Patent application title: METHODS AND MEANS FOR TREATMENT OF OSTEOARTHRITIS
Inventors:
Nick Vandeghinste (Duffel, BE)
Peter Herwig Maria Tomme (Gent, BE)
Frits Michiels (Leiderdorp, NL)
Libin Ma (Oegstgeest, NL)
Blandine Mille-Baker (Oegstgeest, NL)
Helmuth H.g. Van Es (Haarlem, NL)
Assignees:
GALAPAGOS NV
IPC8 Class: AC07H2104FI
USPC Class:
514 44 A
Class name: Nitrogen containing hetero ring polynucleotide (e.g., rna, dna, etc.) antisense or rna interference
Publication date: 2014-04-17
Patent application number: 20140107181
Abstract:
The present invention relates to in vivo and in vitro methods, agents and
compound screening assays for inducing anabolic stimulation of
chondrocytes, including cartilage formation enhancing pharmaceutical
compositions, and the use thereof in treating and/or preventing a disease
involving a systemic or local decrease in mean cartilage thickness in a
subject.Claims:
1. Method for identifying a compound that induces chondrocyte anabolic
stimulation, comprising: contacting a compound with a polypeptide
comprising an amino acid sequence selected from the group consisting of
SEQ ID NO: 55-82 and 198-391, and 480; and measuring a
compound-polypeptide property related to the anabolic stimulation of
chondrocytes.
2. The method according to claim 1, wherein said polypeptide is in an in vitro cell-free preparation.
3. The method according to claim 2, wherein said polypeptide is present in a mammalian cell.
4. The method of claim 1, wherein said property is a binding affinity of said compound to said polypeptide.
5. The method of claim 3, wherein said property is activation of a biological pathway producing a biochemical marker indicative of the anabolic stimulation of chondrocytes.
6. The method of claim 5 wherein said indicator is selected from the group consisting of collagen type II, alpha-1 (col2a1) and aggrecan.
7. The method of claim 6 wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82.
8. The method according to claim 1, wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds having binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391, and 480.
9. The method according to claim 2, wherein said compound is a peptide in a phage display library or an antibody fragment library.
10. An agent for inducing the anabolic stimulation of chondrocytes, selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391, and 480.
11. The agent according to claim 10, wherein polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82.
12. The agent according to claim 11, wherein a vector in a mammalian cell expresses said agent.
13. The agent according to claim 12, wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector.
14. The agent according to claim 10, wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82.
15. The agent according to claim 14, wherein said siRNA further comprises said sense strand.
16. The agent according to claim 15, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-28 and 479.
17. The agent according to claim 16, wherein said siRNA further comprises a loop region connecting said sense and said antisense strand.
18. The agent according to claim 17, wherein said loop region comprises a nucleic acid sequence defined of SEQ ID NO: 83.
19. The agent according to claim 18, wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 84-197 and 410.
20. A chondrocyte anabolic stimulation enhancing pharmaceutical composition comprising a therapeutically effective amount of an agent of claim 10 in admixture with a pharmaceutically acceptable carrier.
21-32. (canceled)
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser. No. 13/360,393 filed Jan. 27, 2012, which a Divisional of U.S. application Ser. No. 12/575,220, filed Oct. 7, 2009, which is a Divisional of U.S. application Ser. No. 11/158,252, filed Jun. 21, 2005, which claims the benefit of priority of U.S. Provisional Application No. 60/581,568, filed Jun. 21, 2004, each of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the field of medicinal research, cartilage physiology and diseases involving the degeneration of cartilage tissue. More specifically, the invention relates to methods and means for identifying compounds that stimulate anabolic processes in chondrocytes and that typically induce the synthesis of cartilage. The invention also relates to the compounds that are useful in the treatment of osteoarthritis.
[0003] Cartilage is an avascular tissue made up largely of cartilage-specific cells, the chondrocytes, proteoglycans and collagen proteins, which are structural proteins that provide structural strength to connective tissue, such as skin, bone and cartilage. Collagen II, together with the protein collagen IX, forms a "biological alloy", which is molded into a fibril-like structure and is arranged in a precise network, providing cartilage with great mechanical strength. The chondrocytes in normal articular cartilage occupy approximately 5% of the tissue volume, while the extra-cellular matrix makes up the remaining 95% of the tissue. The chondrocytes secrete the components of the matrix, which in turn supplies the chondrocytes with an environment suitable for their survival under mechanical stress.
[0004] Breakdown of articular cartilage, which is part of joints and which cushions the ends of the bones, causes the bones to rub against each other leading to pain and loss of movement. Cartilage degradation may also be the result of an imbalance in cartilage synthesizing (anabolic) and cartilage degrading (catabolic) processes. Unlike most tissues, cartilage does not self-repair following injury. The inability of cartilage to self-repair after injury, disease, or surgery is a major limiting factor in rehabilitation of degrading joint surfaces and injury to meniscal cartilage.
[0005] There are many diseases involving the degeneration of cartilage. Rheumatoid arthritis and osteoarthritis are among the most prominent. Osteoarthritis (also referred to as OA, or as wear-and-tear arthritis) is the most common form of arthritis and is characterized by loss of articular cartilage, often associated with hypertrophy of the bone. The disease mainly affects hands and weight-bearing joints such as knees, hips and spines. This process thins the cartilage through a phenomenon called apoptosis, or programmed cell death. When the surface area has disappeared due to the thinning, there is a grade I osteoarthritis; when the tangential surface area has disappeared, there is a grade two osteoarthritis. There are other levels of degeneration and destruction, which affect the deep and the calcified layers that border with the subchondral bone.
[0006] The clinical manifestations of the development of the osteoarthritis condition are: increased volume of the joint, pain, crepitation and functional disability that, gradually and steadily, first hinders the performance of lengthy walks and forced flexion and extension movements, depending on the affected joint, and then pain and limitation of minimum efforts emerge as well as pain at rest which interrupts sleeping. If the condition persists without correction and/or therapy, the joint is destroyed, leading the patient to major replacement surgery with total prosthesis, or to disability.
[0007] Therapeutic methods for the correction of the articular cartilage lesions that appear during the osteoarthritic disease have been developed, but so far none of them have been able to achieve the regeneration of articular cartilage in situ and in vivo.
REPORTED DEVELOPMENTS
[0008] Osteoarthritis is difficult to treat. At present, no cure is available and treatment focuses on relieving pain and preventing the affected joint from becoming deformed. Common treatments include the use of non-steroidal anti-inflammatory drugs (NSAID's), which are often used to relieve pain, while specific COX-2 inhibitors are used to relieve severe pain. Medicines such as chondroitin and glucosamine are thought to improve the cartilage itself. These treatments may be relatively successful, but not a substantive amount of research data is available.
[0009] In severe cases, joint replacement may be necessary. This is especially true for hips and knees. If a joint is extremely painful and cannot be replaced, it may be fused. This procedure stops the pain, but results in the permanent loss of joint function, making walking and bending difficult.
[0010] The treatment that has 74% to 90% effectiveness and produces excellent results is the transplantation of cultured autologous chondrocytes, by taking chondral cellular material from the patient, sending it to a laboratory where it is seeded in a proper medium for its proliferation, and, once enough volume is achieved after a variable period that may last from weeks to months, transporting it in a special container and implanting it in the damaged tissues to cover the tissue's defects.
[0011] Another treatment includes the intraarticular instillation of Hylan G-F 20 (Synvisc, Hyalgan, Artz etc.), a substance that improves temporarily the rheology of the synovial fluid, producing an almost immediate sensation of free movement and a marked reduction of pain. The residual effects of this substance act on the synovial receptors causing a pain reduction that lasts several weeks and even months. However, this isolated effect is counterproductive for the course of the disease and for the viability of the cartilage because, as it masks the symptoms, the joint is used with more intensity and its destruction is accelerated as the original problem is not corrected and the damaged articular cartilage is not restored.
[0012] Other reported methods include application of tendinous, periosteal, fascial, muscular or perichondral grafts; implantation of fibrin or cultured chondrocytes; implantation of synthetic matrices, such as collagen, carbon fiber; administration of electromagnetic fields. All of these have reported minimal and incomplete results with formation of repair, but not regenerative tissue, resulting in a poor quality tissue that can neither support the weighted load nor allow the restoration of an articular function with normal movement.
[0013] Stimulation of the anabolic processes, blocking catabolic processes, or a combination of these two, may result in stabilization of the cartilage, and perhaps even reversion of the damage, and therefore prevent further progression of the disease. Various triggers may stimulate anabolic stimulation of chondrocytes. Insulin-like growth factor-I (IGF-I) is the predominant anabolic growth factor in synovial fluid and stimulates the synthesis of both proteoglycans and collagen. It has also been shown that members of the bone morphogenetic protein (BMP) family, notably BMP2, BMP4, BMP6, and BMP7, and members of the human transforming growth factor-b (TGF-b) family can induce chondrocyte anabolic stimulation (Chubinskaya and Kuettner, 2003). A compound has recently been identified that induces anabolic stimulation of chondrocytes (U.S. Pat. No. 6,500,854; EP 1.391211). However, most of these compounds show severe side effects and, consequently, there is a strong need for compounds that stimulate chondrocyte differentiation without severe side effects.
[0014] The present invention relates to the relationship between the function of selected proteins identified by the present inventors (hereinafter referred to as "TARGETS") and anabolic stimulation of chondrocytes.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a method for identifying compounds that induce cartilage-synthesizing processes, which lead to anabolic stimulation of chondrocytes, comprising contacting the compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391 or a functional fragment or derivative thereof, under conditions that allow said polypeptide to bind to the compound, and measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes.
[0016] The present invention also relates to expression inhibitory agents, pharmaceutical compositions comprising the same, methods for the in vitro production of cartilage tissue, and host cells expressing said agents.
[0017] Aspects of the present method include the in vitro assay of compounds using polypeptide of a TARGET, and cellular assays wherein TARGET inhibition is followed by observing indicators of efficacy, including collagen type II, alpha-1 (col2α1) and aggrecan levels.
[0018] Another aspect of the invention is a method of treatment or prevention of a condition involving de-differentiation of chondrocytes and/or loss of cartilage thickness, in a subject suffering or susceptible thereto, by administering a pharmaceutical composition comprising an effective cartilage formation-enhancing amount of a TARGET inhibitor.
[0019] A further aspect of the present invention is a pharmaceutical composition for use in said method wherein said inhibitor comprises a polynucleotide selected from the group of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said inhibitor comprises a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391 or a fragment thereof.
[0020] Another further aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective cartilage formation-enhancing amount of a TARGET inhibitor or its pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof in admixture with a pharmaceutically acceptable carrier. The present polynucleotides and TARGET inhibitor compounds are also useful for the manufacturing of a medicament for the treatment of conditions involving de-differentiation of chondrocytes and/or cartilage thickness loss.
[0021] Furthermore, the invention relates also to diagnostic methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. Quantification of Col2α1 expression in primary human chondrocytes 12 days post infection with the indicated viruses.
[0023] FIG. 2. Example of duplicate screening results results for Col2α1 expression of part of the SILENCESELECT® library.
[0024] FIG. 3 (A-N): Quantification of Col2α1 expression in primary human chondrocytes 12 days post infection with the indicated viruses.
[0025] FIG. 4. Quantification of alcian blue staining on human primary chondrocytes, 12 days after infection with the indicated viruses, compared to uninfected cells.
[0026] FIG. 5 (A-N): Quantification of aggrecan expression in primary human chondrocytes 12 days post infection with the indicated viruses.
[0027] FIG. 6 (A-K): On target analysis with different constructs targeting the indicated genes. The on target analysis is assessed through detection of the Col2α1 expression in primary human chondrocytes 12 days post infection with the indicated viruses. Data are represented as luminescence units. The different thresholds corresponding to the different infection volumes are indicated as a line. Increasing the infection volumes leads to an increased threshold.
[0028] FIG. 7 (A-L): GAG analysis on chondrocytes in alginate cell culture 10 days post infection. The data are represented as relative GAG levels to the average of KD control 1 and KD control 2, being Ad-siRNA targeting PTGER4 and GRM7 respectively. Two individual data points are shown for every condition.
[0029] FIG. 8 (A-L): Hydroxyproline analysis on chondrocytes in alginate cell culture 10 days post infection. The data are represented as relative hydroxyproline levels to the average of KD control 1 and KD control 2, being Ad-siRNA targeting PTGER4 and GRM7 respectively. Two individual data points are shown for every condition.
[0030] FIG. 9 (A-L): mRNA marker analysis on chondrocytes in alginate cell culture 10 days post infection. The cells are infected with either Ad5/ALPL and Ad5/BMP2 overexpressing viruses, and with an Ad-siRNA targeting the indicated gene. The data are represented as relative mRNA levels to the ALPL control.
DETAILED DESCRIPTION
[0031] The following terms are used herein in accordance with the following definitions:
[0032] The term "agent" means any molecule, including polypeptides, polynucleotides and small molecules.
[0033] The term "anabolic stimulation of chondrocytes" should be understood as inducing chondrogenesis or as inducing or enhancing the anabolic activity of chondrocytes. Anabolic stimulation takes place for instance by stimulating the synthesis of cartilage components, or inducing synthesis of components that are required for cartilage synthesis. "Anabolic stimulation of chondrocytes" may also be understood as a process in which the expression of the matrix Gla protein (MGP) is induced. Anabolic stimulation of chondrocytes may furthermore be understood as inducing the expression of cartilage derived retinoic acid sensitive protein (CD-RAP), as inducing the expression of cartilage oligomeric matrix protein (COMP), as inducing the expression of aggrecan 1 (agc1, also termed chondroitin sulfate proteoglycan core protein 1, or CSPG1), or as inducing synthesis of collagen II, also known as collagen, type II, alpha-1 (col2α1), collagen of cartilage, chondrocalcin, and collagen, type xi, alpha-3 (col11α3).
[0034] The term `antisense nucleic acid` refers to an oligonucleotide that has a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of the target such that the expression of the gene is reduced. Preferably, the specific nucleic acid sequence involved in the expression of the gene is a genomic DNA molecule or mRNA molecule that encodes (a part of) the gene. This genomic DNA molecule can comprise regulatory regions of the gene, or the coding sequence for the mature gene.
[0035] The term "assay" means any process used to measure a specific property of a compound. A "screening assay" means a process used to characterize or select compounds based upon their activity from a collection of compounds.
[0036] The term "binding affinity" is a property that describes how strongly two or more compounds associate with each other in a non-covalent relationship. Binding affinities can be characterized qualitatively, (such as "strong", "weak", "high", or "low") or quantitatively (such as measuring the KD).
[0037] The term "carrier" means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition. A carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution. Examples of physiologically acceptable carriers include aqueous or solid buffer ingredients including 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®.
[0038] The term "complex" means the entity created when two or more compounds bind to each other.
[0039] The term "compound" is used herein in the context of a "test compound" or a "drug candidate compound" described in connection with the assays of the present invention. As such, these compounds comprise organic or inorganic compounds, derived synthetically or from natural sources. The compounds include inorganic or organic compounds such as polynucleotides, lipids or hormone analogs that are characterized by relatively low molecular weights. Other biopolymeric organic test compounds include peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
[0040] The term `complementary to a nucleotide sequence` in the context of antisense oligonucleotides and methods should be understood as sufficiently complementary to such a sequence as to allow hybridization to that sequence in a cell, i.e., under physiological conditions.
[0041] The term "condition" or "disease" means the overt presentation of symptoms (i.e., illness) or the manifestation of abnormal clinical indicators (e.g., biochemical indicators). Alternatively, the term "disease" refers to a genetic or environmental risk of or propensity for developing such symptoms or abnormal clinical indicators.
[0042] The term "contact" or "contacting" means bringing at least two moieties together, whether in an in vitro system or an in vivo system.
[0043] The term "de-differentiation" refers to a general process wherein chondrocytes differentiate away from a cell phenotype that synthesizes cartilage components. Such components include, but are not limited to, collagen II, aggrecan 1, versican, link protein, perlecan, SZP/lubricin, biglycan (DS-PGI), decorin (DS-PGII), epiphycan (DS-PGIII), fibromodulin, lumican, CILP, C-type lectin, fibronectin, PRELP, COMP (thrombospondin-5), thrombospondin-1 and -3, CMP (matrilin-1), matrilin-3, C-type lectin, fibronectin, condroadherin, tenascin-C, fibrillin, elastin, gp-39/YKL-40, matrix Gla protein/MGP, pleiotrophin, chondromodulin-I/SCGP, chondromodulin-II, CD-RAP, chondrocalcin, PARP, lysozyme, and phospholipase A2.
[0044] The term "effective amount" or "therapeutically effective amount" means that amount of a compound or agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to inducing anabolic stimulation of chondrocytes, the term "effective amount" is intended to mean an effective differentiation-promoting amount of a compound or agent that will bring about a biologically meaningful increase in the levels of chondrocyte markers, representative for the process of an increase in chondrocyte anabolism.
[0045] The term "expressible nucleic acid" means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.
[0046] The term "endogenous" shall mean a material that a mammal naturally produces. Endogenous in reference to the term "protease", "kinase", or G-Protein Coupled Receptor ("GPCR") shall mean that which is naturally produced by a mammal (for example, and not limitation, a human). In contrast, the term non-endogenous in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human). Both terms can be utilized to describe both "in vivo" and "in vitro" systems. For example, and not a limitation, in a screening approach, the endogenous or non-endogenous TARGET may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous TARGET, screening of a candidate compound by means of an in vivo system is viable.
[0047] The term "expression" comprises both endogenous expression and overexpression by transduction.
[0048] The term "expression inhibitory agent" means a polynucleotide designed to interfere selectively with the transcription, translation and/or expression of a specific polypeptide or protein normally expressed within a cell. More particularly, "expression inhibitory agent" comprises a DNA or RNA molecule that contains a nucleotide sequence identical to or complementary to at least about 17 sequential nucleotides within the polyribonucleotide sequence coding for a specific polypeptide or protein. Exemplary expression inhibitory molecules include ribozymes, double stranded siRNA molecules, self-complementary single-stranded siRNA molecules, genetic antisense constructs, and synthetic RNA antisense molecules with modified stabilized backbones.
[0049] The term "expressible nucleic acid" means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.
[0050] The term `genetic antisense` as used herein refers to the incorporation of antisense constructs complementary to sequences of genes into the genome of a cell. Such incorporation allows for the continued synthesis of the antisense molecule.
[0051] The term "hybridization" means any process by which a strand of nucleic acid binds with a complementary strand through base pairing. The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C0t or R0t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed). The term "stringent conditions" refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature can increase stringency.
[0052] The term "inhibit" or "inhibiting", in relationship to the term "response" means that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
[0053] The term "inhibition" refers to the reduction, down regulation of a process or the elimination of a stimulus for a process that results in the absence or minimization of the expression of a protein or polypeptide.
[0054] The term "induction" refers to the inducing, up-regulation, or stimulation of a process that results in the expression of a protein or polypeptide, and that may also result in a phenotypical cellular change.
[0055] The term "ligand" means an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
[0056] The term "pharmaceutically acceptable salts" refers to the non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention.
[0057] These salts can be prepared in situ during the final isolation and purification of compounds useful in the present invention.
[0058] The term "polynucleotide" means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pairs, and more preferably 70 percent of its base pairs are in common, most preferably 90 per cent, and in a special embodiment 100 percent of its base pairs. The polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof. The polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, preferably about 100 to about 4000 bases, more preferably about 250 to about 2500 bases. A preferred polynucleotide embodiment comprises from about 10 to about 30 bases in length. A special embodiment of polynucleotide is the polyribonucleotide of from about 10 to about 22 nucleotides, more commonly described as small interfering RNAs (siRNAs). Another special embodiment are nucleic acids with modified backcartilages such as peptide nucleic acid (PNA), polysiloxane, and 2'-O-(2-methoxy)ethylphosphorothioate, or including non-naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection.
[0059] The term "polypeptide" relates to proteins (such as TARGETS), proteinaceous molecules, fractions of proteins peptides and oligopeptides.
[0060] The term `ribozymes` as used herein relates to catalytic RNA molecules capable of cleaving other RNA molecules at phosphodiester bonds in a manner specific to the sequence.
[0061] The term "solvate" means a physical association of a compound useful in this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.
[0062] The term "subject" includes humans and other mammals.
[0063] The term "treating" means an intervention performed with the intention of preventing the development or altering the pathology of, and thereby alleviating a disorder, disease or condition, including one or more symptoms of such disorder or condition. Accordingly, "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treating include those already with the disorder as well as those in which the disorder is to be prevented. The related term "treatment," as used herein, refers to the act of treating a disorder, symptom, disease or condition, as the term "treating" is defined above.
[0064] The term `vectors` also relates to plasmids as well as to viral vectors, such as recombinant viruses, or the nucleic acid encoding the recombinant virus.
[0065] The term "vertebrate cells" means cells derived from animals having vertera structure, including fish, avian, reptilian, amphibian, marsupial, and mammalian species. Preferred cells are derived from mammalian species, and most preferred cells are human cells. Mammalian cells include feline, canine, bovine, equine, caprine, ovine, porcine murine, such as mice and rats, and rabbits.
Applicants' Invention Based on TARGET Relationship to Anabolic Stimulation of Chondrocytes
[0066] As noted above, the present invention is based on the present inventors' discovery that TARGETS are factors in the up-regulation and/or induction of anabolic processes of chondrocytes. The term "TARGET" or "TARGETS" means the proteins identified in accordance with the assay described below to be involved in the induction of the anabolic stimulation of chondrocytes. The present inventors have identified such TARGETS by screening recombinant adenoviruses mediating the expression of a library of shRNAs, referred to herein as "Ad-siRNAs." The collection used herein is further referred to as "adenoviral siRNA library" or SILENCESELECT® collection. These libraries contain recombinant adenoviruses, further referred to as knock-down (KD) viruses or Ad-siRNAs, that mediate the expression in cells of shRNAs which reduce the expression levels of targeted genes by a RNA interference (RNAi)-based mechanism (WO03/020931).
[0067] The preferred TARGETS are identified as SEQ ID NOS. 55-82 and 198-391 in Table 1A. Table 1A lists the polypeptides, polynucleotides and knock-down target sequences of the present invention. Table 1B lists exemplary fragments of the TARGETS, SEQ ID NOS. 198-391. Table 1C lists exemplary KD target sequences useful in the practice of the present expression-inhibitory agent invention.
TABLE-US-00001 TABLE 1A Target DNA KD Hit Gene GenBank or Pro- SEQ ID KD Target Sequence Symbol Accession Name Class mRNA tein NO. H33- CCTGAATGTGACTGTGGAC DGKB- NM_020238 diacylglycerol Kinase 1 55 84-91 025 (SEQ ID NO: 91) INCENP NM_004080 kinase, beta 2 56 NM_145695 90 kDa/inner 3 57 centromere pro- tein antigens 135/155 kDa H33- GACTGACTGGCCTGAAGGC ICK NM_016513 intestinal Kinase 4 58 92-99 032 (SEQ ID NO: 92) NM_014920 cell (MAK- 5 59 like) kinase H33- GATCTACACCACCTTCATC GPR103 AF411117 G protein- GPCR 6 60 100-107 034 (SEQ ID NO: 101) NM_198179 coupled re- 7 61 ceptor 103 H33- GGTGTATGGGCTCATGTAC FZD1 NM_003505 frizzled GPCR 8 62 108-114 041 (SEQ ID NO: 108) homolog 1 (Drosophila) H33- AGAACTGGGTGATGACAGC ELA1 NM_001971 elastase 1, Protease 9 63 115-122 056 (SEQ ID NO: 116) pancreatic H33- ATGAACTCTGTGATCCAGC USP9Y NM_004654 ubiquitin Protease 10 64 123-130 061 (SEQ ID NO: 123) specific pro- tease 9, Y- linked (fat facets-like, Drosophila) H33- TTGGAATTCCAGTGTACCC DUSP11 NM_003584 dual speci- Phospha- 11 65 131-138 082 (SEQ ID NO: 132) ficity phos- tase phatase 11 (RNA/RNP complex 1- interacting) H33- GCTAGTTATCGCCTACCTC DUSP3 NM_004090 dual speci- Phospha- 12 66 139-145 083 (SEQ ID NO: 139) ficity phos- tase phatase 3 (vaccinia virus phos- phatase VH1- related) H33- AGATTCCAGATGCAACCCC JAK1 SK185- Janus kinase Kinase 13 67 146-154 096 (SEQ ID NO: 148) NM_002227 1 (a protein 14 68 tyrosine kinase) H33- CTGAACTACTGGTACAGCC ABCG1 NM_016818 ATP-binding Trans- 15 69 155-162 107 (SEQ ID NO: 157) NM_004915 cassette, porter 16 70 NM_207174 sub-family 17 71 NM_207627 G (WHITE), 18 72 NM_207628 member 1 19 73 NM_207629 20 74 NM_207630 21 75 H33- GCGAATTCCACCAGCATTC SLC26A NM_052961 solute carrier Trans- 22 76 163-175 130 (SEQ ID NO: 165) 8 family porter 26, member 8 H33- ACATTGACCAGGAAGTGAC GGTLA4 NM_178312 gamma- Enzyme 23 77 176-182 192 (SEQ ID NO: 178) NM_178311 glutamyltrans- 24 78 NM_080920 ferase-like 25 79 activity 4 H33- GAAGCTGAATTAGGGCTTC PDE1A NM_005019 phosphodi- PDE 26 80 183-190 217 (SEQ ID NO: 184) NM_00100- esterase 1A, 27 81 3683 calmodulin- dependent H33- GAAGCCATCTCCGACAATC SLC15A NM_021082 solute carrier Trans- 28 82 191-197 279 (SEQ ID NO: 192) 2 family 15 porter (H+/peptide transporter), member 2
TABLE-US-00002 TABLE 1B Seq Accession Name Protein Segment ID protein segment AF411117 GPR103 Extracellular domain 198 AF411117 GPR103 Transmembrane domain 199 AF411117 GPR103 Intracellular domain 200 AF411117 GPR103 Transmembrane domain 201 AF411117 GPR103 Extracellular domain 202 AF411117 GPR103 Transmembrane domain 203 AF411117 GPR103 Intracellular domain 204 AF411117 GPR103 Transmembrane domain 205 AF411117 GPR103 Extracellular domain 206 AF411117 GPR103 Transmembrane domain 207 AF411117 GPR103 Intracellular domain 208 AF411117 GPR103 Transmembrane domain 209 AF411117 GPR103 Extracellular domain 210 NM_198179 GPR103 Extracellular domain 211 NM_198179 GPR103 Transmembrane domain 212 NM_198179 GPR103 Intracellular domain 213 NM_198179 GPR103 Transmembrane domain 214 NM_198179 GPR103 Extracellular domain 215 NM_198179 GPR103 Transmembrane domain 216 NM_198179 GPR103 Intracellular domain 217 NM_198179 GPR103 Transmembrane domain 218 NM_198179 GPR103 Extracellular domain 219 NM_198179 GPR103 Transmembrane domain 220 NM_198179 GPR103 Intracellular domain 221 NM_198179 GPR103 Transmembrane domain 222 NM_198179 GPR103 Extracellular domain 223 NM_198179 GPR103 Transmembrane domain 224 NM_198179 GPR103 Intracellular domain 225 NM_003505 FZD1 Extracellular domain 226 NM_003505 FZD1 Transmembrane domain 227 NM_003505 FZD1 Intracellular domain 228 NM_003505 FZD1 Transmembrane domain 229 NM_003505 FZD1 Extracellular domain 230 NM_003505 FZD1 Transmembrane domain 231 NM_003505 FZD1 Intracellular domain 232 NM_003505 FZD1 Transmembrane domain 233 NM_003505 FZD1 Extracellular domain 234 NM_003505 FZD1 Transmembrane domain 235 NM_003505 FZD1 Intracellular domain 236 NM_003505 FZD1 Transmembrane domain 237 NM_003505 FZD1 Extracellular domain 238 NM_003505 FZD1 Transmembrane domain 239 NM_003505 FZD1 Intracellular domain 240 NM_016818 ABCG1 Extracellular domain 241 NM_016818 ABCG1 Transmembrane domain 242 NM_016818 ABCG1 Intracellular domain 243 NM_016818 ABCG1 Transmembrane domain 244 NM_016818 ABCG1 Extracellular domain 245 NM_016818 ABCG1 Transmembrane domain 246 NM_016818 ABCG1 Intracellular domain 247 NM_016818 ABCG1 Transmembrane domain 248 NM_016818 ABCG1 Extracellular domain 249 NM_016818 ABCG1 Transmembrane domain 250 NM_016818 ABCG1 Intracellular domain 251 NM_016818 ABCG1 Transmembrane domain 252 NM_016818 ABCG1 Extracellular domain 253 NM_016818 ABCG1 Transmembrane domain 254 NM_016818 ABCG1 Intracellular domain 255 NM_004915 ABCG1 Extracellular domain 256 NM_004915 ABCG1 Transmembrane domain 257 NM_004915 ABCG1 Intracellular domain 258 NM_004915 ABCG1 Transmembrane domain 259 NM_004915 ABCG1 Extracellular domain 260 NM_004915 ABCG1 Transmembrane domain 261 NM_004915 ABCG1 Intracellular domain 262 NM_004915 ABCG1 Transmembrane domain 263 NM_004915 ABCG1 Extracellular domain 264 NM_004915 ABCG1 Transmembrane domain 265 NM_004915 ABCG1 Intracellular domain 266 NM_004915 ABCG1 Transmembrane domain 267 NM_004915 ABCG1 Extracellular domain 268 NM_004915 ABCG1 Transmembrane domain 269 NM_004915 ABCG1 Intracellular domain 270 NM_207174 ABCG1 Extracellular domain 271 NM_207174 ABCG1 Transmembrane domain 272 NM_207174 ABCG1 Intracellular domain 273 NM_207174 ABCG1 Transmembrane domain 274 NM_207174 ABCG1 Extracellular domain 275 NM_207174 ABCG1 Transmembrane domain 276 NM_207174 ABCG1 Intracellular domain 277 NM_207174 ABCG1 Transmembrane domain 278 NM_207174 ABCG1 Extracellular domain 279 NM_207174 ABCG1 Transmembrane domain 280 NM_207174 ABCG1 Intracellular domain 281 NM_207174 ABCG1 Transmembrane domain 282 NM_207174 ABCG1 Extracellular domain 283 NM_207174 ABCG1 Transmembrane domain 284 NM_207174 ABCG1 Intracellular domain 285 NM_207627 ABCG1 Extracellular domain 286 NM_207627 ABCG1 Transmembrane domain 287 NM_207627 ABCG1 Intracellular domain 288 NM_207627 ABCG1 Transmembrane domain 289 NM_207627 ABCG1 Extracellular domain 290 NM_207627 ABCG1 Transmembrane domain 291 NM_207627 ABCG1 Intracellular domain 292 NM_207627 ABCG1 Transmembrane domain 293 NM_207627 ABCG1 Extracellular domain 294 NM_207627 ABCG1 Transmembrane domain 295 NM_207627 ABCG1 Intracellular domain 296 NM_207627 ABCG1 Transmembrane domain 297 NM_207627 ABCG1 Extracellular domain 298 NM_207627 ABCG1 Transmembrane domain 299 NM_207627 ABCG1 Intracellular domain 300 NM_207628 ABCG1 Extracellular domain 301 NM_207628 ABCG1 Transmembrane domain 302 NM_207628 ABCG1 Intracellular domain 303 NM_207628 ABCG1 Transmembrane domain 304 NM_207628 ABCG1 Extracellular domain 305 NM_207628 ABCG1 Transmembrane domain 306 NM_207628 ABCG1 Intracellular domain 307 NM_207628 ABCG1 Transmembrane domain 308 NM_207628 ABCG1 Extracellular domain 309 NM_207628 ABCG1 Transmembrane domain 310 NM_207628 ABCG1 Intracellular domain 311 NM_207628 ABCG1 Transmembrane domain 312 NM_207628 ABCG1 Extracellular domain 313 NM_207628 ABCG1 Transmembrane domain 314 NM_207628 ABCG1 Intracellular domain 315 NM_207629 ABCG1 Extracellular domain 316 NM_207629 ABCG1 Transmembrane domain 317 NM_207629 ABCG1 Intracellular domain 318 NM_207629 ABCG1 Transmembrane domain 319 NM_207629 ABCG1 Extracellular domain 320 NM_207629 ABCG1 Transmembrane domain 321 NM_207629 ABCG1 Intracellular domain 322 NM_207629 ABCG1 Transmembrane domain 323 NM_207629 ABCG1 Extracellular domain 324 NM_207629 ABCG1 Transmembrane domain 325 NM_207629 ABCG1 Intracellular domain 326 NM_207629 ABCG1 Transmembrane domain 327 NM_207629 ABCG1 Extracellular domain 328 NM_207629 ABCG1 Transmembrane domain 329 NM_207629 ABCG1 Intracellular domain 330 NM_207630 ABCG1 Extracellular domain 331 NM_207630 ABCG1 Transmembrane domain 332 NM_207630 ABCG1 Intracellular domain 333 NM_207630 ABCG1 Transmembrane domain 334 NM_207630 ABCG1 Extracellular domain 335 NM_207630 ABCG1 Transmembrane domain 336 NM_207630 ABCG1 Intracellular domain 337 NM_207630 ABCG1 Transmembrane domain 338 NM_207630 ABCG1 Extracellular domain 339 NM_207630 ABCG1 Transmembrane domain 340 NM_207630 ABCG1 Intracellular domain 341 NM_207630 ABCG1 Transmembrane domain 342 NM_207630 ABCG1 Extracellular domain 343 NM_207630 ABCG1 Transmembrane domain 344 NM_207630 ABCG1 Intracellular domain 345 NM_052961 SLC26A8 Intracellular domain 346 NM_052961 SLC26A8 Transmembrane domain 347 NM_052961 SLC26A8 Extracellular domain 348 NM_052961 SLC26A8 Transmembrane domain 349 NM_052961 SLC26A8 Intracellular domain 350 NM_052961 SLC26A8 Transmembrane domain 351 NM_052961 SLC26A8 Extracellular domain 352 NM_052961 SLC26A8 Transmembrane domain 353 NM_052961 SLC26A8 Intracellular domain 354 NM_052961 SLC26A8 Transmembrane domain 355 NM_052961 SLC26A8 Extracellular domain 356 NM_052961 SLC26A8 Transmembrane domain 357 NM_052961 SLC26A8 Intracellular domain 358 NM_052961 SLC26A8 Transmembrane domain 359 NM_052961 SLC26A8 Extracellular domain 360 NM_052961 SLC26A8 Transmembrane domain 361 NM_052961 SLC26A8 Intracellular domain 362 NM_052961 SLC26A8 Transmembrane domain 363 NM_052961 SLC26A8 Extracellular domain 364 NM_052961 SLC26A8 Transmembrane domain 365 NM_052961 SLC26A8 Intracellular domain 366 NM_052961 SLC26A8 Transmembrane domain 367 NM_052961 SLC26A8 Extracellular domain 368 NM_021082 SLC15A2 Intracellular domain 369 NM_021082 SLC15A2 Transmembrane domain 370 NM_021082 SLC15A2 Extracellular domain 371 NM_021082 SLC15A2 Transmembrane domain 372 NM_021082 SLC15A2 Intracellular domain 373 NM_021082 SLC15A2 Transmembrane domain 374 NM_021082 SLC15A2 Extracellular domain 375 NM_021082 SLC15A2 Transmembrane domain 376 NM_021082 SLC15A2 Intracellular domain 377 NM_021082 SLC15A2 Transmembrane domain 378 NM_021082 SLC15A2 Extracellular domain 379 NM_021082 SLC15A2 Transmembrane domain 380 NM_021082 SLC15A2 Intracellular domain 381 NM_021082 SLC15A2 Transmembrane domain 382 NM_021082 SLC15A2 Extracellular domain 383 NM_021082 SLC15A2 Transmembrane domain 384 NM_021082 SLC15A2 Intracellular domain 385 NM_021082 SLC15A2 Transmembrane domain 386 NM_021082 SLC15A2 Extracellular domain 387 NM_021082 SLC15A2 Transmembrane domain 388 NM_021082 SLC15A2 Intracellular domain 389 NM_021082 SLC15A2 Transmembrane domain 390 NM_021082 SLC15A2 Extracellular domain 391
TABLE-US-00003 TABLE 1C SEQ ID TARGET Name siRNA_Name KD Target Sequence NO. ABCG1 A150100-ABCG1_v5 NM_004915_idx1797 AGTGGATGTCCTACATCTC 155 A150100-ABCG1_v6 NM_004915_idx500 ATCATGCAGGATGACATGC 156 A150100-ABCG1_v7 NM_004915_idx1481 CTGAACTACTGGTACAGCC 157 A150100-ABCG1_v10 NM_004915_idx872 CAGCTTTACGTCCTGAGTC 158 A150100-ABCG1_v11 NM_004915_idx1067 TCAGACCACAAGAGAGACC 159 A150100-ABCG1_v12 NM_004915_idx1789 GTACCTACAGTGGATGTCC 160 A150100-ABCG1_v8 NM_016818_idx603 TGGTCAAGGAGATACTGAC 161 A150100-ABCG1_v9 NM_016818_idx718 CCCTCCAGTCATGTTCTTC 162 DGKB A150100-DGKB_v1 NM_004080_idx104 TTCCATGGTAATGGTGTGC 84 A150100-DGKB_v2 NM_004080_idx1064 CCTGAATGTGACTGTGGAC 91 A150100-DGKB_v3 NM_004080_idx2398 CCGAAGCAAGGAATAATCC 85 A150100-DGKB_v10 NM_145695_idx466 TATGTTTCGCCTTTATGAC 86 A150100-DGKB_v11 NM_145695_idx654 GGATTCAAGGAGGAATGAC 87 A150100-DGKB_v12 NM_145695_idx870 CTCCCTCTTGCATCAAGAC 88 A150100-DGKB_v13 NM_145695_idx1387 AAATCCTCGTCAGGTTTAC 89 A150100-DGKB_v14 NM_145695_idx1729 AGTGCCTTACAGTATCATC 90 DUSP11 A150100-DUSP11_v1 NM_003584_idx427 CAGAGGATTTGCCAGAAAC 131 A150100-DUSP11_v2 NM_003584_idx743 TTGGAATTCCAGTGTACCC 132 A150100-DUSP11_v3 NM_003584_idx945 CAGAGACACCATCTCCCTC 133 A150100-DUSP11_v4 NM_003584_idx885 ACCCAGACCCAAAGTTTGC 134 A150100-DUSP11_v5 NM_003584_idx221 AAGGTGGAAAGACTATCTC 135 A150100-DUSP11_v6 NM_003584_idx420 TATAAACCAGAGGATTTGC 136 A150100-DUSP11_v7 NM_003584_idx836 GTATAATCTACATCAGATC 137 A150100-DUSP11_v8 NM_003584_idx933 CCACATGTTTACCAGAGAC 138 DUSP3 A150100-DUSP3_v1 NM_004090_idx425 GCTAGTTATCGCCTACCTC 139 A150100-DUSP3_v2 NM_004090_idx300 GACACACAGGAGTTCAACC 140 A150100-DUSP3_v3 NM_004090_idx176 GCTGCAGAAACTAGGCATC 141 A150100-DUSP3_v4 NM_004090_idx248 TGCCAACTTCTACAAGGAC 142 A150100-DUSP3_v5 NM_004090_idx299 CGACACACAGGAGTTCAAC 143 A150100-DUSP3_v6 NM_004090_idx458 GATGGACGTCAAGTCTGCC 144 A150100-DUSP3_v7 NM_004090_idx4305 ACAGGAGTTCAACCTCAGC 145 ELA1 A150100-ELA1_v1 NM_001971_idx754 TAATGTCATCGCCTCCAAC 115 A150100-ELA1_v2 NM_001971_idx162 AGAACTGGGTGATGACAGC 116 A150100-ELA1_v3 NM_001971_idx421 CAACAGTCCCTGCTACATC 117 A150100-ELA1_v4 NM_001971_idx280 GATCGTGGTGCATCCATAC 118 A150100-ELA1_v5 NM_001971_idx230 GCGTGGATTACCAGAAGAC 119 A150100-ELA1_v6 NM_001971_idx459 TAACAACAGTCCCTGCTAC 120 A150100-ELA1_v7 NM_001971_idx669 CCATTGCTTGGTGAATGGC 121 A150100-ELA1_v8 NM_001971_idx692 ATTCTCTCCATGGAGTGAC 122 FZD1 A150100-FZD1_v10 NM_003505_idx1323 GGTGTATGGGCTCATGTAC 108 A150100-FZD1_v11 NM_003505_idx2058 CATCGTCATCGCCTGCTAC 109 A150100-FZD1_v9 NM_003505_idx2007 GCTCATGGTGCGCATTGGC 110 A150100-FZD1_v12 NM_003505_idx2229 GTACCTTATGACGCTGATC 111 A150100-FZD1_v13 NM_003505_idx3317 ACCTGGTATGGGTTTGGCC 112 A150100-FZD1_v14 NM_003505_idx3883 ATGTGTGCAGGTCTACTGC 113 A150100-FZD1_v15 NM_003505_idx2704 TTATTTAGGGCGGTTTAAC 114 GGTLA4 A150100-GGT1_v8 NM_080839_idx451 ACTGGCCATCATCTACAAC 176 A150100-GGTLA4_v5 NM_080920_idx292 TGCTCACCTGTCTGTGGTC 177 A150100-GGTLA4_v6 NM_080920_idx702 ACATTGACCAGGAAGTGAC 178 A150100-GGTLA4_v7 NM_080920_idx411 TGGATGACTTCAGCTCTAC 179 A150100-GGT1_v10 NM_178311_idx629 CTACAACCTCTGGTTCGGC 180 A150100-GGT1_v11 NM_178311_idx707 CACGACAGTGGAGAGAAAC 181 A150100-GGTLA4_v8 NM_178311_idx287 GTTCTACATGCCGGATGAC 182 GPR103 A150100-GPR103_v5 AF411117_idx611 AGGCACCAGGGACTTGTGC 100 A150100-GPR103_v6 AF411117_idx820 GATCTACACCACCTTCATC 101 A150100-GPR103_v7 XM_172359_idx288 TGGTGTTCTACGTGGTGAC 102 A150100-GPR103_v8 AF411117_idx136 TGTTAGGCGCCTGCATTGC 103 A150100-GPR103_v10 AF411117_idx424 CAACATCTTTATCTGCTCC 104 A150100-GPR103_v11 AF411117_idx662 CGAAGGGCTTTCACAATGC 105 A150100-GPR103_v12 AF411117_idx106 GTACTACGTTGTAGCCCAC 106 A150100-GPR103_v9 AF411117_idx186 TGCAGGCGCTTAACATTAC 107 ICK A150100-ICK_v1 NM_016513_idx870 GACTGACTGGCCTGAAGGC 92 A150100-ICK_v2 NM_016513_idx1665 GCAGCACTATTTGAAGCAC 93 A150100-ICK_v3 NM_016513_idx588 GCCTGAGAACCTCCTCTGC 94 A150100-ICK_v10 NM_016513_idx1027 ACAGCTAGTCAGGCACTTC 95 A150100-ICK_v11 NM_016513_idx1707 TATAAGAAATGGCATACTC 96 A150100-ICK_v12 NM_016513_idx1754 CTAATCCATGGTCTAGTTC 97 A150100-ICK_v8 NM_016513_idx504 GTCTGCTATAAGGAATATC 98 A150100-ICK_v9 NM_016513_idx713 AAGTACTCCTGAGGTCTAC 99 JAK1 A150100-JAK1_v1 oKD271 TTGGCATGGAACCAACGAC 146 A150100-JAK1_v2 oKD270 CCTCTTTGCCCTGTATGAC 147 A150100-JAK1_v7 oKD272 AGATTCCAGATGCAACCCC 148 A150100-JAK1_v8 SK185_idx1743 CATGAGCCAGCTGAGTTTC 149 A150100-JAK1_v9 SK185_idx142 GTGGAAGTGATCTTCTATC 150 A150100-JAK1_v12 NM_002227_idx1351 TGGCTGTCATGGTCCAATC 151 A150100-JAK1_v13 NM_002227_idx2512 CCGCTGCATGAACTATGAC 152 A150100-JAK1_v14 NM_002227_idx3093 TTGGAGACTTCGGTTTAAC 153 A150100-JAK1_v15 NM_002227_idx3269 TGTGATTCAGATTCTAGTC 154 PDE1A A150100-PDE1A_v5 NM_005019_idx913 AGGTATCATGCACTGGCTC 183 A150100-PDE1A_v6 NM_005019_idx1382 GAAGCTGAATTAGGGCTTC 184 A150100-PDE1A_v7 NM_005019_idx1709 CTGGTGGACATCATTCAGC 185 A150100-PDE1A_v10 NM_005019_idx1413 TTTGTGATCGGAAGTCAAC 186 A150100-PDE1A_v11 NM_005019_idx1601 ATTGCTGATGCACTAAGAC 187 A150100-PDE1A_v12 NM_005019_idx754 CAGATATGATCTTATCAAC 188 A150100-PDE1A_v13 NM_005019_idx887 ACTGTGCATTACATAATGC 189 A150100-PDE1A_v9 NM_005019_idx1073 CACGTGAGTGCAGCTTATC 190 SLC15A2 A150100-SLC15A2_v1 NM_021082_idx457 AGTCCTATCATTGATCGGC 191 A150100-SLC15A2_v2 NM_021082_idx121 GAAGCCATCTCCGACAATC 192 A150100-SLC15A2_v3 NM_021082_idx1166 ATGGCTGTTGGTATGATCC 193 A150100-SLC15A2_v4 NM_021082_idx1575 CCGTGAGGTTTGTTAACAC 194 A150100-SLC15A2_v5 NM_021082_idx423 TTGGGTGCCTTACCAATAC 195 A150100-SLC15A2_v6 NM_021082_idx1136 CTCCAAGTGTGGAATTAAC 196 A150100-SLC15A2_v7 NM_021082_idx1534 GCATGATGGTAAAGGATAC 197 SLC26A8 A150100- NM_052961_idx1925 TTCTGCAACTGTGATGATC 163 SLC26A8_v10 A150100- NM_052961_idx2288 GTACACTACGTGGATTCAC 164 SLC26A8_v11 A150100-SLC26A8_v2 NM_052961_idx923 GCGAATTCCACCAGCATTC 165 A150100-SLC26A8_v3 NM_052961_idx1761 TCTTCCAGTGCTGCAGCTC 166 A150100-SLC26A8_v4 NM_052961_idx2693 TCAGAACAAGAGGCTGGGC 167 A150100-SLC26A8_v5 NM_052961_idx1228 GAAGATTGCCAGTCTTCAC 168 A150100-SLC26A8_v6 NM_052961_idx457 GATTCCTCCTCTCAACATC 169 A150100-SLC26A8_v7 NM_052961_idx936 GCATTCTAGTATTTCTAAC 170 A150100-SLC26A8_v8 NM_052961_idx1249 TTACAGTGTCAATTCCAAC 171 A150100-SLC26A8_v9 NM_052961_idx1723 TGATTATCGGGAGATCATC 172 A150100- NM_052961_idx338 GAATGGATGTGTATGTATC 173 SLC26A8_v12 A150100- NM_052961_idx1105 TGACATGATTCCTTATAGC 174 SLC26A8_v13 A150100- NM_052961_idx1446 TCTACACACTGCCAAATGC 175 SLC26A8_v14 USP9Y A150100-USP9Y_v1 NM_004654_idx5651 ATGAACTCTGTGATCCAGC 123 A150100-USP9Y_v2 NM_004654_idx1600 AGGTTGGCTAGTGGATCTC 124 A150100-USP9Y_v3 NM_004654_idx2636 AAGTGGGTAATTCCTGCTC 125 A150100-USP9X_v4 NM_021906_idx1189 CGAATGGCAGAATGGATAC 126 A150100-USP9X_v5 NM_004654_idx7911 TCTGGCAGGTTGCATATTC 127 A150100-USP9Y_v4 NM_004654_idx1489 CTGCAAGTTTCATATCTAC 128 A150100-USP9Y_v5 NM_004654_idx2820 ATAGCATCAGATTGTATGC 129 A150100-USP9Y_v6 NM_004654_idx5731 TTTACACGATGATATGTTC 130
[0068] The present invention relates to a method for assaying for compounds that induce anabolic stimulation of chondrocytes, comprising contacting the compound with a polypeptide comprising an amino acid sequence of the polypeptides of SEQ ID NO: 55-82 ("TARGETS") or a functional fragment thereof under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound. One preferred means of measuring the complex formation is to determine the binding affinity of said compound to said polypeptide.
