaa
1250gaggagtgag aggaaggcgg ggggcgcagc aacgcatcgt gtggccctcc
1300cctcccacct ccctgtgtat aaatatttac atgtgatgtc tggtctgaat
1350gcacaagcta agagagcttg caaaaaaaaa aagaaaaaag aaaaaaaaaa
1400accacgtttc tttgttgagc tgtgtcttga aggcaaaaga aaaaaaattt
1450ctacagtagt ctttcttgtt tctagttgag ctgcgtgcgt gaatgcttat
1500tttcttttgt ttatgataat ttcacttaac tttaaagaca tatttgcaca
1550aaacctttgt ttaaagatct gcaatattat atatataaat atatataaga
1600taagagaaac tgtatgtgcg agggcaggag tatttttgta ttagaagagg
1650cctattaaaa aaaaaagttg ttttctgaac tagaagagga aaaaaatggc
1700aatttttgag tgccaagtca gaaagtgtgt attaccttgt aaagaaaaaa
1750attacaaagc aggggtttag agttatttat ataaatgttg agattttgca
1800ctatttttta atataaatat gtcagtgctt gcttgatgga aacttctctt
1850gtgtctgttg agactttaag ggagaaatgt cggaatttca gagtcgcctg
1900acggcagagg gtgagccccc gtggagtctg cagagaggcc ttggccagga
1950gcggcgggct ttcccgaggg gccactgtcc ctgcagagtg gatgcttctg
2000cctagtgaca ggttatcacc acgttatata ttccctaccg aaggagacac
2050cttttccccc ctgacccaga acagccttta aatcacaagc aaaataggaa
2100agttaaccac ggaggcaccg agttccaggt agtggttttg cctttcccaa
2150aaatgaaaat aaactgttac cgaaggaatt agtttttcct cttctttttt
2200ccaactgtga aggtccccgt ggggtggagc atggtgcccc tcacaagccg
2250cagcggctgg tgcccgggct accagggaca tgccagaggg ctcgatgact
2300tgtctctgca gggcgctttg gtggttgttc agctggctaa aggttcaccg
2350gtgaaggcag gtgcggtaac tgccgcactg gaccctagga agccccaggt
2400attcgcaatc tgacctcctc ctgtctgttt cccttcacgg atcaattctc
2450acttaagagg ccaataaaca acccaacatg aaaaggtgac aagcctgggt
2500ttctcccagg ataggtgaaa gggttaaaat gagtaaagca gttgagcaaa
2550caccaacccg agcttcgggc gcagaattct tcaccttctc ttcccctttc
2600catctccttt ccccgcggaa acaacgcttc ccttctggtg tgtctgttga
2650tctgtgtttt catttacatc tctcttagac tccgctcttg ttctccaggt
2700tttcaccaga tagatttggg gttggcggga cctgctggtg acgtgcaggt
2750gaaggacagg aaggggcatg tgagcgtaaa tagaggtgac cagaggagag
2800catgaggggt ggggctttgg gacccaccgg ggccagtggc tggagcttga
2850cgtctttcct ccccatgggg gtgggagggc ccccagctgg aagagcagac
2900tcccagctgc taccccctcc cttcccatgg gagtggcttt ccattttggg
2950cagaatgctg actagtagac taacataaaa gatataaaag gcaataacta
3000ttgtttgtga gcaacttttt tataacttcc aaaacaaaaa cctgagcaca
3050gttttgaagt tctagccact cgagctcatg catgtgaaac gtgtgcttta
3100cgaaggtggc agctgacaga cgtgggctct gcatgccgcc agcctagtag
3150aaagttctcg ttcattggca acagcagaac ctgcctctcc gtgaagtcgt
3200cagcctaaaa tttgtttctc tcttgaagag gattctttga aaaggtcctg
3250cagagaaatc agtacaggtt atcccgaaag gtacaaggac gcacttgtaa
3300agatgattaa aacgtatctt tcctttatgt gacgcgtctc tagtgcctta
3350ctgaagaagc agtgacactc ccgtcgctcg gtgaggacgt tcccggacag
3400tgcctcactc acctgggact ggtatcccct cccagggtcc accaagggct
3450cctgcttttc agacacccca tcatcctcgc gcgtcctcac cctgtctcta
3500ccagggaggt gcctagcttg gtgaggttac tcctgctcct ccaacctttt
3550tttgccaagg tttgtacacg actcccatct aggctgaaaa cctagaagtg
3600gaccttgtgt gtgtgcatgg tgtcagccca aagccaggct gagacagtcc