[0069] More particularly, the invention relates to a method for identifying an agent that induces anabolic stimulation of chondrocytes, the method comprising further:
[0070] (a) contacting a population of chondrocyte cells with one or more of said compound that exhibits binding affinity for said TARGETS, and
[0071] (b) measuring a compound-polypeptide property related to the anabolic stimulation of chondrocytes.
[0072] The compound-polypeptide property referred to above is related to the anabolic stimulation of chondrocytes, and is a measurable phenomenon chosen by the person of ordinary skill in the art. The measurable property may e.g. be the binding affinity for a peptide domain of the polypeptide TARGET or the level of any one of a number of biochemical marker levels of increased chondrocyte anabolism. Anabolic stimulation of chondrocytes can e.g. be measured by measuring the level of proteins and other molecules that are induced during the differentiation process, such as key components of normal cartilage. In particular, the induction of the major protein component of cartilage, collagen II, is measured.
[0073] In addition, compound-polypeptide properties related to the anabolic stimulation of chondrocytes are measured in C20/A4; T/C-28a2; T/C-28a4; C-28/12; Ch-4,8,N; Ch-8-OA; TC6; MCT; MC615; IRC; RCS2; Hig82; and D1 ORL UVA (D1) cells. However, such porterties are also measured in non-chondrocyte cell systems. For example, in situ binding assays that determine the affinity of compounds to bind to polypeptides of the invention are performed using any cell type that expresses the polypeptide. Expression of the polypeptide is exogenous or endogenous. Furthermore, when the compound-polypeptide property is activation of a biological pathway, any cell that contains the pathway cellular components is used to measure the compound-polypeptide property. For example, induction of col2α1 or aggrecan in chondrocytes is indicative of anabolic stimulation of chondrocytes. Specifically, non-chondrocyte cells can be engineered to contain a reporter molecule activated by the col2α1 or aggrecan promoters. In this way a non-chondrocyte can be used to measure a property indicative of anabolic stimulation of chondrocytes.
[0074] The invention relates to a method for identifying a compound that induces and/or increases anabolic stimulation of chondrocytes, said method comprising the steps of: culturing a population of cells expressing a polypeptide of any one of those listed in Table 1A, or a functional fragment or derivative thereof; determining a first level of chondrogenic differentiation in said population of cells; exposing said population of cells to a compound, or a mixture of compounds; determining the level of chondrogenic differentiation in said population of cells during or after exposure of said population of cells to the compound, or the mixture of compounds; and identifying the compound that induces and/or increases chondrogenic differentiation.
[0075] The invention also relates to a method for identifying a compound that decreases the expression and/or activity of any one of the polypeptides listed in Table 1A, said method comprising the steps of: culturing a population of cells expressing said polypeptide, or a fragment, or a derivative thereof; determining a first level of expression and/or activity of said polypeptide; exposing said population of cells to a compound, or a mixture of compounds; determining the level of expression and/or activity of said polypeptide during or after exposure of said population of cells to the compound, or the mixture of compounds; and identifying the compound that decreases the expression and/or activity of said polypeptide. If the polypeptide activity is not readily measurable, the identification of the compound may benefit from an extra step comprising exposing said population of cells to an agonist of said polypeptide. Furthermore, the methods of the present invention may comprise the step of introducing a gene encoding any one of the polypeptides listed in Table 1A, in said population of cells. For high-throughput purposes it may be beneficial to have the gene stably integrated in the genome of said cells.
[0076] In a preferred embodiment, the level of chondrocyte (re-)differentiation is determined by measuring the expression level of a marker gene, wherein a preferred marker gene encodes collagen type II, alpha-1 (col2α1) or aggrecan. For proper anabolic stimulation it is preferred that the expression and/or activity of col2α1 or aggrecan is increased.
[0077] The present invention provides in one particular embodiment methods for identifying novel compounds, wherein the polypeptide is a GPCR. If so, the expression and/or activity of said GPCR is preferably determined by measuring the level of a second messenger. Preferred second messengers are cyclic AMP, Ca2+ or both. Typically, the level of the second messenger is determined with a reporter gene under the control of a promoter that is responsive to the second messenger, wherein it is preferred that the promoter is a cyclic AMP-responsive promoter, an NF-KB responsive promoter, or a NF-AT responsive promoter, and wherein the reporter gene is selected from the group consisting of: alkaline phosphatase, GFP, eGFP, dGFP, luciferase and β-α galactosidase.
[0078] In another particular embodiment, the invention provides methods for identifying novel compounds, wherein the polypeptide is a kinase or a phosphatase. Preferably, the activity of said kinase or phosphatase is determined by measuring the level of phosphorylation of a substrate of said kinase or phosphatase.
[0079] In yet another particular embodiment, the invention provides methods for identifying novel compounds, wherein the polypeptide is a protease. Preferably, the activity of said protease is measured by determining the level of cleavage of a substrate of said protease.
[0080] Methods for determining second messenger levels, use of the reporter genes and second-messenger responsive promoters as well as phosphatase assays and protease assays are well known in the art and not further elaborated upon herein.
[0081] In a preferred embodiment, the compound that inhibits the polypeptide exhibits a binding affinity to the polypeptide of at most 10 micromolar.
[0082] In a preferred embodiment of the invention, the polypeptide TARGET comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391 (Tables 1A and 1B). In an especially preferred embodiment of the invention, the polypeptide TARGET comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 (Table 1A).
[0083] Depending on the choice of the skilled artisan, the present assay method may be designed to function as a series of measurements, each of which is designed to determine whether the drug candidate compound is indeed acting on the polypeptide to thereby induce the anabolic stimulation of chondrocytes. For example, an assay designed to determine the binding affinity of a compound to the polypeptide, or fragment thereof, may be necessary, but not sufficient, to ascertain whether the test compound would be useful for increasing mean cartilage thickness when administered to a subject. Nonetheless, such binding information would be useful in identifying a set of test compounds for use in an assay that would measure a different property, further up the biochemical pathway, such as cartilage component synthesis, assayed by measuring the amount of collagen II. Such second assay may be designed to confirm that the test compound, having binding affinity for the polypeptide, actually induces the anabolic stimulation of chondrocytes. Suitable controls should always be in place to insure against false positive readings.
[0084] The order of taking these measurements is not believed to be critical to the practice of the present invention, which may be practiced in any order. For example, one may first perform a screening assay of a set of compounds for which no information is known respecting the compounds' binding affinity for the polypeptide. Alternatively, one may screen a set of compounds identified as having binding affinity for a polypeptide domain, or a class of compounds identified as being an inhibitor of the polypeptide. However, for the present assay to be meaningful to the ultimate use of the drug candidate compounds, a measurement of collagen II levels or aggrecan is necessary. Validation studies including controls, and measurements of binding affinity to the polypeptides of the invention are nonetheless useful in identifying a compound useful in any therapeutic or diagnostic application.
[0085] The binding affinity of the compound with the polypeptide TARGET can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis), by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures TARGET function. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range.
[0086] The present assay method may also be practiced in a cellular assay, A host cell expressing TARGET can be a cell with endogenous expression or a cell over-expressing the TARGET e.g. by transduction. When the endogenous expression of the polypeptide is not sufficient to determine a baseline that can easily be measured, one may use using host cells that over-express TARGET. Over-expression has the advantage that the level of the TARGET substrate end products is higher than the activity level by endogenous expression. Accordingly, measuring such levels using presently available techniques is easier. In such cellular assay, the biological activity of TARGET may be measured by following the production of cartilage component synthesis.
[0087] The present invention further relates to a method for identifying a compound that induces anabolic stimulation of chondrocytes, comprising:
[0088] (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82 and 198-391;
[0089] (b) determining the binding affinity of the compound to the polypeptide;
[0090] (c) contacting a population of mammalian cells expressing said polypeptide with the compound that exhibits a binding affinity of at least 10 micromolar; and
[0091] (d) identifying the compound that induces the synthesis of proteins that are a constituent of normal cartilage and/or that are required for the formation of cartilage.
[0092] For high-throughput purposes, libraries of compounds may be used such as antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g. LOPAP®, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g. LOPAC®, Sigma Aldrich) or natural compound libraries (Specs, TimTec).
[0093] Preferred drug candidate compounds are low molecular weight compounds. Low molecular weight compounds, i.e. with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et al. (1997)). Peptides comprise another preferred class of drug candidate compounds. Peptides may be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. Natural compounds are another preferred class of drug candidate compound. Such compounds are found in and extracted from natural sources, and which may thereafter be synthesized. The lipids are another preferred class of drug candidate compound.
[0094] Another preferred class of drug candidate compounds is an antibody. The present invention also provides antibodies directed against a TARGET. These antibodies may be endogenously produced to bind to the TARGET within the cell, or added to the tissue to bind to TARGET polypeptide present outside the cell. These antibodies may be monoclonal antibodies or polyclonal antibodies. The present invention includes chimeric, single chain, and humanized antibodies, as well as FAb fragments and the products of a FAb expression library, and Fv fragments and the products of an Fv expression library.
[0095] In certain embodiments, polyclonal antibodies may be used in the practice of the invention. The skilled artisan knows methods of preparing polyclonal antibodies. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. Antibodies may also be generated against the intact TARGET protein or polypeptide, or against a fragment, derivatives including conjugates, or other epitope of the TARGET protein or polypeptide, such as the TARGET embedded in a cellular membrane, or a library of antibody variable regions, such as a phage display library.
[0096] It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). One skilled in the art without undue experimentation may select the immunization protocol.
[0097] In some embodiments, the antibodies may be monoclonal antibodies. Monoclonal antibodies may be prepared using methods known in the art. The monoclonal antibodies of the present invention may be "humanized" to prevent the host from mounting an immune response to the antibodies. A "humanized antibody" is one in which the complementarity determining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins. Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. The humanization of antibodies may be accomplished by methods known in the art (see, e.g. Mark and Padlan, (1994) "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer-Verlag, New York). Transgenic animals may be used to express humanized antibodies.
[0098] Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol. 222:581-97). The techniques of Cole, et al. and Boerner, et al. are also available for the preparation of human monoclonal antibodies (Cole, et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner, et al (1991). J. Immunol., 147(1):86-95).
[0099] Techniques known in the art for the production of single chain antibodies can be adapted to produce single chain antibodies to the TARGET polypeptides and proteins of the present invention. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively; the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.
[0100] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens and preferably for a cell-surface protein or receptor or receptor subunit. In the present case, one of the binding specificities is for one domain of the TARGET; the other one is for another domain of the same or different TARGET.
[0101] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, (1983) Nature 305:537-9). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps usually accomplish the purification of the correct molecule. Similar procedures are disclosed in Trauneeker, et al. (1991) EMBO J. 10:3655-9.
[0102] According to another preferred embodiment, the assay method uses a drug candidate compound identified as having a binding affinity for a TARGET, and/or has already been identified as having down-regulating activity such as antagonist activity vis-a-vis one or more TARGET.
[0103] The present invention further relates to a method for inducing anabolic stimulation of chondrocytes comprising contacting said cells with an expression inhibitory agent comprising a polynucleotide sequence that complements at least about 17 nucleotides of the polyribonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-28. In a preferred embodiment the expression-inhibitory agent comprises a polynucleotide sequence that complements a nucleotide sequence selected from the group consisting of SEQ ID NO: 84-197 and 410.
[0104] Another aspect of the present invention relates to a method for inducing the anabolic stimulation of chondrocytes, comprising by contacting said cell with an expression-inhibiting agent that inhibits the translation in the cell of a polyribonucleotide encoding a TARGET polypeptide. A particular embodiment relates to a composition comprising a polynucleotide including at least one antisense strand that functions to pair the agent with the TARGET TARGET mRNA, and thereby down-regulate or block the expression of TARGET polypeptide. The inhibitory agent preferably comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence encoding a portion of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82. In a preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 84-197 and 410.
[0105] An embodiment of the present invention relates to a method wherein the expression-inhibiting agent is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 55-82, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 55-82, such that the siRNA, preferably shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide. Preferably the expression-inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA, complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 84-197 and 410.
[0106] A special embodiment of the present invention relates to a method wherein the expression-inhibiting agent is a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 55-82, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 55-82, such that the siRNA, preferably shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide. Preferably the nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 1-28. In an especially preferred embodiment nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 84-197 and 410.
[0107] The down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding a TARGET polypeptide or the corresponding messenger RNA. In addition, antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding a TARGET polypeptide by inhibiting splicing of its primary transcript. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for a TARGET. Preferably, the antisense sequence is at least about 17 nucleotides in length. The preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is known in the art.
[0108] One embodiment of expression-inhibitory agent is a nucleic acid that is antisense to a nucleic acid comprising SEQ ID NO: 1-28. For example, an antisense nucleic acid (e.g. DNA) may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of nucleic acids comprising SEQ ID NO: 1-28. Antisense oligonucleotides preferably comprise a sequence containing from about 17 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 18 to about 30 nucleotides. Antisense nucleic acids may be prepared from about 10 to about 30 contiguous nucleotides complementary to a nucleic acid sequence selected from the sequences of SEQ ID NO: 1-28.
[0109] The antisense nucleic acids are preferably oligonucleotides and may consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both. The antisense nucleic acids can be synthetic oligonucleotides. The oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage. Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases. In addition, when a phosphorothioate antisense oligonucleotide hybridizes to its TARGET site, the RN202-315NA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule.
[0110] In addition, antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation. Furthermore, chemical groups can be added to the 2' carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its TARGET site. Modifications may include 2'-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates, modified bases, as well as other modifications known to those of skill in the art.
[0111] Another type of expression-inhibitory agent that reduces the levels of TARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains. The substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its TARGET sequence. The catalytic portion cleaves the TARGET RNA at a specific site. The substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence. The ribozyme recognizes and then binds a TARGET mRNA through complementary base pairing. Once it is bound to the correct TARGET site, the ribozyme acts enzymatically to cut the TARGET mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its TARGET sequence, it is released and can repeatedly bind and cleave at other mRNAs.
[0112] Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).
[0113] Ribozymes may be chemically synthesized by combining an oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the TARGET mRNA after transcription. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers. The amplification product is cloned into a eukaryotic expression vector.
[0114] Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21:2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15).
[0115] A particularly preferred inhibitory agent is a small interfering RNA (siRNA, preferably shRNA). siRNA, preferably shRNA, mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA. siRNA according to the present invention comprises a sense strand of 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence selected from the group of sequences described in SEQ ID NO: 1-28, preferably from the group of sequences described in SEQ ID No: 84-197 or 410, and an antisense strand of 17-23 nucleotides complementary to the sense strand. Exemplary sequences are described as sequences complementary to SEQ ID NO: 84-197 or 410. The most preferred siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the TARGET polynucleotide sequence. Preferably the siRNA further comprises a loop region linking the sense and the antisense strand.
[0116] A self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region linker. Preferably, the loop region sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long. In a most preferred embodiment the linker sequence is UUGCUAUA (SEQ ID NO: 83). Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors.
[0117] Analogous to antisense RNA, the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified internucleoside linkages, modified nucleic acid bases, modified sugars and/or chemical linkage the siRNA to one or more moieties or conjugates. The nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the TARGET sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO2004094636, published Nov. 4, 2004, and UA20030198627, are hereby incorporated by reference).
[0118] The present invention also relates to compositions, and methods using said compositions, comprising a DNA expression vector capable of expressing a polynucleotide capable of inducing anabolic stimulation of chondrocytes and described hereinabove as an expression inhibition agent.
[0119] A special aspect of these compositions and methods relates to the down-regulation or blocking of the expression of a TARGET polypeptide by the induced expression of a polynucleotide encoding an intracellular binding protein that is capable of selectively interacting with the TARGET polypeptide. An intracellular binding protein includes any protein capable of selectively interacting, or binding, with the polypeptide in the cell in which it is expressed and neutralizing the function of the polypeptide. Preferably, the intracellular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody having binding affinity to an epitope of the TARGET polypeptide of SEQ ID NO: 55-82, 198-391. More preferably, the intracellular binding protein is a single chain antibody.
[0120] A special embodiment of this composition comprises the expression-inhibiting agent selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 55-82, and a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide coding for SEQ ID NO: 55-82, such that the siRNA interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
[0121] The polynucleotide expressing the expression-inhibiting agent is preferably included within a vector. The polynucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell. A variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral or a sendaviral vector systems, and all may be used to introduce and express polynucleotide sequence for the expression-inhibiting agents in TARGET cells.
[0122] Preferably, the viral vectors used in the methods of the present invention are replication defective. Such replication defective vectors will usually pack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome, which are necessary for encapsidating, the viral particles.
[0123] In a preferred embodiment, the viral element is derived from an adenovirus. Preferably, the vehicle includes an adenoviral vector packaged into an adenoviral capsid, or a functional part, derivative, and/or analogue thereof. Adenovirus biology is also comparatively well known on the molecular level. Many tools for adenoviral vectors have been and continue to be developed, thus making an adenoviral capsid a preferred vehicle for incorporating in a library of the invention. An adenovirus is capable of infecting a wide variety of cells. However, different adenoviral serotypes have different preferences for cells. To combine and widen the TARGET cell population that an adenoviral capsid of the invention can enter in a preferred embodiment, the vehicle includes adenoviral fiber proteins from at least two adenoviruses. Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51. Techniques or construction and expression of these chimeric vectors are disclosed in US Published Patent Applications 20030180258 and 20040071660, hereby incorporated by reference.
[0124] In a preferred embodiment, the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof. An adenoviral late protein, for instance an adenoviral fiber protein, may be favorably used to TARGET the vehicle to a certain cell or to induce enhanced delivery of the vehicle to the cell. Preferably, the nucleic acid derived from an adenovirus encodes for essentially all adenoviral late proteins, enabling the formation of entire adenoviral capsids or functional parts, analogues, and/or derivatives thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and/or analogue thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and/or analogue thereof, which facilitates, at least in part, replication of an adenoviral derived nucleic acid in a cell. The adenoviral vectors used in the examples of this application are exemplary of the vectors useful in the present method of treatment invention.
[0125] Certain embodiments of the present invention use retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV ("murine Moloney leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Lentiviral vector systems may also be used in the practice of the present invention.
[0126] In other embodiments of the present invention, adeno-associated viruses ("AAV") are utilized. The AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
[0127] In the vector construction, the polynucleotide agents of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc.
[0128] Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are lac, lacZ, T3, T7, lambda Pr, P1, and trp promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g. actin promoter in smooth muscle cells, or Flt and Flk promoters active in endothelial cells), including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, (1985) Nature 315:115-22), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature 318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95), albumin gene control region which is active in liver (Pinkert, et al. (1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control region which is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol., 5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey, et al. (1987) Genes and Devel., 1: 161-71), beta-globin gene control region which is active in myeloid cells (Mogram, et al. (1985) Nature 315:338-40; Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead, et al. (1987) Cell 48:703-12), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, (1985) Nature 314.283-6), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason, et al. (1986) Science 234:1372-8).
[0129] Other promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, Ela, and MLP promoters.
[0130] Additional vector systems include the non-viral systems that facilitate introduction of polynucleotide agents into a patient. For example, a DNA vector encoding a desired sequence can be introduced in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7); see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer, et al. (1993) Science 259:1745-8). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold, (1989) Nature 337:387-8). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, for example, a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO 95/21931).
[0131] It is also possible to introduce a DNA vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Naked DNA vectors for therapeutic purposes can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem. 262:4429-32).
[0132] The present invention also provides biologically compatible, cartilage formation-enhancing compositions comprising an effective amount of one or more compounds identified as TARGET inhibitors, and/or the expression-inhibiting agents as described hereinabove.
[0133] A biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the compound, polynucleotide, vector, and antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity. For example, a compound of the invention would have inverse agonist or antagonist activity on the TARGET; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary mRNA of a TARGET; a vector would be able to transfect a TARGET cell and expression the antisense, antibody, ribozyme or siRNA as described hereinabove; an antibody would bind a TARGET polypeptide domain.
[0134] A preferred biologically compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels. Biologically compatible solutions may include stabilizing agents and preservatives. In a more preferred embodiment, the biocompatible composition is a pharmaceutically acceptable composition. Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques. The composition may be administered parenterally in dosage unit formulations containing standard, well-known non-toxic physiologically acceptable carriers, adjuvants and vehicles as desired.
[0135] A particularly preferred embodiment of the present composition invention is a cartilage formation-enhancing pharmaceutical composition comprising a therapeutically effective amount of an expression-inhibiting agent as described hereinabove, in admixture with a pharmaceutically acceptable carrier. Another preferred embodiment is a pharmaceutical composition for the treatment or prevention of a condition a systemic or local decrease in mean cartilage thickness, or a susceptibility to the condition, comprising an effective cartilage formation-enhancing amount of a TARGET antagonist or inverse agonist, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier.
[0136] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
[0137] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
[0138] Preferred sterile injectable preparations can be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium; calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
[0139] The composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available. A hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.
[0140] Embodiments of pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector encoding the polynucleotide inhibitory agent of the present invention and a transfection enhancer, such as poloxamer. An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N.J.) and is a non-toxic, biocompatible polyol. A poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.
[0141] The active expression-inhibiting agents may also be entrapped in microcapsules prepared, for example, 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 (1980) 16th edition, Osol, A. Ed.
[0142] 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(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-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. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
[0143] As defined above, therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
[0144] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
[0145] The pharmaceutical compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to TARGET tissues, complexed with cationic lipids, packaged within liposomes, or delivered to TARGET cells by other methods known in the art. Localized administration to the desired tissues may be done by direct injection, transdermal absorption, catheter, infusion pump or stent. The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and administration are provided in Sullivan et al. WO 94/02595.
[0146] Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time. Those skilled in the art may employ different formulations for polynucleotides than for proteins. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
[0147] As discussed hereinabove, recombinant viruses may be used to introduce DNA encoding polynucleotide agents useful in the present invention. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 104 and about 1014 pfu. In the case of AAVs and adenoviruses, doses of from about 106 to about 1011 pfu are preferably used. The term pfu ("plaque-forming unit") corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art.
[0148] The present invention also provides methods of enhancing cartilage formation, which comprise the administration to said subject a therapeutically effective amount of an expression-inhibiting agent of the invention. A further aspect of the invention relates to a method of treating or preventing a disease involving chondrocyte anabolic stimulation, comprising administering to said subject a cartilage formation-enhancing pharmaceutical composition as described herein.
[0149] Examples of diseases involving anabolic stimulation of chondrocytes that are treatable using the means and methods of the present invention include, but are not limited to osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis like osteoarthritis deformans endemica, Mseleni disease, and Handigodu disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis. Furthermore, people suffering from congenital cartilage malformations, including hereditary chondrolysis, chondrodysplasias and pseudoachondrodysplasias, are likely to benefit from programs that result in anabolic stimulation of chondrocytes, and these diseases therefore may also be treated by using the methods and means of the present invention. Non-limiting examples of congenital cartilage malformation related diseases are microtia, anotia, and metaphyseal chondrodysplasia.
[0150] The polypeptides or the polynucleotides employed in the methods of the present invention may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. To perform the methods it is feasible to immobilize either the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay. Interaction (e.g., binding of) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, the polypeptide of the present invention can be "His" tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions with the polypeptides of the present invention can be adsorbed to IgG, which are then combined with the cell lysates (e.g., (35S-labelled) and the candidate compound, and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix is immobilized. The amount of radioactivity can be determined directly, or in the supernatant after dissociation of the complexes. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the protein of the present invention quantitated from the gel using standard electrophoretic techniques.
[0151] Other techniques for immobilizing protein on matrices can also be used in the method of identifying compounds. For example, either the polypeptide of the present invention or the compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated protein molecules of the present invention can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptides of the present invention but which do not interfere with binding of the polypeptide to the compound can be derivatized to the wells of the plate, and the polypeptide of the present invention can be trapped in the wells by antibody conjugation. As described above, preparations of a labeled candidate compound are incubated in the wells of the plate presenting the polypeptide of the present invention, and the amount of complex trapped in the well can be quantitated.
[0152] Another embodiment of the present invention relates to a method for in vitro production of cartilage tissue, comprising the steps of contacting chondrocyte cells with a polynucleotide sequence comprising a sequence selected from the group consisting of sequences complementary to SEQ ID No: 1-28, preferably selected from the group consisting of sequences complementary to SEQ ID NO: 84-197 for a time sufficient to re-differentiate the chondrocytes thereby producing a cartilaginous matrix.
[0153] In a preferred embodiment, the method comprises the steps of:
[0154] (a) applying chondrocyte cells on a substrate to form a cellular substrate,
[0155] (b) introducing a polynucleotide comprising a nucleotide sequence selected from the group consisting of sequences complementary to SEQ ID No: 1-28, preferably selected from the group consisting of sequences complementary to SEQ ID NO: 84-197, for a time sufficient to re-differentiate the chondrocyte cells, thereby producing a cartilaginous matrix.
[0156] The invention thus provides a method for producing a substrate with a matrix grown thereon, which matrix may be used for the provision of load-bearing implants, including joint prostheses, such as artificial knee joints and finger joints, and maxillofacial implants. It can also be used for special surgery devices, such as spacers, or cartilage fillers, and for use in augmentation, obliteration or reconstitution of cartilage defects and damaged or lost cartilage.
[0157] The present invention also relates to a combination of a load-bearing implant (preferably coated with a matrix as described above) with a cartilage filler comprising a matrix as described.
[0158] The method of the invention is also very suitable in relation to revision surgery, i.e., when previous surgical devices require replacement.
[0159] Suitable cells are stem cells cells, including mesenchymal stem cells cells and in particular chondrosyte precursor cells. The mesenchymal stem cells, and especially the chondrosyte precursor cells are found to be very effective in the cartilage producing process when taken from their original environment. In addition, cells derived from cartilage biopsies of a subject may be cultured and utilized with the present invention.
[0160] The mesenchymal stem cells can be directly applied on the substrate or they can advantageously be multiplied in the absence of the substrate before being applied on the substrate. In the latter mode, the cells are still largely multipotent after multiplication and, for the purpose of the invention, they are still referred to as undifferentiated. Subsequently, the cells are allowed to differentiate. Differentiation can be induced or enhanced by the presence of suitable inductors, such as bone morphogenic proteins (BMP2; BM4; BMP7), transforming growth factor beta (TGFbeta), CDMP1 and CDMP2. Especially suitable inductors of differentiation are the expression inhibitory agents of the present invention.
[0161] The use of mesenchymal stem cells provides several advantages. Firstly, their lower differentiation implies a higher proliferation rate and allows the eventual functionality to be better directed and controlled. Moreover, culturing these cells not only produces the required cartilage matrix containing organic and inorganic components, but also results in the presence, in the culture medium and in the matrix, of several factors which are essential for growth of the tissue and for adaptation to existing living tissue. Also, the culture medium can be a source of active factors such as growth factors, to be used in connection with the implanting process. Furthermore, such undifferentiated cells are often available in large quantities and more conveniently than e.g., mature cartilage cells, and exhibit a lower morbidity during recovery. Moreover, the undifferentiated cells can be obtained from the patient for whom the implant is intended. The cartilage resulting from these cells is autologous to the patient and thus no immune response will be induced. Matrices as thick as 100 μm can be produced as a result of the use of undifferentiated cells.
[0162] The substrate on which the undifferentiated cells can be applied and cultured can be a metal, such as titanium, cobalt/chromium alloy or stainless steel, a bioactive surface such as a calcium phosphate, polymer surfaces such as polyethylene, and the like. Although less preferred, siliceous material such as glass ceramics, can also be used as a substrate. Most preferred are metals, such as titanium, and calcium phosphates, even though calcium phosphate is not an indispensable component of the substrate. The substrate may be porous or non-porous. The cells can be applied at a rate of e.g., 103-106 per cm2, in particular 104-2×105 cells per cm2.
[0163] The culture medium to be used in the method according to the invention can be a commonly known culture medium such as MEM (minimum essential medium). Advantageously, the medium can be a conditioned medium. In this context, a conditioned medium is understood to be a medium wherein similar cells have previously been incubated, causing the medium to contain factors such as polypeptides, secreted by the cells which are important for cell growth and cell differentiation.
[0164] The cells are cultured for a time sufficient to produce a matrix layer, e.g., a matrix layer having a thickness of at least 0.5 um, in particular from 1 up to 100 um, more in particular of 10-50 um. The cells may be contacted with the culture medium for e.g. 2-15 weeks, in particular 4-10 weeks.
[0165] The production of the matrix, when applied on a substrate, results in a continuous or quasi-continuous coating covering the substrate for at least 50%, in particular at least 80% of its surface area.
[0166] In yet another aspect of the invention, the invention provides a method for diagnosing a pathological condition involving chondrocyte de-differentiation, said method comprising the steps of: determining the nucleic acid sequence of any one of the genes encoding the polypeptides listed in Table 1A in a genomic DNA sample; comparing the sequence from step (a) with the nucleic acid sequence of a healthy subject; and identifying any difference(s) related to the pathological condition. Such differences may be further checked in in vitro assays applying similar marker genes as disclosed herein. Such assays will reveal the role of the gene or its encoded polypeptide in anabolic stimulation processes of chondrocytes. If such mutations are identified this knowledge can be further exploited in test-kits for diagnosis of similar diseases.
[0167] Still another aspect or the invention relates to a method for diagnosing a pathological condition involving chondrocyte anabolic stimulation or a susceptibility to the condition in a subject, comprising determining the amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55-82, 198-391 in a biological sample, and comparing the amount with the amount of the polypeptide in a healthy subject, wherein an increase of the amount of polypeptide compared to the healthy subject is indicative of the presence of the pathological condition. Clearly, the activity and/or expression levels of the target genes as disclosed herein may have an effect on anabolic stimulation of chondrocytes. It remains to be determined to what level the activity should be elevated to diagnose for the disease. However, by comparing levels found in patients, individuals without symptoms and clearly healthy individuals the skilled person may easily determine these relevant levels. Since the skilled person is now aware which polypeptides should be monitored, the present invention provides novel tools for test assays for such diagnostics. A prominent disease that may be controlled, checked and diagnosed by using the knowledge provided by the present invention is osteoarthritis.
[0168] The rate of chondrocyte anabolic stimulation can typically be measured by determining the deposition of cartilage, or cartilage components, or cartilage-containing extra-cellular matrix produced by the chondrocytes, in the medium. A cell-based ELISA, enzymatic assays, or other general techniques known in the art can be used to measure cartilage components, like the ones described in Walsh G., Proteins: Biotechnology and Biochemistry. John Wiley and Sons, 2001.
[0169] The invention is further illustrated in the following figures and examples.
EXAMPLES
Example 1
Development of a High-Throughput Screening Method for the Detection of Endogenous Collagen Type II, Alpha-1 (Col2α1)
Principle of the Assay:
[0170] Normal human articular chondrocytes (NHAC's) that are grown in two-dimensional cultures become dedifferentiated and gradually cease to synthesize cartilage. They can be re-differentiated into anabolic, active chondrocytes in the presence of appropriate factors (e.g. BMP2). An assay to screen for such factors was developed by monitoring the levels of collagen type ii, alpha-1 (col2α1), a major constituent of normal cartilage. NHAC's are seeded in 384 well plates and 1 day after plating infected with individual siRNA adenoviruses (Ad-siRNA) from the SILENCESELECT® collection (see WO03/020931). Col2α1 deposition is determined at 14 days after the start of the infection (14 dpi.
Control Viruses
[0171] Ad-BMP2; described in WO 03/018799
[0172] BMP4; Ad5 dE1/E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP--570912).
[0173] Ad-LacZ; referred to as pIPspAdApt6-lacZ in WO 02/070744
[0174] Ad-eGFP; referred to as pIPspAdApt6-eGFP in WO 02/070744
[0175] Ad-Empty; described in WO 02/070744
Development of the Assay
[0176] NHAC's were isolated from donors who died from unrelated causes, and were obtained after informed consent (Cambrex, Verviers, Belgium).
[0177] In a series of experiments, carried out in 384-well plates, several parameters are optimized: cell seeding density, multiplicities of infection (MOI) of control viruses (Ad-BMP2 or Ad-eGFP), duration of infection, toxicity, infection efficiency (using Ad-eGFP) and the day of readout.
[0178] Using Ad-BMP2 (BMP2 over-expression) as a positive control for assay development, the following protocol resulted in the highest dynamic range for the assay with the lowest standard deviation on the background signal:
[0179] NHAC's are seeded on day 0 at 1500 cells/well of a 384-well plate in 60 μl of DMEM/F12 (InVitrogen), containing 10% heat-inactivated fetal calf serum (FBS-HI) and Pen/Strep) and infected the next day with 2.5 μl of Ad-control-virus (Ad-BMP2 or Ad-eGFP; this corresponds to an assumed MOI of 2000). After 7 days, 10 μl of a 50 μg/ml 2-Phospho-L-ascorbic acid in assay culture medium is added to each well. Up-regulation of Col2α1 is read at 10 dpi: The medium is removed with a VacuSafe; 50 μl ice-cold MeOH is added with a multidrop and removed immediately with a VacuSafe; 80 μl of ice-cold MeOH is added with a multidrop to fixate the cells, and plates are incubated for 20 min at -20° C.; MeOH is removed with a VacuSafe; plates are air-dried for 20 min, followed by 2× washing with 80 μl of phosphate buffered saline (PBS); 75 μl of blocking buffer (0.1% casein in PBS) is added and plates are incubated for at least 2 h at room temperature (RT); blocking buffer is removed; cells are washed with 25 μl of EC buffer (20 mM sodium phosphate, 2 mM EDTA, 400 mM NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% casein, 0.05% NaN3, pH 7) and 35 μl of the primary antibody (Collagen II Ab-2 Neomarkers. Catalogus number MS-235-P) diluted 1/450, 1/225 in buffer C (20 mM sodium phosphate, 2 mM EDTA, 400 mM NaCl, 1% BSA, pH 7)) is added with a multichannel pipette; plates are incubated overnight at 4° C.; primary antibody is removed; cells are washed twice with 80 μl of PBST (0.5% Tween 20 in PBS) and once with 80 μl PBS; 35 μl of the secondary antibody (Goat-anti-mouse Immunoglobulins/HRP. DAKO. Catalogus number PO477; diluted 1/2000 in buffer C) is added with multichannel pipette; plates are incubated at RT for 1 h; secondary antibody is removed and cells are washed twice with 80 μl PBST and once with 80 μl PBS; 50 μl of luminol substrate is added and after 5 minutes read-out is determined on a luminometer.
[0180] After optimization of the assay (see FIG. 1), a 384 well control plate is prepared that contains positive control viruses (BMP2 and BMP4) and neutral viruses (eGFP; lacZ and empty, see FIG. 1). Aliquots of the control plate are prepared and frozen at -20° C. A control plate is thawed and taken along in every screening batch.
Example 2
Screening of 9216 Adenoviral siRNA Vectors in the Chondrogenesis Assay
[0181] The optimized protocol for screening the SILENCESELECT® library runs as follows: on day 0, propagated human primary chondrocytes are seeded in Greiner white, flat bottom, TC-treated 384 well plates with clear bottom (Catalogue number 781080) in 60 μl medium at a density of 1500 cells per well. One day later, 2.5 μl Ad-siRNA virus from each well of the SILENCESELECT® collection (WO 03/020931), stored in 384 well plates (estimated titer of 1×109 viral particles per ml) is transferred with the aid of a 96/384 channel dispenser (Tecan Freedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan AG, Switzerland) to individual wells of the 384 well plates containing chondrocytes. The control plate is run under the same conditions as the aliquot plates from the SILENCESELECT® collection. All Ad-siRNA viruses are screened in duplicate on independent assay plates. After infection, plates are incubated at 37° C. Seven days post infection the medium containing the adenoviruses is replaced by fresh medium. Thirteen days post infection, the amounts of col2α1 depositions per well is determined with the cELISA method. A typical result of a 384 well screening plate is depicted in FIG. 2.
[0182] The duplicate screen is repeated once. Ad-siRNA viruses are nominated as hits if at least 2 data points of the four tested (two times screened in duplicate) score above threshold. Threshold is set at average plus 2.5 times standard deviation of all data points per plate.
[0183] A total of 282 hits are isolated that scored above the threshold, representing 274 independent genes. A representative example is provided in FIG. 2, in which the "times standard deviation" of duplicate data points are indicated on the X-axis and Y-axis. The threshold (2.5 times standard deviation) is indicated by dotted lines. Negative values indicate data points that scored below average.
[0184] The results for some of the genes are shown in FIG. 3. A clear induction of the collagen II levels is observed upon infection of the Ad-siRNA targeting the indicated gene. The data are represented as relative light units (rlu) correlating to collagen II levels.
Example 3
Propagation of Hits
[0185] The 282 Ad-siRNA hits are subjected to further analysis to establish their therapeutic potential to induce chondrocyte anabolic stimulation. A first step entails a quality control on the Ad-siRNA selected for further analysis (this example). Next steps are the screening of the targets in other assays to validate their role in chondrocyte anabolic stimulation such as the induction of aggrecan, another main constituent of cartilage besides collagen II (Example 4), the ability to induce chondrocyte anabolic stimulation in chondrocytes from other donors (Example 5), the induction of a correct marker profile in three-dimensional chondrocyte cultures (example 11), the presence of posttranslational modifications on aggrecan (example 9) and collagen II (example 10) in three-dimensional chondrocyte cultures, the development of additional Ad-siRNAs targeting the identified transcripts (example 7), and confirmation that the corresponding genes are indeed expressed in residing chondrocytes (example 12).
[0186] To propagate the 282 hits of the chondrogenesis assay, 2.25×104 PerC6.E2A cells are seeded in 200 μl of DMEM containing 10% non-heat inactivated FCS into each well of a 96 well plate and incubated overnight at 39° C. in a humidified incubator at 10% CO2. Subsequently, 1 μl of crude lysate from the siRNA adenovirus stocks in matrix tubes is added and incubation proceeds at 34° C. in a humidified incubator at 10% CO2 for 7 days. All hits are propagated in duplicate on two independent plates. The two lysates are pooled and aliquots are frozen at -20° C.
[0187] The propagated Ad-siRNAs are re-screened at three MOI's in the chondrogenesis assay in duplicate (see Example 1). The Ad-siRNAs have to score at least once above threshold (average+2.5 times standard deviation) to pass this quality control step.
Example 4
Aggrecan Induction
[0188] A second assay to screen for chondrocyte anabolic factors is developed by monitoring the levels of aggrecan, another major constituent of cartilage. In this assay, glycosaminoglycans on aggrecan are stained by Alcian blue. NHACs are seeded in 384 well plates and 1 day after plating infected with individual Ad-siRNA from the SILENCESELECT® collection. Aggrecan deposition is determined at 14 days post infection. Using Ad-BMP2 as a positive control, we confirm in a series of experiments that several parameters optimized for the ColII cELISA assay are also applicable for the Alcian blue stain assay for aggrecan. These parameters include cell seeding density, MOIs of control viruses, duration of infection, and the day of readout.
[0189] NHAC's are seeded on day 0 at 1500 cells/well of a 384 well black-plate with clear bottom in 60 μl of DMEM/F12, containing 10% FBS-HI and Pen/Strep and infected the next day with 2.5 μl of Ad-BMP2 or Ad-eGFP; at an MOI of 2000. After 7 days, 10 μl of a 50 μg/ml 2-Phospho-L-ascorbic acid in assay culture medium is added to each well. Up-regulation of aggrecan is read at 10 dpi with Alcian blue staining: The medium is removed with a VacuSafe; 50 μl ice-cold MeOH is added with a multidrop and removed immediately with a VacuSafe; 80 μl of ice-cold MeOH is added with a multidrop to fixate the cells, and plates are incubated for 20 min at -20° C.; MeOH is removed with a VacuSafe; plates are air dried for 20 min. After washing once with 80 μl of PBS; 80 μl of 0.05% Alcian blue stain buffer (0.05% Alcian blue, Sigma, catalog number S-2889; 0.4 M MgCl2/25 mM sodium acetate, pH5.5) is added and plates are incubated overnight at RT. The next day cells are washed subsequently in 80 μl of 3% acetic acid, 25% ethanol/3% acetic acid, and 50% ethanol/3% acetic acid. Solutions are added with a multidrop and removed with a Vacusafe. After replacing 50% ethanol/3% acetic acid with 70% ethanol/3% acetic acid, each individual well is photographed with a SONY CCD camera, images are analyzed using a Galapagos Alcian blue quantification algorithm based on the separation of the blue signal through a color threshold procedure after a noise reduction filter. The amount of blue staining, which is proportional to aggrecan content, is expressed in pixel unit (see FIG. 4).
[0190] The propagated hits from Example 3 are used to transduce NHAc cells at three MOI's in duplicate in the chondrogenesis assay (see Example 1). The Ad-siRNAs have to score in duplicate in at least one MOI above threshold (average+2.5× standard deviation) to pass this secondary assay. In total, 101 out of 282 hits passed the Alcian blue assay for aggrecan (see Table 1). The results for some of the genes are shown in FIG. 5. Values represent the numerical output of the described algorithm and are correlated to Alcian blue staining levels. A clear induction of the aggrecan levels is observed upon infection of the Ad-siRNA targeting the indicated gene. The Ad-siRNA targeting FZD1 does not seem to induce Alcian blue staining.
TABLE-US-00004 TABLE 1 Overview of the 101 target sequences and their respective KD target sequences corresponding to the genes encoding the different polypeptides involved in chondrogenic differentiation. The GenBank numbers for the polypeptides and target gene symbols (general names) are also given. KD Target Sequence Hit KD Target Gene GenBank SEQ ID ID Sequence Symbol Accession Name Class NO H33- ATAAGCGGTTATCA PCTK2 NM_002595 PCTAIRE protein Kinase 392 006 CTGCC kinase 2 H33- GCTGGGATTCCAAG RYK SK340- RYK receptor-like Kinase 393 007 TGGAC NM_002958 tyrosine kinase H33- AACTGTGCAGGGC NTRK1 NM_002529 neurotrophic Kinase 394 008 CTCTCC tyrosine kinase, receptor, type 1 H33- GCTGCTGGATGTCA CDK2 NM_052827- cyclin-dependent Kinase 395 009 TTCAC NM_001798 kinase 2 H33- AGAGACACAGTGC PCK1 NM_002591 phosphoenolpyru- Kinase 396 010 CCATCC vate carboxykinase 1 (soluble) H33- ACTGAACCTCCGAA NEK4 SK256- NIMA (never in Kinase 397 011 ATGCC NM_003157 mitosis gene a)- related kinase 4 H33- GTGCTGGAGTGCTT MAPK NM_004635 mitogen-activated Kinase 398 013 CCATC APK3 protein kinase- activated protein kinase 3 H33- TTCAGACCTACCTT UMP- NM_016308 UMP-CMP kinase Kinase 399 020 CAGTC CMPK H33- CCTGAATGTGACTG DGKB- NM_020238- diacylglycerol Kinase 91 025 TGGAC INCEN NM_004080- kinase, beta P NM_145695 90 kDa/inner centromere protein antigens 135/155 kDa H33- GAGTCACACAGAG ROCK1 NM_005406 Rho-associated, Kinase 400 027 ATGAGC coiled-coil containing protein kinase 1 H33- CGATGTGCCTTCAA PRKCN SK489- protein kinase C, Kinase 401 028 GATTC NM_005813 nu H33- CAGTGGTTTGGGAA PLK4- SK341- serine/threonine Kinase 402 031 TCTGC STK18 NM_014264 kinase 18 (STK18)/polo- like kinase 4 (Drosophila) H33- GACTGACTGGCCTG ICK NM_016513- intestinal cell Kinase 92 032 AAGGC NM_014920 (MAK-like) kinase H33- GATCTACACCACCT GPR10 AF411117- G protein-coupled GPCR 101 034 TCATC 3 NM_198179 receptor 103 H33- GTGACTACACAAG CCR2 NM_000647 chemokine (C-C GPCR 403 036 GACTCC motif) receptor 2 H33- GACTGATTCGCTCT FPRL2 NM_002030 formyl peptide GPCR 404 040 TTGCC receptor-like 2 H33- GGTGTATGGGCTCA FZD1 NM_003505 frizzled homolog 1 GPCR 108 041 TGTAC (Drosophila) H33- AGTGCAGCCTTGTG P2RY1 NM_198333- purinergic GPCR 405 042 GGTTC 0 NM_014499 receptor P2Y, G- protein coupled, 10 H33- TAACACTCACTGCA EMR3 NM_152939- egf-like module GPCR 406 044 CCTGC NM_032571 containing, mucin- like, hormone receptor-like 3 H33- TAACTGAAACTCAG PROZ NM_003891 protein Z, vitamin Protease 407 049 CTAGC K-dependent plasma glycoprotein H33- ACTGAAGTAGCCCT THRB NM_000461 thyroid hormone NHR 408 054 CCTTC receptor, beta (erythroblastic leukemia viral (v- erb-a) oncogene homolog 2, avian) H33- AGAACTGGGTGAT ELA1 NM_001971 elastase 1, Protease 116 056 GACAGC pancreatic H33- AGTGCAGTACAGC COL7A NM_000094 collagen, type VII, Not 409 058 GATGAC 1 alpha 1 classified (epidermolysis bullosa, dystrophic, dominant and recessive) H33- TTCACATCGCTGAG CPZ NM_003652 carboxypeptidase Z Protease 410 059 CACCC H33- ATGAACTCTGTGAT USP9Y NM_004654 ubiquitin specific Protease 123 061 CCAGC protease 9, Y- linked (fat facets- like, Drosophila) H33- AGCCAGCAACGAC CST3 NM_000099 cystatin C Not 411 063 ATGTAC (amyloid classified angiopathy and cerebral hemorrhage) H33- GCTGCTGGGCATGT LNPEP NM_005575 leucyl/cystinyl Protease 412 065 CCTTC aminopeptidase H33- TGTGATCGTCATCA NLGN1 NM_014932 neuroligin 1 Enzyme 413 066 CAGTC H33- AACATGATATGTGC KLK10 NM_145888- kallikrein 10 Protease 414 068 TGGAC NM_002776 H33- CTGAGAAGGCTTCC LOC11 XM_061692 similar to Protease 415 069 ACTGC 9795 glutamyl aminopeptidase (aminopeptidase A); gp160 H33- TGATACGTGGATCC LOC12 NM_178453 similar to distal Protease 416 070 AGGCC 4221- intestinal serine MGC52 protease 282 H33- CTACAGTGACAAG OVTN- XM_089945- (similar to) Protease 417 072 GCTAAC LOC15 oviductin protease 9938 NM_198185 H33- GAACTGGATAGCC LOC20 XM_116274- similar to Protease 418 073 CTCATC 6008- XM_371709 KIAA1214 protein/ RNF15 ring finger 0 protein 150 H33- CCCTGGTAAAGCTG LOC22 XM_166659 similar to Protease 419 074 CATTC 0213 evidence: NAS/ pothetical hyprotein/putative H33- GATGAAGGCTTCG XYLB NM_005108 xylulokinase Kinase 420 076 GGCTTC homolog (H. influenzae) H33- TGTAAAGCTGGAA PTEN NM_000314- putative protein Phosphatase 421 080 AGGGAC BC038293- tyrosine AF017999 phosphatase homologue H33- CTGAAGAAGCTGG PTPN2 NM_015466 protein tyrosine Phosphatase 422 081 AGTTGC 3 phosphatase, non- receptor type 23/ protein tyrosine phosphatase TD14 H33- TTGGAATTCCAGTG DUSP1 NM_003584 dual specificity Phosphatase 132 082 TACCC 1 phosphatase 11 (RNA/RNP complex 1- interacting) H33- GCTAGTTATCGCCT DUSP3 NM_004090 dual specificity Phosphatase 139 083 ACCTC phosphatase 3 (vaccinia virus phosphatase VH1- related) H33- TCCTTGCAGCAGGC SLC24 NM_004727 solute carrier Ion Channel 423 084 ACATC A1 family 24 (sodium/potassium/ calcium exchanger), member 1 H33- TCTGTGCGTGGACT GABRP NM_014211 gamma- Ion Channel 424 092 GGAAC aminobutyric acid (GABA) A receptor, pi H33- CTTTGCTCGGAAGA RAF1 NM_002880 v-raf-1 murine Kinase 425 095 CGTTC leukemia viral oncogene homolog 1 H33- AGATTCCAGATGCA JAK1 SK185- Janus kinase 1 (a Kinase 148 096 ACCCC NM_002227 protein tyrosine kinase) H33- GAAGGCTTTGGAA LOC16 XM_094437- hypothetical Kinase 426 098 AGTGTC 7359- NM_153361 protein MGC42 MGC42105 105 H33- GTGAACTCTGCTGC PKD1L XM_091397- similar to GPCR 427 102 GACTC 3- NM_181536 KIAA1879 protein/ LOC16 polycystic 2163 kidney disease 1- like 3 H33- GACAAGGCTATGA RPS6K NM_004586 ribosomal protein Kinase 428 104 TGCTGC A3 S6 kinase, 90 kDa, polypeptide 3 H33- GGATGTGTGGTGCT RBKS NM_022128 ribokinase Kinase 429 105 GTCAC H33- CTGAACTACTGGTA ABCG1 NM_016818- ATP-binding Transporter 157 107 CAGCC NM_004915 cassette, sub- family G (WHITE), member 1 H33- CTCTGTGTTCCACT DPYD NM_000110 dihydropyrimidine Enzyme 430 108 TCGGC dehydrogenase H33- CAGCAATGCAGAG TNFRS NM_001561 tumor necrosis Other 431 110 TGTGAC F9 factor receptor drugable or superfamily, secreted member 9 H33- CAAAGCTGGCTACT TNFSF NM_172014- tumor necrosis Other 432 114 ACTAC 14 NM_003807 factor (ligand) drugable or superfamily, secreted member 14 H33- CAGTGCAAAGAGC GAPDS NM_014364 glyceraldehyde-3- Enzyme 433 117 CCAAAC phosphate dehydrogenase, testis-specific H33- GTATTCTGTACACC RDH11 NM_016026 retinol Enzyme 434
118 CTGGC dehydrogenase 11 (all-trans and 9- cis) H33- GTGATCGACAGGA PRKAG NM_017431 protein kinase, Kinase 435 120 TTGCTC 3 AMP-activated, gamma 3 non- catalytic subunit H33- GCGAATTCCACCAG SLC26 NM_052961 solute carrier Transporter 165 130 CATTC A8 family 26, member 8 H33- CACAGTGAAACCTT B4GAL NM_004776 UDP- Enzyme 436 138 CCTGC T5 Gal: betaGlcNAc beta 1,4- galactosyl- transferase, polypeptide 5 H33- ATCTGTGACACTGG LOC12 XM_064820 similar to Aldose Enzyme 437 145 ATCGC 5836 Reductase (E.C.1.1.1.21) H33- AGAGACTGGAGTT GNPN NM_198066 glucosamine- Enzyme 438 147 GTCAGC AT1 phosphate N- acetyltransferase 1 H33- CCTGAGTTGAATGT CYP17 NM_000102 cytochrome P450, Cytochrome 39 152 CATAC A1 family 17, P450 subfamily A, polypeptide 1 H33- CTGAACTAGTGACT MAGI- NM_152900- membrane- Kinase 440 158 ATCCC 3 NM_020965 associated guanylate kinase- related H33- ATAAGCACCGTGA LOC13 XM_071222 similar to Cytochrome 441 161 GCGACC 8967 cytochrome P450 P450 1A1 H33- CATTGGGCCACAG ADOR NM_000674 adenosine A1 GPCR 442 167 ACCTAC A1 receptor H33- GATGAAGACAGCA OPRK1 NM_000912 opioid receptor, GPCR 443 168 ACCAAC kappa 1 H33- AGCATATGATGACC CTSC NM_148170- cathepsin C Protease 444 175 TTGGC NM_001814 H33- ATTCCACTACTACA H105E3 NM_015922 NAD(P) Enzyme 445 180 GCTGC dependent steroid dehydrogenase- like H33- GAAACTGTGGCAG LOC25 XM_171056 similar to Putative Kinase 446 182 GCTAAC 6519 serine/threonine- protein kinase D1044.3 in chromosome III H33- CTGATGAAGGCCTT LOC12 XM_063593 similar to NADH- Enzyme 447 186 CGACC 3326 ubiquinone oxidoreductase PDSW subunit (Complex I- PDSW) (CI- PDSW) H33- TTGAAACAAGAGG ACYP1 XM_370768- (similar to) Phosphatase 448 188 AAGTCC NM_203488- acylphosphatase 1, NM_001107 erythrocyte (common) type H33- TGAACTTGCTCTGA KCNJ1 NM_170720- potassium Ion Channel 449 190 GCTGC 4 NM_013348 inwardly- rectifying channel, subfamily J, member 14 H33- ATCTGTAACCTCAG PPP3C S46622- protein Phosphatase 450 191 CACAC C NM_005605 phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform (calcineurin A gamma) H33- ACATTGACCAGGA GGTLA NM_178312- gamma- Enzyme 178 192 AGTGAC 4 NM_178311- glutamyltransferase- NM080920 like activity 4 H33- GAAGCTAAGCCTC PIK4C NM_058004 phosphatidylinositol Kinase 451 202 GGTTAC A 4-kinase, catalytic, alpha polypeptide H33- TAACCGTGGCATCT TPP2 NM_003291 tripeptidyl Protease 452 204 ACCTC peptidase II H33- TGACCACCTGGAGT CST11 NM_130794 cystatin 11 Not 453 205 ATCAC classified H33- GTGGACATCTTTGA GRIK4 NM_014619 glutamate Ion Channel 454 208 GCTTC receptor, ionotropic, kainate 4 H33- GCTGAGAAGTACTT ARHG NM_014448 Rho guanine Other 455 209 CCACC EF16 exchange factor drugable or (GEF) 16 secreted H33- AGACTACTGCAAG STK23 NM_014370 serine/threonine Kinase 456 210 GGCGGC kinase 23 H33- GAGTATTTGCTGGC SLCO1 XM_372282- solute carrier Protease 457 213 ATTCC A2- NM_134431- organic anion CRLF2 NM_022148- transporter family, NM_021094 member 1A2/ cytokine receptor- like factor 2 H33- GAAGCTGAATTAG PDE1A NM_005019 phosphodiesterase PDE 184 217 GGCTTC 1A, calmodulin- dependent H33- GGAGACACGGAAT PPP1R1 NM_032105- protein Phosphatase 458 219 AAACTC 2B NM_002481 phosphatase 1, regulatory (inhibitor) subunit 12B H33- CCGAGACCACCTCA ACAD8 NM_014384 acyl-Coenzyme A Enzyme 459 222 ATGTC dehydrogenase family, member 8 H33- ATGGACATCTCCAC PTPRN NM_002846 protein tyrosine Phosphatase 460 223 GGGAC phosphatase, receptor type, N H33- TATCCTGACCTTCC KCNG1 NM_172318- potassium voltage- Ion Channel 461 230 TGCGC NM_002237 gated channel, subfamily G, member 1 H33- CACATGATCAAGCT LOC22 XM_055551 similar to Heat Kinase 462 236 AGGTC 0763 shock protein HSP 90-beta (HSP 84) (HSP 90) H33- GAAGCCAGGCATC SPOCK NM_014767 sparc/osteonectin, Enzyme 463 237 TTCATC 2 cwcv and kazal- like domains proteoglycan (testican) 2 H33- GCTGAAGTTATCCA PTPN1 NM_080685- protein tyrosine Phosphatase 464 238 GTCTC 3 NM_080684- phosphatase, non- NM_080683- receptor type 13 NM_006264 (APO-1/CD95 (Fas)-associated phosphatase) H33- AGCATTGGACCAGT GABR NM_173536 gamma- Ion Channel 465 239 TGATC G1 aminobutyric acid (GABA) A receptor, gamma 1 H33- GTGATCTACGTGAA DPP3 NM_005700 dipeptidylpeptidase Protease 466 243 CTGGC 3 H33- GCCGACAGTGGTG LYPLA NM_012320 lysophospholipase Enzyme 467 245 CACTAC 3 3 (lysosomal phospholipase A2) H33- AACATGATGGCTCA CTSE NM_148964- cathepsin E Protease 468 251 GAACC NM_001910 H33- TACAGTGATGGATC SULT1 NM_014465 sulfotransferase Enzyme 469 253 ATAGC B1 family, cytosolic, 1B, member 1 H33- ACCAATATGCCTAC KLKB1 NM_000892 kallikrein B, Protease 470 255 CTTCC plasma (Fletcher factor) 1 H33- ACTGTATCCCAGCA SENP7 NM_020654 sentrin/SUMO- Protease 471 258 GTCCC specific protease H33- AAGCTGAACATAA PTPRR NM_002849 protein tyrosine Phosphatase 472 259 CCTTGC phosphatase, receptor type, R H33- TTGAATAGCTCGGT LOC16 XM_095455 similar to Kinase 473 261 GTCCC 9014 Mitogen-activated protein kinase 6 (Extracellular signal-regulated kinase 3) (ERK-3) (MAP kinase isoform p97) (p97-MAPK) H33- GTGGAAGGCAAGA ABCD1- XM_372940- ATP-binding Transporter 474 263 TCTTCC LOC38 XM_370972- cassette, sub- 8253- NM_000033 family D (ALD), LOC39 member 1/similar 1403 to Adrenoleukodys- trophy protein (ALDP) H33- TGTATGGCTGGTCG ABCA7 NM_033308- ATP-binding Transporter 475 264 ATCAC NM_019112 cassette, sub- family A (ABC1), member 7 H33- GCTGCGACAACTTC GPR11 NM_153840 G protein-coupled GPCR 476 269 TGTTC 0 receptor 110 H33- GCCCACGGTCTTCC ACPT NM_080791- acid phosphatase, Phosphatase 477 276 ACTAC NM_080789- testicular NM_033068 H33- GAAGCCATCTCCGA SLC15 NM_021082 solute carrier Transporter 192 279 CAATC A2 family 15 (H+/peptide transporter), member 2 H33- GACTGAATCAGGC PPIH NM_006347 peptidyl prolyl Enzyme 478 295 CTTCCC isomerase H (cyclophilin H)
Example 5
Donor Dependency
[0191] The 282 hits identified by the ColII cELISA assay are further subject to a donor dependency test to demonstrate that the induction of ColII production by a given hit is not restricted to a single donor. In addition to the 11-year-old donor (donor I) previously used, NHAC's from multiple donors with ages of 24 (donor II), 41 (donor III), and 50 (donor IV), are obtained after informed consent (Cambrex, Verviers, Belgium). Cells are seeded as described in Examples 1 and 3. The propagated Ad-siRNAs are used to transduce NHAc cells from these different donors at three MOI's in duplicate in the chondrogenesis assay (see Examples 1 and 3). The Ad-siRNAs have to score at least once above threshold (average+2.5 times standard deviation) to pass this donor dependency test.
[0192] Out of 101 hits that passed the Alcian blue assay for aggrecan, 97 score positive in Donor IV. Of the remaining 4, 1 score positive in Donor II. The other 3 did not score in the other donors tested. In addition, 40 of the 101 hits score positive in all three additional donors. These results demonstrate that 98 out of these 101 hits function in a non-donor-dependent manner. Exceptions are H33-145; H33-182; and H33-263 (see Table 1). Indicated in this Table 1 are the Target Gene Symbol, Gene Bank Accession Number, and drugability class of the genes that correspond to the target sequences. The results for some of the genes are shown in Table 2. These data show that knocking down the RNA levels of the indicated genes induces collagen II levels in at least 2 donors. The values represent times standard deviation of the background.
TABLE-US-00005 TABLE 2 overview donor dependency data. N = 2 for every condition. All data points are represented as fold standard deviation of the background. All values above 2.5 are considered to be positive and are shaded grey. ##STR00001## ##STR00002##
Example 6
Quality Control of Target Ad-siRNAs
[0193] Target Ad-siRNAs are propagated using derivatives of PER.C6© cells (Crucell, Leiden, The Netherlands) at a 96-well plate level, followed by re-screening these viruses at several MOI's in the primary assay (see Example 1) and by sequencing the siRNAs encoded by the target Ad-siRNA viruses. PER.E2A cells are seeded in 96 well plates at a density of 40,000 cells per well in 180 μl PER.E2A medium. Cells are then incubated overnight at 39° C. in a 10% CO2 humidified incubator. One day later, cells are infected with 1 μl of crude cell lysate from SILENCESELECT® stocks containing target Ad-siRNAs. Cells are incubated further at 34° C., 10% CO2 until appearance of cytopathic effect (as revealed by the swelling and rounding up of the cells, typically 7 days post infection). The supernatant is collected and the virus crude lysate is treated with proteinase K: 12 μl crude lysate is added to 4 μl Lysis buffer (1× Expand High Fidelity buffer with MgCl2 (Roche Molecular Biochemicals, Cat. No 1332465) supplemented with 1 mg/ml proteinase K (Roche Molecular Biochemicals, Cat No 745 723) and 0.45% Tween-20 (Roche Molecular Biochemicals, Cat No 1335465) in sterile PCR tubes. These are incubated at 55° C. for 2 h followed by a 15 min inactivation step at 95° C. For the PCR reaction, 1 μl lysate is added to a PCR master mix composed of 5 μl 10× Expand High Fidelity buffer with MgCl2, 0.5 μl of dNTP mix (10 mM for each dNTP), 1 μl of `Forward primer` (10 mM stock, sequence: 5' CCG TTT ACG TGG AGA CTC GCC, SEQ ID NO: 29), 1 μl of `Reverse Primer` (10 mM stock, sequence: 5' CCC CCA CCT TAT ATA TAT TCT TTC C, SEQ ID NO: 30), 0.2 μl of Expand High Fidelity DNA polymerase (3.5 U/μl, Roche Molecular Biochemicals) and 41.3 μl of H2O. PCR is performed in a PE Biosystems GeneAmp PCR system 9700 as follows: the PCR mixture (50 μl in total) is incubated at 95° C. for 5 min; each cycle runs at 95° C. for 15 sec, 55° C. for 30 sec, 68° C. for 4 min, and is repeated for 35 cycles. A final incubation at 68° C. is performed for 7 min. 5 μl of the PCR mixture is mixed with 2 μl of 6× gel loading buffer, loaded on a 0.8% agarose gel containing 0.5 μg/μl ethidium bromide to resolve the amplification products. The size of the amplified fragments is estimated from a standard DNA ladder loaded on the same gel. The expected size is ˜500 bp. For sequencing analysis, the siRNA constructs expressed by the target adenoviruses are amplified by PCR using primers complementary to vector sequences flanking the SapI site of the pIPspAdapt6-U6 plasmid. The sequence of the PCR fragments is determined and compared with the expected sequence. All sequences are found to be identical to the expected sequence.
Example 7
Evaluation of the on Target Effect of the Identified siRNA Sequences
[0194] To evaluate whether the identified siRNA sequences really increase collagen II and aggrecan levels through the knock down of the target mRNA, a second siRNA sequence is identified that exerts the same effect.
[0195] A number of additional siRNA sequences targeting the HIT sequences are designed and incorporated in adenoviruses according to WO 03/020931. After production of these adenoviruses, the Ad-siRNAs are infected at different volumes (1.5 μl, 5 μl and 15 μl) in the chondrocytes and their effect on collagen II is assessed as described in example 1. The threshold (average+2.5 standard deviation) is calculated for every volume. If a virus scores above the threshold for one or more of the different infection volumes, it is considered to be positive. The on target results are shown in FIG. 6. These results indicate that at least one additional siRNA, which targets the HIT sequence, can be identified. This underscores the on target effect of the siRNA sequences identified during the screening of the Silence Select library.
Example 8
Development of a Three-Dimensional-Alginate Culture System for a Quantitative Marker Analysis for Stable Cartilage and the Assessment of Glycosaminoglycans (GAG) and Hydroxyprolines (Hyp) Synthesis
Principle of the Assay:
[0196] Normal human articular chondrocytes (NHAC's) grown in three-dimensional cultures are able to maintain a "differentiated state" as measured by the expression of Collagen type II and aggrecan. De-differentiated chondrocytes cultured in a two-dimensional system for a limited amount of passages can revert to a differentiated state when transferred into a three-dimensional culture system. This system was established to test the capability of siRNA adenoviruses (Ad-siRNA) 1) to induce a mRNA expression pattern that correlates with anabolic active chondrocytes and 2) to induce protein modification of collagen type II (hydroxyprolines) and aggrecan (glycosaminoglycans) involved in the stability of cartilage. Normal cartilage NHAC's are cultured in a two-dimensional culture system for two or three passages for cell expansion purposes. Cells are transduced with individual siRNA adenoviruses (Ad-siRNA) in the two-dimensional culture system and three days later are transferred into the three-dimensional alginate culture system. After 10 days in the alginate culture, various parameters can be assessed (e.g. mRNA marker analysis and protein modifications).
Assay Procedure
[0197] Using Ad-BMP2 (BMP2: "strong" collagen II inducer) and Ad-BMP7 (BMP7: "weak" collagen II inducer) as positive controls and Ad-ALPL as negative control the following protocol is set up (for both mRNA marker analysis and protein modification assessment): After two or three passages in monolayer culture conditions NHAC's are seeded at 2.10E+06 cells/T175 flask in 30 ml of chondrocyte growing medium (Cambrex) and transduced the following day with control-viruses (Ad-BMP2, Ad-BMP4, Ad-BMP7, Ad-ALPL) using an MOI of 2000. After three days, cells are trypsinised using the chondrocyte reagent pack (Cambrex) and washed once with 155 mM sodium chloride/20 mM Hepes ph 7.4 (Cambrex). Cells are re-suspended at a density of 2×106 cells/ml in 1.2% sodium alginate (Cambrex). The cell suspension is transferred into a syringe attached to a 21-22 gauge needle and expelled in a drop-wise fashion into 102 mM calcium chloride/5 mM Hepes pH 7.4 (1 ml and 5 ml respectively in 24-well and 6-well plate) Five and 50 beads per well are produced respectively in the 24-well and 6-well plates. Plates containing the alginate beads are incubated for 10 minutes with gentle shaking every 2 minutes. The calcium chloride solution is aspirated with a Vacusafe system and beads are washed three times with the sodium chloride solution using the Vacusafe and once with DMEMF/12 supplemented with 10% heat inactivated fetal bovine serum (FBS-HI) and 1% penicillin/streptomycin. Alginate beads are finally re-suspended into 0.5 ml and 3 ml (respectively in 24-well and 6-well plate) of DMEMF/12 supplemented with 10% heat inactivated fetal calf serum (FBS-HI), 1% penicillin/streptomycin and 25 μg/ml of ascorbic acid (Fluka, Sigm101-128 and 401-594ldrich). The alginate cultures are incubated in a humidified incubator at 37° C. and 5% CO2 during 10 days with a medium refreshment every 48/72 h.
[0198] For each adenoviral transduction, 60 alginate beads are generated: 2×5 beads were cultured in 24-well plates for the GAGs/Hyps assessments and 50 beads are cultured in a single well of a 6-well plate for the mRNA expression pattern determination.
Control Viruses
[0199] Ad-BMP2; described in WO 03/018799
[0200] Ad-BMP7; Ad5 dE1/E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 7 pre-protein (NP--001710).
[0201] Ad-BMP4; Ad5 dE1/E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP--570912).
[0202] Ad-ALPL; Ad5 dE1/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP--000469).
Example 9
Effect of Knock Down of Target Genes in Chondrocytes Embedded in Alginate Beads, on the Glycosaminoglycans (GAGs) Levels
[0203] Chondrocytes are infected and embedded in alginate beads according to example 8. After 10 days in culture the alginate beads are treated with papain in order to solubilise the glycosaminoglycans prior to quantification: Beads cultured in the 24-well plates are washed once with a 50 mM Phosphate buffer pH 6.5 and incubated for 3 to 4 h at 65° C. with 250 μl/well of the same buffer containing 2 mM EDTA, 2 mM L-cystein and 126 μg/ml papain (Sigma). Complete digestion of the beads is assessed by microscopic observation. Papain digests are frozen at -20° C. until glycosaminoglycans quantification is performed. The GAGs produced by the primary chondrocytes in the alginate culture system are measured using the Blyscan® assay (Biocolor Ltd, Newtownabbey, Northern Ireland).
Principle of the Blyscan Assay
[0204] The Blyscan Assay is a quantitative dye-binding method for the analysis of sulfated GAGs. The dye label used in the assay is 1,9-dimethylmethylene blue employed under conditions that produce a specific label for the sulfated polysaccharides component of proteoglycans and/or the protein-free sulfated glycosaminoglycan chains. Aggrecan is the predomninant proteoglycan in articular cartilage, representing ±90% of the cartilage proteoglycans. It is composed of a central core protein attached to ±50 keratan sulfate and ±100 chondroitin sulfate chains known as GAGs required for the biological function and the stability of aggrecan.
Assay Description
[0205] The papain digests generated after culturing the chondrocytes for 10 days in alginate beads (Example 7) are diluted in water 1:100, 1:200 if cells were originally transduced with the positives controls and 1:50, 1:100 if transduced with the negative controls. This dilution step allows readout values within the standard range of the assay. The GAG standard provided by the manufacturer contains 100 μg/ml of chondroitin 4-sulfate purified from bovine trachea. This standard is run in duplicate at four concentrations corresponding to 1, 2, 3 and 5 μg of GAGs. Standard and controls are individually diluted in 100 μl final volume in eppendorf tubes. One milliliter of the Blyscan Dye Reagent is added to each tube and incubated for 30 minutes at room temperature with continuous shaking. When formed, the GAG-dye complex becomes insoluble and is then separated from the remaining excess soluble unbound dye by centrifugation (10000×g for 10 minutes). Supernatant are discarded by inverting and careful draining of the tube contents. One milliliter of Blyscan Dissociation Reagent is added to each tube and incubated for one hour to one and a half hour with continuous shaking. This reagent brings the GAG-bound dye back into solution. The GAG content of the assayed samples is spectrophotometrically determined by the amount of dye recovered from the GAGs in the test sample. Two hundred microliters of the dissociation dye solutions are added to the wells of a 96-well plate and reading is performed on an automatic plate reader set at a dual wavelength (656 and 450 nm).
[0206] The GAGs concentrations measured in the Blyscan assay are normalized to DNA content by performing a fluorimetric Hoechst assay on the same papain digests. Hoechst 33342 dye reagent (Molecular Probes), a bisbenzimidasole dye that binds to adenine/thymine rich regions on DNA. Papain digests were diluted 1:1.7 and 1:3.3 in TE buffer, this dilution step allows readout values within the standard range of the assay. Purified calf thymus DNA (Sigm101-128 and 401-594ldrich) is used as standard DNA: the initial stock solution (2 μg/ml) is sequentially diluted in TE buffer in order to obtain the following concentration range: 1.5, 1.0, 0.75, 0.50, 0.2 μg/ml. Standard and test samples are diluted in TE to a final volume of 100 μl and added to a 96-well plate. One hundred microliters of Hoechst 33342 dye reagent are added to the wells, reading is performed on a multifunctional microplate reader (Fluostar Galaxy, BMG Labtechnologies GmbH) with an excitation wavelength set at 360 nm and an emission wavelength set at 440 nm.
[0207] When GAG concentrations are normalized to DNA content the resulting values expressed as the ratio GAG concentration (μg/ml)/DNA concentration (μg/ml) are used to calculate the assay window. This window is calculated as the ratio normalized GAG Ad-BMP2 (or Ad-BMP4)/normalized GAG Ad-ALPL.
[0208] The effect of the knock down of the 14 target genes on the GAG levels is assessed as described. The results are shown in FIG. 7. The results are expressed as fold induction of the GAG levels compared to the average of the normalized GAG values obtained for two negative control knock-down adenoviruses (Ad-PTGER4 and Ad-GRM7). Knock down of the mRNA of the respective genes results in an increase of the GAG levels.
Control Viruses
[0209] Ad-BMP2: described in WO 03/018799
[0210] Ad-BMP4: Ad5 dE1/E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP--570912).
[0211] Ad-ALPL: Ad5 dE1/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP--000469).
[0212] Ad-PTGER4: Ad5 dE1/E2A adenoviruses that comprise the siRNA sequence CCATGCCTATTTCTACAGC (SEQ ID NO: 31) to knock down the prostaglandin E receptor 4 mRNA.
[0213] Ad-GRM7: Ad5 dE1/E2A adenoviruses that comprise the siRNA sequence TCAGTAACAGCTCCCAGAC (SEQ ID NO: 32) to knock down the metabotropic glutamate receptor 7 mRNA.
Example 10
Quantitative Analysis of Hydroxyprolines (Hyps)
[0214] In articular cartilage, approximately 95% of the collagen is type II collagen. Its polymers are the fibrils that form the basic cohesive framework of the tissue. The collagen biosynthesis involves several unique posttranslational modifications including hydroxylation of proline and lysine residues. These modifications are crucial for collagen stability and resistance to proteolytic enzymes.
[0215] Chondrocytes are infected and embedded in alginate beads according to example 8. After 10 days in culture the alginate beads are treated with papain: Beads cultured in the 24-well plates are washed once with a 50 mM Phosphate buffer pH 6.5 and incubated for 3 to 4 h at 65° C. with 250 μl/well of the same buffer containing 2 mM EDTA, 2 mM L-cystein and 126 μg/ml papain (Sigma). Complete digestion of the beads is assessed by microscopic observation. Papain digests are frozen at -20° C. until hydroxyproline quantification is performed.
[0216] Hydroxyproline assessment in the papain digests is performed by HPLC after acid hydrolysis and FMOC (9-fluorenylmethyl chloroformate) derivatisation of the samples. This method is described in "Bank R A, Jansen E J, Beekman B and Te Koppele J M. (1996) Amino acid analysis by reverse-phase high performance liquid chromatography: Improved derivatisation and detection conditions with 9-fluorenylmethyl chloroformate. Anal Biochem. 240 (2): 167-176.
[0217] The effect of the knock down of the 14 target genes on the hydroxyproline levels is assessed as described. The results are shown in FIG. 8. The results are expressed as fold induction compared to the average of Hyp concentrations measured for the two KD controls (Ad-PTGER4 and AD-GRM7). Knock down of the mRNA of the respective genes results in an increase of the hydroxyproline levels.
Control Viruses
[0218] Ad-BMP2; described in WO 03/018799
[0219] Ad-BMP4; Ad5 dE1/E2A adenoviruses that mediate the expression of full length bone morphogenetic protein 4 pre-protein (see NP--570912).
[0220] Ad-ALPL: Ad5 dE1/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP--000469).
[0221] Ad-PTGER4: Ad5 dE1/E2A adenoviruses that comprise the siRNA sequence CCATGCCTATTTCTACAGC (SEQ ID NO: 33) to knock down the prostaglandin E receptor 4 mRNA.
[0222] Ad-GRM7: Ad5 dE1/E2A adenoviruses that comprise the siRNA sequence TCAGTAACAGCTCCCAGAC (SEQ ID NO: 34) to knock down the metabotropic glutamate receptor 7 mRNA.
Example 11
Quantitative Analysis of Markers for Stable Cartilage
Assay General Principle:
[0223] Chondrocyte phenotypes can be categorized by characteristic patterns of gene expression. Quantitative RT-PCR techniques are used to monitor the expression pattern of a set of key marker molecules to define which phenotype is induced on the chondrocytes. Positive markers included collagen type II and FGFR3, typically expressed by cartilage chondrocytes. Negative makers included (1) collagens types I and III for dedifferentiated or fibroblast-like chondrocytes, a phenotype that can also be induced by retinoic acid or interleukin-1; (2) collagen X, PTHLH and ALK-1 for hypertrophic chondrocytes that are found in the calcified zone of adult cartilage and the lower hypertrophic zone of the fetal growth-plate cartilage; and (3) MMP13 as proteolytic enzyme involved in cartilage degradation. Functional cartilage chondrocytes should express high levels of positive markers but low or not detectable levels of negative markers.
Assay Description
[0224] Chondrocytes are infected and embedded in alginate beads according to example 8. After 10 days in culture the alginate beads are treated with 55 mM sodium citrate (Cambrex) in order to recover the chondrocytes from the alginate beads and harvest RNA: Incubation medium was removed from the 6-well plates with the Vacusafe and 5 ml of 55 mM sodium citrate are added to each well and incubated for 15 minutes at room temperature. The partially solubilised beads are gently mixed and transferred to a FALCON tube, wells are rinsed once with 2 ml of sodium citrate solution to collect the remaining beads and released cells. Tubes are laid on their side and gently mixed every 2-3 minutes until beads have completely solubilised (+/-15 minutes). Tubes are centrifuged at 1000 rpm for 10 minutes. Supernatant is discarded and the cell pellet is re-suspended in 2 ml of sodium citrate solution and left for 5 minutes. Six milliliters of 155 mM sodium chloride/20 mM Hepes ph 7.4 (Cambrex) are added to each tube prior to a 5 minutes centrifugation at 210 g. Cell pellet is lysed in 180 μl SV40 lysing buffer (Promega SV40 total RNA extraction kit) and frozen at -20° C. until RNA isolation is performed with the Promega SV40 total RNA extraction kit according to the manufacturer's instructions. Purified RNA is quantified using ribogreen reagent (Molecular Probes) and yeast RNA (Ambion) as standard RNA.
[0225] Purified RNA from chondrocytes transduced with control adenoviruses and cultured for 10 days in the alginate 3-dimensional culture system is used in a reverse transcription (RT) reaction. RNA is first diluted in water (Life Technologies-Invitrogen, Breda, The Netherlands) depending on the initial concentration of the sample: For RNA concentration below 25 ng/ml, samples are used undiluted, for RNA concentration between 25 and 50 ng/ml samples are diluted 1:2.5, For RNA concentration between 50 and 100 ng/ml samples are diluted 1:5 and for RNA concentration between 100 and 160 ng/ml samples are diluted 1:10. Two microliters of diluted/undiluted RNA are added to 5 μl of a reaction mix consisting of: 1× Taqman RT buffer, 5 mM MgCl2, 500 μM dNTPs (2.5 mM each), 2.5 μM Random hexamers, 0.4 U/μl of RNAse inhibitor and 1.25 U/μl of MultiScribe Reverse Transcriptase (all reagents purchased from Applied Biosystems). The PCR reaction is performed in a Peltier Thermal Cycler-200 (BIOzym, Landgraaf, The Netherlands) as followed: the PCR total mixture (60 μl) is incubated 10 minutes at 25° C. followed by 30 minutes at 48° C., followed by 5 minutes at 95° C. Each reaction is run in parallel with a control that does not contain any RNAse inhibitor or Reverse Transcriptase.
[0226] Reverse transcription is followed by a quantitative PCR for specific amplification of the selected positive and negative marker genes, GAPDH is included as the endogenous control. Five microliters of cDNA (from RT reaction) are added to 20 μl of a PCR reaction mix consisting of: 1× Brilliant® SYBR® Green QPCR Master Mix (Sratagene Europe, Amsterdam, The Netherlands), 300 μM of each forward and reverse primers (table 3) (except for GAPDH and ALK-1, used at 100 μM)(Life Technologies-Invitrogen, Breda, The Netherlands) and 300 nM of reference dye (Stratagene) (diluted 1:100 in H2O). The PCR mixture (25 μl) is incubated for 10 minutes at 95° C. followed by 40 cycles: 15 seconds at 95° C. followed by 1 minute at 60° C. in the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems, Nieuwerkerk A/D Ijssel, The Netherlands).