3650tcatatcctc ttgagccaaa ctgtttgggt ctcgttgctt catggtatgg
3700tctggatttg tgggaatggc tttgcgtgag aaaggggagg agagtggttg
3750ctgccctcag ccggcttgag gacagagcct gtccctctca tgacaactca
3800gtgttgaagc ccagtgtcct cagcttcatg tccagtggat ggcagaagtt
3850catggggtag tggcctctca aaggctgggc gcatcccaag acagccagca
3900ggttgtctct ggaaacgacc agagttaagc tctcggcttc tctgctgagg
3950gtgcaccctt tcctctagat ggtagttgtc acgttatctt tgaaaactct
4000tggactgctc ctgaggaggc cctcttttcc agtaggaagt tagatggggg
4050ttctcagaag tggctgattg gaaggggaca agcttcgttt caggggtctg
4100ccgttccatc ctggttcaga gaaggccgag cgtggctttc tctagccttg
4150tcactgtctc cctgcctgtc aatcaccacc tttcctccag aggaggaaaa
4200ttatctcccc tgcaaagccc ggttctacac agatttcaca aattgtgcta
4250agaaccgtcc gtgttctcag aaagcccagt gtttttgcaa agaatgaaaa
4300gggaccccat atgtagcaaa aatcagggct gggggagagc cgggttcatt
4350ccctgtcctc attggtcgtc cctatgaatt gtacgtttca gagaaatttt
4400ttttcctatg tgcaacacga agcttccaga accataaaat atcccgtcga
4450taaggaaaga aaatgtcgtt gttgttgttt ttctggaaac tgcttgaaat
4500cttgctgtac tatagagctc agaaggacac agcccgtcct cccctgcctg
4550cctgattcca tggctgttgt gctgattcca atgctttcac gttggttcct
4600ggcgtgggaa ctgctctcct ttgcagcccc atttcccaag ctctgttcaa
4650gttaaactta tgtaagcttt ccgtggcatg cggggcgcgc acccacgtcc
4700ccgctgcgta agactctgta tttggatgcc aatccacagg cctgaagaaa
4750ctgcttgttg tgtatcagta atcattagtg gcaatgatga cattctgaaa
4800agctgcaata cttatacaat aaattttaca attctttgg
4839120287PRTHomo Sapien 120Met His Arg Leu Met Gly Val Asn Ser Thr Ala
Ala Ala Ala Ala1 5 10
15Gly Gln Pro Asn Val Ser Cys Thr Cys Asn Cys Lys Arg Ser Leu20
25 30Phe Gln Ser Met Glu Ile Thr Glu Leu Glu Phe
Val Gln Ile Ile35 40 45Ile Ile Val Val
Val Met Met Val Met Val Val Val Ile Thr Cys50 55
60Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg65
70 75His Ser Gln Gly Arg Arg Arg Glu Asp
Ala Leu Ser Ser Glu Gly80 85 90Cys Leu
Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile Pro95 100
105Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg
Leu Ala110 115 120Val Pro Pro Phe Ala Gln
Arg Glu Arg Phe His Arg Phe Gln Pro125 130
135Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro Pro Thr Ile140
145 150Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro
Tyr Gln Gly Pro Cys155 160 165Thr Leu Gln
Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg170 175
180Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser
Asp185 190 195Leu Met Asp Ser Ala Arg Leu
Gly Gly Pro Cys Pro Pro Ser Ser200 205
210Asn Ser Gly Ile Ser Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met215
220 225Glu Gly Pro Pro Pro Thr Tyr Ser Glu Val
Ile Gly His Tyr Pro230 235 240Gly Ser Ser
Phe Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu245 250
255Leu Glu Gly Thr Arg Leu His His Thr His Ile Ala Pro Leu
Glu260 265 270Ser Ala Ala Ile Trp Ser Lys
Glu Lys Asp Lys Gln Lys Gly His275 280
285Pro Leu
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