TABLE-US-00006 TABLE 3 primer sequences for the positive and negative markers: SEQ ID Primer Gene Primer sequence NO Forward Collagen GGCAATAGCAGGTTCACGTACA 35 2a1 L10347 Reverse Collagen CGATAACAGTCTTGCCCCACTT 36 2a1 L10347 Forward FGFR3 ACGGCACACCCTACGTTACC 37 NM_000142 Reverse FGFR3 TGTGCAAGGAGAGAACCTCTAGCT 38 NM_000142 Forward BMP-2 CCAACACTGTGCGCAGCTT 39 NM_001200 Reverse BMP-2 AAGAATCTCCGGGTTGTTTTCC 40 NM_001200 Forward ALK-1 CAGTCTCATCCTGAAAGCATCTGA 41 NM_000020 Reverse ALK-1 TTTCCCACACACTCCACCAA 42 NM_000020 Forward collagen TGGAGTGTTTTACGCTGAACGAT 43 10a1 NM_000493 Reverse collagen CCTCTTACTGCTATACCTTTACTCTT 44 10a1 TATGG NM_000493 Forward Collagen TGCCATCAAAGTCTTCTGCAA 45 1A1 NM_000088 Reverse Collagen CGCCATACTCGAACTGGAATC 46 1A1 NM_000088 Forward Collagen CACTATTATTTTGGCACAACAGGAA 47 3a1 NM_000090 Reverse Collagen AGACACATATTTGGCATGGTTCTG 48 3a1 NM_000090 Forward MMP13 CAAGGGATCCAGTCTCTCTATGGT 49 NM_002427 Reverse MMP13 GGATAAGGAAGGGTCACATTTGTC 50 NM_002427 Forward PTHLH GCTCGGTGGAGGGTCTCA 51 NM_002820 Reverse PTHLH CTGTGTGGATTTCTGCGATCA 52 NM_002820 Forward GAPDH CATCCATGACAACTTTGGTATCG 53 NM_002046 Reverse GAPDH AGTCTTCTGGGTGGCAGTGAT 54 NM_002046
[0227] Results are expressed for each tested marker gene as the relative expression in the sample (transduction with tested adenovirus) versus control (knock-down control adenoviruses) (Relative expression=2ddCt, where ddCt=dCtKD sample-dCtKD control and dCt=Ctsample-CtGAPDH). The results for the 14 genes are shown in FIG. 9. The knock down of the mRNA of the respective genes results in expression of the positive markers, while the levels of the negative markers are either low or not detectable.
Control Viruses
[0228] Ad-BMP2; described in WO 03/018799
[0229] Ad-ALPL: Ad5 dE1/E2A adenoviruses that mediate the expression of full length liver/bone/kidney alkaline phosphatase (NP--000469).
Example 12
Expression of cDNA's in Human Cartilage
[0230] Upon identification of a modulator of cartilage synthesis, it is of the highest importance to evaluate whether the modulator is expressed in the tissue and the cells of interest. This can be achieved by measuring the RNA and/or protein levels. In recent years, RNA levels are being quantified through real time PCR technologies, whereby the RNA is first transcribed to cDNA and then the amplification of the cDNA of interest is monitored during a quantitative PCR reaction. The amplification plot and the resulting Ct value are indicators for the amount of a specific RNA transcript present in the sample. Ct values are determined in the presence or absence of the reverse transcriptase step (+RT versus -RT). An amplification signal in the -RT condition indicates the occurrence of non-specific PCR products originating from the genomic DNA. If the +RT Ct value is 3 Ct values higher than the -RT Ct value, then the investigated RNA is present in the sample.
[0231] To assess whether the polypeptides of the genes identified in the above assays are expressed in human cartilage, real time PCR with specific primers for the polynucleotides ("Assay on Demand" Applied Biosystems) is performed on human cartilage total RNA (Clinomics Biosciences). 2 samples of non-osteoarthritis and 2 of osteoarthritis patients are analyzed.
[0232] In short, 40 ng of RNA is transcribed to DNA using the MultiScribe Reverse Transcriptase (50 U/μl) enzyme (Applied BioSystems). The resulting cDNA is amplified with AmpliTaq Gold DNA polymerase (Applied BioSystems) during 40 cycles using an ABI PRISM® 7000 Sequence Detection System. Amplification of the transcript is detected via SybrGreen which results in a fluorescent signal upon intercalation in double stranded DNA.
[0233] Total RNA isolated from human cartilage is analyzed for the presence of transcripts listed in Table 4 via quantitative real time PCR.
[0234] For the genes listed in Table 4 the obtained Ct values indicate that they are detected in all RNA samples. ELA1 RNA is not detected in the real time PCR analysis, underscoring the need for additional patient analysis.
TABLE-US-00007 TABLE 4 Ct values Normal Cartilage OA Cartilage TARGET Ct Sample 1 Ct Sample 2 Ct Sample 1 Ct Sample 2 PDE1A 30.8 28.5 30.7 30.2 GPR103 33.1 33.3 38.4 37.4 JAK1 26.2 24.1 27 26 ICK 36.3 33.8 36.1 35.5 DGKB 28.5 26.7 29.7 28.7 DUSP3 27.1 24.2 27 27.1 DUSP11 28.8 27.5 29.9 29.2 SLC26A8 36.13 34.04 37.12 37.22 SLC15A2 32.5 28.1 34.7 32.9 ABCG1 29.7 28 31.1 29.5 FZD1 28.1 25.4 34.1 27.1 ELA1 40 40 40 39.1
Example 13
Identification of Small Molecules that Inhibit TARGET Kinase Activity
[0235] Compounds are screened for inhibition of the activity of the TARGETS that are kinase polypeptides. The affinity of the compounds to the polypeptides is determined in an experiment detecting changed reaction conditions after phosphorylation. The TARGET kinase polypeptides are incubated with its substrate and ATP in an appropriate buffer. The combination of these components results in the in vitro phosphorylation of the substrate. Sources of compounds include commercially available screening library, peptides in a phage display library or an antibody fragment library, and compounds that have been demonstrated to have binding affinity for a TARGET kinase.
[0236] The TARGET kinase polypeptides can be prepared in a number of ways depending on whether the assay will be run using cells, cell fractions or biochemically, on purified proteins. The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise TARGET kinase catalytic activity.
[0237] Identification of small molecules inhibiting the activity of the TARGET kinase polypeptides is performed by measuring changes in levels of phosphorylated substrate or ATP. Since ATP is consumed during the phosphorylation of the substrate, its levels correlate with the kinase activity. Measuring ATP levels via chemiluminescent reactions therefore represents a method to measure kinase activity in vitro (Perkin Elmer). In a second type of assay, changes in the levels of phosphorylated substrate are detected with phosphospecific agents and are correlated to kinase activity. These levels are detected in solution or after immobilization of the substrate on a microtiter plate or other carrier. In solution, the phosphorylated substrate is detected via fluorescence resonance energy transfer (FRET) between the Eu labeled substrate and an APC labeled phosphospecific antibody (Perkin Elmer), via fluorescence polarization (FP) after binding of a phosphospecific antibody to the fluorescently labeled phosphorylated substrate (Panvera), via an Amplified Luminescent Proximity Homogeneous Assay (ALPHA) using the phosphorylated substrate and phosphospecific antibody, both coupled to ALPHA beads (Perkin Elmer) or using the IMAP binding reagent that specifically detects phosphate groups and thus alleviates the use of the phosphospecific antibody (Molecular Devices). Alternatively, the substrate is immobilized directly or by using biotin-streptavidin on a microtiter plate. After immobilization, the level of phosphorylated substrate is detected using a classical ELISA where binding of the phosphospecific antibody is either monitored via an enzyme such as horseradish peroxidase (HRP) or alkaline phospahtase (AP) which are either directly coupled to the phosphospecific antibody or are coupled to a secondary antibody. Enzymatic activity correlates to phosphorylated substrate levels. Alternatively, binding of the Eu-labeled phosphospecific antibody to the immobilized phosphorylated substrate is determined via time resolved fluorescence energy (TRF) (Perkin Elmer). In addition, the substrate can be coated on FLASH plates (Perkin Elmer) and phosphorylation of the substrate is detected using 33P labeled ATP or 125I labeled phosphospecific antibody.
[0238] Small molecules are randomly screened or are preselected based upon drug class, (i.e. known kinase inhibitors), or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the kinase reaction and their effect on levels of phosphorylated substrate is measured with one or more of the above-described technologies.
[0239] Small molecules that inhibit the kinase activity are identified and are subsequently tested at different concentrations. IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in collagen II assay to check for their effect on the induction of chondrocyte anabolic stimulation.
Example 14
Ligand Screens for TARGET GPCRs
Example 14 A
Reporter Gene Screen
[0240] Mammalian cells such as Hek293 or CHO-K1 cells are either stably transfected with a plasmid harboring the luciferase gene under the control of a cAMP dependent promoter (CRE elements) or transduced with an adenovirus harboring a luciferase gene under the control of a cAMP dependent promoter. In addition reporter constructs can be used with the luciferase gene under the control of a Ca2+ dependent promoter (NF-AT elements) or a promoter that is controlled by activated NF-κB. These cells, expressing the reporter construct, are then transduced with an adenovirus harboring the cDNA of a TARGET GPCR. Forty (40) hours after transduction the cells are treated with the following:
[0241] a) an agonist for the receptor and screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of the SEQ ID NOs of the TARGET GPCRs; or
[0242] b) a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs of the TARGET GPCRs.
[0243] Compounds, which decrease the agonist induced increase in luciferase activity or the constitutive activity, are considered to be antagonists or inverse agonists for a TARGET GPCR. These compounds are screened again for verification and screened against their effect on anabolic stimulation of chondrocytes. The compounds are also screened to verify binding to the GPCR. The binding and reporter activity assays can be performed in essentially any order to screen compounds.
[0244] In addition, cells expressing the NF-AT reporter gene can be transduced with an adenovirus harboring the cDNA encoding the α-subunit of G15 or chimerical Gα subunits. G15 is a promiscuous G protein of the Gq class that couples to many different GPCRs and as such re-directs their signaling towards the release of intracellular Ca2+ stores. The chimerical G alpha subunits are members of the Gs and Gi/o family by which the last 5 C-terminal residues are replaced by those of G.sub.αq, these chimerical G-proteins also redirect cAMP signaling to Ca2+ signaling.
Example 14 B
FLIPR Screen
[0245] Mammalian cells such as Hek293 or CHO-K1 cells are stably transfected with an expression plasmid construct harboring the cDNA of a TARGET GPCR. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. Cells are loaded with a Ca2+ dependent fluorophore such as Fura3 or Fura4. After washing away the excess of fluorophore the cells are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs of the TARGET GPCRs, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells. Activation of the receptor is measured as an almost instantaneously increase in fluorescence due to the interaction of the fluorophore and the Ca2+ that is released. Compounds that reduce or inhibit the agonist induced increase in fluorescence (or constitutive fluorescence) are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds are screened again to measure the amount of anabolic stimulation of chondrocytes as well as binding to a TARGET GPCR.
Example 14 C
AequoScreen
[0246] CHO cells, stably expressing Apoaequorin are stably transfected with a plasmid construct harboring the cDNA of a TARGET GPCR. Cells are seeded, grown, and selected until sufficient stable cells can be obtained. The cells are loaded with coelenterazine, a cofactor for apoaequorin. Upon receptor activation intracellular Ca2+ stores are emptied and the aequorin will react with the coelenterazine in a light emitting process. The emitted light is a measure for receptor activation. The CHO, stable expressing both the apoaequorin and the receptor are screened against a large collection of reference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs, TimTech), small chemical compounds (Tocris), commercially available screening libraries, and compounds that have been demonstrated to have binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs of the TARGET GPCRs, by simultaneously adding an agonist (alternatively no agonist need be added if the constitutive activity of the receptor is used) and a compound to the cells. Activation of the receptor is measured as an almost instantaneously light flash due to the interaction of the apoaequorin, coelenterazine, and the Ca2+ that is released. Compounds that reduce or inhibit the agonist induced increase in light or the constitutive activity are considered to be antagonists or inverse agonists for the receptor they are screened against. These compounds are screened again to measure the amount of anabolic stimulation of chondrocytes as well as binding to a TARGET GPCR.
[0247] In addition, CHO cells stable expressing the apoaequorin gene are stably transfected with a plasmid construct harboring the cDNA encoding the α-subunit of G15 or chimerical G.sub.α subunits. G15 is a promiscuous G protein of the Gq class that couples to many different GPCRs and as such redirects their signaling towards the release of intracellular Ca2+ stores. The chimerical G alpha subunits are members of the Gs and Gi/o family by which the last 5 C-terminal residues are replaced by those of G.sub.αq, these chimerical G-proteins also redirect cAMP signaling to Ca2+ signaling.
Example 14 D
Screening for Compounds that Bind to the GPCR Polypeptides (Displacement Experiment)
[0248] Compounds are screened for binding to the TARGET GPCR polypeptides. The affinity of the compounds to the polypeptides is determined in a displacement experiment. In brief, the GPCR polypeptides are incubated with a labeled (radiolabeled, fluorescent labeled) ligand that is known to bind to the polypeptide and with an unlabeled compound. The displacement of the labeled ligand from the polypeptide is determined by measuring the amount of labeled ligand that is still associated with the polypeptide. The amount associated with the polypeptide is plotted against the concentration of the compound to calculate IC50 values. This value reflects the binding affinity of the compound to its TARGET, i.e. the TARGET GPCR polypeptides. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied an anabolic stimulation of chondrocytes assay to check for their effect on osteogenesis. The TARGET GPCR polypeptides can be prepared in a number of ways depending on whether the assay are run on cells, cell fractions or biochemically, on purified proteins.
Example 14 E
Screening for Compounds that Bind to a TARGET GPCR (Generic GPCR Screening Assay)
[0249] When a G protein receptor becomes constitutively active, it binds to a G protein (Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyses the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [35S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [35S]GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. Moreover, a preferred approach is the use of a GPCR-G protein fusion protein. The strategy to generate a TARGET GPCR-G protein fusion protein is well known for those known in the art. Membranes expressing TARGET GPCR-G protein fusion protein are prepared for use in the direct identification of candidate compounds such as inverse agonist. Homogenized membranes with TARGET GPCR-G protein fusion protein are transferred in a 96-well plate. A pin-tool is used to transfer a candidate compound in each well plus [35S]GTPγS, followed by incubation on a shaker for 60 minutes at room temperature. The assay is stopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C. The plates are then aspirated and radioactivity is then read.
Example 14 F
Receptor Ligand Binding Study on Cell Surface
[0250] The receptor is expressed in mammalian cells (Hek293, CHO, COS7) by adenoviral transducing the cells (see U.S. Pat. No. 6,340,595). The cells are incubated with both labeled ligand (iodinated, tritiated, or fluorescent) and the unlabeled compound at various concentrations, ranging from 10 pM to 10 μM (3 hours at 4° C.: 25 mM HEPES, 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2 and 0.2% BSA, adjusted to pH 7.4). Reactions mixtures are aspirated onto PEI-treated GF/B glass filters using a cell harvester (Packard). The filters are washed twice with ice cold wash buffer (25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, adjusted to pH 7.4). Scintillant (MicroScint-10; 35 μl) is added to dried filters and the filters counted in a (Packard Topcount) scintillation counter. Data are analyzed and plotted using Prism software (GraphPad Software, San Diego, Calif.). Competition curves are analyzed and IC50 values calculated. If one or more data points do not fall within the sigmoidal range of the competition curve or close to the sigmoidal range the assay is repeated and concentrations of labeled ligand and unlabeled compound adapted to have more data points close to or in the sigmoidal range of the curve.
Example 14 G
Receptor Ligand Binding Studies on Membrane Preparations
[0251] Membranes preparations are isolated from mammalian cells (Hek293, CHO, COS7) cells over expressing the receptor is done as follows: Medium is aspirated from the transduced cells and cells are harvested in 1×PBS by gentle scraping. Cells are pelleted (2500 rpm 5 min) and resuspended in 50 mM Tris pH 7.4 (10×106 cells/ml). The cell pellet is homogenized by sonicating 3×5 sec (UP50H; sonotrode MS1; max amplitude: 140 um; max Sonic Power Thickness: 125 W/cm2). Membrane fractions are prepared by centrifuging 20 min at maximal speed (13,000 rpm˜15,000 to 20,000 g or rcf). The resulting pellet is resuspended in 500 μl 50 mM Tris pH 7.4 and sonicated again for 3×5 sec. The membrane fraction is isolated by centrifugation and finally resuspended in PBS. Binding competition and derivation of IC50 values are determined as described above.
Example 14 H
Internalization Screen (1)
[0252] Activation of a GPCR-associated signal transduction pathway commonly leads to translocation of specific signal transduction molecules from the cytoplasm to the plasma membrane or from the cytoplasm to the nucleus. Norak has developed their transfluor assay based on agonist-induced translocation of receptor-β-arrestin-GFP complex from the cytosol to the plasma membrane and subsequent internalization of this complex, which occurs during receptor desensitization. A similar assay uses GFP tagged receptor instead of β-arrestin. Hek293 cells are transduced with a TARGET GPCR vector that translates for a TARGET GPCR-eGFP fusion protein. 48 hours after transduction, the cells are set to fresh serum-free medium for 60 minutes and treated with a ligand for 15, 30, 60 or 120 minutes at 37° C. and 5% CO2. After indicated exposure times, cells are washed with PBS and fixed with 5% paraformaldehyde for 20 minutes at RT. GFP fluorescence is visualized with a Zeiss microscope with a digital camera. This method aims for the identification of compounds that inhibit a ligand-mediated (constitutive activity-mediated) translocation of the fusion protein to intracellular compartments.
Example 14 I
Internalization Screen (2)
[0253] Various variations on translocation assays exists using β-arrestin and β-galactosidase enzyme complementation and BRET based assays with receptor as energy donor and β-arrestin as energy acceptor. Also the use of specific receptor antibodies labeled with pH sensitive dyes are used to detect agonist induced receptor translocation to acidic lysosomes. All of the translocation assays are used for screening for both agonistic and antagonistic acting ligands.
Example 14 J
Melanophore Assay (Arena Pharmaceutical)
[0254] The melanophore assay is based on the ability of GPCRs to alter the distribution of melanin containing melanosomes in Xenopus melanophores. The distribution of the melanosomes depends on the exogenous receptor that is either Gi/o or Gs/q coupled. The distribution of the melanosomes (dispersed or aggregated) is easily detected by measuring light absorption. This type of assay is used for both agonist as well as antagonist compound screens.
Example 15
Identification of Small Molecules that Inhibit Protease Activity
[0255] Compounds are screened for inhibition of the activity of the polypeptides of the present invention. The affinity of the compounds to the polypeptides is determined in an experiment detecting changes in levels of cleaved substrate. In brief, the polypeptides of the present invention are incubated with its substrate in an appropriate buffer. The combination of these components results in the cleavage of the substrate.
[0256] The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise the catalytic activity of the polypeptide of the invention.
[0257] Cleavage of the substrate can be followed in several ways. In a first method, the substrate protein is heavily labeled with a fluorescent dye, like fluorescein, resulting in a complete quenching of the fluorescent signal. Cleavage of the substrate however, releases individual fragments, which contain less fluorescent labels. This results in the loss of quenching and the generation of a fluorescent signal, which correlates to the levels of cleaved substrate. Cleavage of the protein, which results in smaller peptide fragments, can also be measured using fluorescent polarization (FP). Alternatively, cleavage of the substrate can also be detected using fluorescence resonance energy transfer (FRET): a peptide substrate is labeled on both sides with either a quencher and fluorescent molecule, like DABCYL and EDANS. Upon cleavage of the substrate both molecules are separated resulting in fluorescent signal correlating to the levels of cleaved substrate. In addition, cleavage of a peptide substrate can also generate a new substrate for another enzymatic reaction, which is then detected via a fluorescent, chemiluminescent or colorimetric method.
[0258] Small molecules are randomly screened or are preselected based upon drug class, i.e. protease, or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the proteolytic reaction and their effect on levels of cleaved substrate is measured with the described technologies.
[0259] Small molecules that inhibit the protease activity are identified and are subsequently tested at different concentrations. IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in amyloid beta secretion assay to check for their effect on the beta amyloid secretion and processing.
Example 16
Identification of Small Molecules that Inhibit Phosphodiesterase Activity
[0260] Compounds are screened for inhibition of the activity of the polypeptides of the present invention. The affinity of the compounds to the polypeptides is determined in an experiment detecting changes in levels of substrate or product. In brief, the polypeptides of the present invention are incubated with its substrate in an appropriate buffer. The combination of these components results in the conversion of the substrate into its product.
[0261] The polypeptides can be applied as complete polypeptides or as polypeptide fragments, which still comprise the catalytic activity of the polypeptide of the invention.
[0262] Conversion of cAMP or cGMP in AMP or GMP can be followed 1) by determining the cAMP or cGMP levels using e.g. ELISA. Alpha screen technology, Time resolved fluorescent technology, IMAP 2) by determining the levels of the products AMP and GMP using a colorimetric assay. The basis for the latter assay is the cleavage of cAMP or
[0263] cGMP by a cyclic nucleotide phosphodiesterase. The 5'-nucleotide released is further cleaved into the nucleoside and phosphate by the enzyme 5'-nucleotidase. The phosphate released due to enzymatic cleavage is quantified using BIOMOL GREEN® reagent in a modified Malachite Green assay.
[0264] Small molecules are randomly screened or are preselected based upon drug class, i.e. PDE, or upon virtual ligand screening (VLS) results. VLS uses virtual docking technology to test large numbers of small molecules in silico for their binding to the polypeptide of the invention. Small molecules are added to the PDE reaction and their effect on cyclic nucleotide levels is measured with the described technologies.
[0265] Small molecules that inhibit the PDE activity are identified and are subsequently tested at different concentrations. IC50 values are calculated from these dose response curves. Strong binders have an IC50 in the nanomolar and even picomolar range. Compounds that have an IC50 of at least 10 micromol or better (nmol to pmol) are applied in assays evaluating the anabolic activity of chondrocytes. This can be achieved by determining col2α1 and aggrecan levels produced by the chondrocytes.
Sequence CWU
1
1
48013536DNAHomo sapiens 1atggggacga cggccccagg gcccattcac ctgctggagc
tatgtgacca gaagctcatg 60gagtttctct gcaacatgga taataaggac ttggtgtggc
ttgaggagat ccaagaggag 120gccgagcgca tgttcaccag agaattcagc aaagagccag
agctgatgcc caaaacacct 180tctcagaaga accgacggaa gaagagacgg atttcttatg
ttcaggatga aaacagagat 240cccatcagga gaaggttatc ccgcagaaag tctcggagca
gccagctgag ctcccgacgc 300ctccgcagca aggacagtgt agagaagctg gctacagtgg
tcggggagaa cggctccgtt 360ctgcggcgtg tgacccgtgc tgcggctgca gctgccgcgg
ctaccatggc attggctgca 420ccttcttcac ccacccctga gtctcccacg atgctgacta
agaagcccga ggataaccac 480acccagtgcc agctggtgcc tgtggtggag atcggcatca
gtgagcgcca gaatgctgag 540cagcatgtca cccagctcat gtccaccgag cctctgcccc
gcactctgtc cccgactcca 600gcttcagcca cagctccaac ctcccagggc atcccgacat
cagatgagga atcaacacct 660aagaagtcga aggccaggat actggagtcc atcacagtga
gctccctgat ggctacaccc 720caggacccca agggtcaagg ggtcgggacg gggcggtctg
cgtctaagct caggattgcg 780caggtctccc ctggcccacg ggactcgcca gcctttccag
attctccatg gcgggagcgg 840gtgctggctc ccatcctgcc ggataacttc tccacgccca
cgggctctcg cacggactct 900caatcggtgc ggcacagccc gatcgccccg tcttccccga
gtccccaagt cttagcccag 960aagtactctc tggtggccaa acaggaaagt gttgtccgca
gggcgagcag aaggcttgcc 1020aagaagactg ccgaagagcc agctgcctct ggccgcatca
tctgtcacag ttacctggag 1080aggctcctga atgttgaggt gccccagaaa gttggttctg
agcagaagga accccccgag 1140gaggctgagc ctgtggcggc agctgagcca gaggtccctg
agaacaacgg aaataactcg 1200tggccccaca atgacacgga gattgccaac agcacaccca
acccgaagcc tgcagccagc 1260agcccggaaa caccctctgc agggcagcaa gaggccaaga
cggaccaagc agatggaccc 1320agagagccac cgcagagtgc caggaggaag cgcagctaca
agcaggccgt gagtgagctg 1380gacgaggagc agcacctgga ggatgaggag ctgcagcccc
ccaggagcaa gaccccttcc 1440tcaccctgcc cagccagcaa ggtggtacgg cccctccgga
cctttctgca cacagtgcag 1500aggaaccaga tgctcatgac ccctacctca gccccacgca
gcgtcatgaa gtcctttatt 1560aagcgcaaca ctcccctgcg catggacccc aaggagaagg
agcggcagcg cctggagaat 1620ctgcggcgga aggaggaggc cgagcagctg cgcaggcaga
aggtggagga ggacaagcgg 1680cggcggctgg aggaggtgaa gctgaagcgt gaggaacgcc
tccgcaaggt gctgcaggcc 1740cgcgagcggg tggagcagat gaaggaggag aagaagaagc
agattgagca gaagtttgct 1800cagatcgacg agaagactga gaaggccaag gaggagcggc
tggcagagga gaaggccaag 1860aaaaaggcgg cggccaagaa gatggaggag gtggaagcac
gcaggaagca ggaagaggat 1920gcacgtaggc tcaggtggct gcagcaggag gaggaagagc
ggcggcacca agagctgctg 1980cagaagaaga aggaagagga gcaggagcgg ctgcggaagg
cggccgaggc taagcggctg 2040gcagagcagc gggagcagga gcggcgggag caggagcggc
gggagcagga gcggcgcgag 2100caggagcggc gcgagcagga gcggcgggag caggagcggc
gcgagcagga gcgacagctg 2160gcagagcagg agcgtcggcg ggagcaggag cggctccaag
ccgagaggga gctgcaggag 2220cgggagaagg ccctgcggct gcagaaggag cagctgcaga
gggaactgga ggagaagaag 2280aagaaggaag agcagcagcg tctggctgag cggcagctgc
aggaggagca agagaagaaa 2340gccaaggagg cagcaggggc cagcaaggcc ctgaatgtga
ctgtggacgt gcagtctcca 2400gcttgtacct catctcccat cactccgcaa gggcacaagg
cccctcccca gatcaacccc 2460cacaactacg ggatggatct gaatagcgac gactccaccg
atgatgaggc ccatccccgg 2520aagcccatcc ccacctgggc ccgaggcacc ccgctcagcc
aggctatcat tcaccagtac 2580taccagccac cgaaccttct ggagctcttt ggaaccattc
tcccactgga cttggaggat 2640atcttcaaga agagcaagcc ccgctatcac aagcgcacca
gctctgctgt ctggaactca 2700ccgcccctgc agggcgccag ggtccccagc agcctggcct
acagcctgaa gaagcactga 2760ggctggcctg cggccttctt ggcagcctcg cctcctgtcc
atgtctatct gtctgtctgt 2820cggtctgtgt cttggtctgt tgccctcctt cttggcatgc
cattgtggag ggcttggcca 2880ggtgtatata aacgtcctct gtgctgggtg tttctgctgc
aggtggcagg tggccccagg 2940cctgtttgga ggatgggctg ggtgggtggg tggggaagaa
atgggcccag ccccacatgg 3000cctgcagaca gtgctctgta aatagttgtt ttaatttagc
tgaatgttag cattttagtc 3060tttggcattt tagcgtttgg gaggtagatt aataaagtat
attccttcaa gcctgctgtt 3120gataccatga agactgggcg cctcagtccc agccctgtag
ctgtgtgtct tgggccacca 3180gtggcctgca ggacgaaggt actgttccat cacctgcggt
gtgcctcagg atcaccaggt 3240gcaggccccc accctcggag atgctgctgc agtgagtggt
tccactgcct ggataaccct 3300tgaggaacac gtcagttact gtcacgatgg ggcaggtgga
gctccttcct attttttggg 3360gtgctccctg tttgtaaagg ggagtttgtt cattgggaaa
gacctgggtc ttgacacggc 3420cctgccactt agtcccctac cctctccatt ccccaggctc
cacccgtgct gctcaagtgc 3480aaatggactt gagagtattt atgtgctggt gaagtatgag
gtctgagtag aaaagg 353623926DNAHomo sapiens 2atgacaaacc aggaaaaatg
ggcccacctc agcccttcgg aattttccca acttcagaaa 60tatgctgagt attctacaaa
gaaattaaag gatgttcttg aagaattcca tggtaatggt 120gtgcttgcaa agtataatcc
tgaagggaaa caagacattc ttaaccaaac aatagatttt 180gaaggtttca aactattcat
gaagacattc ctggaagccg agcttcctga tgatttcact 240gcacaccttt tcatgtcatt
tagcaacaag tttcctcatt ctagtccaat ggtaaaaagt 300aagcctgctc tcctatcagg
cggtctgaga atgaataaag gtgccatcac ccctccccga 360actacttctc ctgcaaatac
gtgttcccca gaagtaatcc atctgaagga cattgtctgt 420tacctgtctc tgcttgaaag
aggaagacct gaggataagc ttgagtttat gtttcgcctt 480tatgacacgg atgggaatgg
cttcctggac agctcggagc tagaaaatat catcagtcag 540atgatgcatg ttgcagaata
ccttgagtgg gatgtcactg aacttaatcc aatcctccat 600gaaatgatgg aagaaattga
ctatgatcat gatggaaccg tgtctctgga ggaatggatt 660caaggaggaa tgacaacgat
tccacttctt gtgctcctgg gcttagaaaa taacgtgaag 720gatgatggac agcacgtgtg
gcgactgaag cactttaaca aacctgccta ttgcaacctt 780tgcctgaaca tgctgattgg
cgtggggaag cagggcctct gctgttcctt ctgcaagtac 840acagtccatg agcgctgtgt
ggctcgagca cctccctctt gcatcaagac ctatgtgaag 900tccaaaagga acactgatgt
catgcaccat tactgggttg aaggtaactg cccaaccaag 960tgtgataagt gccacaaaac
tgttaaatgt taccagggcc tgacaggact gcattgtgtt 1020tggtgtcaga tcacactgca
taataaatgt gcttctcatc taaaacctga atgtgactgt 1080ggacctttga aggaccatat
tttaccaccc acaacaatct gtccagtggt actgcagact 1140ctgcccactt caggagtttc
agttcctgag gaaagacaat caacagtgaa aaaggaaaag 1200agtggttccc agcagccaaa
caaagtgatt gacaagaata aaatgcaaag agccaactct 1260gttactgtag atggacaagg
cctgcaggtc actcctgtgc ctggtactca cccactttta 1320gtttttgtga accccaaaag
tggtggaaaa caaggagaac gaatttacag aaaattccag 1380tatctattaa atcctcgtca
ggtttacagt ctttctggaa atggaccaat gccagggtta 1440aactttttcc gtgatgttcc
tgacttcaga gtgttagcct gtggtggaga tggaaccgtg 1500ggctgggttt tggattgcat
agaaaaggcc aatgtaggca agcatcctcc agttgcgatt 1560ctgcctcttg ggactggcaa
tgatctagca agatgcctgc gatggggagg aggttacgaa 1620ggtgagaatc tgatgaaaat
tctaaaagac attgaaaaca gcacagaaat catgttggac 1680aggtggaagt ttgaagtcat
acctaatgac aaagatgaga aaggagaccc agtgccttac 1740agtatcatca ataattactt
ttccattggc gtggatgcct ccattgcaca cagattccac 1800atcatgagag aaaaacaccc
agagaaattc aacagtagaa tgaagaacaa attttggtat 1860tttgagtttg gcacatctga
aactttctca gccacctgca agaagctaca tgaatctgta 1920gaaatagaat gtgatggagt
acagatagat ttaataaaca tctctctgga aggaattgct 1980attttgaata taccaagcat
gcatggagga tccaatcttt ggggagagtc taagaaaaga 2040cgaagccatc gacgaataga
gaaaaaaggg tctgacaaaa ggaccaccgt cacagatgcc 2100aaagagttga agtttgcaag
tcaagatctc agtgaccagc tgctggaggt ggtcggcttg 2160gaaggagcca tggagatggg
gcaaatatac acaggcctga aaagtgctgg ccggcggctg 2220gctcagtgct cctgcgtggt
catcaggacg agcaagtctc tgccaatgca aattgatggg 2280gagccatgga tgcagacccc
atgcacaata aaaattacac acaagaacca agccccaatg 2340ctgatgggcc cgcctccaaa
aaccggttta ttctgctccc tcgtcaaaag gacaagaaac 2400cgaagcaagg aataatcctg
tgttgtttca ctcttagaaa ttgaattagc ataattgggc 2460catggaacac atatgctgga
aatctttgaa ccatttcaag tctcctgctc atgcaaaatc 2520atggaagtgg tttaacagtt
tttgttacta agctaatgta aaattcagct attagaaaat 2580ttattgtctc agtttttata
ggcatctttg catgaagaaa gcagaagttt acctgaagtg 2640atactgcata tttttggtgc
atgcattccc atagattttt acatctccca cccaactctt 2700ccccaatttc cttttactaa
cctgtgagaa aaacccgtga aacatgaaaa aggaaatacc 2760atgggaaacg tgattctcag
tgtgattcca attattacga agcactaatc agtaacgcta 2820caatgatcat aattgcagat
tgctatacgt ttccctttta gaatcagtgt atcagtgacc 2880tatgacttga ggagaaactt
ttaattcgaa gattttatta aatagttgac tacaatacct 2940tgctatatat acatagtttt
tcttcaacat cttaactctt ctgagtggaa ataaaaatat 3000caggcataag gttttctcat
gctgaaaaat agaacgcggt ttttattttg cttagttttc 3060tttttaattc cagaaataag
tgaaaacatg ttacttgaca gtcaagtgtg gtaatatggc 3120aagccttgtt cctttctgca
tgagaatcta ggagagaatt cataaccaca ccaataacga 3180aatagaagtt ttaaactatg
tgcctaatca atgtgtttcc caccaaagat tcagaaaaca 3240atgcttgaga gaaatgggtt
aatgcataat taattaagca ttgtggagca aatttagggt 3300tcctgtgatt aattttgtga
tgactaaaat gctggaaagc aagtgagttg cccattaatt 3360atgattaaaa ttctcacctt
tcacagacag acaataagcc agacaacaca atcaaagctc 3420aatagatgat ttcttgcttt
tttcagtcat ttataaatat aggtgtaatt tttcatggat 3480cagttaagta cacttgaagg
aagtaaatga ttgtatcagt ttatttctag tataaatggg 3540tacctgtaat aatactgagc
tcttggaagc gaatcatgca tgcaattagc tccctcctcc 3600tcacctactc cactcccatc
tttatgacat ttcaaatgtt tatttggaaa caacagccta 3660gatcactgtt gaaggtgttc
atggcatagt tggagtctct gactgtttaa agaaatcaca 3720gaacagtact tttcttttag
tgtttcatta agcctatgat gtaaaatgaa atgcttctga 3780gcagtcttgt aatattgttc
attcatattg acctgcatct catcattgca tgttttatgt 3840tttcaaacat gccataagga
aaacgagtgc ctgaactgca tgatttatta gtttctctcc 3900actctgcatt aaagtgctaa
tgattt 392633172DNAHomo sapiens
3atgacaaacc aggaaaaatg ggcccacctc agcccttcgg aattttccca acttcagaaa
60tatgctgagt attctacaaa gaaattaaag gatgttcttg aagaattcca tggtaatggt
120gtgcttgcaa agtataatcc tgaagggaaa caagacattc ttaaccaaac aatagatttt
180gaaggtttca aactattcat gaagacattc ctggaagccg agcttcctga tgatttcact
240gcacaccttt tcatgtcatt tagcaacaag tttcctcatt ctagtccaat ggtaaaaagt
300aagcctgctc tcctatcagg cggtctgaga atgaataaag gtgccatcac ccctccccga
360actacttctc ctgcaaatac gtgttcccca gaagtaatcc atctgaagga cattgtctgt
420tacctgtctc tgcttgaaag aggaagacct gaggataagc ttgagtttat gtttcgcctt
480tatgacacgg atgggaatgg cttcctggac agctcggagc tagaaaatat catcagtcag
540atgatgcatg ttgcagaata ccttgagtgg gatgtcactg aacttaatcc aatcctccat
600gaaatgatgg aagaaattga ctatgatcat gatggaaccg tgtctctgga ggaatggatt
660caaggaggaa tgacaacgat tccacttctt gtgctcctgg gcttagaaaa taacgtgaag
720gatgatggac agcacgtgtg gcgactgaag cactttaaca aacctgccta ttgcaacctt
780tgcctgaaca tgctgattgg cgtggggaag cagggcctct gctgttcctt ctgcaagtac
840acagtccatg agcgctgtgt ggctcgagca cctccctctt gcatcaagac ctatgtgaag
900tccaaaagga acactgatgt catgcaccat tactgggttg aaggtaactg cccaaccaag
960tgtgataagt gccacaaaac tgttaaatgt taccagggcc tgacaggact gcattgtgtt
1020tggtgtcaga tcacactgca taataaatgt gcttctcatc taaaacctga atgtgactgt
1080ggacctttga aggaccatat tttaccaccc acaacaatct gtccagtggt actgcagact
1140ctgcccactt caggagtttc agttcctgag gaaagacaat caacagtgaa aaaggaaaag
1200agtggttccc agcagccaaa caaagtgatt gacaagaata aaatgcaaag agccaactct
1260gttactgtag atggacaagg cctgcaggtc actcctgtgc ctggtactca cccactttta
1320gtttttgtga accccaaaag tggtggaaaa caaggagaac gaatttacag aaaattccag
1380tatctattaa atcctcgtca ggtttacagt ctttctggaa atggaccaat gccagggtta
1440aactttttcc gtgatgttcc tgacttcaga gtgttagcct gtggtggaga tggaaccgtg
1500ggctgggttt tggattgcat agaaaaggcc aatgtaggca agcatcctcc agttgcgatt
1560ctgcctcttg ggactggcaa tgatctagca agatgcctgc gatggggagg aggttacgaa
1620ggtgagaatc tgatgaaaat tctaaaagac attgaaaaca gcacagaaat catgttggac
1680aggtggaagt ttgaagtcat acctaatgac aaagatgaga aaggagaccc agtgccttac
1740agtatcatca ataattactt ttccattggc gtggatgcct ccattgcaca cagattccac
1800atcatgagag aaaaacaccc agagaaattc aacagtagaa tgaagaacaa attttggtat
1860tttgagtttg gcacatctga aactttctca gccacctgca agaagctaca tgaatctgta
1920gaaatagaat gtgatggagt acagatagat ttaataaaca tctctctgga aggaattgct
1980attttgaata taccaagcat gcatggagga tccaatcttt ggggagagtc taagaaaaga
2040cgaagccatc gacgaataga gaaaaaaggg tctgacaaaa ggaccaccgt cacagatgcc
2100aaagagttga agtttgcaag tcaagatctc agtgaccagc tgctggaggt ggtcggcttg
2160gaaggagcca tggagatggg gcaaatatac acaggcctga aaagtgctgg ccggcggctg
2220gctcagtgct cctgcgtggt catcaggacg agcaagtctc tgccaatgca aattgatggg
2280gagccatgga tgcagacccc atgcacagtg agtacagagt agttgatatg ctatgtcaat
2340ctcagttttg ctttcctctt tgactaaata accacaataa ctgatttttt tctttatttc
2400ttttcaacct atcagcaaat agtctttttg ttgttgttgt tatgtgtgtg tcagagccac
2460tacatttagg ctgtagacat tatataccct tggcaatgat ttagctcttg aatgtttgtg
2520ctagcctaag tataaataga tcttttaaat agatcaatta taaaccatag atcaattata
2580aactatggag ctaaacaaaa tattaataaa agtttatctg aaactttttt gtttatttca
2640gagcacatta ttagaatatt atttgcgaga aatgcagacc taagcttata tgtgaactta
2700tttctcagct tttctatgcc tccatttggg gatttgaggg ctttcttctc cataagaaaa
2760aaatttctct ccagtttcta ccataattaa ttgtgttttc cagaatgagg tattatttaa
2820ggcagacact gcccctctca aaaaaaatca gttttcattt gcatagtgaa tattttattg
2880catttcaaaa acatgctagg aactgctttt ggcactggga gtagacacat gaacaagacc
2940aacagtgtaa tttccttcaa gttacttaca ttcctataat agaggaccga ataaataaac
3000aactacatga taaatataac ttcagactgt gagagttatt aaaaaataag gtgaaatgat
3060gataagaagc tggattaggt gtggagaata aatactactt gagataaggg agacctcttt
3120gaaaggacat agccaaaagc ttagtataaa attaaaaaaa ataaaaaaaa aa
317246228DNAHomo sapiens 4cgggttcctg ctgcactacc atgcgccgtg cggcccgtgc
gactcgccgg acctcgcggg 60cgtccctgta cggagccctc ggccggtcct agcagggatt
gtccccattt ccagctccgg 120agcgggcggc tgcgccccgc tcgtcgagga gctgcgctca
cctcaggggc gggcccccgc 180ctgcgttcgc ggcgccaaga agagtattcc tttccactta
caactaaata agacctgctt 240ttgctggagc tgtgctaggc tgagggaatt ccacactgaa
ttttacaagc gggatggatt 300tctctaaggc agaagactga tttttggaaa tatgtatttg
ggagacagtc acgtcctatt 360gaataccttg tgctggtgct gccatcgaaa aatctggtta
cactctgggg aggactgcta 420ccactgcaga actgaaccac ttcggccgtg agatgagtgt
ccggcctgag caggcacacc 480atgaatagat acacaacaat caggcagctc ggggatggaa
cctacggttc cgtcctgctg 540ggaagaagca ttgagtctgg ggagctgatc gctattaaaa
aaatgaaaag aaaattttat 600tcctgggagg aatgcatgaa ccttcgggag gttaagtctt
taaagaagct caaccatgcc 660aatgtagtca aattaaaaga agttatcagg gaaaatgatc
atctttattt tatcttcgag 720tacatgaagg aaaatcttta ccagctcatt aaagagagaa
ataagttgtt tcctgagtct 780gctataagga atatcatgta tcagatatta caaggactcg
catttattca caaacacggc 840ttctttcatc gagacttaaa gcctgagaac ctcctctgca
tgggaccaga acttgtgaaa 900attgcagact ttggtttggc ccgagaaata cgatcaaaac
ctccatatac agattatgta 960tctaccagat ggtacagggc tccagaagta ctcctgaggt
ctaccaacta cagctccccc 1020attgacgtct gggcggtggg ctgcatcatg gcagaagttt
acaccctcag gccactcttc 1080cctggagcca gtgaaattga cacaatattc aaaatttgcc
aagtgctggg gacaccaaaa 1140aagactgact ggcctgaagg ctatcaactt tcaagtgcaa
tgaacttccg ttggccacag 1200tgtgtaccca ataacttaaa gaccttgatt cccaatgcta
gcagtgaagc agtccagctc 1260ctgagagaca tgcttcagtg ggatcccaag aaacgaccaa
cagctagtca ggcacttcga 1320tatccttact tccaagttgg acacccacta ggcagcacca
cacaaaacct tcaggattca 1380gaaaaaccac agaaaggcat cctggaaaag gcaggcccac
ctccttatat taagccagtc 1440ccacctgccc agccaccagc caagccacac acacgaattt
cttcacgaca gcatcaagcc 1500agccagcccc ctctgcatct cacgtacccc tacaaagcag
aggtctccag gacagatcac 1560ccaagccatc tccaggagga caagccaagc ccgttgcttt
tcccatccct ccacaacaag 1620catccacagt cgaaaatcac agctggcctg gagcacaaaa
atggtgagat aaagccaaag 1680agtaggagaa ggtggggtct tatttccagg tcaacaaagg
attcagatga ttgggctgac 1740ttggatgact tggatttcag tccatccctc agcaggattg
acctgaaaaa caagaaaaga 1800cagagtgatg acactctctg caggtttgag agtgttttgg
acctgaagcc ctctgagcct 1860gtgggcacag gaaacagtgc ccccacccag acgtcatatc
agcggcgaga cacgcccacc 1920ctgagatctg cagccaagca gcactatttg aagcactctc
gatacttgcc tgggatcagt 1980ataagaaatg gcatactctc gaatccaggc aaggaattta
ttccacctaa tccatggtct 2040agttctggct tgtctggaaa atcttcaggg acaatgtcag
taatcagcaa agtaaattca 2100gttggttcca gctctacaag ttctagtgga ctgactggaa
actatgtccc ttcctttctg 2160aaaaaagaaa tcggttctgc tatgcagagg gtacacctag
cacctattcc agacccttcc 2220cctggttatt cctccctgaa ggccatgaga cctcatcctg
ggcgaccatt cttccacacc 2280cagcctagaa gcactcctgg gttgatacca cggcctccag
ccgcccagcc agtgcatggc 2340cggacagact gggcttccaa gtacgcatct cggcgatgac
tgtctgcctt ggtgatgaat 2400ctcttcctag ggagaagcag gatactttcc ctcagctgac
tggtgttcta cctgcaagat 2460gtgcagaggg cataaaagca aatcaacact ttatagttat
tcttctgaac taagacatgt 2520caatattctt ttttaaagtt tttttttaaa atattgattt
gaatgcagta ggcttttttg 2580tataaaatta ttttattcta aaactgggtc ccattatttt
cttaaacaac agcattttgt 2640atatatggat tatgttttag cattttatac agtcaacttt
gtaatgaact ttttaaaaat 2700taattgattt tcctttgggg ttccagataa tattttctac
agattttgaa aaatgtaata 2760atattaatgc agtattgcaa caggggtgca atttaaggct
atgtgataga gggttattta 2820ctcagtgtgt gcagatattt atgaagtggt gaaatttcaa
gtgtggctca ctaggtactt 2880caggccttct tggactgttg ttagaaaagt gatcctctgc
ttttcttagt aggtcattgg 2940tttgattttt ggataccact ctgctgttct aaaaggacta
ttatattata taattcactt 3000tgttttactt ttgttcccca gatgaaagaa ctctaagtaa
atacatttta aaaaattttt 3060ctgacaccct ttaatgtggt tgcagatctc agatgaaacc
aagcttaatt atactatgcc 3120attatattct aatttattcc atttttgaaa tcaagttgta
tgtgtaccaa taaaagagat 3180ttctgcttca aaaggctctc aacatgaagg ttaacacagt
caatcaaact tacattcctg 3240ccaagatgca tggccaaaaa actaagtatc aaagcagcag
aaggtttttg attatagtaa 3300ctgagatgga attttgtgcc tagctcagtt ctccagatct
ggctaggagc agtcaatgac 3360taatgttctg tcctagccaa attctcagga caatttgggg
agcagaaaga gttatggcag 3420aggttccact catctacaaa gtcacagtca catgccacat
ttgatctcct aaccctggtg 3480tagtttcttt caagagtgag aactttattt gttgggcaga
ggctgttcca ttgagaggaa 3540tgtttacagc agtttcaaaa atgacaaagt cagtttggag
acagaaaaag acaaaaggtc 3600cagtctcatc catctctata tggtacattt gcctcactta
tggttgcctt aaaggcaaga 3660gggaaggtca ccatcagtga acgcaatgca atctcaacag
tgtattgatt catattctcc 3720tagggctcaa actactctct attggttcca ggataatgac
aaattgaacc atatgtaagt 3780aatcttttat tttttatttt ttttttgaga cagagtctca
ctctgtcacc caggctggag 3840tgcagtggcg cgatcttagc tctctgcaac ctctgcctcc
caggttcaag cctcctgagt 3900aactgggact acaggcgccc gccaccacgc ccagctaatt
ttttgtattt ttagtagaga 3960cggggtttca ctgtgttagc caggacggcc tcgatctcct
gacctcgtga tccaccctcc 4020tccacctccc aaagtactgg gattacaggc atgagccact
gcacccagcc aagtgatcat 4080ttttataggt taaaatgata ggtgaaatga atatagacac
tttcatatgg ttcaacctaa 4140tgacttggta aattattgcc ttggtgtatt aataatatgt
tgcattctga acaaataacc 4200atggcttcca aagggcccta acctaaaatc ggagagtaat
ttatgctttg gagaatttga 4260ctcaaatata tacttgacca agcaccatga tccctagggg
catgagaaaa gcacataatg 4320gatgtggatg tgataggtgg tcttttcctg ttaacaagct
ggcagcaaag cttcagaaaa 4380tatatatgca agcacaactt gaagctgaat tcatttctgt
attatattct caactcgtta 4440tctaaagcat cagaacatgt gttttcagag atgagtcctt
tactataagg ttaatattta 4500ttttcatttt ctgtattata tatgaaaagt aaattaatgt
gaaacctggc ccagcttgct 4560ggaaagcagg ttttaaattg taaatattcc ttagaggagc
aaatggattg tttaatacca 4620tagtctcagt aatctagctt atataaggtc attacatttt
ttaactgaaa aacctagtta 4680cctgattatt gcacattata aaattgtttt tctaatactt
tatagggccc aacttcagaa 4740aatacttcgc ttttttcttt ttatgctttc gtttgtttac
cagcaagcaa cttccctggg 4800gaagccaaac acatattcat aaaaaaaatc aagtagctga
tgtgcagttg agaaaactag 4860aggactgaaa aaacaaattt taactagcaa atgctgtgaa
ttactcttcc tccccttctc 4920tgaaatgggt aaaggacaaa ttgtgtaaaa aaacctatgc
actatagaag ggaatagtaa 4980ccatttcttt tgtctctctg tttctgttct gactgagaac
ctgcagccat ttcttgttac 5040atgaaaacaa aatgctactt gttacctcta ttttttgtta
ctatacaatt atgaaatgta 5100atgtaagaca ccaacagaaa tgatatacct gtaactgtac
ctatcaggac tatacctcat 5160ttacagtcag aaagcttact gggatgtcag gaaatgatac
agggttggtt ctcatttcgt 5220gccgaaatga gacagaaatt cagtgacgaa ggtgcgttgt
aggggtattg atgtgcccca 5280ggtagtgcca gcagagtagg gaaaactgca tttgcataaa
aactactctt gacatgattg 5340ttcattttac aaaaaaattc cattaattac caagccctca
cccagcccat gtgtgatagg 5400atttatgtag gaagaaactt gattttcaaa taatttttta
aatgtatctc ttgcctaaag 5460gactatatac atctaataaa gtaacactgt gtcatcttct
ggagttatca aaaattgtat 5520acaatcaaga caacacaaga attattttat ttttgagtgc
aaatacaggt actgttggag 5580ttgatgggca ccatgctttc tcatgaagta gcatttccct
accatcaagc cattgttttg 5640tgccattcag gagaggaaaa aaaggaattt atgctgtaca
tttcagttca gtgtatgacc 5700aaaagcaata tgtttataag aagatgtttg acatactaat
tattttatat catttaaacc 5760atactgtagc aacataatat atggagctaa tttgtagaat
tatttttacg atttccaaac 5820aaatgtactg tactgttata taatttattg tgaggacctt
ctcatggaag ccattaggaa 5880aacaaactag aggtaaatat cacattaatc tgtattatca
atttctcata gacactgtgc 5940taatgtgaat tttaaatgac ctgcatcaag tcttctgatc
tcagataact cagtacagat 6000agcaattagt cagctgattt gattacaatg gagtaaccga
caatatattt atttataaag 6060cacatattca taataacgag aagaattcag aaaaccactt
aagcaagacc cttctgaaat 6120aaaaaatgtt gctttttaaa tagtttgtcc taaggtgttt
aaaacatgtc aaccttatgt 6180aaggaaaaat ttcctggtcc aaataaagtt gaagtttaag
aaaaattg 622856095DNAHomo sapiens 5gcgccgtgcg gcccgtgcga
ctcgccggac ctcgcgggcg tccctgtacg gagccctcgg 60ccggtcctag cagggattgt
ccccatttcc agctccggag cgggcggctg cgccccgctc 120gtcgaggagc tgcgctcacc
tcaggggcgg gcccccgcct gcgttcgcgg cgccagcaga 180agactgattt ttggaaatat
gtatttggga gacagtcacg tcctattgaa taccttgtgc 240tggtgctgcc atcgaaaaat
ctggttacac tctggggagg actgctacca ctgcagaact 300gaaccacttc ggccgtgaga
tgagtgtccg gcctgagcag gcacaccatg aatagataca 360caacaatcag gcagctcggg
gatggaacct acggttccgt cctgctggga agaagcattg 420agtctgggga gctgatcgct
attaaaaaaa tgaaaagaaa attttattcc tgggaggaat 480gcatgaacct tcgggaggtt
aagtctttaa agaagctcaa ccatgccaat gtagtcaaat 540taaaagaagt tatcagggaa
aatgatcatc tttattttat cttcgagtac atgaaggaaa 600atctttacca gctcattaaa
gagagaaata agttgtttcc tgagtctgct ataaggaata 660tcatgtatca gatattacaa
ggactcgcat ttattcacaa acacggcttc tttcatcgag 720acttaaagcc tgagaacctc
ctctgcatgg gaccagaact tgtgaaaatt gcagactttg 780gtttggcccg agaaatacga
tcaaaacctc catatacaga ttatgtatct accagatggt 840acagggctcc agaagtactc
ctgaggtcta ccaactacag ctcccccatt gacgtctggg 900cggtgggctg catcatggca
gaagtttaca ccctcaggcc actcttccct ggagccagtg 960aaattgacac aatattcaaa
atttgccaag tgctggggac accaaaaaag actgactggc 1020ctgaaggcta tcaactttca
agtgcaatga acttccgttg gccacagtgt gtacccaata 1080acttaaagac cttgattccc
aatgctagca gtgaagcagt ccagctcctg agagacatgc 1140ttcagtggga tcccaagaaa
cgaccaacag ctagtcaggc acttcgatat ccttacttcc 1200aagttggaca cccactaggc
agcaccacac aaaaccttca ggattcagaa aaaccacaga 1260aaggcatcct ggaaaaggca
ggcccacctc cttatattaa gccagtccca cctgcccagc 1320caccagccaa gccacacaca
cgaatttctt cacgacagca tcaagccagc cagccccctc 1380tgcatctcac gtacccctac
aaagcagagg tctccaggac agatcaccca agccatctcc 1440aggaggacaa gccaagcccg
ttgcttttcc catccctcca caacaagcat ccacagtcga 1500aaatcacagc tggcctggag
cacaaaaatg gtgagataaa gccaaagagt aggagaaggt 1560ggggtcttat ttccaggtca
acaaaggatt cagatgattg ggctgacttg gatgacttgg 1620atttcagtcc atccctcagc
aggattgacc tgaaaaacaa gaaaagacag agtgatgaca 1680ctctctgcag gtttgagagt
gttttggacc tgaagccctc tgagcctgtg ggcacaggaa 1740acagtgcccc cacccagacg
tcatatcagc ggcgagacac gcccaccctg agatctgcag 1800ccaagcagca ctatttgaag
cactctcgat acttgcctgg gatcagtata agaaatggca 1860tactctcgaa tccaggcaag
gaatttattc cacctaatcc atggtctagt tctggcttgt 1920ctggaaaatc ttcagggaca
atgtcagtaa tcagcaaagt aaattcagtt ggttccagct 1980ctacaagttc tagtggactg
actggaaact atgtcccttc ctttctgaaa aaagaaatcg 2040gttctgctat gcagagggta
cacctagcac ctattccaga cccttcccct ggttattcct 2100ccctgaaggc catgagacct
catcctgggc gaccattctt ccacacccag cctagaagca 2160ctcctgggtt gataccacgg
cctccagccg cccagccagt gcatggccgg acagactggg 2220cttccaagta cgcatctcgg
cgatgactgt ctgccttggt gatgaatctc ttcctaggga 2280gaagcaggat actttccctc
agctgactgg tgttctacct gcaagatgtg cagagggcat 2340aaaagcaaat caacacttta
tagttattct tctgaactaa gacatgtcaa tattcttttt 2400taaagttttt ttttaaaata
ttgatttgaa tgcagtaggc ttttttgtat aaaattattt 2460tattctaaaa ctgggtccca
ttattttctt aaacaacagc attttgtata tatggattat 2520gttttagcat tttatacagt
caactttgta atgaactttt taaaaattaa ttgattttcc 2580tttggggttc cagataatat
tttctacaga ttttgaaaaa tgtaataata ttaatgcagt 2640attgcaacag gggtgcaatt
taaggctatg tgatagaggg ttatttactc agtgtgtgca 2700gatatttatg aagtggtgaa
atttcaagtg tggctcacta ggtacttcag gccttcttgg 2760actgttgtta gaaaagtgat
cctctgcttt tcttagtagg tcattggttt gatttttgga 2820taccactctg ctgttctaaa
aggactatta tattatataa ttcactttgt tttacttttg 2880ttccccagat gaaagaactc
taagtaaata cattttaaaa aatttttctg acacccttta 2940atgtggttgc agatctcaga
tgaaaccaag cttaattata ctatgccatt atattctaat 3000ttattccatt tttgaaatca
agttgtatgt gtaccaataa aagagatttc tgcttcaaaa 3060ggctctcaac atgaaggtta
acacagtcaa tcaaacttac attcctgcca agatgcatgg 3120ccaaaaaact aagtatcaaa
gcagcagaag gtttttgatt atagtaactg agatggaatt 3180ttgtgcctag ctcagttctc
cagatctggc taggagcagt caatgactaa tgttctgtcc 3240tagccaaatt ctcaggacaa
tttggggagc agaaagagtt atggcagagg ttccactcat 3300ctacaaagtc acagtcacat
gccacatttg atctcctaac cctggtgtag tttctttcaa 3360gagtgagaac tttatttgtt
gggcagaggc tgttccattg agaggaatgt ttacagcagt 3420ttcaaaaatg acaaagtcag
tttggagaca gaaaaagaca aaaggtccag tctcatccat 3480ctctatatgg tacatttgcc
tcacttatgg ttgccttaaa ggcaagaggg aaggtcacca 3540tcagtgaacg caatgcaatc
tcaacagtgt attgattcat attctcctag ggctcaaact 3600actctctatt ggttccagga
taatgacaaa ttgaaccata tgtaagtaat cttttatttt 3660ttattttttt tttgagacag
agtctcactc tgtcacccag gctggagtgc agtggcgcga 3720tcttagctct ctgcaacctc
tgcctcccag gttcaagcct cctgagtaac tgggactaca 3780ggcgcccgcc accacgccca
gctaattttt tgtattttta gtagagacgg ggtttcactg 3840tgttagccag gacggcctcg
atctcctgac ctcgtgatcc accctcctcc acctcccaaa 3900gtactgggat tacaggcatg
agccactgca cccagccaag tgatcatttt tataggttaa 3960aatgataggt gaaatgaata
tagacacttt catatggttc aacctaatga cttggtaaat 4020tattgccttg gtgtattaat
aatatgttgc attctgaaca aataaccatg gcttccaaag 4080ggccctaacc taaaatcgga
gagtaattta tgctttggag aatttgactc aaatatatac 4140ttgaccaagc accatgatcc
ctaggggcat gagaaaagca cataatggat gtggatgtga 4200taggtggtct tttcctgtta
acaagctggc agcaaagctt cagaaaatat atatgcaagc 4260acaacttgaa gctgaattca
tttctgtatt atattctcaa ctcgttatct aaagcatcag 4320aacatgtgtt ttcagagatg
agtcctttac tataaggtta atatttattt tcattttctg 4380tattatatat gaaaagtaaa
ttaatgtgaa acctggccca gcttgctgga aagcaggttt 4440taaattgtaa atattcctta
gaggagcaaa tggattgttt aataccatag tctcagtaat 4500ctagcttata taaggtcatt
acatttttta actgaaaaac ctagttacct gattattgca 4560cattataaaa ttgtttttct
aatactttat agggcccaac ttcagaaaat acttcgcttt 4620tttcttttta tgctttcgtt
tgtttaccag caagcaactt ccctggggaa gccaaacaca 4680tattcataaa aaaaatcaag
tagctgatgt gcagttgaga aaactagagg actgaaaaaa 4740caaattttaa ctagcaaatg
ctgtgaatta ctcttcctcc ccttctctga aatgggtaaa 4800ggacaaattg tgtaaaaaaa
cctatgcact atagaaggga atagtaacca tttcttttgt 4860ctctctgttt ctgttctgac
tgagaacctg cagccatttc ttgttacatg aaaacaaaat 4920gctacttgtt acctctattt
tttgttacta tacaattatg aaatgtaatg taagacacca 4980acagaaatga tatacctgta
actgtaccta tcaggactat acctcattta cagtcagaaa 5040gcttactggg atgtcaggaa
atgatacagg gttggttctc atttcgtgcc gaaatgagac 5100agaaattcag tgacgaaggt
gcgttgtagg ggtattgatg tgccccaggt agtgccagca 5160gagtagggaa aactgcattt
gcataaaaac tactcttgac atgattgttc attttacaaa 5220aaaattccat taattaccaa
gccctcaccc agcccatgtg tgataggatt tatgtaggaa 5280gaaacttgat tttcaaataa
ttttttaaat gtatctcttg cctaaaggac tatatacatc 5340taataaagta acactgtgtc
atcttctgga gttatcaaaa attgtataca atcaagacaa 5400cacaagaatt attttatttt
tgagtgcaaa tacaggtact gttggagttg atgggcacca 5460tgctttctca tgaagtagca
tttccctacc atcaagccat tgttttgtgc cattcaggag 5520aggaaaaaaa ggaatttatg
ctgtacattt cagttcagtg tatgaccaaa agcaatatgt 5580ttataagaag atgtttgaca
tactaattat tttatatcat ttaaaccata ctgtagcaac 5640ataatatatg gagctaattt
gtagaattat ttttacgatt tccaaacaaa tgtactgtac 5700tgttatataa tttattgtga
ggaccttctc atggaagcca ttaggaaaac aaactagagg 5760taaatatcac attaatctgt
attatcaatt tctcatagac actgtgctaa tgtgaatttt 5820aaatgacctg catcaagtct
tctgatctca gataactcag tacagatagc aattagtcag 5880ctgatttgat tacaatggag
taaccgacaa tatatttatt tataaagcac atattcataa 5940taacgagaag aattcagaaa
accacttaag caagaccctt ctgaaataaa aaatgttgct 6000ttttaaatag tttgtcctaa
ggtgtttaaa acatgtcaac cttatgtaag gaaaaatttc 6060ctggtccaaa taaagttgaa
gtttaagaaa aattg 609561368DNAHomo sapiens
6atgatctgct gcagtgctct gagccctagg attcatcttt cttttcaccg tagcctgact
60ggcattgtat tagcaaactc atcactagac atcgtactac acgacacgta ctacgttgta
120gcccactgcg ggggaaatgt taggcgcctg cattgcggtg gccccgcgtc ccgggagcgc
180acagcaatgc aggcgcttaa cattaccccg gagcagttct ctcggctgct gcgggaccac
240aacctgacgc gggagcagtt catcgctctg taccggctgc gaccgctcgt ctacacccca
300gagctgccgg gacgcgccaa gctggccctc gtgctcaccg gcgtgctcat cttcgccctg
360gcactctttg gcaatgctct ggtgttctac gtggtgaccc gcagcaaggc catgcgcacc
420gtcaccaaca tctttatctg ctccttggcg ctcagtgacc tgctcatcac cttcttctgc
480attcccgtca ccatgctcca gaacatttcc gacaactggc tggggggtgc tttcatttgc
540aagatggtgc catttgtcca gtctaccgct gttgtgacag aaatcctcac tatgacctgc
600attgctgtgg aaaggcacca gggacttgtg catcctttta aaatgaagtg gcaatacacc
660aaccgaaggg ctttcacaat gctaggtgtg gtctggctgg tggcagtcat cgtaggatca
720cccatgtggc acgtgcaaca acttgagatc aaatatgact tcctatatga aaaggaacac
780atctgctgct tagaagagtg gaccagccct gtgcaccaga agatctacac caccttcatc
840ctgtcatcct cttcctcctg cctcttatgg aagaagaaac gagctgtcat tatgatggtg
900acagtggtgg ctctctttgc tgtgtgctgg gcaccattcc atgttgtcca tatgatgatt
960gaatacagta attttgaaaa ggaatatgat gatgtcacaa tcaagatgat ttttgctatc
1020gtgcaaatta ttggattttc caactccatc tgtaatccca ttgtctatgc atttatgaat
1080gaaaacttca aaaaaaatgt tttgtctgca gtttgttatt gcatagtaaa taaaaccttc
1140tctccagcac aaaggcatgg aaattcagga attacaatga tgcggaagaa agcaaagttt
1200tccctcagag agaatccagt ggaggaaacc aaaggagaag cattcagtga tggcaacatt
1260gaagtcaaat tgtgtgaaca gacagaggag aagaaaaagc tcaaacgaca tcttgctctc
1320tttaggtctg aactggctga gaattctcct ttagacagtg ggcattaa
136871296DNAHomo sapiens 7atgcaggcgc ttaacattac cccggagcag ttctctcggc
tgctgcggga ccacaacctg 60acgcgggagc agttcatcgc agttcatcgc ctgcgaccgc
tcgtctacac cccagagctg 120ccgggacgcg ccaagctggc cctcgtgctc accggcgtgc
tcatcttcgc cctggcgctc 180tttggcaatg ctctggtgtt ctacgtggtg acccgcagca
aggccatgcg caccgtcacc 240aacatcttta tctgctcctt ggcgctcagt gacctgctca
tcaccttctt ctgcattccc 300gtcaccatgc tccagaacat ttccgacaac tggctggggg
gtgctttcat ttgcaagatg 360gtgccatttg tccagtctac cgctgttgtg acagaaatcc
tcactatgac ctgcattgct 420gtggaaaggc accagggact tgtgcatcct tttaaaatga
agtggcaata caccaaccga 480agggctttca caatgctagg tgtggtctgg ctggtggcag
tcatcgtagg atcacccatg 540tggcacgtgc aacaacttga gatcaaatat gacttcctat
atgaaaagga acacatctgc 600tgcttagaag agtggaccag ccctgtgcac cagaagatct
acaccacctt catccttgtc 660atcctcttcc tcctgcctct tatggtgatg cttattctgt
acagtaaaat tggttatgaa 720ctttggataa agaaaagagt tggggatggt tcagtgcttc
gaactattca tggaaaagaa 780atgtccaaaa tagccaggaa gaagaaacga gctgtcatta
tgatggtgac agtggtggct 840ctctttgctg tgtgctgggc accattccat gttgtccata
tgatgattga atacagtaat 900tttgaaaagg aatatgatga tgtcacaatc aagatgattt
ttgctatcgt gcaaattatt 960ggattttcca actccatctg taatcccatt gtctatgcat
ttatgaatga aaacttcaaa 1020aaaaatgttt tgtctgcagt ttgttattgc atagtaaata
aaaccttctc tccagcacaa 1080aggcatggaa attcaggaat tacaatgatg cggaagaaag
caaagttttc cctcagagag 1140aatccagtgg aggaaaccaa aggagaagca ttcagtgatg
gcaacattga agtcaaattg 1200tgtgaacaga cagaggagaa gaaaaagctc aaacgacatc
ttgctctctt taggtctgaa 1260ctggctgaga attctccttt agacagtggg cattaa
129684350DNAHomo sapiens 8agttgaggga ttgacacaaa
tggtcaggcg gcggcggcgg agaaggaggc ggaggcgcag 60gggggagccg agcccgctgg
gctgcggaga gttgcgctct ctacggggcc gcggccacta 120gcgcggcgcc gccagccggg
agccagcgag ccgagggcca ggaaggcggg acacgacccc 180ggcgcgccct agccacccgg
gttctccccg ccgcccgcgc ttcatgaatc gcaagtttcc 240gcggcggcgg cggctgcggt
acgcagaaca ggagccgggg gagcgggccg aaagcggctt 300gggctcgacg gagggcaccc
gcgcagaggt ctccctggcc gcagggggag ccgccgccgg 360ccgtgcccct ggcagcccca
gcggagcggc gccaagagag gagccgagaa agtatggctg 420aggaggaggc gcctaagaag
tcccgggccg ccggcggtgg cgcgagctgg gaactttgtg 480ccggggcgct ctcggcccgg
ctggcggagg agggcagcgg ggacgccggt ggccgccgcc 540gcccgccagt tgacccccgg
cgattggcgc gccagctgct gctgctgctt tggctgctgg 600aggctccgct gctgctgggg
gtccgggccc aggcggcggg ccaggggcca ggccaggggc 660ccgggccggg gcagcaaccg
ccgccgccgc ctcagcagca acagagcggg cagcagtaca 720acggcgagcg gggcatctcc
gtcccggacc acggctattg ccagcccatc tccatcccgc 780tgtgcacgga catcgcgtac
aaccagacca tcatgcccaa cctgctgggc cacacgaacc 840aggaggacgc gggcctggag
gtgcaccagt tctaccctct agtgaaagtg cagtgttccg 900ctgagctcaa gttcttcctg
tgctccatgt acgcgcccgt gtgcaccgtg ctagagcagg 960cgctgccgcc ctgccgctcc
ctgtgcgagc gcgcgcgcca gggctgcgag gcgctcatga 1020acaagttcgg cttccagtgg
ccagacacgc tcaagtgtga gaagttcccg gtgcacggcg 1080ccggcgagct gtgcgtgggc
cagaacacgt ccgacaaggg caccccgacg ccctcgctgc 1140ttccagagtt ctggaccagc
aaccctcagc acggcggcgg agggcaccgt ggcggcttcc 1200cggggggcgc cggcgcgtcg
gagcgaggca agttctcctg cccgcgcgcc ctcaaggtgc 1260cctcctacct caactaccac
ttcctggggg agaaggactg cggcgcacct tgtgagccga 1320ccaaggtgta tgggctcatg
tacttcgggc ccgaggagct gcgcttctcg cgcacctgga 1380ttggcatttg gtcagtgctg
tgctgcgcct ccacgctctt cacggtgctt acgtacctgg 1440tggacatgcg gcgcttcagc
tacccggagc ggcccatcat cttcttgtcc ggctgttaca 1500cggccgtggc cgtggcctac
atcgccggct tcctcctgga agaccgagtg gtgtgtaatg 1560acaagttcgc cgaggacggg
gcacgcactg tggcgcaggg caccaagaag gagggctgca 1620ccatcctctt catgatgctc
tacttcttca gcatggccag ctccatctgg tgggtgatcc 1680tgtcgctcac ctggttcctg
gcggctggca tgaagtgggg ccacgaggcc atcgaagcca 1740actcacagta ttttcacctg
gccgcctggg ctgtgccggc catcaagacc atcaccatcc 1800tggcgctggg ccaggtggac
ggcgatgtgc tgagcggagt gtgcttcgtg gggcttaaca 1860acgtggacgc gctgcgtggc
ttcgtgctgg cgcccctctt cgtgtacctg tttatcggca 1920cgtcctttct gctggccggc
tttgtgtcgc tcttccgcat ccgcaccatc atgaagcacg 1980atggcaccaa gaccgagaag
ctggagaagc tcatggtgcg cattggcgtc ttcagcgtgc 2040tgtacactgt gccagccacc
atcgtcatcg cctgctactt ctacgagcag gccttccggg 2100accagtggga acgcagctgg
gtggcccaga gctgcaagag ctacgctatc ccctgccctc 2160acctccaggc gggcggaggc
gccccgccgc acccgcccat gagcccggac ttcacggtct 2220tcatgattaa gtaccttatg
acgctgatcg tgggcatcac gtcgggcttc tggatctggt 2280ccggcaagac cctcaactcc
tggaggaagt tctacacgag gctcaccaac agcaaacaag 2340gggagactac agtctgagac
ccggggctca gcccatgccc aggcctcggc cggggcgcag 2400cgatccccca aagccagcgc
cgtggagttc gtgccaatcc tgacatctcg aggtttcctc 2460actagacaac tctctttcgc
aggctccttt gaacaactca gctcctgcaa aagcttccgt 2520ccctgaggca aaaggacacg
agggcccgac tgccagaggg aggatggaca gacctcttgc 2580cctcacactc tggtaccagg
actgttcgct tttatgattg taaatagcct gtgtaagatt 2640tttgtaagta tatttgtatt
taaatgacga ccgatcacgc gtttttcttt ttcaaaagtt 2700tttaattatt tagggcggtt
taaccatttg aggcttttcc ttcttgccct tttcggagta 2760ttgcaaagga gctaaaactg
gtgtgcaacc gcacagcgct cctggtcgtc ctcgcgcgcc 2820tctccctacc acgggtgctc
gggacggctg ggcgccagct ccggggcgag ttcagcactg 2880cggggtgcga ctagggctgc
gctgccaggg tcacttcccg cctcctcctt ttgccccctc 2940cccctccttc tgtcccctcc
ctttctttcc tggcttgagg taggggctct taaggtacag 3000aactccacaa accttccaaa
tctggaggag ggcccccata cattacaatt cctcccttgc 3060tcggcggtgg attgcgaagg
cccgtccctt cgacttcctg aagctggatt tttaactgtc 3120cagaactttc ctccaacttc
atgggggccc acgggtgtgg gcgctggcag tctcagcctc 3180cctccacggt caccttcaac
gcccagacac tcccttctcc caccttagtt ggttacaggg 3240tgagtgagat aaccaatgcc
aaactttttg aagtctaatt tttgaggggt gagctcattt 3300cattctctag tgtctaaaac
ctggtatggg tttggccagc gtcatggaaa gatgtggtta 3360ctgagatttg ggaagaagca
tgaagctttg tgtgggttgg aagagactga agatatgggt 3420tataaaatgt taattctaat
tgcatacgga tgcctggcaa ccttgccttt gagaatgaga 3480cagcctgcgc ttagatttta
ccggtctgta aaatggaaat gttgaggtca cctggaaagc 3540tttgttaagg agttgatgtt
tgctttcctt aacaagacag caaaacgtaa acagaaattg 3600aaaacttgaa ggatatttca
gtgtcatgga cttcctcaaa atgaagtgct attttcttat 3660ttttaatcaa ataactagac
atatatcaga aactttaaaa tgtaaaagtt gtacactttc 3720aacattttat tacgattatt
attcagcagc acattctgag gggggaacaa ttcacaccac 3780caataataac ctggtaagat
ttcaggaggt aaagaaggtg gaataattga cggggagata 3840gcgcctgaaa taaacaaaat
atgggcatgc atgctaaagg gaaaatgtgt gcaggtctac 3900tgcattaaat cctgtgtgct
cctcttttgg atttacagaa atgtgtcaaa tgtaaatctt 3960tcaaagccat ttaaaaatat
tcactttagt tctctgtgaa gaagaggaga aaagcaatcc 4020tcctgattgt attgttttaa
actttaagaa tttatcaaaa tgccggtact taggacctaa 4080atttatctat gtctgtcata
cgctaaaatg atattggtct ttgaatttgg tatacattta 4140ttctgttcac tatcacaaaa
tcatctatat ttatagagga atagaagttt atatatatat 4200aataccatat ttttaatttc
acaaataaaa aattcaaagt tttgtacaaa attatatgga 4260ttttgtgcct gaaaataata
gagcttgagc tgtctgaact attttacatt ttatggtgtc 4320tcatagccaa tcccacagtg
taaaaattca 43509952DNAHomo sapiens
9ttggtccaag caagaaggca gtggtctact ccatcggcaa catgctggtc ctttatggac
60acagcaccca ggaccttccg gaaaccaatg cccgcgtagt cggagggact gaggccggga
120ggaattcctg gccctctcag atttccctcc agtaccggtc tggaggttcc cggtatcaca
180cctgtggagg gacccttatc agacagaact gggtgatgac agctgctcac tgcgtggatt
240accagaagac tttccgcgtg gtggctggag accataacct gagccagaat gatggcactg
300agcagtacgt gagtgtgcag aagatcgtgg tgcatccata ctggaacagc gataacgtgg
360ctgccggcta tgacatcgcc ctgctgcgcc tggcccagag cgttaccctc aatagctatg
420tccagctggg tgttctgccc caggagggag ccatcctggc taacaacagt ccctgctaca
480tcacaggctg gggcaagacc aagaccaatg ggcagctggc ccagaccctg cagcaggctt
540acctgccctc tgtggactac gccatctgct ccagctcctc ctactggggc tccactgtga
600agaacaccat ggtgtgtgct ggtggagatg gagttcgctc tggatgccag ggtgactctg
660ggggccccct ccattgcttg gtgaatggca agtattctgt ccatggagtg accagctttg
720tgtccagccg gggctgtaat gtctccagga agcctacagt cttcacccag gtctctgctt
780acatctcctg gataaataat gtcatcgcct ccaactgaac attttcctga gtccaacgac
840cttcccaaaa tggttcttag atctgcaata ggacttgcga tcaaaaagta aaacacattc
900tgaaagacta ttgagccatt gatagaaaag caaataaaac tagatataca tt
952109372DNAHomo sapiens 10tgaataattg aactttgttt atttctccat atttttgcag
tggtaattcc attataaaac 60ctaatgaaac aatgttttta tagatggtgt ggaaagactt
ttctgggctc agaggtgaaa 120ctgacccttg tgtatcagca gcatttctga ctgactgaga
gagtgtagtg attaacagag 180ttgtgatgtt agttaagaaa cttagatttg ccattgtagc
ttttctacca attagcagat 240tgtttaactc actgaaattg taaagtggta gacgtggact
tagtcattac tgggcagctt 300atgaattgta ttcatttact catgatgtaa aaatggttag
tctccacttt taaggctcta 360gttctagtgg ctaaataggt acttatttat acagtatgat
aactgctgta ttaaaataca 420tgtctcaaat gtggaatagt agaagaggtg aagaaaatca
tagtttgagg tagaatactg 480tttgctggtc ttaaaaactg tggtattttg gtgattccat
aaattaggtc agatacttcc 540actggaggga aacagtttaa aggatatatg tgatactatt
aatagaatga ggaagacaca 600ccagatattt aggagggaat tagcgagctt gaaactaaga
gctggtttga atgagactgg 660gtcataagtg atttcaagta ccagattaag gcactgagat
tttattttta agcactgaag 720tcagattttt tccttttaaa agaaaggatt catgatgaaa
tctgcttttt gttttgcaga 780gagcttggag ataattctgg tggctgtgtg gagtatgtgt
tggaggtatt aaattttcac 840agtatatata aggcagcaat tgataggcct ttcacagatt
cttctgataa ctacataaag 900agacaaaaaa aagaaaaaag agcaaagatc tgtgctgtgt
caagtatgac agccatcact 960catggctctc cagtaggagg gaacgacagc cagggccagg
ttcttgatgg ccagtctcag 1020catctcttcc aacagaacca gacttcatca cctgattctt
ccaatgagaa ttccgtagca 1080actcctcctc cagaggaaca agggcaaggt gatgccccac
cacagcatga agatgaagag 1140cctgcatttc cacatactga gctggcaaac ctggatgaca
tgatcaacag gcctcgatgg 1200gtggttcctg ttttgccaaa aggggaatta gaagtgcttt
tagaagctgc tattgatctt 1260agtgtaaaag gccttgatgt taaaagtgaa gcatgccaac
gtttttttcg agatggacta 1320acaatatctt tcactaaaat tcttatggat gaggctgtga
gtggctggaa gtttgaaatt 1380catagatgta ttattaacaa tactcatcgc ctagtggagc
tttgtgtggc caagttgtcc 1440caagattggt ttccacttct agaacttctc gccatggcct
taaatcctca ctgcaagttt 1500catatctaca atggtacacg tccgtgtgaa ttaatttcct
caaatgctca gttgcctgaa 1560gaagaattat ttgctcgttc ttcagatcct cgatcaccaa
aaggttggct agtggatctc 1620atcaataaat ttggcacatt aaatgggttc cagattttgc
atgatcgttt ttttaatgga 1680tcagcattaa atattcaaat aattgcagct cttattaaac
catttggaca atgctatgag 1740tttctcagtc aacatacact gaaaaagtac ttcattccag
ttatagaaat agttccacat 1800ttattggaaa acttaactga tgaagaactg aaaaaggagg
caaagaatga agccaaaaat 1860gatgcccttt caatgattat taaatctttg aagaacttag
cttcaagaat ttcaggacaa 1920gatgagacta taaaaaattt ggaaattttt aggttaaaga
tgatactcag attgttgcaa 1980atttcctctt ttaatggaaa gatgaatgca ctgaatgaaa
taaataaggt tatatctagt 2040gtatcatatt atactcatcg gcatagtaat cctgaggagg
aagaatggct gacagctgag 2100cgaatggcag aatggataca gcaaaataat atcttatcca
tagtcttgca agacagtctt 2160catcaaccac aatatgtaga aaagctagag aaaattcttc
gttttgtgat taaagaaaag 2220gctcttacat tacaggacct tgataatatc tgggcagcac
aggcaggaaa acatgaagcc 2280attgtgaaga atgtacatga tctgctagca aagttggctt
gggatttttc tcctggacaa 2340cttgatcatc tttttgattg ctttaaggca agttggacaa
atgcaagtaa aaagcaacgt 2400gaaaagctcc ttgagttgat acgccgtctt gcagaagatg
ataaagatgg tgtgatggca 2460cacaaagtgt tgaaccttct ttggaacctg gctcagagtg
atgatgtgcc tgtagacatc 2520atggaccttg ctcttagtgc ccacataaaa atactagatt
atagttgttc ccaggatcga 2580gatgcacaga agatccagtg gatagatcac tttatagaag
aacttcgcac aaatgacaag 2640tgggtaattc ctgctctgaa acaaataaga gaaatttgta
gtttgtttgg tgaagcatct 2700caaaatttga gtcaaactca gcgaagtccc cacatatttt
atcgccatga tttaatcaac 2760cagcttcaac aaaatcatgc tttagttact ttggtagcag
aaaaccttgc aacctacatg 2820aatagcatca gattgtatgc tggagatcat gaagactatg
atccacaaac agtgaggctt 2880ggaagtcgat acagtcatgt tcaagaagtt caagaacgac
taaacttcct tagattttta 2940ctgaaggatg gccaactgtg gctctgtgct cctcaggcaa
aacaaatatg gaagtgctta 3000gcagaaaatg cagtttatct ttgtgatcgt gaagcctgtt
ttaagtggta ttccaagtta 3060atgggggatg aaccagactt ggatcctgat attaataagg
acttctttga aagtaatgta 3120cttcagcttg atccttccct tttaactgaa aatggaatga
aatgctttga aagatttttc 3180aaagctgtca attgtcgaga aaggaaacta atagcaaaaa
gaagatccta tatgatggat 3240gatttggaat taattggact agactacctt tggagggttg
tgattcagag tagtgacgag 3300attgctaaca gagctataga tcttcttaaa gagatataca
caaaccttgg cccaagatta 3360aaagccaatc aggtggttat ccatgaagac ttcattcagt
cttgctttga tcgtttaaaa 3420gcatcatatg atacactgtg tgtttttgat ggtgacaaaa
acagcattaa ttgtgcaaga 3480caagaagcca ttcgaatggt tagagtatta actgttataa
aagagtacat taatgaatgt 3540gacagtgatt atcacaagga aagaatgatt ttacctatgt
cgagagcatt ttgtggcaaa 3600cacctctctc ttatagttcg gtttccaaac cagggcagac
aggttgatga gttggatata 3660tggtttcata cgaatgacac aattggttca gtacggcgat
gtattgttaa tcgtattaaa 3720gccaatgtag cccacaaaaa aattgaactt tttgtgggtg
gtgagctgat agattctgaa 3780aatgacagaa agctaattgg acaattaaac ttaaaagata
aatctctaat tacagccaaa 3840cttacacaaa taaatttcaa tatgccatca agtcctgata
gctcttccga ttcctcaact 3900gcatctcctg gaaaccaccg taatcattac aatgatggtc
ccaatctaaa ggtggaaagt 3960tgtttgcctg gggtgataat gtcagtgcat cccaaataca
tctctttcct ttggcaattt 4020gcaaacttag gtagcaacct gaatatgcca cctcttaaaa
atggagcaag agtacttatg 4080aaacttatgc caccagatag aacagctgta gaaaaattac
gaactgtttg tttggaccat 4140gcaaaccttg gagaaggcaa acttagtcca ccccttgact
cccttttctt tggtccttct 4200gcctcccaag ttctatacct aacagaggta gtttatgcct
tgttaatgcc tgctggtgtg 4260cctctaactg atgggtcctc tgactttcaa gttcacttct
tgaaaagtgg tggcttacct 4320cttgtactga gtatgctaat aagaaataac ttcttgccaa
atacagatat ggaaactcga 4380aggggtgctt atttaaatgc tcttaaaata gccaaactgt
tgttaactgc gattggctat 4440ggccatgttc gagctgtagc agaagcttgt cagccagttg
tagatggtac agaccccata 4500acacagatta accaagttac tcatgatcaa gcagtggtgc
tacaaagtgc ccttcagagc 4560attcctaatc cctcatccga gtgcgtactt agaaatgagt
ccatacttct tgctcaggaa 4620atatctaatg aggcttcaag atatatgcct gatatttgtg
taattagggc tatacagaaa 4680attatctggg catcagcatg tggggcatta ggactatttt
ttagcccaaa tgaagaaata 4740actaaaattt atcagatgac caccaatgga agcaataagc
tggaggtgga agatgaacaa 4800gtttgttgtg aagcactgga agtgatgacc ttatgttttg
ctttacttcc aacagcgttg 4860gatgcactta gtaaagaaaa agcctggcag accttcatca
ttgacttatt attgcactgt 4920ccaagcaaaa ctgttcgtca gttggcacag gagcagttct
ttttaatgtg caccagatgt 4980tgcatgggac acaggcctct gcttttcttc attactttac
tctttaccat actggggagc 5040acagcaagag agaagggtaa atattcaggt gattatttca
cacttttacg gcaccttctc 5100aattatgctt acaatggcaa tattaacata cccaatgctg
aagttcttct tgtcagtgaa 5160attgattggc tcaaaaggat tagggataat gttaaaaaca
caggtgaaac aggtgtcgaa 5220gagccaatac tggaaggcca ccttggggta acaaaagagt
tattggcctt tcaaacttct 5280gagaaaaagt atcactttgg ttgtgaaaaa ggaggtgcta
atctcattaa agaattaatt 5340gatgatttca tctttcccgc atccaaagtt tacctgcagt
atttaagaag tggagaacta 5400ccagctgagc aggctattcc agtctgtagt tcacccgtta
ccatcaatgc cggttttgag 5460ctacttgtag cattagctat tggctgtgtg aggaatctca
aacagatagt agactgtttg 5520actgaaatgt attacatggg cacagcaatt actacttgtg
aagcacttac tgagtgggaa 5580tatctgcccc ctgttggacc ccgcccacca aaaggatttg
tgggactcaa aaatgctggt 5640gctacgtgtt acatgaactc tgtgatccag cagctataca
tgattccttc tatcaggaac 5700agtattcttg caattgaagg cacaggtagt gatttacacg
atgatatgtt cggggatgag 5760aagcaggaca gtgagagtaa tgttgatccc cgagatgatg
tatttggata tcctcatcaa 5820tttgaagaca agccagcatt aagtaagaca gaagatagga
aagagtataa tattggtgtc 5880ctaagacacc ttcaggtcat ctttggtcat ttagctgctt
cccaactaca atactatgta 5940cccagaggat tttggaaaca gttcaggctt tggggtgaac
ctgttaatct ccgtgaacaa 6000catgatgcct tagagttttt taattctttg gtggatagtt
tagatgaagc tttaaaagct 6060ttaggacacc cggctatact aagtaaagtc ctaggaggct
cctttgctga tcagaagatc 6120tgccagggct gcccacatag gtttgaatgt gaagaatctt
ttacaacttt gaatgtggat 6180attagaaatc atcaaaatct tcttgactct ttggaacagt
atatcaaagg agatttattg 6240gaaggtgcaa atgcatatca ttgtgaaaaa tgtgataaaa
aggttgacac agtaaagcgc 6300ctgctaatta aaaaattgcc tcgggttctt gctatccaac
tcaaacgatt tgactatgac 6360tgggaaagag aatgtgcaat taaattcaat gattattttg
aatttcctcg agagctggat 6420atgggacctt acacagtagc aggtgttgca aacctggaaa
gggataatgt aaactcagaa 6480aatgagttga ttgaacagaa agagcagtct gacaatgaaa
ctgcaggagg cacaaagtac 6540agacttgtag gagtgcttgt acacagtggt caagcaagcg
gtgggcatta ttattcttac 6600atcattcaaa ggaatggtaa agatgatcag acagatcact
ggtataaatt tgatgatgga 6660gatgtaacag aatgcaaaat ggatgatgat gaagaaatga
aaaatcagtg ttttggtgga 6720gagtacatgg gagaagtatt tgatcacatg atgaagcgca
tgtcatatag gcgacagaag 6780aggtggtgga atgcttacat acctttttat gaacaaatgg
atatgataga tgaagatgat 6840gagatgataa gatacatatc agagctaact attgcaagac
cccatcagat cattatgtca 6900ccagccattg agagaagtgt acggaaacaa aatgtgaaat
ttatgcataa ccgattgcaa 6960tatagtttag agtattttca gtttgtgaaa aaactgctta
catgtaatgg tgtttattta 7020aaccctgctc cagggcagga ttatttgttg cctgaagcag
aagaaattac tatgattagt 7080attcagcttg ctgctagatt cctctttacc actggatttc
acaccaagaa aatagttcgt 7140ggtcctgcca gtgactggta tgatgcactg tgcgttcttc
tccgtcacag caaaaatgta 7200cgtttttggt ttactcataa tgtccttttt aatgtatcaa
atcgcttctc tgaatacctc 7260ctggagtgcc ctagtgcaga agtgaggggt gcatttgcaa
aacttatagt gtttattgca 7320cacttttcct tgcaagatgg gtcttgtcct tctccttttg
catctccagg accttctagt 7380caggcatgtg ataacttgag cttgagtgac cacttactaa
gagccacact aaatctcttg 7440agaagggaag tttcagagca tggacatcat ttacagcaat
attttaattt gtttgtaatg 7500tatgccaatt taggtgtggc agaaaaaaca cagcttctga
aattgaatgt acctgctacc 7560tttatgcttg tgtctttaga cgagggacca ggtcctccaa
tcaaatatca gtatgctgaa 7620ttaggcaagt tatattcagt agtgtctcag ctgattcgtt
gttgcaatgt gtcatcaaca 7680atgcagtctt caatcaatgg taatccccct ctccccaatc
ctttcggtga ccttaattta 7740tcacagccta taatgccaat tcagcagaat gtgttagaca
ttttatttgt gagaacaagt 7800tatgtgaaga aaattattga agactgcagt aactcagagg
ataccatcaa attacttcgc 7860ttttgctctt gggagaatcc tcagttctca tctactgtcc
tcagcgaact tctctggcag 7920gttgcatatt catataccta tgaacttcgg ccatatttag
atctactttt ccaaatttta 7980ctgattgagg actcctggca gactcacaga attcataatg
cacttaaagg aattccagat 8040gacagagatg ggctgttcga tacaatacag cgctcgaaga
atcactatca aaaacgagca 8100tatcagtgca taaaatgtat ggtagctcta tttagcagtt
gtcctgttgc ttaccagatc 8160ttacagggta acggagatct taaaagaaaa tggacctggg
cagtggaatg gctaggagat 8220gaacttgaaa gaagaccata tactggcaat cctcagtata
gttacaacaa ttggtctcct 8280ccagtacaaa gcaatgaaac agcaaatggt tatttcttag
aaagatcaca tagtgctagg 8340atgacacttg caaaagcttg tgaactctgt ccagaagagg
agccagatga ccaggatgcc 8400ccagatgagc atgagccctc tccatcagaa gatgccccat
tatatcctca ttcacctgcc 8460tctcagtatc aacagaataa tcatgtacat ggacagccat
atacaggacc agcagcacat 8520cacttgaaca accctcagaa aacaggccaa cgaacacaag
aaaattatga aggcaatgaa 8580gaagtatcct cacctcagat gaaggatcag tgaaaagcaa
taattaactg cttcctttat 8640gactatgcac taaggtctta tagtccaaac tttctctgtg
tctggctagt attgaaaact 8700agataaactg ctccaaacca acatggagta aagagcatat
tcactggttt atttgcagta 8760atttgcaatt tgtcagtgta taagacacat gcagggtgaa
gtgtacagag ttttgtaaca 8820aatgactggt cctaatctgt aaatgagaaa ggtatatata
ctatgttaat gtctgactgt 8880taattcttaa gcaagaaact ttttttgatg aaaacaagtc
agatctacac agtcacacaa 8940ttattttttg ttgtgttcac tacattgtgc aattgatatt
gcctgctttg agcagtttgg 9000tcaacttacc aacttccccc caaaaaaggg aacataaaag
agcccatctt tgtcagttta 9060caccaatagt ttcttgttaa tccttctttc ctggatatat
aaggctggtg gtaacttttg 9120aattatatgg ttgatgtgga aaattggcag tgtaacattt
ctagatactt ttcattacct 9180ttttattctg gtatataggc taaccacttt aaagctattc
ttatgctgta acagttagca 9240tggcttcaca ctgtttgtgt agccaagagg acagaattac
atgaatgaca gtgcccagag 9300tgacagctgt atattgctca gagcttttat ttcttatacc
tagaataaat ataaaatggg 9360ggaaaaaaaa aa
9372111593DNAHomo sapiens 11ctgggagcgc ggcgtaggtg
gctgccgagt cttttcctgt ttagggtctt atcctggcat 60tgagggcgcc ggactggcgc
ttttggccgg cttggcattg ggtgggcggc ttcttgggac 120ccacatgagc cagtggcatc
atccccgcag tggctggggc cggagacgcg acttttcagg 180acgctcctca gccaagaaga
agggcggaaa ccacatcccc gaaaggtgga aagactatct 240cccagttgga cagcggatgc
ctgggactcg tttcattgct ttcaaagttc ctttgcaaaa 300gagttttgaa aagaaacttg
ctccagaaga atgcttttcc cctttggatc tttttaacaa 360aatccgagaa caaaatgaag
aacttggact gattattgat ttaacatata ctcaacgcta 420ttataaacca gaggatttgc
cagaaactgt tccttactta aaaattttta cagttggaca 480tcaagtgcct gatgatgaga
ctatttttaa attcaaacac gctgttaatg ggtttttgaa 540agaaaataaa gataatgata
aacttattgg tgtccactgt acccatggtt taaacaggac 600tggctacctc atttgcagat
atttgattga tgtagaaggc gtgaggccag atgatgcaat 660tgaattattc aataggtgcc
ggggacattg cttagaaaga caaaactaca ttgaagacct 720tcagaatggt cctatcagaa
agaattggaa ttccagtgta cccaggtcaa gtgattttga 780agactcagca catctcatgc
aaccagtcca caataagcct gttaaacaag gacctaggta 840taatctacat cagatccagg
gtcactcagc tcctcgacat ttccacaccc agacccaaag 900tttgcaacaa tcagtcagaa
aattttcaga gaatccacat gtttaccaga gacaccatct 960ccctcctcct ggtccccctg
gagaggacta ttcacacagg aggtattctt ggaatgtgaa 1020gcccaatgcc agtcgggcag
cccaggatag aagaaggtgg tatccttata attactccag 1080actctcctat ccagcctgtt
gggaatggac ccagtgatac aaacctgtcc tggaattcta 1140cctggagacc agagctggcc
tgaaaattac tggtgtgact tttaattagt tcaggtctaa 1200tcaggtttct ttattgttcc
cttatgtatt caagcttaag gaaaaattgc attgctgttt 1260acctctttgc tgataaattt
gcagtaatta cagcattgca ggaaaaacaa tctgttattc 1320cagtcttaaa tttttctaaa
agaagacaat attttagaac tgaagcattg agaacttccc 1380ttgcaaatta tttttaaaat
tctatcttgt ttttctatgt atttctttct gactagactt 1440gtgatatgcg tgtgtttatg
tacagaaatt tttagtgttt ttgttatgtt ctgttattga 1500cccaaaggcc atctttattt
tctataactg ttcaaaattt atattaaaat ctacttagga 1560gataatttct ttagaaaaaa
aaaaaaaaaa aaa 1593124116DNAHomo sapiens
12cgccgctctc cgcctcgctt gctcctgccg ggcgtgcagg gccccgccgc cgccatgtcg
60ggctcgttcg agctctcggt gcaggatctc aacgacctgc tctcggacgg cagcggctgc
120tacagcctcc cgagccagcc ctgcaacgag gtcaccccgc ggatctacgt gggcaacgcg
180tctgtggctc aggacatccc caagctgcag aaactaggca tcacccatgt gctgaacgcg
240gctgagggca ggtccttcat gcacgtcaac accaatgcca acttctacaa ggactccggc
300atcacatacc tgggcatcaa ggccaacgac acacaggagt tcaacctcag cgcttacttt
360gaaagggctg ccgacttcat tgaccaggct ttggctcaaa agaatggccg ggtgctcgtc
420cactgccggg aaggttatag ccgctcccca acgctagtta tcgcctacct catgatgcgg
480cagaagatgg acgtcaagtc tgccctgagc atcgtgaggc agaaccgtga gatcggcccc
540aacgatggct tcctggccca gctctgccag ctcaatgaca gactagccaa ggaggggaag
600ttgaaaccct agggcacccc caccgcctct gctcgagagg tccgtggggg aggccgtggg
660caaaggtgtc ccgagctgcc atgtttagga aacacactgt accctgctcc cagcatcaca
720aggcacttgt ctacaagtgt gtcccaacac agtcctgggc cactttcccc accctgggga
780gcacataaag aagcttgcca aggggggcgt ccttgctccc cagttgtcct gtttctgtaa
840cttatgatgt cttttccctg agatgggggc tcagaggggg aaggcctgtg gcctgcatgc
900ttcccgatgg cccacggcag gaggtgtgtg gaagtgtaag gcctaagatg ctcacagagg
960tccctcatga cctcccttcc ccaactcccg aatcctctct tgagtgtgga cctcaacacc
1020ttgagcccta gtaaaggaac tatgcaaatg caggccactc tccccaccac gtctgtgccc
1080cgcactgtcc ccacagcctt ccacaccctg tgcataggca gccctctcac gtcttgaggt
1140ccgaagctgg ggtgggggtg tccgtcagtt attagtggat ggagattccc acagcaaggc
1200tgcatttgaa tgatttcctt aggatgaatg gtccctacac aaagaggcct tgtgggcaaa
1260cctggagaac cctcctaaat ccatagagtt ttcaaaatgt gaatctttgg aagccttgag
1320ttcagaatct gctgctctgg aatatttccc ttcgatctta tctcagtcac ttcgtttttg
1380agaagagtga tgccttgggc atgctttttt ttttttcttt tttagaaaac agggagttga
1440agtccaacct atttaaaaac cccaccattt ggagaattac aagggttttg tcctgaattg
1500tagtgttggc aagcccaagc cactcgtgct aactgctttt tgtctcggtt gctattccaa
1560gaacagaagg aggaagttgg ccaattacag cgtgtgtgca tggatgtgtg tggggggcgt
1620gcctctcaga aacgcggcca gaagacaagc agggaagtga aaggtcccag gcacacaccc
1680tgcccattgc aggtggctct tacagctctc tggtgccagc acgggatccc tgaagtgact
1740cagccaggca gacatgagac atggcggagt gtccaaatgg atcctttatt ggtggtagag
1800caaaaaaacc caaacacgat aaacctttca aaagactttc taaggatgat attggaatgc
1860accagccctc acatgtgtat gcacatttgc cagaatataa gagttttgtt ttaaatacag
1920tcttgttagg attttacgtt attgttatta tggaaagtga ttgtgatgct atttatcttc
1980agggtcactc tgggcaaaga gaaggtcctc agccatgccc ccagcacctt gcacataggt
2040gtctgataaa agtttaagaa attaaacact ttttgagcac caaatatata tagggcattg
2100ttctggtggg tgtgtcacgc tcccagaaga ctgaatttat ggtaggatca ctcgcaaggc
2160cttgtgaagg agtcttacct aaaacaaaag aaatatcagg gacttttgtt gactatttac
2220aactcagttt tacatttaaa ttcaggcagt gttaatatgc caaggtaggg aatgtgcctt
2280tttcagagtt ggccaggagc tcctggctgg gacacggaga ggcaggtgtg gcgtaaggcc
2340tcactcccgg ctgtgaaggt ctctgatcac acagaagcag ccctgcccag cctggtcatt
2400tgctgtccgc ttttctctgt gaccacagca gccctgaaca accagtatgt gtcttcttct
2460ccagatagtg aaaaaggtgt ccagataaac ccacctaagt gaaatggcca tcctctaaac
2520tgggtacctc actgcacagc ttctaggtag ccttccaact taatctaact tgagcctcac
2580agtaaccctg taaagttagt agagcttgtt cttgtattgt gacctttttt aaaaaaaagg
2640aactgaggtt cagaatgatt aagggcctgg cccccagggt tgtccagctc cataaggtgg
2700agctgggcaa gattttgggt ttgctgctcc ctgaagctgg attctttcat acgatactct
2760ttctcaagaa gggggctccc tgggatctcc aggtgtactg cacttaccct caatccagcc
2820ccggagaagc aagtgaaaag ggtgggtccc tcataggcta gaatgtgcag ctctttctcc
2880aggtgggatg tagcacccca aagtagagct ttctgctctg ctcctggaaa aggctaggga
2940gctggggctg gggctcccct cccatgacca ggcagtggtc accccatggg acaggcacag
3000ctacttacgc gaacacagca ggttggtgtg gctggctaac taggacctct cgaaagtctc
3060tgtgggggca tgagggagaa aaggccattg ggagaattac tgcctttact ttgggactac
3120ttttatgctg ataacttggg atttcttgat agtccttcac ccctgaaacc ccgtatttac
3180ttaacaagat ttagctctta gttcttcaag taaaattaaa gtctcttgtg taagagccaa
3240cacatgccca gctgcggatg ggagctgttc ctggacagcc ttctactgcc tgggaagtga
3300tggaacagga actcagggtg cccttacccc ctccccagac ctgttccctt tctttgactg
3360acagagcacc atccaggcaa aattagagcg ccaaatggtt ttcttctcaa tcttaaagca
3420gtataccttt ccacaggctc gtctgtgtcc ctgccactct gagttatcca gaaaccacca
3480cctacaaatg aggggactca tctagaagac ctctaaggtc cccttttggc tctgaggggt
3540ctctaataat ccccacttgg aattcagcac cgcaaggaaa ttatgggtat gtgagccata
3600atatgatggc cagcaggtgg cgctgccttc cacccatggt gatggatggt ttggaaaggg
3660aatgttggtg ccttttgtgc cacaagttaa gatgctactg ttttaaagga aaaaaaaaaa
3720aaaaagtact gatcttcaat atgaagacat gagcttttct cgcaggaaat tttctttttc
3780acagaactgg tgtcaggaat cactgaaggg ctaaccgtga tagtccttgc aagtaagtca
3840aggttttatc ctgattggaa atagaagaca tttccggttg agagaacaga ttcgttggaa
3900gcttaacttt tgttgcctct taacgccacc aaattttagg gtaatttgat tatgaaagag
3960tgaatttttc tggacagaaa agggagagct accaaattgt ttttttcttt ttaaaaggaa
4020gtttaatgtc cgttgtatca caaatcagtg ttaaaacacc agaactttag ccaaaataaa
4080tgtcttacat tacaaaggta aaaaaaaaaa aaaaaa
4116133540DNAHomo sapiens 13ccagtttgct tcttggagaa cactggacag ctgaataaat
gcagtatcta aatataaaag 60aggactgcaa tgccatggct ttctgtgcta aaatgaggag
ctccaagaag actgaggtga 120acctggaggc ccctgagcca ggggtggaag tgatcttcta
tctgtcggac agggagcccc 180tccggctggg cagtggagag tacacagcag aggaactgtg
catcagggct gcacaggcat 240gccgtatctc tcctctttgt cacaacctct ttgccctgta
tgacgagaac accaagctct 300ggtatgctcc aaatcgcacc atcaccgttg atgacaagat
gtccctccgg ctccactacc 360ggatgaggtt ctatttcacc aattggcatg gaaccaacga
caatgagcag tcagtgtggc 420gtcattctcc aaagaagcag aaaaatggct acgagaaaaa
aaagattcca gatgcaaccc 480ctctccttga tgccagctca ctggagtatc tgtttgctca
gggacagtat gatttggtga 540aatgcctggc tcctattcga gaccccaaga ccgagcagga
tggacatgat attgagaacg 600agtgtctagg gatggctgtc ctggccatct cacactatgc
catgatgaag aagatgcagt 660tgccagaact gcccaaggac atcagctaca agcgatatat
tccagaaaca ttgaataagt 720ccatcagaca gaggaacctt ctcaccagga tgcggataaa
taatgttttc aaggatttcc 780taaaggaatt taacaacaag accatttgtg acagcagcgt
gtccacgcat gacctgaagg 840tgaaatactt ggctaccttg gaaactttga caaaacatta
cggtgctgaa atatttgaga 900cttccatgtt actgatttca tcagaaaatg agatgaattg
gtttcattcg aatgacggtg 960gaaacgttct ctactacgaa gtgatggtga ctgggaatct
tggaatccag tggaggcata 1020aaccaaatgt tgtttctgtt gaaaaggaaa aaaataaact
gaagcggaaa aaactggaaa 1080ataaagacaa gaaggatgag gagaaaaaca agatccggga
agagtggaac aatttttcat 1140tcttccctga aatcactcac attgtaataa aggagtctgt
ggtcagcatt aacaagcagg 1200acaacaagaa aatggaactg aagctctctt cccacgagga
ggccttgtcc tttgtgtccc 1260tggtagatgg ctacttccgg ctcacagcag atgcccatca
ttacctctgc accgacgtgg 1320cccccccgtt gatcgtccac aacatacaga atggctgtca
tggtccaatc tgtacagaat 1380acgccatcaa taaattgcgg caagaaggaa gcgaggaggg
gatgtacgtg ctgaggtgga 1440gctgcaccga ctttgacaac atcctcatga ccgtcacctg
ctttgagaag tctgagcagg 1500tgcagggtgc ccagaagcag ttcaagaact ttcagatcga
ggtgcagaag ggccgctaca 1560gtctgcacgg ttcggaccgc agcttcccca gcttgggaga
cctcatgagc cacctcaaga 1620agcagatcct gcgcacggat aacatcagct tcatgctaaa
acgctgctgc cagcccaagc 1680cccgagaaat ctccaacctg ctggtggcta ctaagaaagc
ccaggagtgg cagcccgtct 1740accccatgag ccagctgagt ttcgatcgga tcctcaagaa
ggatctggtg cagggcgagc 1800accttgggag aggcacgaga acacacatct attctgggac
cctgatggat tacaaggatg 1860acgaaggaac ttctgaagag aagaagataa aagtgatcct
caaagtctta gaccccagcc 1920acagggatat ttccctggcc ttcttcgagg cagccagcat
gatgagacag gtctcccaca 1980aacacatcgt gtacctctat ggcgtctgtg tccgcgacgt
ggagaatatc atggtggaag 2040agtttgtgga agggggtcct ctggatctct tcatgcaccg
gaaaagtgat gtccttacca 2100caccatggaa attcaaagtt gccaaacagc tggccagtgc
cctgagctac ttggaggata 2160aagacctggt ccatggaaat gtgtgtacta aaaacctcct
cctggcccgt gagggaatcg 2220acagtgagtg tggcccattc atcaagctca gtgaccccgg
catccccatt acggtgctgt 2280ctaggcaaga atgcattgaa cgaatcccat ggattgctcc
tgagtgtgtt gaggactcca 2340agaacctgag tgtggctgct gacaagtgga gctttggaac
cacgctctgg gaaatctgct 2400acaatggcga gatccccttg aaagacaaga cgctgattga
gaaagagaga ttctatgaaa 2460gccggtgcag gccagtgaca ccatcatgta aggagctggc
tgacctcatg acccgctgca 2520tgaactatga ccccaatcag aggcctttct tccgagccat
catgagagac attaataagc 2580ttgaagagca gaatccagat attgtttcca gaaaaaaaaa
ccagccaact gaagtggacc 2640ccacacattt tgagaagcgc ttcctaaaga ggatccgtga
cttgggagag ggccactttg 2700ggaaggttga gctctgcagg tatgaccccg aagacaatac
aggggagcag gtggctgtta 2760aatctctgaa gcctgagagt ggaggtaacc acatagctga
tctgaaaaag gaaatcgaga 2820tcttaaggaa cctctatcat gagaacattg tgaagtacaa
aggaatctgc acagaagacg 2880gaggaaatgg tattaagctc atcatggaat ttctgccttc
gggaagcctt aaggaatatc 2940ttccaaagaa taagaacaaa ataaacctca aacagcagct
aaaatatgcc gttcagattt 3000gtaaggggat ggactatttg ggttctcggc aatacgttca
ccgggacttg gcagcaagaa 3060atgtccttgt tgagagtgaa caccaagtga aaattggaga
cttcggttta accaaagcaa 3120ttgaaaccga taaggagtat tacaccgtca aggatgaccg
ggacagccct gtgttttggt 3180atgctccaga atgtttaatg caatctaaat tttatattgc
ctctgacgtc tggtcttttg 3240gagtcactct gcatgagctg ctgacttact gtgattcaga
ttctagtccc atggctttgt 3300tcctgaaaat gataggccca acccatggcc agatgacagt
cacaagactt gtgaatacgt 3360taaaagaagg aaaacgcctg ccgtgcccac ctaactgtcc
agatgaggtt tatcagctta 3420tgagaaaatg ctgggaattc caaccatcca atcggacaag
ctttcagaac cttattgaag 3480gatttgaagc acttttaaaa taagaagcat gaataacatt
taaattccac agattatcaa 3540143541DNAHomo sapiens 14tccagtttgc ttcttggaga
acactggaca gctgaataaa tgcagtatct aaatataaaa 60gaggactgca atgccatggc
tttctgtgct aaaatgagga gctccaagaa gactgaggtg 120aacctggagg cccctgagcc
aggggtggaa gtgatcttct atctgtcgga cagggagccc 180ctccggctgg gcagtggaga
gtacacagca gaggaactgt gcatcagggc tgcacaggca 240tgccgtatct ctcctctttg
tcacaacctc tttgccctgt atgacgagaa caccaagctc 300tggtatgctc caaatcgcac
catcaccgtt gatgacaaga tgtccctccg gctccactac 360cggatgaggt tctatttcac
caattggcat ggaaccaacg acaatgagca gtcagtgtgg 420cgtcattctc caaagaagca
gaaaaatggc tacgagaaaa aaaagattcc agatgcaacc 480cctctccttg atgccagctc
actggagtat ctgtttgctc agggacagta tgatttggtg 540aaatgcctgg ctcctattcg
agaccccaag accgagcagg atggacatga tattgagaac 600gagtgtctag ggatggctgt
cctggccatc tcacactatg ccatgatgaa gaagatgcag 660ttgccagaac tgcccaagga
catcagctac aagcgatata ttccagaaac attgaataag 720tccatcagac agaggaacct
tctcaccagg atgcggataa ataatgtttt caaggatttc 780ctaaaggaat ttaacaacaa
gaccatttgt gacagcagcg tgtccacgca tgacctgaag 840gtgaaatact tggctacctt
ggaaactttg acaaaacatt acggtgctga aatatttgag 900acttccatgt tactgatttc
atcagaaaat gagatgaatt ggtttcattc gaatgacggt 960ggaaacgttc tctactacga
agtgatggtg actgggaatc ttggaatcca gtggaggcat 1020aaaccaaatg ttgtttctgt
tgaaaaggaa aaaaataaac tgaagcggaa aaaactggaa 1080aataaagaca agaaggatga
ggagaaaaac aagatccggg aagagtggaa caatttttca 1140ttcttccctg aaatcactca
cattgtaata aaggagtctg tggtcagcat taacaagcag 1200gacaacaaga aaatggaact
gaagctctct tcccacgagg aggccttgtc ctttgtgtcc 1260ctggtagatg gctacttccg
gctcacagca gatgcccatc attacctctg caccgacgtg 1320gcccccccgt tgatcgtcca
caacatacag aatggctgtc atggtccaat ctgtacagaa 1380tacgccatca ataaattgcg
gcaagaagga agcgaggagg ggatgtacgt gctgaggtgg 1440agctgcaccg actttgacaa
catcctcatg accgtcacct gctttgagaa gtctgagcag 1500gtgcagggtg cccagaagca
gttcaagaac tttcagatcg aggtgcagaa gggccgctac 1560agtctgcacg gttcggaccg
cagcttcccc agcttgggag acctcatgag ccacctcaag 1620aagcagatcc tgcgcacgga
taacatcagc ttcatgctaa aacgctgctg ccagcccaag 1680ccccgagaaa tctccaacct
gctggtggct actaagaaag cccaggagtg gcagcccgtc 1740taccccatga gccagctgag
tttcgatcgg atcctcaaga aggatctggt gcagggcgag 1800caccttggga gaggcacgag
aacacacatc tattctggga ccctgatgga ttacaaggat 1860gacgaaggaa cttctgaaga
gaagaagata aaagtgatcc tcaaagtctt agaccccagc 1920cacagggata tttccctggc
cttcttcgag gcagccagca tgatgagaca ggtctcccac 1980aaacacatcg tgtacctcta
tggcgtctgt gtccgcgacg tggagaatat catggtggaa 2040gagtttgtgg aagggggtcc
tctggatctc ttcatgcacc ggaaaagtga tgtccttacc 2100acaccatgga aattcaaagt
tgccaaacag ctggccagtg ccctgagcta cttggaggat 2160aaagacctgg tccatggaaa
tgtgtgtact aaaaacctcc tcctggcccg tgagggaatc 2220gacagtgagt gtggcccatt
catcaagctc agtgaccccg gcatccccat tacggtgctg 2280tctaggcaag aatgcattga
acgaatccca tggattgctc ctgagtgtgt tgaggactcc 2340aagaacctga gtgtggctgc
tgacaagtgg agctttggaa ccacgctctg ggaaatctgc 2400tacaatggcg agatcccctt
gaaagacaag acgctgattg agaaagagag attctatgaa 2460agccggtgca ggccagtgac
accatcatgt aaggagctgg ctgacctcat gacccgctgc 2520atgaactatg accccaatca
gaggcctttc ttccgagcca tcatgagaga cattaataag 2580cttgaagagc agaatccaga
tattgtttcc agaaaaaaaa accagccaac tgaagtggac 2640cccacacatt ttgagaagcg
cttcctaaag aggatccgtg acttgggaga gggccacttt 2700gggaaggttg agctctgcag
gtatgacccc gaagacaata caggggagca ggtggctgtt 2760aaatctctga agcctgagag
tggaggtaac cacatagctg atctgaaaaa ggaaatcgag 2820atcttaagga acctctatca
tgagaacatt gtgaagtaca aaggaatctg cacagaagac 2880ggaggaaatg gtattaagct
catcatggaa tttctgcctt cgggaagcct taaggaatat 2940cttccaaaga ataagaacaa
aataaacctc aaacagcagc taaaatatgc cgttcagatt 3000tgtaagggga tggactattt
gggttctcgg caatacgttc accgggactt ggcagcaaga 3060aatgtccttg ttgagagtga
acaccaagtg aaaattggag acttcggttt aaccaaagca 3120attgaaaccg ataaggagta
ttacaccgtc aaggatgacc gggacagccc tgtgttttgg 3180tatgctccag aatgtttaat
gcaatctaaa ttttatattg cctctgacgt ctggtctttt 3240ggagtcactc tgcatgagct
gctgacttac tgtgattcag attctagtcc catggctttg 3300ttcctgaaaa tgataggccc
aacccatggc cagatgacag tcacaagact tgtgaatacg 3360ttaaaagaag gaaaacgcct
gccgtgccca cctaactgtc cagatgaggt ttatcagctt 3420atgagaaaat gctgggaatt
ccaaccatcc aatcggacaa gctttcagaa ccttattgaa 3480ggatttgaag cacttttaaa
ataagaagca tgaataacat ttaaattcca cagattatca 3540a
3541152982DNAHomo sapiens
15cgcaccccgc gcagcggctg agccgggagc cagcgcagcc tcggccccgc agctcaagcc
60tcgtccccgc cgccgccgcc gccgccgccg ccgccgcccc cggggcatgg cctgtctgat
120ggccgctttc tcggtcggca ccgccatgaa tgccagcagt tactctgcag agatgacgga
180gcccaagtcg gtgtgtgtct cggtggatga ggtggtgtcc agcaacatgg aggccactga
240gacggacctg ctgaatggac atctgaaaaa agtagataat aacctcacgg aagcccagcg
300cttctcctcc ttgcctcgga gggcagctgt gaacattgaa ttcagggacc tttcctattc
360ggttcctgaa ggaccctggt ggaggaagaa aggatacaag accctcctga aaggaatttc
420cgggaagttc aatagtggtg agttggtggc cattatgggt ccttccgggg ccgggaagtc
480cacgctgatg aacatcctgg ctggatacag ggagacgggc atgaaggggg ccgtcctcat
540caacggcctg ccccgggacc tgcgctgctt ccggaaggtg tcctgctaca tcatgcagga
600tgacatgctg ctgccgcatc tcactgtgca ggaggccatg atggtgtcgg cacatctgaa
660gcttcaggag aaggatgaag gcagaaggga aatggtcaag gagatactga cagcgctggg
720cttgctgtct tgcgccaaca cgcggaccgg gagcctgtca ggtggtcagc gcaagcgcct
780ggccatcgcg ctggagctgg tgaacaaccc tccagtcatg ttcttcgatg agcccaccag
840cggcctggac agcgcctcct gcttccaggt ggtctcgctg atgaaagggc tcgctcaagg
900gggtcgctcc atcatttgca ccatccacca gcccagcgcc aaactcttcg agctgttcga
960ccagctttac gtcctgagtc aaggacaatg tgtgtaccgg ggaaaagtct gcaatcttgt
1020gccatatttg agggatttgg gtctgaactg cccaacctac cacaacccag cagattttgt
1080catggaggtt gcatccggcg agtacggtga tcagaacagt cggctggtga gagcggttcg
1140ggagggcatg tgtgactcag accacaagag agacctcggg ggtgatgccg aggtgaaccc
1200ttttctttgg caccggccct ctgaagagga ctcctcgtcc atggaaggct gccacagctt
1260ctctgccagc tgcctcacgc agttctgcat cctcttcaag aggaccttcc tcagcatcat
1320gagggactcg gtcctgacac acctgcgcat cacctcgcac attgggatcg gcctcctcat
1380tggcctgctg tacttgggga tcgggaacga agccaagaag gtcttgagca actccggctt
1440cctcttcttc tccatgctgt tcctcatgtt cgcggccctc atgcctactg ttctgacatt
1500tcccctggag atgggagtct ttcttcggga acacctgaac tactggtaca gcctgaaggc
1560ctactacctg gccaagacca tggcagacgt gccctttcag atcatgttcc cagtggccta
1620ctgcagcatc gtgtactgga tgacgtcgca gccgtccgac gccgtgcgct ttgtgctgtt
1680tgccgcgctg ggcaccatga cctccctggt ggcacagtcc ctgggcctgc tgatcggagc
1740cgcctccacg tccctgcagg tggccacttt cgtgggccca gtgacagcca tcccggtgct
1800cctgttctcg gggttcttcg tcagcttcga caccatcccc acgtacctac agtggatgtc
1860ctacatctcc tatgtcaggt atgggttcga aggggtcatc ctctccatct atggcttaga
1920ccgggaagat ctgcactgtg acatcgacga gacgtgccac ttccagaagt cggaggccat
1980cctgcgggag ctggacgtgg aaaatgccaa gctgtacctg gacttcatcg tactcgggat
2040tttcttcatc tccctccgcc tcattgccta ttttgtcctc aggtacaaaa tccgggcaga
2100gaggtaaaac acctgaatgc caggaaacag gaagattaga cactgtggcc gagggcacgt
2160ctagaatcga ggaggcaagc ctgtgcccga ccgacgacac agagactctt ctgatccaac
2220ccctagaacc gcgttgggtt tgtgggtgtc tcgtgctcag ccactctgcc cagctgggtt
2280ggatcttctc tccattcccc tttctagctt taactaggaa gatgtaggca gattggtggt
2340tttttttttt ttaacataca gaattttaaa taccacaact ggggcagaat ttaaagctgc
2400aacacagctg gtgatgagag gcttcctcag tccagtcgct ccttagcacc aggcaccgtg
2460ggtcctggat ggggaactgc aagcagcctc tcagctgatg gctgcacagt cagatgtctg
2520gtggcagaga gtccgagcat ggagcgattc cattttatga ctgttgtttt tcacattttc
2580atctttctaa ggtgtgtctc ttttccaatg agaagtcatt tttgcaagcc aaaagtcgat
2640caatcgcatt cattttaaga aattatacct ttttagtact tgctgaagaa tgattcaggg
2700taaatcacat actttgttta gagaggcgag gggtttaacc gagtcaccca gctggtctca
2760tacatagaca gcacttgtga aggattgaat gcaggttcca ggtggaggga agacgtggac
2820accatctcca ctgagccatg cagacatttt taaaagctat acaaaaaatt gtgagaagac
2880attggccaac tctttcaaag tctttctttt tccacgtgct tcttatttta agcgaaatat
2940attgtttgtt tcttcctaaa aaaaaaaaaa aaaaaaaaaa aa
2982163018DNAHomo sapiens 16cgcaccccgc gcagcggctg agccgggagc cagcgcagcc
tcggccccgc agctcaagcc 60tcgtccccgc cgccgccgcc gccgccgccg ccgccgcccc
cggggcatgg cctgtctgat 120ggccgctttc tcggtcggca ccgccatgaa tgccagcagt
tactctgcag agatgacgga 180gcccaagtcg gtgtgtgtct cggtggatga ggtggtgtcc
agcaacatgg aggccactga 240gacggacctg ctgaatggac atctgaaaaa agtagataat
aacctcacgg aagcccagcg 300cttctcctcc ttgcctcgga gggcagctgt gaacattgaa
ttcagggacc tttcctattc 360ggttcctgaa ggaccctggt ggaggaagaa aggatacaag
accctcctga aaggaatttc 420cgggaagttc aatagtggtg agttggtggc cattatgggt
ccttccgggg ccgggaagtc 480cacgctgatg aacatcctgg ctggatacag ggagacgggc
atgaaggggg ccgtcctcat 540caacggcctg ccccgggacc tgcgctgctt ccggaaggtg
tcctgctaca tcatgcagga 600tgacatgctg ctgccgcatc tcactgtgca ggaggccatg
atggtgtcgg cacatctgaa 660gcttcaggag aaggatgaag gcagaaggga aatggtcaag
gagatactga cagcgctggg 720cttgctgtct tgcgccaaca cgcggaccgg gagcctgtca
ggtggtcagc gcaagcgcct 780ggccatcgcg ctggagctgg tgaacaaccc tccagtcatg
ttcttcgatg agcccaccag 840cggcctggac agcgcctcct gcttccaggt ggtctcgctg
atgaaagggc tcgctcaagg 900gggtcgctcc atcatttgca ccatccacca gcccagcgcc
aaactcttcg agctgttcga 960ccagctttac gtcctgagtc aaggacaatg tgtgtaccgg
ggaaaagtct gcaatcttgt 1020gccatatttg agggatttgg gtctgaactg cccaacctac
cacaacccag cagattttgt 1080catggaggtt gcatccggcg agtacggtga tcagaacagt
cggctggtga gagcggttcg 1140ggagggcatg tgtgactcag accacaagag agacctcggg
ggtgatgccg aggtgaaccc 1200ttttctttgg caccggccct ctgaagaggt aaagcagaca
aaacgattaa aggggttgag 1260aaaggactcc tcgtccatgg aaggctgcca cagcttctct
gccagctgcc tcacgcagtt 1320ctgcatcctc ttcaagagga ccttcctcag catcatgagg
gactcggtcc tgacacacct 1380gcgcatcacc tcgcacattg ggatcggcct cctcattggc
ctgctgtact tggggatcgg 1440gaacgaagcc aagaaggtct tgagcaactc cggcttcctc
ttcttctcca tgctgttcct 1500catgttcgcg gccctcatgc ctactgttct gacatttccc
ctggagatgg gagtctttct 1560tcgggaacac ctgaactact ggtacagcct gaaggcctac
tacctggcca agaccatggc 1620agacgtgccc tttcagatca tgttcccagt ggcctactgc
agcatcgtgt actggatgac 1680gtcgcagccg tccgacgccg tgcgctttgt gctgtttgcc
gcgctgggca ccatgacctc 1740cctggtggca cagtccctgg gcctgctgat cggagccgcc
tccacgtccc tgcaggtggc 1800cactttcgtg ggcccagtga cagccatccc ggtgctcctg
ttctcggggt tcttcgtcag 1860cttcgacacc atccccacgt acctacagtg gatgtcctac
atctcctatg tcaggtatgg 1920gttcgaaggg gtcatcctct ccatctatgg cttagaccgg
gaagatctgc actgtgacat 1980cgacgagacg tgccacttcc agaagtcgga ggccatcctg
cgggagctgg acgtggaaaa 2040tgccaagctg tacctggact tcatcgtact cgggattttc
ttcatctccc tccgcctcat 2100tgcctatttt gtcctcaggt acaaaatccg ggcagagagg
taaaacacct gaatgccagg 2160aaacaggaag attagacact gtggccgagg gcacgtctag
aatcgaggag gcaagcctgt 2220gcccgaccga cgacacagag actcttctga tccaacccct
agaaccgcgt tgggtttgtg 2280ggtgtctcgt gctcagccac tctgcccagc tgggttggat
cttctctcca ttcccctttc 2340tagctttaac taggaagatg taggcagatt ggtggttttt
ttttttttaa catacagaat 2400tttaaatacc acaactgggg cagaatttaa agctgcaaca
cagctggtga tgagaggctt 2460cctcagtcca gtcgctcctt agcaccaggc accgtgggtc
ctggatgggg aactgcaagc 2520agcctctcag ctgatggctg cacagtcaga tgtctggtgg
cagagagtcc gagcatggag 2580cgattccatt ttatgactgt tgtttttcac attttcatct
ttctaaggtg tgtctctttt 2640ccaatgagaa gtcatttttg caagccaaaa gtcgatcaat
cgcattcatt ttaagaaatt 2700ataccttttt agtacttgct gaagaatgat tcagggtaaa
tcacatactt tgtttagaga 2760ggcgaggggt ttaaccgagt cacccagctg gtctcataca
tagacagcac ttgtgaagga 2820ttgaatgcag gttccaggtg gagggaagac gtggacacca
tctccactga gccatgcaga 2880catttttaaa agctatacaa aaaattgtga gaagacattg
gccaactctt tcaaagtctt 2940tctttttcca cgtgcttctt attttaagcg aaatatattg
tttgtttctt cctaaaaaaa 3000aaaaaaaaaa aaaaaaaa
3018172983DNAHomo sapiens 17gcgaggggca agcccggatt
cctgccggcc gcctttctgc gcgcgccgga gagagagacg 60cggtggggac agggatgcgc
atttcacttc cccgagctcc ggagagggat ggcggggtgt 120cggcgagttc actgctggac
acagttacta atgccagcag ttactctgca gagatgacgg 180agcccaagtc ggtgtgtgtc
tcggtggatg aggtggtgtc cagcaacatg gaggccactg 240agacggacct gctgaatgga
catctgaaaa aagtagataa taacctcacg gaagcccagc 300gcttctcctc cttgcctcgg
agggcagctg tgaacattga attcagggac ctttcctatt 360cggttcctga aggaccctgg
tggaggaaga aaggatacaa gaccctcctg aaaggaattt 420ccgggaagtt caatagtggt
gagttggtgg ccattatggg tccttccggg gccgggaagt 480ccacgctgat gaacatcctg
gctggataca gggagacggg catgaagggg gccgtcctca 540tcaacggcct gccccgggac
ctgcgctgct tccggaaggt gtcctgctac atcatgcagg 600atgacatgct gctgccgcat
ctcactgtgc aggaggccat gatggtgtcg gcacatctga 660agcttcagga gaaggatgaa
ggcagaaggg aaatggtcaa ggagatactg acagcgctgg 720gcttgctgtc ttgcgccaac
acgcggaccg ggagcctgtc aggtggtcag cgcaagcgcc 780tggccatcgc gctggagctg
gtgaacaacc ctccagtcat gttcttcgat gagcccacca 840gcggcctgga cagcgcctcc
tgcttccagg tggtctcgct gatgaaaggg ctcgctcaag 900ggggtcgctc catcatttgc
accatccacc agcccagcgc caaactcttc gagctgttcg 960accagcttta cgtcctgagt
caaggacaat gtgtgtaccg gggaaaagtc tgcaatcttg 1020tgccatattt gagggatttg
ggtctgaact gcccaaccta ccacaaccca gcagattttg 1080tcatggaggt tgcatccggc
gagtacggtg atcagaacag tcggctggtg agagcggttc 1140gggagggcat gtgtgactca
gaccacaaga gagacctcgg gggtgatgcc gaggtgaacc 1200cttttctttg gcaccggccc
tctgaagagg actcctcgtc catggaaggc tgccacagct 1260tctctgccag ctgcctcacg
cagttctgca tcctcttcaa gaggaccttc ctcagcatca 1320tgagggactc ggtcctgaca
cacctgcgca tcacctcgca cattgggatc ggcctcctca 1380ttggcctgct gtacttgggg
atcgggaacg aagccaagaa ggtcttgagc aactccggct 1440tcctcttctt ctccatgctg
ttcctcatgt tcgcggccct catgcctact gttctgacat 1500ttcccctgga gatgggagtc
tttcttcggg aacacctgaa ctactggtac agcctgaagg 1560cctactacct ggccaagacc
atggcagacg tgccctttca gatcatgttc ccagtggcct 1620actgcagcat cgtgtactgg
atgacgtcgc agccgtccga cgccgtgcgc tttgtgctgt 1680ttgccgcgct gggcaccatg
acctccctgg tggcacagtc cctgggcctg ctgatcggag 1740ccgcctccac gtccctgcag
gtggccactt tcgtgggccc agtgacagcc atcccggtgc 1800tcctgttctc ggggttcttc
gtcagcttcg acaccatccc cacgtaccta cagtggatgt 1860cctacatctc ctatgtcagg
tatgggttcg aaggggtcat cctctccatc tatggcttag 1920accgggaaga tctgcactgt
gacatcgacg agacgtgcca cttccagaag tcggaggcca 1980tcctgcggga gctggacgtg
gaaaatgcca agctgtacct ggacttcatc gtactcggga 2040ttttcttcat ctccctccgc
ctcattgcct attttgtcct caggtacaaa atccgggcag 2100agaggtaaaa cacctgaatg
ccaggaaaca ggaagattag acactgtggc cgagggcacg 2160tctagaatcg aggaggcaag
cctgtgcccg accgacgaca cagagactct tctgatccaa 2220cccctagaac cgcgttgggt
ttgtgggtgt ctcgtgctca gccactctgc ccagctgggt 2280tggatcttct ctccattccc
ctttctagct ttaactagga agatgtaggc agattggtgg 2340tttttttttt tttaacatac
agaattttaa ataccacaac tggggcagaa tttaaagctg 2400caacacagct ggtgatgaga
ggcttcctca gtccagtcgc tccttagcac caggcaccgt 2460gggtcctgga tggggaactg
caagcagcct ctcagctgat ggctgcacag tcagatgtct 2520ggtggcagag agtccgagca
tggagcgatt ccattttatg actgttgttt ttcacatttt 2580catctttcta aggtgtgtct
cttttccaat gagaagtcat ttttgcaagc caaaagtcga 2640tcaatcgcat tcattttaag
aaattatacc tttttagtac ttgctgaaga atgattcagg 2700gtaaatcaca tactttgttt
agagaggcga ggggtttaac cgagtcaccc agctggtctc 2760atacatagac agcacttgtg
aaggattgaa tgcaggttcc aggtggaggg aagacgtgga 2820caccatctcc actgagccat
gcagacattt ttaaaagcta tacaaaaaat tgtgagaaga 2880cattggccaa ctctttcaaa
gtctttcttt ttccacgtgc ttcttatttt aagcgaaata 2940tattgtttgt ttcttcctaa
aaaaaaaaaa aaaaaaaaaa aaa 2983183142DNAHomo sapiens
18agacactgaa tcatcatttg tagtttgggg ggctttacat gcctgcagtg gtgaaaactg
60aaattttgtc ccacttaagg gagtttcttc ttccctttat taattgcaaa ataaatatat
120gtcacttcag agggcagcag ctggactacc tatgtttgtg gcgctacacc aacctgaact
180tcgcttcctg agacctaaga ttcagccccg tgctcagcag acatcaggga tcaccgactc
240tgtgccagga gctgttcttg atgctgggaa cgcaggggtg gacaaaacag agaaagccct
300gccctcagaa tgccagcagt tactctgcag agatgacgga gcccaagtcg gtgtgtgtct
360cggtggatga ggtggtgtcc agcaacatgg aggccactga gacggacctg ctgaatggac
420atctgaaaaa agtagataat aacctcacgg aagcccagcg cttctcctcc ttgcctcgga
480gggcagctgt gaacattgaa ttcagggacc tttcctattc ggttcctgaa ggaccctggt
540ggaggaagaa aggatacaag accctcctga aaggaatttc cgggaagttc aatagtggtg
600agttggtggc cattatgggt ccttccgggg ccgggaagtc cacgctgatg aacatcctgg
660ctggatacag ggagacgggc atgaaggggg ccgtcctcat caacggcctg ccccgggacc
720tgcgctgctt ccggaaggtg tcctgctaca tcatgcagga tgacatgctg ctgccgcatc
780tcactgtgca ggaggccatg atggtgtcgg cacatctgaa gcttcaggag aaggatgaag
840gcagaaggga aatggtcaag gagatactga cagcgctggg cttgctgtct tgcgccaaca
900cgcggaccgg gagcctgtca ggtggtcagc gcaagcgcct ggccatcgcg ctggagctgg
960tgaacaaccc tccagtcatg ttcttcgatg agcccaccag cggcctggac agcgcctcct
1020gcttccaggt ggtctcgctg atgaaagggc tcgctcaagg gggtcgctcc atcatttgca
1080ccatccacca gcccagcgcc aaactcttcg agctgttcga ccagctttac gtcctgagtc
1140aaggacaatg tgtgtaccgg ggaaaagtct gcaatcttgt gccatatttg agggatttgg
1200gtctgaactg cccaacctac cacaacccag cagattttgt catggaggtt gcatccggcg
1260agtacggtga tcagaacagt cggctggtga gagcggttcg ggagggcatg tgtgactcag
1320accacaagag agacctcggg ggtgatgccg aggtgaaccc ttttctttgg caccggccct
1380ctgaagagga ctcctcgtcc atggaaggct gccacagctt ctctgccagc tgcctcacgc
1440agttctgcat cctcttcaag aggaccttcc tcagcatcat gagggactcg gtcctgacac
1500acctgcgcat cacctcgcac attgggatcg gcctcctcat tggcctgctg tacttgggga
1560tcgggaacga agccaagaag gtcttgagca actccggctt cctcttcttc tccatgctgt
1620tcctcatgtt cgcggccctc atgcctactg ttctgacatt tcccctggag atgggagtct
1680ttcttcggga acacctgaac tactggtaca gcctgaaggc ctactacctg gccaagacca
1740tggcagacgt gccctttcag atcatgttcc cagtggccta ctgcagcatc gtgtactgga
1800tgacgtcgca gccgtccgac gccgtgcgct ttgtgctgtt tgccgcgctg ggcaccatga
1860cctccctggt ggcacagtcc ctgggcctgc tgatcggagc cgcctccacg tccctgcagg
1920tggccacttt cgtgggccca gtgacagcca tcccggtgct cctgttctcg gggttcttcg
1980tcagcttcga caccatcccc acgtacctac agtggatgtc ctacatctcc tatgtcaggt
2040atgggttcga aggggtcatc ctctccatct atggcttaga ccgggaagat ctgcactgtg
2100acatcgacga gacgtgccac ttccagaagt cggaggccat cctgcgggag ctggacgtgg
2160aaaatgccaa gctgtacctg gacttcatcg tactcgggat tttcttcatc tccctccgcc
2220tcattgccta ttttgtcctc aggtacaaaa tccgggcaga gaggtaaaac acctgaatgc
2280caggaaacag gaagattaga cactgtggcc gagggcacgt ctagaatcga ggaggcaagc
2340ctgtgcccga ccgacgacac agagactctt ctgatccaac ccctagaacc gcgttgggtt
2400tgtgggtgtc tcgtgctcag ccactctgcc cagctgggtt ggatcttctc tccattcccc
2460tttctagctt taactaggaa gatgtaggca gattggtggt tttttttttt ttaacataca
2520gaattttaaa taccacaact ggggcagaat ttaaagctgc aacacagctg gtgatgagag
2580gcttcctcag tccagtcgct ccttagcacc aggcaccgtg ggtcctggat ggggaactgc
2640aagcagcctc tcagctgatg gctgcacagt cagatgtctg gtggcagaga gtccgagcat
2700ggagcgattc cattttatga ctgttgtttt tcacattttc atctttctaa ggtgtgtctc
2760ttttccaatg agaagtcatt tttgcaagcc aaaagtcgat caatcgcatt cattttaaga
2820aattatacct ttttagtact tgctgaagaa tgattcaggg taaatcacat actttgttta
2880gagaggcgag gggtttaacc gagtcaccca gctggtctca tacatagaca gcacttgtga
2940aggattgaat gcaggttcca ggtggaggga agacgtggac accatctcca ctgagccatg
3000cagacatttt taaaagctat acaaaaaatt gtgagaagac attggccaac tctttcaaag
3060tctttctttt tccacgtgct tcttatttta agcgaaatat attgtttgtt tcttcctaaa
3120aaaaaaaaaa aaaaaaaaaa aa
3142193060DNAHomo sapiens 19acttcagagg gcagcagctg gactacctat gtttgtggcg
ctacaccaac ctgaacttcg 60cttcctgaga cctaagattc agccccgtgc tcagcagaca
tcagggatca ccgactctgt 120gccaggagct gttcttgatg ctgggaacgc aggggtggac
aaaacagaga aagccctgcc 180ctcagtgaga aatatgctgt catgtaaatt gctttttccc
ctatagaatg ccagcagtta 240ctctgcagag atgacggagc ccaagtcggt gtgtgtctcg
gtggatgagg tggtgtccag 300caacatggag gccactgaga cggacctgct gaatggacat
ctgaaaaaag tagataataa 360cctcacggaa gcccagcgct tctcctcctt gcctcggagg
gcagctgtga acattgaatt 420cagggacctt tcctattcgg ttcctgaagg accctggtgg
aggaagaaag gatacaagac 480cctcctgaaa ggaatttccg ggaagttcaa tagtggtgag
ttggtggcca ttatgggtcc 540ttccggggcc gggaagtcca cgctgatgaa catcctggct
ggatacaggg agacgggcat 600gaagggggcc gtcctcatca acggcctgcc ccgggacctg
cgctgcttcc ggaaggtgtc 660ctgctacatc atgcaggatg acatgctgct gccgcatctc
actgtgcagg aggccatgat 720ggtgtcggca catctgaagc ttcaggagaa ggatgaaggc
agaagggaaa tggtcaagga 780gatactgaca gcgctgggct tgctgtcttg cgccaacacg
cggaccggga gcctgtcagg 840tggtcagcgc aagcgcctgg ccatcgcgct ggagctggtg
aacaaccctc cagtcatgtt 900cttcgatgag cccaccagcg gcctggacag cgcctcctgc
ttccaggtgg tctcgctgat 960gaaagggctc gctcaagggg gtcgctccat catttgcacc
atccaccagc ccagcgccaa 1020actcttcgag ctgttcgacc agctttacgt cctgagtcaa
ggacaatgtg tgtaccgggg 1080aaaagtctgc aatcttgtgc catatttgag ggatttgggt
ctgaactgcc caacctacca 1140caacccagca gattttgtca tggaggttgc atccggcgag
tacggtgatc agaacagtcg 1200gctggtgaga gcggttcggg agggcatgtg tgactcagac
cacaagagag acctcggggg 1260tgatgccgag gtgaaccctt ttctttggca ccggccctct
gaagaggact cctcgtccat 1320ggaaggctgc cacagcttct ctgccagctg cctcacgcag
ttctgcatcc tcttcaagag 1380gaccttcctc agcatcatga gggactcggt cctgacacac
ctgcgcatca cctcgcacat 1440tgggatcggc ctcctcattg gcctgctgta cttggggatc
gggaacgaag ccaagaaggt 1500cttgagcaac tccggcttcc tcttcttctc catgctgttc
ctcatgttcg cggccctcat 1560gcctactgtt ctgacatttc ccctggagat gggagtcttt
cttcgggaac acctgaacta 1620ctggtacagc ctgaaggcct actacctggc caagaccatg
gcagacgtgc cctttcagat 1680catgttccca gtggcctact gcagcatcgt gtactggatg
acgtcgcagc cgtccgacgc 1740cgtgcgcttt gtgctgtttg ccgcgctggg caccatgacc
tccctggtgg cacagtccct 1800gggcctgctg atcggagccg cctccacgtc cctgcaggtg
gccactttcg tgggcccagt 1860gacagccatc ccggtgctcc tgttctcggg gttcttcgtc
agcttcgaca ccatccccac 1920gtacctacag tggatgtcct acatctccta tgtcaggtat
gggttcgaag gggtcatcct 1980ctccatctat ggcttagacc gggaagatct gcactgtgac
atcgacgaga cgtgccactt 2040ccagaagtcg gaggccatcc tgcgggagct ggacgtggaa
aatgccaagc tgtacctgga 2100cttcatcgta ctcgggattt tcttcatctc cctccgcctc
attgcctatt ttgtcctcag 2160gtacaaaatc cgggcagaga ggtaaaacac ctgaatgcca
ggaaacagga agattagaca 2220ctgtggccga gggcacgtct agaatcgagg aggcaagcct
gtgcccgacc gacgacacag 2280agactcttct gatccaaccc ctagaaccgc gttgggtttg
tgggtgtctc gtgctcagcc 2340actctgccca gctgggttgg atcttctctc cattcccctt
tctagcttta actaggaaga 2400tgtaggcaga ttggtggttt tttttttttt aacatacaga
attttaaata ccacaactgg 2460ggcagaattt aaagctgcaa cacagctggt gatgagaggc
ttcctcagtc cagtcgctcc 2520ttagcaccag gcaccgtggg tcctggatgg ggaactgcaa
gcagcctctc agctgatggc 2580tgcacagtca gatgtctggt ggcagagagt ccgagcatgg
agcgattcca ttttatgact 2640gttgtttttc acattttcat ctttctaagg tgtgtctctt
ttccaatgag aagtcatttt 2700tgcaagccaa aagtcgatca atcgcattca ttttaagaaa
ttataccttt ttagtacttg 2760ctgaagaatg attcagggta aatcacatac tttgtttaga
gaggcgaggg gtttaaccga 2820gtcacccagc tggtctcata catagacagc acttgtgaag
gattgaatgc aggttccagg 2880tggagggaag acgtggacac catctccact gagccatgca
gacattttta aaagctatac 2940aaaaaattgt gagaagacat tggccaactc tttcaaagtc
tttctttttc cacgtgcttc 3000ttattttaag cgaaatatat tgtttgtttc ttcctaaaaa
aaaaaaaaaa aaaaaaaaaa 3060202946DNAHomo sapiens 20gctttataaa ggggagtttc
cctgcacaag ctctctctct tgtctgccgc catgtgagac 60atgcctttca ccttccgcca
tgatcatgag gcttccccag ccacatggaa ctaatgccag 120cagttactct gcagagatga
cggagcccaa gtcggtgtgt gtctcggtgg atgaggtggt 180gtccagcaac atggaggcca
ctgagacgga cctgctgaat ggacatctga aaaaagtaga 240taataacctc acggaagccc
agcgcttctc ctccttgcct cggagggcag ctgtgaacat 300tgaattcagg gacctttcct
attcggttcc tgaaggaccc tggtggagga agaaaggata 360caagaccctc ctgaaaggaa
tttccgggaa gttcaatagt ggtgagttgg tggccattat 420gggtccttcc ggggccggga
agtccacgct gatgaacatc ctggctggat acagggagac 480gggcatgaag ggggccgtcc
tcatcaacgg cctgccccgg gacctgcgct gcttccggaa 540ggtgtcctgc tacatcatgc
aggatgacat gctgctgccg catctcactg tgcaggaggc 600catgatggtg tcggcacatc
tgaagcttca ggagaaggat gaaggcagaa gggaaatggt 660caaggagata ctgacagcgc
tgggcttgct gtcttgcgcc aacacgcgga ccgggagcct 720gtcaggtggt cagcgcaagc
gcctggccat cgcgctggag ctggtgaaca accctccagt 780catgttcttc gatgagccca
ccagcggcct ggacagcgcc tcctgcttcc aggtggtctc 840gctgatgaaa gggctcgctc
aagggggtcg ctccatcatt tgcaccatcc accagcccag 900cgccaaactc ttcgagctgt
tcgaccagct ttacgtcctg agtcaaggac aatgtgtgta 960ccggggaaaa gtctgcaatc
ttgtgccata tttgagggat ttgggtctga actgcccaac 1020ctaccacaac ccagcagatt
ttgtcatgga ggttgcatcc ggcgagtacg gtgatcagaa 1080cagtcggctg gtgagagcgg
ttcgggaggg catgtgtgac tcagaccaca agagagacct 1140cgggggtgat gccgaggtga
acccttttct ttggcaccgg ccctctgaag aggactcctc 1200gtccatggaa ggctgccaca
gcttctctgc cagctgcctc acgcagttct gcatcctctt 1260caagaggacc ttcctcagca
tcatgaggga ctcggtcctg acacacctgc gcatcacctc 1320gcacattggg atcggcctcc
tcattggcct gctgtacttg gggatcggga acgaagccaa 1380gaaggtcttg agcaactccg
gcttcctctt cttctccatg ctgttcctca tgttcgcggc 1440cctcatgcct actgttctga
catttcccct ggagatggga gtctttcttc gggaacacct 1500gaactactgg tacagcctga
aggcctacta cctggccaag accatggcag acgtgccctt 1560tcagatcatg ttcccagtgg
cctactgcag catcgtgtac tggatgacgt cgcagccgtc 1620cgacgccgtg cgctttgtgc
tgtttgccgc gctgggcacc atgacctccc tggtggcaca 1680gtccctgggc ctgctgatcg
gagccgcctc cacgtccctg caggtggcca ctttcgtggg 1740cccagtgaca gccatcccgg
tgctcctgtt ctcggggttc ttcgtcagct tcgacaccat 1800ccccacgtac ctacagtgga
tgtcctacat ctcctatgtc aggtatgggt tcgaaggggt 1860catcctctcc atctatggct
tagaccggga agatctgcac tgtgacatcg acgagacgtg 1920ccacttccag aagtcggagg
ccatcctgcg ggagctggac gtggaaaatg ccaagctgta 1980cctggacttc atcgtactcg
ggattttctt catctccctc cgcctcattg cctattttgt 2040cctcaggtac aaaatccggg
cagagaggta aaacacctga atgccaggaa acaggaagat 2100tagacactgt ggccgagggc
acgtctagaa tcgaggaggc aagcctgtgc ccgaccgacg 2160acacagagac tcttctgatc
caacccctag aaccgcgttg ggtttgtggg tgtctcgtgc 2220tcagccactc tgcccagctg
ggttggatct tctctccatt cccctttcta gctttaacta 2280ggaagatgta ggcagattgg
tggttttttt ttttttaaca tacagaattt taaataccac 2340aactggggca gaatttaaag
ctgcaacaca gctggtgatg agaggcttcc tcagtccagt 2400cgctccttag caccaggcac
cgtgggtcct ggatggggaa ctgcaagcag cctctcagct 2460gatggctgca cagtcagatg
tctggtggca gagagtccga gcatggagcg attccatttt 2520atgactgttg tttttcacat
tttcatcttt ctaaggtgtg tctcttttcc aatgagaagt 2580catttttgca agccaaaagt
cgatcaatcg cattcatttt aagaaattat acctttttag 2640tacttgctga agaatgattc
agggtaaatc acatactttg tttagagagg cgaggggttt 2700aaccgagtca cccagctggt
ctcatacata gacagcactt gtgaaggatt gaatgcaggt 2760tccaggtgga gggaagacgt
ggacaccatc tccactgagc catgcagaca tttttaaaag 2820ctatacaaaa aattgtgaga
agacattggc caactctttc aaagtctttc tttttccacg 2880tgcttcttat tttaagcgaa
atatattgtt tgtttcttcc taaaaaaaaa aaaaaaaaaa 2940aaaaaa
2946213498DNAHomo sapiens
21ttctttccaa gggtctctgg gtgaggcccg tgaccttccc aagcctctcc ctgtcttgtg
60aaacctgggc gtgatatacc tcccttttag ggctgctgcg atcatttagg cagattaaac
120ctcataagtg gtttcccata caagaaagat gctagcagtg caacagacag aacacttacc
180tgcctgccct cccgccagga ggtggtcttc caacttttgc ccggagtcta cagagggtgg
240gccctctctg ctggggctcc gggacatggt caggagaggt tggtctgtct gtaccgccat
300tctcttggcc agactgtggt gtctggtccc tactcacacc ttcctgtcag agtatccaga
360ggccgcagag tatccacacc ctggctgggt gtactggcta cagatggctg tggctccagg
420tcacctgcgt gcctgggtga tgagaaataa tgtcacaaca aatatcccat ctgcattctc
480tgggacactg acccatgaag agaaagcagt tctcacagtt tttacaggca cagccacagc
540cgtgcatgta caggtggcag ctttagcttc tgctaaactg gagagctcag tgtttgtgac
600agactgcgtg tcctgcaaaa tcgaaaatgt ctgtgattca gctcttcagg gaaaaagggt
660gccgatgtct ggcctacagg gctcaagcat tgtcatcatg cccccatcca accgtccact
720cgccagtgcg gcatcctgca cgtggtcagt ccaagtccag ggagggcccc atcacctggg
780ggtggtcgct atcagtggca aagtcttgtc agcagctcat ggggcaggaa gggcctatgg
840ttgggggttt cctggcgatc ccatggagga aggatacaag accctcctga aaggaatttc
900cgggaagttc aatagtggtg agttggtggc cattatgggt ccttccgggg ccgggaagtc
960cacgctgatg aacatcctgg ctggatacag ggagacgggc atgaaggggg ccgtcctcat
1020caacggcctg ccccgggacc tgcgctgctt ccggaaggtg tcctgctaca tcatgcagga
1080tgacatgctg ctgccgcatc tcactgtgca ggaggccatg atggtgtcgg cacatctgaa
1140gcttcaggag aaggatgaag gcagaaggga aatggtcaag gagatactga cagcgctggg
1200cttgctgtct tgcgccaaca cgcggaccgg gagcctgtca ggtggtcagc gcaagcgcct
1260ggccatcgcg ctggagctgg tgaacaaccc tccagtcatg ttcttcgatg agcccaccag
1320cggcctggac agcgcctcct gcttccaggt ggtctcgctg atgaaagggc tcgctcaagg
1380gggtcgctcc atcatttgca ccatccacca gcccagcgcc aaactcttcg agctgttcga
1440ccagctttac gtcctgagtc aaggacaatg tgtgtaccgg ggaaaagtct gcaatcttgt
1500gccatatttg agggatttgg gtctgaactg cccaacctac cacaacccag cagattttgt
1560catggaggtt gcatccggcg agtacggtga tcagaacagt cggctggtga gagcggttcg
1620ggagggcatg tgtgactcag accacaagag agacctcggg ggtgatgccg aggtgaaccc
1680ttttctttgg caccggccct ctgaagaggt aaagcagaca aaacgattaa aggggttgag
1740aaaggactcc tcgtccatgg aaggctgcca cagcttctct gccagctgcc tcacgcagtt
1800ctgcatcctc ttcaagagga ccttcctcag catcatgagg gactcggtcc tgacacacct
1860gcgcatcacc tcgcacattg ggatcggcct cctcattggc ctgctgtact tggggatcgg
1920gaacgaagcc aagaaggtct tgagcaactc cggcttcctc ttcttctcca tgctgttcct
1980catgttcgcg gccctcatgc ctactgttct gacatttccc ctggagatgg gagtctttct
2040tcgggaacac ctgaactact ggtacagcct gaaggcctac tacctggcca agaccatggc
2100agacgtgccc tttcagatca tgttcccagt ggcctactgc agcatcgtgt actggatgac
2160gtcgcagccg tccgacgccg tgcgctttgt gctgtttgcc gcgctgggca ccatgacctc
2220cctggtggca cagtccctgg gcctgctgat cggagccgcc tccacgtccc tgcaggtggc
2280cactttcgtg ggcccagtga cagccatccc ggtgctcctg ttctcggggt tcttcgtcag
2340cttcgacacc atccccacgt acctacagtg gatgtcctac atctcctatg tcaggtatgg
2400gttcgaaggg gtcatcctct ccatctatgg cttagaccgg gaagatctgc actgtgacat
2460cgacgagacg tgccacttcc agaagtcgga ggccatcctg cgggagctgg acgtggaaaa
2520tgccaagctg tacctggact tcatcgtact cgggattttc ttcatctccc tccgcctcat
2580tgcctatttt gtcctcaggt acaaaatccg ggcagagagg taaaacacct gaatgccagg
2640aaacaggaag attagacact gtggccgagg gcacgtctag aatcgaggag gcaagcctgt
2700gcccgaccga cgacacagag actcttctga tccaacccct agaaccgcgt tgggtttgtg
2760ggtgtctcgt gctcagccac tctgcccagc tgggttggat cttctctcca ttcccctttc
2820tagctttaac taggaagatg taggcagatt ggtggttttt ttttttttaa catacagaat
2880tttaaatacc acaactgggg cagaatttaa agctgcaaca cagctggtga tgagaggctt
2940cctcagtcca gtcgctcctt agcaccaggc accgtgggtc ctggatgggg aactgcaagc
3000agcctctcag ctgatggctg cacagtcaga tgtctggtgg cagagagtcc gagcatggag
3060cgattccatt ttatgactgt tgtttttcac attttcatct ttctaaggtg tgtctctttt
3120ccaatgagaa gtcatttttg caagccaaaa gtcgatcaat cgcattcatt ttaagaaatt
3180ataccttttt agtacttgct gaagaatgat tcagggtaaa tcacatactt tgtttagaga
3240ggcgaggggt ttaaccgagt cacccagctg gtctcataca tagacagcac ttgtgaagga
3300ttgaatgcag gttccaggtg gagggaagac gtggacacca tctccactga gccatgcaga
3360catttttaaa agctatacaa aaaattgtga gaagacattg gccaactctt tcaaagtctt
3420tctttttcca cgtgcttctt attttaagcg aaatatattg tttgtttctt cctaaaaaaa
3480aaaaaaaaaa aaaaaaaa
3498223400DNAHomo sapiens 22ggaaccgccg ccggtatccg cgtccgcagc gccgccagcc
aggcgagagc cgtgtgggat 60cccagcgccc gcactcccgc ccccgccaag gagccaggaa
tggcacaact agagaggagc 120gccatctctg gcttcagctc taagtccagg cgaaactcat
tcgcatatga tgttaagcgt 180gaagtataca atgaggagac ctttcaacag gaacacaaaa
ggaaggcctc ctcttctggg 240aacatgaaca tcaacatcac caccttcaga caccacgtcc
agtgccgctg ctcatggcac 300aggttcctac gatgcgtgct tacaatcttt cccttcctag
aatggatgtg tatgtatcga 360ttaaaggatt ggcttctggg agacttactt gctggtataa
gtgttggcct tgtgcaagtt 420ccccaaggcc tgacacttag tttgctggca aggcaactga
ttcctcctct caacatcgct 480tatgcagctt tctgttcttc ggtaatctat gtaatttttg
gatcgtgtca tcaaatgtcc 540attggttcct tcttcctggt gagtgctctg ctgatcaacg
ttctgaaagt gagcccattc 600aacaacggtc aactggtcat gggatctttc gtcaagaatg
agttttcggc cccctcctac 660cttatgggct ataataaatc cttgagtgtg gtggcaacca
caacttttct gactgggatt 720attcagctaa taatgggcgt attgggtttg ggcttcattg
ccacttacct tccggagtct 780gcaatgagtg cttacctggc tgctgtggca cttcatatca
tgctgtccca gctgactttc 840atctttggga ttatgattag tttccatgcc ggtcccatct
ccttcttcta tgacataatt 900aattactgtg tagctctccc aaaagcgaat tccaccagca
ttctagtatt tctaactgtt 960gttgttgctc tgcgaatcaa caaatgtatc agaatttctt
tcaatcagta tcccattgag 1020tttcccatgg aattatttct gattattggc ttcactgtga
ttgcaaacaa gataagcatg 1080gccacagaaa ccagccagac gcttattgac atgattcctt
atagctttct gcttcctgta 1140acaccagatt tcagccttct tcccaagata attttacaag
ccttctcctt atctttggtg 1200agctcctttc tgctcatatt tctgggcaag aagattgcca
gtcttcacaa ttacagtgtc 1260aattccaacc aggatttaat agccatcggc ctttgcaatg
tcgtcagttc atttttcaga 1320tcttgtgtgt ttactggtgc tattgctagg actattatcc
aggataaatc tggaggaaga 1380caacagtttg catctctggt aggcgcaggt gtgatgctgc
tcctgatggt gaagatggga 1440cactttttct acacactgcc aaatgctgtg ctggctggta
ttattctgag caacgtcatt 1500ccctaccttg aaaccatttc taacctaccc agcctgtgga
ggcaggacca atatgactgt 1560gctctttgga tgatgacatt ctcatcttca attttcctgg
gactggacat tggactaatt 1620atctcagtag tttctgcttt cttcatcacc actgttcgtt
cacacagagc taagattctt 1680ctcctgggtc aaatccctaa caccaacatt tatagaagca
tcaatgatta tcgggagatc 1740atcaccattc ctggggtgaa aatcttccag tgctgcagct
caattacatt tgtaaatgtt 1800tactacctaa agcataagct gttaaaagag gttgatatgg
taaaggtgcc tcttaaagaa 1860gaagaaattt tcagcttgtt taattcaagt gacaccaatc
tacaaggagg aaagatttgc 1920aggtgtttct gcaactgtga tgatctggag ccgctgccca
ggattcttta cacagagcga 1980tttgaaaata aactggatcc cgaagcatcc tccattaacc
tgattcactg ctcacatttt 2040gagagcatga acacaagcca aactgcatcc gaagaccaag
tgccatacac agtatcgtcc 2100gtgtctcaga aaaatcaagg gcaacagtat gaggaggtgg
aggaagtttg gcttcctaat 2160aactcatcaa gaaacagctc accaggactg cctgatgtgg
cggaaagcca ggggaggaga 2220tcactcatcc cttactcaga tgcgtctcta ctgcccagtg
tccacaccat catcctggat 2280ttctccatgg tacactacgt ggattcacgg gggttagtcg
tattaagaca gatatgcaat 2340gcctttcaaa acgccaacat tttgatactc attgcagggt
gtcactcttc catagtcagg 2400gcatttgaga ggaatgattt ctttgacgct ggcatcacca
agacccagct gttcctcagc 2460gttcacgacg ccgtgctgtt tgccttgtca aggaaggtca
taggctcctc tgagttaagc 2520atcgatgaat ccgagacagt gatacgggaa acctactcag
aaacagacaa gaatgacaat 2580tcaagatata aaatgagcag cagttttcta ggaagccaaa
aaaatgtaag tccaggcttc 2640atcaagatcc aacagcctgt agaagaggag tcggagttgg
atttggagct ggaatcagaa 2700caagaggctg ggctgggtct ggacctagac ctggatcggg
agctggagcc tgaaatggag 2760cccaaggctg agaccgagac caagacccag accgagatgg
agccccagcc tgagactgag 2820cctgagatgg agcccaaccc caaatctagg ccaagagctc
acacttttcc tcagcagcgt 2880tactggccta tgtatcatcc gtctatggct tccacccagt
ctcagactca gactcggaca 2940tggtcagtgg agaggagacg ccatcctatg gattcatact
caccagaggg caacagcaat 3000gaagatgtct aggagatgaa ctagaaataa ggggtcagat
aatgctggca aatcctccta 3060cccaaaaagg ggtcaattgt ccagagacct agactggata
cgaactagca gtacttcctt 3120cctgactgtg actcctacta cctgccagcc ttcttccttg
ctctgcgctg ggatcatact 3180cccaaatcac attactaaat gccaacaatt atctctgaat
tccctatcca ggctcccctc 3240atttcacctt cagcatatat tctagtcatg aatttccttc
ttcacacacc ccacatctct 3300gggctttgtg ccagaccatc tctaacttaa tcctctcatc
cctgttcccc tttctccaaa 3360gagatgaagc tcaaataaaa tgtataactc tagtaaaaaa
340023977DNAHomo sapiens 23attgaatgca gcaagggtct
ggaggctgag gaccaggcag acaaacattc agagttgctg 60gaatgcgaca gagacaggga
gtcagactgg tcatgcaagg ccagctctgg ggtctcggca 120ggtggtccgc gacatgacct
ccgagttctt ctctgcccag ctccgggccc agatctctga 180cgacaccact cacccgatct
cctactacaa gcccgagttc tacatgccgg atgacggggg 240cactgctcac ctgtctgtgg
tcgcagagga cggcagtgct gtgtccgcca ccagcaccat 300caacctctac tttggctcca
aggtgcgctc cccagtcagc gggatcctgc tcaataatga 360aatggatgac ttcagctcta
ccagcatcac caacgagttt ggggtacccc cctcacctgc 420caatttcatc cagccaggga
agcagccgct ctcgtccatg tgcccgacga tcatggtggg 480ccaggacggc caggtccgga
tggtggtggg agctgccggg ggcacgcaga tcaccatggc 540cactgcactg gccatcatct
acaacctctg gttcggctat gacgtgaagt gggccgtgga 600ggagccccgg ctgcacaacc
agcttctgcc caacgtcacg acagtggaga gaaacattga 660ccaggaagtg actgcagccc
tggagacccg gcaccatcac acccagatca cgtccacctt 720cattgctgtg gtgcaagcca
tcgtccgcat ggctggtggc tgggcagctg cctcggactc 780caggaaaggt ggggaacctg
ctggctactg attgctccag gcggacaagg ctgacaagca 840atccaggaac aaaatactca
ccaggacgag gaagaggact ttgggggaca ggcttctcct 900gtgagcagca gagcagcaca
ataaatgagg ccactgtgcc aggctccagg tggcctccct 960ggcctgtctc cccactc
977241049DNAHomo sapiens
24ctcgagagct gggctctgcg tcctcgtcca gccgccaact cggccaaagg cgaagccagc
60agtttcttct gctgccgggc aacgcgcctt ttaaacctga gggagtgggc gcgtgagcac
120ttaatggcgc cggtgacaga gtgagcttaa cggattaata agcgcagcca ggccagctct
180ggggtctcgg caggtggtcc gcgacatgac ctccgagttc ttctctgccc agctccgggc
240ccagatctct gacgacacca ctcacccgat ctcctactac aagcccgagt tctacatgcc
300ggatgacggg ggcactgctc acctgtctgt ggtcgcagag gacggcagtg ctgtgtccgc
360caccagcacc atcaacctct actttggctc caaggtgcgc tccccagtca gcgggatcct
420gctcaataat gaaatggatg acttcagctc taccagcatc accaacgagt ttggggtacc
480cccctcacct gccaatttca tccagccagg gaagcagccg ctctcgtcca tgtgcccgac
540gatcatggtg ggccaggacg gccaggtccg gatggtggtg ggagctgccg ggggcacgca
600gatcaccatg gccactgcac tggccatcat ctacaacctc tggttcggct atgacgtgaa
660gtgggccgtg gaggagcccc ggctgcacaa ccagcttctg cccaacgtca cgacagtgga
720gagaaacatt gaccaggaag tgactgcagc cctggagacc cggcaccatc acacccagat
780cacgtccacc ttcattgctg tggtgcaagc catcgtccgc atggctggtg gctgggcagc
840tgcctcggac tccaggaaag gtggggaacc tgctggctac tgattgctcc aggcggacaa
900ggctgacaag caatccagga acaaaatact caccaggacg aggaagagga ctttggggga
960caggcttctc ctgtgagcag cagagcagca caataaatga ggccactgtg ccaggctcca
1020ggtggcctcc ctggcctgtc tccccactc
1049251108DNAHomo sapiens 25attgaatgca gcaagggtct ggaggctgag gaccaggcag
acaaacattc agagttgctg 60gaatgcgaca gagacaggga gtcagactgg tcatgcaagg
tcctgggcct gcccttgggt 120cctggggagc cacggaaggt tgtgggtgcc agagggttgt
ggtcagagcc acagtcaggg 180gccttctgag acctgtgccc cctccccacc ctccctcccc
acctccctag gccagctctg 240gggtctcggc aggtggtccg cgacatgacc tccgagttct
tctctgccca gctccgggcc 300cagatctctg acgacaccac tcacccgatc tcctactaca
agcccgagtt ctacatgccg 360gatgacgggg gcactgctca cctgtctgtg gtcgcagagg
acggcagtgc tgtgtccgcc 420accagcacca tcaacctcta ctttggctcc aaggtgcgct
ccccagtcag cgggatcctg 480ctcaataatg aaatggatga cttcagctct accagcatca
ccaacgagtt tggggtaccc 540ccctcacctg ccaatttcat ccagccaggg aagcagccgc
tctcgtccat gtgcccgacg 600atcatggtgg gccaggacgg ccaggtccgg atggtggtgg
gagctgccgg gggcacgcag 660atcaccatgg ccactgcact ggccatcatc tacaacctct
ggttcggcta tgacgtgaag 720tgggccgtgg aggagccccg gctgcacaac cagcttctgc
ccaacgtcac gacagtggag 780agaaacattg accaggaagt gactgcagcc ctggagaccc
ggcaccatca cacccagatc 840acgtccacct tcattgctgt ggtgcaagcc atcgtccgca
tggctggtgg ctgggcagct 900gcctcggact ccaggaaagg tggggaacct gctggctact
gattgctcca ggcggacaag 960gctgacaagc aatccaggaa caaaatactc accaggacga
ggaagaggac tttgggggac 1020aggcttctcc tgtgagcagc agagcagcac aataaatgag
gccactgtgc caggctccag 1080gtggcctccc tggcctgtct ccccactc
1108262414DNAHomo sapiens 26gtcagagtct tccctcagct
ttgtacatca gcactgtttt gatagataag agaggctact 60gtataagtgt gtaagatttg
taatggactg aggaatgctt gttctagctg ctgaaactga 120ctgtaactgt attgtcttag
gagcatcatc atggggtcta gtgccacaga gattgaagaa 180ttggaaaaca ccacttttaa
gtatcttaca ggagaacaga ctgaaaaaat gtggcagcgc 240ctgaaaggaa tactaagatg
cttggtgaag cagctggaaa gaggtgatgt taacgtcgtc 300gacttaaaga agaatattga
atatgcggca tctgtgctgg aagcagttta tatcgatgaa 360acaagaagac ttctggatac
tgaagatgag ctcagtgaca ttcagactga ctcagtccca 420tctgaagtcc gggactggtt
ggcttctacc tttacacgga aaatggggat gacaaaaaag 480aaacctgagg aaaaaccaaa
atttcggagc attgtgcatg ctgttcaagc tggaattttt 540gtggaaagaa tgtaccgaaa
aacatatcat atggttggtt tggcatatcc agcagctgtc 600atcgtaacat taaaggatgt
tgataaatgg tctttcgatg tatttgccct aaatgaagca 660agtggagagc atagtctgaa
gtttatgatt tatgaactgt ttaccagata tgatcttatc 720aaccgtttca agattcctgt
ttcttgccta atcacctttg cagaagcttt agaagttggt 780tacagcaagt acaaaaatcc
atatcacaat ttgattcatg cagctgatgt cactcaaact 840gtgcattaca taatgcttca
tacaggtatc atgcactggc tcactgaact ggaaatttta 900gcaatggtct ttgctgctgc
cattcatgat tatgagcata cagggacaac aaacaacttt 960cacattcaga caaggtcaga
tgttgccatt ttgtataatg atcgctctgt ccttgagaat 1020caccacgtga gtgcagctta
tcgacttatg caagaagaag aaatgaatat cttgataaat 1080ttatccaaag atgactggag
ggatcttcgg aacctagtga ttgaaatggt tttatctaca 1140gacatgtcag gtcacttcca
gcaaattaaa aatataagaa acagtttgca gcagcctgaa 1200gggattgaca gagccaaaac
catgtccctg attctccacg cagcagacat cagccaccca 1260gccaaatcct ggaagctgca
ttatcggtgg accatggccc taatggagga gtttttcctg 1320cagggagata aagaagctga
attagggctt ccattttccc cactttgtga tcggaagtca 1380accatggtgg cccagtcaca
aataggtttc atcgatttca tagtagagcc aacattttct 1440cttctgacag actcaacaga
gaaaattgtt attcctctta tagaggaagc ctcaaaagcc 1500gaaacttctt cctatgtggc
aagcagctca accaccattg tggggttaca cattgctgat 1560gcactaagac gatcaaatac
aaaaggctcc atgagtgatg ggtcctattc cccagactac 1620tcccttgcag cagtggacct
gaagagtttc aagaacaacc tggtggacat cattcagcag 1680aacaaagaga ggtggaaaga
gttagctgca caaggtgaat ctgatcttca taagaactca 1740gaagacttag taaatgctga
agaaaaacat gatgagacac attcataggc ccgaaacacc 1800ttaaagactt ctgtcatttt
aaacatgaga ggacaatgaa atcagcatga aaacatccta 1860aattctcaac tttccacaaa
gctatggctc ttctttcaac atagaattgg attgggccat 1920tttaattgac tcctatacaa
ggaattaaga agaacataaa ttttgagcta gtaactctgg 1980ccaaataaat acactcaagt
ttttatcaga gtttttggcc agtgcttctg ccattttttt 2040ccctccacaa tttggccttc
ttcaatcaag ccagataaat ttttgagaca aaagtcagac 2100agtttttaat ttttcttgct
ttgaaccctg tcataatgac tgtgcaatac atgtgcagaa 2160gatgaggtat tttaaaattt
acttccttgc actgtcttac acagagtgct ataactataa 2220atttttcaag gtcttaaata
aaaggaagca aaaacaaaat tattgaaaaa ttttttttgt 2280tgtgctgggg aatatactat
ttagattgtc cttcttattt taaatgcatg ggaacagaat 2340gacagggggg atgctgagga
gctggttgaa gcatcagagc aatgctacag tccaacaatg 2400gagcattaga tccc
2414272009DNAHomo sapiens
27gaattctgat gtgcttcagt gcacagaaca gtaacagatg agctgctttt ggggagagct
60tgagtactca gtcggagcat catcatgggg tctagtgcca cagagattga agaattggaa
120aacaccactt ttaagtatct tacaggagaa cagactgaaa aaatgtggca gcgcctgaaa
180ggaatactaa gatgcttggt gaagcagctg gaaagaggtg atgttaacgt cgtcgactta
240aagaagaata ttgaatatgc ggcatctgtg ctggaagcag tttatatcga tgaaacaaga
300agacttctgg atactgaaga tgagctcagt gacattcaga ctgactcagt cccatctgaa
360gtccgggact ggttggcttc tacctttaca cggaaaatgg ggatgacaaa aaagaaacct
420gaggaaaaac caaaatttcg gagcattgtg catgctgttc aagctggaat ttttgtggaa
480agaatgtacc gaaaaacata tcatatggtt ggtttggcat atccagcagc tgtcatcgta
540acattaaagg atgttgataa atggtctttc gatgtatttg ccctaaatga agcaagtgga
600gagcatagtc tgaagtttat gatttatgaa ctgtttacca gatatgatct tatcaaccgt
660ttcaagattc ctgtttcttg cctaatcacc tttgcagaag ctttagaagt tggttacagc
720aagtacaaaa atccatatca caatttgatt catgcagctg atgtcactca aactgtgcat
780tacataatgc ttcatacagg tatcatgcac tggctcactg aactggaaat tttagcaatg
840gtctttgctg ctgccattca tgattatgag catacaggga caacaaacaa ctttcacatt
900cagacaaggt cagatgttgc cattttgtat aatgatcgct ctgtccttga gaatcaccac
960gtgagtgcag cttatcgact tatgcaagaa gaagaaatga atatcttgat aaatttatcc
1020aaagatgact ggagggatct tcggaaccta gtgattgaaa tggttttatc tacagacatg
1080tcaggtcact tccagcaaat taaaaatata agaaacagtt tgcagcagcc tgaagggatt
1140gacagagcca aaaccatgtc cctgattctc cacgcagcag acatcagcca cccagccaaa
1200tcctggaagc tgcattatcg gtggaccatg gccctaatgg aggagttttt cctgcaggga
1260gataaagaag ctgaattagg gcttccattt tccccacttt gtgatcggaa gtcaaccatg
1320gtggcccagt cacaaatagg tttcatcgat ttcatagtag agccaacatt ttctcttctg
1380acagactcaa cagagaaaat tgttattcct cttatagagg aagcctcaaa agccgaaact
1440tcttcctatg tggcaagcag ctcaaccacc attgtggggt tacacattgc tgatgcacta
1500agacgatcaa atacaaaagg ctccatgagt gatgggtcct attccccaga ctactccctt
1560gcagcagtgg acctgaagag tttcaagaac aacctggtgg acatcattca gcagaacaaa
1620gagaggtgga aagagttagc tgcacaagaa gcaagaacca gttcacagaa gtgtgagttt
1680attcatcagt aaacaccttt aagtaaaacc tcgtgcatgg tggcagctct aatttgacca
1740aaagacttgg agattttgat tatgcttgct ggaaatctac cctgtcctgt gtgagacagg
1800aaatctattt ttgcagattg ctcaataagc atcatgagcc acataaataa cagctgtaaa
1860ctccttaatt caccgggctc aactgctacc gaacagattc atctagtggc tacatcagca
1920ccttgtgctt tcagatatct gtttcaatgg cattttgtgg catttgtctt taccgagtgc
1980caataaattt tctttgagca gctaaaaaa
2009282846DNAHomo sapiens 28aaatgcttga ggagagagag agagtaagga gccagccatg
aatcctttcc agaaaaatga 60gtccaaggaa actctttttt cacctgtctc cattgaagag
gtaccacctc gaccacctag 120ccctccaaag aagccatctc cgacaatctg tggctccaac
tatccactga gcattgcctt 180cattgtggtg aatgaattct gcgagcgctt ttcctattat
ggaatgaaag ctgtgctgat 240cctgtatttc ctgtatttcc tgcactggaa tgaagatacc
tccacatcta tataccatgc 300cttcagcagc ctctgttatt ttactcccat cctgggagca
gccattgctg actcgtggtt 360gggaaaattc aagacaatca tctatctctc cttggtgtat
gtgcttggcc atgtgatcaa 420gtccttgggt gccttaccaa tactgggagg acaagtggta
cacacagtcc tatcattgat 480cggcctgagt ctaatagctt tggggacagg aggcatcaaa
ccctgtgtgg cagcttttgg 540tggagaccag tttgaagaaa aacatgcaga ggaacggact
agatacttct cagtcttcta 600cctgtccatc aatgcaggga gcttgatttc tacatttatc
acacccatgc tgagaggaga 660tgtgcaatgt tttggagaag actgctatgc attggctttt
ggagttccag gactgctcat 720ggtaattgca cttgttgtgt ttgcaatggg aagcaaaata
tacaataaac caccccctga 780aggaaacata gtggctcaag ttttcaaatg tatctggttt
gctatttcca atcgtttcaa 840gaaccgttct ggagacattc caaagcgaca gcactggcta
gactgggcgg ctgagaaata 900tccaaagcag ctcattatgg atgtaaaggc actgaccagg
gtactattcc tttatatccc 960attgcccatg ttctgggctc ttttggatca gcagggttca
cgatggactt tgcaagccat 1020caggatgaat aggaatttgg ggttttttgt gcttcagccg
gaccagatgc aggttctaaa 1080tccccttctg gttcttatct tcatcccgtt gtttgacttt
gtcatttatc gtctggtctc 1140caagtgtgga attaacttct catcacttag gaaaatggct
gttggtatga tcctagcatg 1200cctggcattt gcagttgcgg cagctgtaga gataaaaata
aatgaaatgg ccccagccca 1260gccaggtccc caggaggttt tcctacaagt cttgaatctg
gcagatgatg aggtgaaggt 1320gacagtggtg ggaaatgaaa acaattctct gttgatagag
tccatcaaat cctttcagaa 1380aacaccacac tattccaaac tgcacctgaa aacaaaaagc
caggattttc acttccacct 1440gaaatatcac aatttgtctc tctacactga gcattctgtg
caggagaaga actggtacag 1500tcttgtcatt cgtgaagatg ggaacagtat ctccagcatg
atggtaaagg atacagaaag 1560cagaacaacc aatgggatga caaccgtgag gtttgttaac
actttgcata aagatgtcaa 1620catctccctg agtacagata cctctctcaa tgttggtgaa
gactatggtg tgtctgctta 1680tagaactgtg caaagaggag aataccctgc agtgcactgt
agaacagaag ataagaactt 1740ttctctgaat ttgggtcttc tagactttgg tgcagcatat
ctgtttgtta ttactaataa 1800caccaatcag ggtcttcagg cctggaagat tgaagacatt
ccagccaaca aaatgtccat 1860tgcgtggcag ctaccacaat atgccctggt tacagctggg
gaggtcatgt tctctgtcac 1920aggtcttgag ttttcttatt ctcaggctcc ctctggcatg
aaatctgtgc tccaggcagc 1980ttggctattg acaattgcag ttgggaatat catcgtgctt
gttgtggcac agttcagtgg 2040cctggtacag tgggccgaat tcattttgtt ttcctgcctc
ctgctggtga tctgcctgat 2100cttctccatc atgggctact actatgttcc tgtaaagaca
gaggatatgc ggggtccagc 2160agataagcac attcctcaca tccaggggaa catgatcaaa
ctagagacca agaagacaaa 2220actctgatga ctccctagat tctgtcctga ccccaattcc
tggccctgtc ttgaagcatt 2280ttttttcttc tactggatta gacaagagag atagcagcat
atcagagctg atctcctcca 2340cctttctcca atgacagaag ttccaggact ggttttccag
tacatcttta aacaaggccc 2400cagagactct atgtctgccc gtccatcagt gaactcatta
aaacttgtgc agtgttgctg 2460gagctggcct ggtgtctcca aatgaccatg aaaatacaca
cgtataatgg agatcattct 2520ctgtgggtat gcaaagttat gggaattcct ttataggtaa
ctgccattta ggactgatgg 2580ccctaatttt tgaggtgctg atttagaggc aaaattgcag
aataacaaag aaatggtatt 2640tcaagttttt ttttttataa gcaatgtaat tatgctattc
acaggggcct caagaattgg 2700tatgtatgat gtgatctggt ccagccaggg cctggcttgt
cagctctcta ggtttgatat 2760gactttagta aatttgtcaa tatagatggt aggaagcaga
atgccatttt attaaaacac 2820aggagaagtt aaaaaaaaaa aaaaaa
28462921DNAArtificial SequenceSynthetic
oligonucleotide 29ccgtttacgt ggagactcgc c
213025DNAArtificial SequenceSynthetic oligonucleotide
30cccccacctt atatatattc tttcc
253119DNAArtificial SequenceSynthetic oligonucleotide 31ccatgcctat
ttctacagc
193219DNAArtificial SequenceSynthetic oligonucleotide 32tcagtaacag
ctcccagac
193319DNAArtificial SequenceSynthetic oligonucleotide 33ccatgcctat
ttctacagc
193419DNAArtificial SequenceSynthetic oligonucleotide 34tcagtaacag
ctcccagac
193522DNAArtificial SequenceSynthetic oligonucleotide 35ggcaatagca
ggttcacgta ca
223622DNAArtificial SequenceSynthetic oligonucleotide 36cgataacagt
cttgccccac tt
223720DNAArtificial SequenceSynthetic oligonucleotide 37acggcacacc
ctacgttacc
203824DNAArtificial SequenceSynthetic oligonucleotide 38tgtgcaagga
gagaacctct agct
243919DNAArtificial SequenceSynthetic oligonucleotide 39ccaacactgt
gcgcagctt
194022DNAArtificial SequenceSynthetic oligonucleotide 40aagaatctcc
gggttgtttt cc
224124DNAArtificial SequenceSynthetic oligonucleotide 41cagtctcatc
ctgaaagcat ctga
244220DNAArtificial SequenceSynthetic oligonucleotide 42tttcccacac
actccaccaa
204323DNAArtificial SequenceSynthetic oligonucleotide 43tggagtgttt
tacgctgaac gat
234431DNAArtificial SequenceSynthetic oligonucleotide 44cctcttactg
ctataccttt actctttatg g
314521DNAArtificial SequenceSynthetic oligonucleotide 45tgccatcaaa
gtcttctgca a
214621DNAArtificial SequenceSynthetic oligonucleotide 46cgccatactc
gaactggaat c
214725DNAArtificial SequenceSynthetic oligonucleotide 47cactattatt
ttggcacaac aggaa
254824DNAArtificial SequenceSynthetic oligonucleotide 48agacacatat
ttggcatggt tctg
244924DNAArtificial SequenceSynthetic oligonucleotide 49caagggatcc
agtctctcta tggt
245024DNAArtificial SequenceSynthetic oligonucleotide 50ggataaggaa
gggtcacatt tgtc
245118DNAArtificial SequenceSynthetic oligonucleotide 51gctcggtgga
gggtctca
185221DNAArtificial SequenceSynthetic oligonucleotide 52ctgtgtggat
ttctgcgatc a
215323DNAArtificial SequenceSynthetic oligonucleotide 53catccatgac
aactttggta tcg
235421DNAArtificial SequenceSynthetic oligonucleotide 54agtcttctgg
gtggcagtga t 2155919PRTHomo
sapiens 55Met Gly Thr Thr Ala Pro Gly Pro Ile His Leu Leu Glu Leu Cys Asp
1 5 10 15 Gln Lys
Leu Met Glu Phe Leu Cys Asn Met Asp Asn Lys Asp Leu Val 20
25 30 Trp Leu Glu Glu Ile Gln Glu
Glu Ala Glu Arg Met Phe Thr Arg Glu 35 40
45 Phe Ser Lys Glu Pro Glu Leu Met Pro Lys Thr Pro
Ser Gln Lys Asn 50 55 60
Arg Arg Lys Lys Arg Arg Ile Ser Tyr Val Gln Asp Glu Asn Arg Asp 65
70 75 80 Pro Ile Arg
Arg Arg Leu Ser Arg Arg Lys Ser Arg Ser Ser Gln Leu 85
90 95 Ser Ser Arg Arg Leu Arg Ser Lys
Asp Ser Val Glu Lys Leu Ala Thr 100 105
110 Val Val Gly Glu Asn Gly Ser Val Leu Arg Arg Val Thr
Arg Ala Ala 115 120 125
Ala Ala Ala Ala Ala Ala Thr Met Ala Leu Ala Ala Pro Ser Ser Pro 130
135 140 Thr Pro Glu Ser
Pro Thr Met Leu Thr Lys Lys Pro Glu Asp Asn His 145 150
155 160 Thr Gln Cys Gln Leu Val Pro Val Val
Glu Ile Gly Ile Ser Glu Arg 165 170
175 Gln Asn Ala Glu Gln His Val Thr Gln Leu Met Ser Thr Glu
Pro Leu 180 185 190
Pro Arg Thr Leu Ser Pro Thr Pro Ala Ser Ala Thr Ala Pro Thr Ser
195 200 205 Gln Gly Ile Pro
Thr Ser Asp Glu Glu Ser Thr Pro Lys Lys Ser Lys 210
215 220 Ala Arg Ile Leu Glu Ser Ile Thr
Val Ser Ser Leu Met Ala Thr Pro 225 230
235 240 Gln Asp Pro Lys Gly Gln Gly Val Gly Thr Gly Arg
Ser Ala Ser Lys 245 250
255 Leu Arg Ile Ala Gln Val Ser Pro Gly Pro Arg Asp Ser Pro Ala Phe
260 265 270 Pro Asp Ser
Pro Trp Arg Glu Arg Val Leu Ala Pro Ile Leu Pro Asp 275
280 285 Asn Phe Ser Thr Pro Thr Gly Ser
Arg Thr Asp Ser Gln Ser Val Arg 290 295
300 His Ser Pro Ile Ala Pro Ser Ser Pro Ser Pro Gln Val
Leu Ala Gln 305 310 315
320 Lys Tyr Ser Leu Val Ala Lys Gln Glu Ser Val Val Arg Arg Ala Ser
325 330 335 Arg Arg Leu Ala
Lys Lys Thr Ala Glu Glu Pro Ala Ala Ser Gly Arg 340
345 350 Ile Ile Cys His Ser Tyr Leu Glu Arg
Leu Leu Asn Val Glu Val Pro 355 360
365 Gln Lys Val Gly Ser Glu Gln Lys Glu Pro Pro Glu Glu Ala
Glu Pro 370 375 380
Val Ala Ala Ala Glu Pro Glu Val Pro Glu Asn Asn Gly Asn Asn Ser 385
390 395 400 Trp Pro His Asn Asp
Thr Glu Ile Ala Asn Ser Thr Pro Asn Pro Lys 405
410 415 Pro Ala Ala Ser Ser Pro Glu Thr Pro Ser
Ala Gly Gln Gln Glu Ala 420 425
430 Lys Thr Asp Gln Ala Asp Gly Pro Arg Glu Pro Pro Gln Ser Ala
Arg 435 440 445 Arg
Lys Arg Ser Tyr Lys Gln Ala Val Ser Glu Leu Asp Glu Glu Gln 450
455 460 His Leu Glu Asp Glu Glu
Leu Gln Pro Pro Arg Ser Lys Thr Pro Ser 465 470
475 480 Ser Pro Cys Pro Ala Ser Lys Val Val Arg Pro
Leu Arg Thr Phe Leu 485 490
495 His Thr Val Gln Arg Asn Gln Met Leu Met Thr Pro Thr Ser Ala Pro
500 505 510 Arg Ser
Val Met Lys Ser Phe Ile Lys Arg Asn Thr Pro Leu Arg Met 515
520 525 Asp Pro Lys Glu Lys Glu Arg
Gln Arg Leu Glu Asn Leu Arg Arg Lys 530 535
540 Glu Glu Ala Glu Gln Leu Arg Arg Gln Lys Val Glu
Glu Asp Lys Arg 545 550 555
560 Arg Arg Leu Glu Glu Val Lys Leu Lys Arg Glu Glu Arg Leu Arg Lys
565 570 575 Val Leu Gln
Ala Arg Glu Arg Val Glu Gln Met Lys Glu Glu Lys Lys 580
585 590 Lys Gln Ile Glu Gln Lys Phe Ala
Gln Ile Asp Glu Lys Thr Glu Lys 595 600
605 Ala Lys Glu Glu Arg Leu Ala Glu Glu Lys Ala Lys Lys
Lys Ala Ala 610 615 620
Ala Lys Lys Met Glu Glu Val Glu Ala Arg Arg Lys Gln Glu Glu Asp 625
630 635 640 Ala Arg Arg Leu
Arg Trp Leu Gln Gln Glu Glu Glu Glu Arg Arg His 645
650 655 Gln Glu Leu Leu Gln Lys Lys Lys Glu
Glu Glu Gln Glu Arg Leu Arg 660 665
670 Lys Ala Ala Glu Ala Lys Arg Leu Ala Glu Gln Arg Glu Gln
Glu Arg 675 680 685
Arg Glu Gln Glu Arg Arg Glu Gln Glu Arg Arg Glu Gln Glu Arg Arg 690
695 700 Glu Gln Glu Arg Arg
Glu Gln Glu Arg Arg Glu Gln Glu Arg Gln Leu 705 710
715 720 Ala Glu Gln Glu Arg Arg Arg Glu Gln Glu
Arg Leu Gln Ala Glu Arg 725 730
735 Glu Leu Gln Glu Arg Glu Lys Ala Leu Arg Leu Gln Lys Glu Gln
Leu 740 745 750 Gln
Arg Glu Leu Glu Glu Lys Lys Lys Lys Glu Glu Gln Gln Arg Leu 755
760 765 Ala Glu Arg Gln Leu Gln
Glu Glu Gln Glu Lys Lys Ala Lys Glu Ala 770 775
780 Ala Gly Ala Ser Lys Ala Leu Asn Val Thr Val
Asp Val Gln Ser Pro 785 790 795
800 Ala Cys Thr Ser Ser Pro Ile Thr Pro Gln Gly His Lys Ala Pro Pro
805 810 815 Gln Ile
Asn Pro His Asn Tyr Gly Met Asp Leu Asn Ser Asp Asp Ser 820
825 830 Thr Asp Asp Glu Ala His Pro
Arg Lys Pro Ile Pro Thr Trp Ala Arg 835 840
845 Gly Thr Pro Leu Ser Gln Ala Ile Ile His Gln Tyr
Tyr Gln Pro Pro 850 855 860
Asn Leu Leu Glu Leu Phe Gly Thr Ile Leu Pro Leu Asp Leu Glu Asp 865
870 875 880 Ile Phe Lys
Lys Ser Lys Pro Arg Tyr His Lys Arg Thr Ser Ser Ala 885
890 895 Val Trp Asn Ser Pro Pro Leu Gln
Gly Ala Arg Val Pro Ser Ser Leu 900 905
910 Ala Tyr Ser Leu Lys Lys His 915
56804PRTHomo sapiens 56Met Thr Asn Gln Glu Lys Trp Ala His Leu Ser
Pro Ser Glu Phe Ser 1 5 10
15 Gln Leu Gln Lys Tyr Ala Glu Tyr Ser Thr Lys Lys Leu Lys Asp Val
20 25 30 Leu Glu
Glu Phe His Gly Asn Gly Val Leu Ala Lys Tyr Asn Pro Glu 35
40 45 Gly Lys Gln Asp Ile Leu Asn
Gln Thr Ile Asp Phe Glu Gly Phe Lys 50 55
60 Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu Pro
Asp Asp Phe Thr 65 70 75
80 Ala His Leu Phe Met Ser Phe Ser Asn Lys Phe Pro His Ser Ser Pro
85 90 95 Met Val Lys
Ser Lys Pro Ala Leu Leu Ser Gly Gly Leu Arg Met Asn 100
105 110 Lys Gly Ala Ile Thr Pro Pro Arg
Thr Thr Ser Pro Ala Asn Thr Cys 115 120
125 Ser Pro Glu Val Ile His Leu Lys Asp Ile Val Cys Tyr
Leu Ser Leu 130 135 140
Leu Glu Arg Gly Arg Pro Glu Asp Lys Leu Glu Phe Met Phe Arg Leu 145
150 155 160 Tyr Asp Thr Asp
Gly Asn Gly Phe Leu Asp Ser Ser Glu Leu Glu Asn 165
170 175 Ile Ile Ser Gln Met Met His Val Ala
Glu Tyr Leu Glu Trp Asp Val 180 185
190 Thr Glu Leu Asn Pro Ile Leu His Glu Met Met Glu Glu Ile
Asp Tyr 195 200 205
Asp His Asp Gly Thr Val Ser Leu Glu Glu Trp Ile Gln Gly Gly Met 210
215 220 Thr Thr Ile Pro Leu
Leu Val Leu Leu Gly Leu Glu Asn Asn Val Lys 225 230
235 240 Asp Asp Gly Gln His Val Trp Arg Leu Lys
His Phe Asn Lys Pro Ala 245 250
255 Tyr Cys Asn Leu Cys Leu Asn Met Leu Ile Gly Val Gly Lys Gln
Gly 260 265 270 Leu
Cys Cys Ser Phe Cys Lys Tyr Thr Val His Glu Arg Cys Val Ala 275
280 285 Arg Ala Pro Pro Ser Cys
Ile Lys Thr Tyr Val Lys Ser Lys Arg Asn 290 295
300 Thr Asp Val Met His His Tyr Trp Val Glu Gly
Asn Cys Pro Thr Lys 305 310 315
320 Cys Asp Lys Cys His Lys Thr Val Lys Cys Tyr Gln Gly Leu Thr Gly
325 330 335 Leu His
Cys Val Trp Cys Gln Ile Thr Leu His Asn Lys Cys Ala Ser 340
345 350 His Leu Lys Pro Glu Cys Asp
Cys Gly Pro Leu Lys Asp His Ile Leu 355 360
365 Pro Pro Thr Thr Ile Cys Pro Val Val Leu Gln Thr
Leu Pro Thr Ser 370 375 380
Gly Val Ser Val Pro Glu Glu Arg Gln Ser Thr Val Lys Lys Glu Lys 385
390 395 400 Ser Gly Ser
Gln Gln Pro Asn Lys Val Ile Asp Lys Asn Lys Met Gln 405
410 415 Arg Ala Asn Ser Val Thr Val Asp
Gly Gln Gly Leu Gln Val Thr Pro 420 425
430 Val Pro Gly Thr His Pro Leu Leu Val Phe Val Asn Pro
Lys Ser Gly 435 440 445
Gly Lys Gln Gly Glu Arg Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450
455 460 Pro Arg Gln Val
Tyr Ser Leu Ser Gly Asn Gly Pro Met Pro Gly Leu 465 470
475 480 Asn Phe Phe Arg Asp Val Pro Asp Phe
Arg Val Leu Ala Cys Gly Gly 485 490
495 Asp Gly Thr Val Gly Trp Val Leu Asp Cys Ile Glu Lys Ala
Asn Val 500 505 510
Gly Lys His Pro Pro Val Ala Ile Leu Pro Leu Gly Thr Gly Asn Asp
515 520 525 Leu Ala Arg Cys
Leu Arg Trp Gly Gly Gly Tyr Glu Gly Glu Asn Leu 530
535 540 Met Lys Ile Leu Lys Asp Ile Glu
Asn Ser Thr Glu Ile Met Leu Asp 545 550
555 560 Arg Trp Lys Phe Glu Val Ile Pro Asn Asp Lys Asp
Glu Lys Gly Asp 565 570
575 Pro Val Pro Tyr Ser Ile Ile Asn Asn Tyr Phe Ser Ile Gly Val Asp
580 585 590 Ala Ser Ile
Ala His Arg Phe His Ile Met Arg Glu Lys His Pro Glu 595
600 605 Lys Phe Asn Ser Arg Met Lys Asn
Lys Phe Trp Tyr Phe Glu Phe Gly 610 615
620 Thr Ser Glu Thr Phe Ser Ala Thr Cys Lys Lys Leu His
Glu Ser Val 625 630 635
640 Glu Ile Glu Cys Asp Gly Val Gln Ile Asp Leu Ile Asn Ile Ser Leu
645 650 655 Glu Gly Ile Ala
Ile Leu Asn Ile Pro Ser Met His Gly Gly Ser Asn 660
665 670 Leu Trp Gly Glu Ser Lys Lys Arg Arg
Ser His Arg Arg Ile Glu Lys 675 680
685 Lys Gly Ser Asp Lys Arg Thr Thr Val Thr Asp Ala Lys Glu
Leu Lys 690 695 700
Phe Ala Ser Gln Asp Leu Ser Asp Gln Leu Leu Glu Val Val Gly Leu 705
710 715 720 Glu Gly Ala Met Glu
Met Gly Gln Ile Tyr Thr Gly Leu Lys Ser Ala 725
730 735 Gly Arg Arg Leu Ala Gln Cys Ser Cys Val
Val Ile Arg Thr Ser Lys 740 745
750 Ser Leu Pro Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro
Cys 755 760 765 Thr
Ile Lys Ile Thr His Lys Asn Gln Ala Pro Met Leu Met Gly Pro 770
775 780 Pro Pro Lys Thr Gly Leu
Phe Cys Ser Leu Val Lys Arg Thr Arg Asn 785 790
795 800 Arg Ser Lys Glu 57773PRTHomo sapiens 57Met
Thr Asn Gln Glu Lys Trp Ala His Leu Ser Pro Ser Glu Phe Ser 1
5 10 15 Gln Leu Gln Lys Tyr Ala
Glu Tyr Ser Thr Lys Lys Leu Lys Asp Val 20
25 30 Leu Glu Glu Phe His Gly Asn Gly Val Leu
Ala Lys Tyr Asn Pro Glu 35 40
45 Gly Lys Gln Asp Ile Leu Asn Gln Thr Ile Asp Phe Glu Gly
Phe Lys 50 55 60
Leu Phe Met Lys Thr Phe Leu Glu Ala Glu Leu Pro Asp Asp Phe Thr 65
70 75 80 Ala His Leu Phe Met
Ser Phe Ser Asn Lys Phe Pro His Ser Ser Pro 85
90 95 Met Val Lys Ser Lys Pro Ala Leu Leu Ser
Gly Gly Leu Arg Met Asn 100 105
110 Lys Gly Ala Ile Thr Pro Pro Arg Thr Thr Ser Pro Ala Asn Thr
Cys 115 120 125 Ser
Pro Glu Val Ile His Leu Lys Asp Ile Val Cys Tyr Leu Ser Leu 130
135 140 Leu Glu Arg Gly Arg Pro
Glu Asp Lys Leu Glu Phe Met Phe Arg Leu 145 150
155 160 Tyr Asp Thr Asp Gly Asn Gly Phe Leu Asp Ser
Ser Glu Leu Glu Asn 165 170
175 Ile Ile Ser Gln Met Met His Val Ala Glu Tyr Leu Glu Trp Asp Val
180 185 190 Thr Glu
Leu Asn Pro Ile Leu His Glu Met Met Glu Glu Ile Asp Tyr 195
200 205 Asp His Asp Gly Thr Val Ser
Leu Glu Glu Trp Ile Gln Gly Gly Met 210 215
220 Thr Thr Ile Pro Leu Leu Val Leu Leu Gly Leu Glu
Asn Asn Val Lys 225 230 235
240 Asp Asp Gly Gln His Val Trp Arg Leu Lys His Phe Asn Lys Pro Ala
245 250 255 Tyr Cys Asn
Leu Cys Leu Asn Met Leu Ile Gly Val Gly Lys Gln Gly 260
265 270 Leu Cys Cys Ser Phe Cys Lys Tyr
Thr Val His Glu Arg Cys Val Ala 275 280
285 Arg Ala Pro Pro Ser Cys Ile Lys Thr Tyr Val Lys Ser
Lys Arg Asn 290 295 300
Thr Asp Val Met His His Tyr Trp Val Glu Gly Asn Cys Pro Thr Lys 305
310 315 320 Cys Asp Lys Cys
His Lys Thr Val Lys Cys Tyr Gln Gly Leu Thr Gly 325
330 335 Leu His Cys Val Trp Cys Gln Ile Thr
Leu His Asn Lys Cys Ala Ser 340 345
350 His Leu Lys Pro Glu Cys Asp Cys Gly Pro Leu Lys Asp His
Ile Leu 355 360 365
Pro Pro Thr Thr Ile Cys Pro Val Val Leu Gln Thr Leu Pro Thr Ser 370
375 380 Gly Val Ser Val Pro
Glu Glu Arg Gln Ser Thr Val Lys Lys Glu Lys 385 390
395 400 Ser Gly Ser Gln Gln Pro Asn Lys Val Ile
Asp Lys Asn Lys Met Gln 405 410
415 Arg Ala Asn Ser Val Thr Val Asp Gly Gln Gly Leu Gln Val Thr
Pro 420 425 430 Val
Pro Gly Thr His Pro Leu Leu Val Phe Val Asn Pro Lys Ser Gly 435
440 445 Gly Lys Gln Gly Glu Arg
Ile Tyr Arg Lys Phe Gln Tyr Leu Leu Asn 450 455
460 Pro Arg Gln Val Tyr Ser Leu Ser Gly Asn Gly
Pro Met Pro Gly Leu 465 470 475
480 Asn Phe Phe Arg Asp Val Pro Asp Phe Arg Val Leu Ala Cys Gly Gly
485 490 495 Asp Gly
Thr Val Gly Trp Val Leu Asp Cys Ile Glu Lys Ala Asn Val 500
505 510 Gly Lys His Pro Pro Val Ala
Ile Leu Pro Leu Gly Thr Gly Asn Asp 515 520
525 Leu Ala Arg Cys Leu Arg Trp Gly Gly Gly Tyr Glu
Gly Glu Asn Leu 530 535 540
Met Lys Ile Leu Lys Asp Ile Glu Asn Ser Thr Glu Ile Met Leu Asp 545
550 555 560 Arg Trp Lys
Phe Glu Val Ile Pro Asn Asp Lys Asp Glu Lys Gly Asp 565
570 575 Pro Val Pro Tyr Ser Ile Ile Asn
Asn Tyr Phe Ser Ile Gly Val Asp 580 585
590 Ala Ser Ile Ala His Arg Phe His Ile Met Arg Glu Lys
His Pro Glu 595 600 605
Lys Phe Asn Ser Arg Met Lys Asn Lys Phe Trp Tyr Phe Glu Phe Gly 610
615 620 Thr Ser Glu Thr
Phe Ser Ala Thr Cys Lys Lys Leu His Glu Ser Val 625 630
635 640 Glu Ile Glu Cys Asp Gly Val Gln Ile
Asp Leu Ile Asn Ile Ser Leu 645 650
655 Glu Gly Ile Ala Ile Leu Asn Ile Pro Ser Met His Gly Gly
Ser Asn 660 665 670
Leu Trp Gly Glu Ser Lys Lys Arg Arg Ser His Arg Arg Ile Glu Lys
675 680 685 Lys Gly Ser Asp
Lys Arg Thr Thr Val Thr Asp Ala Lys Glu Leu Lys 690
695 700 Phe Ala Ser Gln Asp Leu Ser Asp
Gln Leu Leu Glu Val Val Gly Leu 705 710
715 720 Glu Gly Ala Met Glu Met Gly Gln Ile Tyr Thr Gly
Leu Lys Ser Ala 725 730
735 Gly Arg Arg Leu Ala Gln Cys Ser Cys Val Val Ile Arg Thr Ser Lys
740 745 750 Ser Leu Pro
Met Gln Ile Asp Gly Glu Pro Trp Met Gln Thr Pro Cys 755
760 765 Thr Val Ser Thr Glu 770
58632PRTHomo sapiens 58Met Asn Arg Tyr Thr Thr Ile Arg Gln Leu
Gly Asp Gly Thr Tyr Gly 1 5 10
15 Ser Val Leu Leu Gly Arg Ser Ile Glu Ser Gly Glu Leu Ile Ala
Ile 20 25 30 Lys
Lys Met Lys Arg Lys Phe Tyr Ser Trp Glu Glu Cys Met Asn Leu 35
40 45 Arg Glu Val Lys Ser Leu
Lys Lys Leu Asn His Ala Asn Val Val Lys 50 55
60 Leu Lys Glu Val Ile Arg Glu Asn Asp His Leu
Tyr Phe Ile Phe Glu 65 70 75
80 Tyr Met Lys Glu Asn Leu Tyr Gln Leu Ile Lys Glu Arg Asn Lys Leu
85 90 95 Phe Pro
Glu Ser Ala Ile Arg Asn Ile Met Tyr Gln Ile Leu Gln Gly 100
105 110 Leu Ala Phe Ile His Lys His
Gly Phe Phe His Arg Asp Leu Lys Pro 115 120
125 Glu Asn Leu Leu Cys Met Gly Pro Glu Leu Val Lys
Ile Ala Asp Phe 130 135 140
Gly Leu Ala Arg Glu Ile Arg Ser Lys Pro Pro Tyr Thr Asp Tyr Val 145
150 155 160 Ser Thr Arg
Trp Tyr Arg Ala Pro Glu Val Leu Leu Arg Ser Thr Asn 165
170 175 Tyr Ser Ser Pro Ile Asp Val Trp
Ala Val Gly Cys Ile Met Ala Glu 180 185
190 Val Tyr Thr Leu Arg Pro Leu Phe Pro Gly Ala Ser Glu
Ile Asp Thr 195 200 205
Ile Phe Lys Ile Cys Gln Val Leu Gly Thr Pro Lys Lys Thr Asp Trp 210
215 220 Pro Glu Gly Tyr
Gln Leu Ser Ser Ala Met Asn Phe Arg Trp Pro Gln 225 230
235 240 Cys Val Pro Asn Asn Leu Lys Thr Leu
Ile Pro Asn Ala Ser Ser Glu 245 250
255 Ala Val Gln Leu Leu Arg Asp Met Leu Gln Trp Asp Pro Lys
Lys Arg 260 265 270
Pro Thr Ala Ser Gln Ala Leu Arg Tyr Pro Tyr Phe Gln Val Gly His
275 280 285 Pro Leu Gly Ser
Thr Thr Gln Asn Leu Gln Asp Ser Glu Lys Pro Gln 290
295 300 Lys Gly Ile Leu Glu Lys Ala Gly
Pro Pro Pro Tyr Ile Lys Pro Val 305 310
315 320 Pro Pro Ala Gln Pro Pro Ala Lys Pro His Thr Arg
Ile Ser Ser Arg 325 330
335 Gln His Gln Ala Ser Gln Pro Pro Leu His Leu Thr Tyr Pro Tyr Lys
340 345 350 Ala Glu Val
Ser Arg Thr Asp His Pro Ser His Leu Gln Glu Asp Lys 355
360 365 Pro Ser Pro Leu Leu Phe Pro Ser
Leu His Asn Lys His Pro Gln Ser 370 375
380 Lys Ile Thr Ala Gly Leu Glu His Lys Asn Gly Glu Ile
Lys Pro Lys 385 390 395
400 Ser Arg Arg Arg Trp Gly Leu Ile Ser Arg Ser Thr Lys Asp Ser Asp
405 410 415 Asp Trp Ala Asp
Leu Asp Asp Leu Asp Phe Ser Pro Ser Leu Ser Arg 420
425 430 Ile Asp Leu Lys Asn Lys Lys Arg Gln
Ser Asp Asp Thr Leu Cys Arg 435 440
445 Phe Glu Ser Val Leu Asp Leu Lys Pro Ser Glu Pro Val Gly
Thr Gly 450 455 460
Asn Ser Ala Pro Thr Gln Thr Ser Tyr Gln Arg Arg Asp Thr Pro Thr 465
470 475 480 Leu Arg Ser Ala Ala
Lys Gln His Tyr Leu Lys His Ser Arg Tyr Leu 485
490 495 Pro Gly Ile Ser Ile Arg Asn Gly Ile Leu
Ser Asn Pro Gly Lys Glu 500 505
510 Phe Ile Pro Pro Asn Pro Trp Ser Ser Ser Gly Leu Ser Gly Lys
Ser 515 520 525 Ser
Gly Thr Met Ser Val Ile Ser Lys Val Asn Ser Val Gly Ser Ser 530
535 540 Ser Thr Ser Ser Ser Gly
Leu Thr Gly Asn Tyr Val Pro Ser Phe Leu 545 550
555 560 Lys Lys Glu Ile Gly Ser Ala Met Gln Arg Val
His Leu Ala Pro Ile 565 570
575 Pro Asp Pro Ser Pro Gly Tyr Ser Ser Leu Lys Ala Met Arg Pro His
580 585 590 Pro Gly
Arg Pro Phe Phe His Thr Gln Pro Arg Ser Thr Pro Gly Leu 595
600 605 Ile Pro Arg Pro Pro Ala Ala
Gln Pro Val His Gly Arg Thr Asp Trp 610 615
620 Ala Ser Lys Tyr Ala Ser Arg Arg 625
630 59632PRTHomo sapiens 59Met Asn Arg Tyr Thr Thr Ile Arg
Gln Leu Gly Asp Gly Thr Tyr Gly 1 5 10
15 Ser Val Leu Leu Gly Arg Ser Ile Glu Ser Gly Glu Leu
Ile Ala Ile 20 25 30
Lys Lys Met Lys Arg Lys Phe Tyr Ser Trp Glu Glu Cys Met Asn Leu
35 40 45 Arg Glu Val Lys
Ser Leu Lys Lys Leu Asn His Ala Asn Val Val Lys 50
55 60 Leu Lys Glu Val Ile Arg Glu Asn
Asp His Leu Tyr Phe Ile Phe Glu 65 70
75 80 Tyr Met Lys Glu Asn Leu Tyr Gln Leu Ile Lys Glu
Arg Asn Lys Leu 85 90
95 Phe Pro Glu Ser Ala Ile Arg Asn Ile Met Tyr Gln Ile Leu Gln Gly
100 105 110 Leu Ala Phe
Ile His Lys His Gly Phe Phe His Arg Asp Leu Lys Pro 115
120 125 Glu Asn Leu Leu Cys Met Gly Pro
Glu Leu Val Lys Ile Ala Asp Phe 130 135
140 Gly Leu Ala Arg Glu Ile Arg Ser Lys Pro Pro Tyr Thr
Asp Tyr Val 145 150 155
160 Ser Thr Arg Trp Tyr Arg Ala Pro Glu Val Leu Leu Arg Ser Thr Asn
165 170 175 Tyr Ser Ser Pro
Ile Asp Val Trp Ala Val Gly Cys Ile Met Ala Glu 180
185 190 Val Tyr Thr Leu Arg Pro Leu Phe Pro
Gly Ala Ser Glu Ile Asp Thr 195 200
205 Ile Phe Lys Ile Cys Gln Val Leu Gly Thr Pro Lys Lys Thr
Asp Trp 210 215 220
Pro Glu Gly Tyr Gln Leu Ser Ser Ala Met Asn Phe Arg Trp Pro Gln 225
230 235 240 Cys Val Pro Asn Asn
Leu Lys Thr Leu Ile Pro Asn Ala Ser Ser Glu 245
250 255 Ala Val Gln Leu Leu Arg Asp Met Leu Gln
Trp Asp Pro Lys Lys Arg 260 265
270 Pro Thr Ala Ser Gln Ala Leu Arg Tyr Pro Tyr Phe Gln Val Gly
His 275 280 285 Pro
Leu Gly Ser Thr Thr Gln Asn Leu Gln Asp Ser Glu Lys Pro Gln 290
295 300 Lys Gly Ile Leu Glu Lys
Ala Gly Pro Pro Pro Tyr Ile Lys Pro Val 305 310
315 320 Pro Pro Ala Gln Pro Pro Ala Lys Pro His Thr
Arg Ile Ser Ser Arg 325 330
335 Gln His Gln Ala Ser Gln Pro Pro Leu His Leu Thr Tyr Pro Tyr Lys
340 345 350 Ala Glu
Val Ser Arg Thr Asp His Pro Ser His Leu Gln Glu Asp Lys 355
360 365 Pro Ser Pro Leu Leu Phe Pro
Ser Leu His Asn Lys His Pro Gln Ser 370 375
380 Lys Ile Thr Ala Gly Leu Glu His Lys Asn Gly Glu
Ile Lys Pro Lys 385 390 395
400 Ser Arg Arg Arg Trp Gly Leu Ile Ser Arg Ser Thr Lys Asp Ser Asp
405 410 415 Asp Trp Ala
Asp Leu Asp Asp Leu Asp Phe Ser Pro Ser Leu Ser Arg 420
425 430 Ile Asp Leu Lys Asn Lys Lys Arg
Gln Ser Asp Asp Thr Leu Cys Arg 435 440
445 Phe Glu Ser Val Leu Asp Leu Lys Pro Ser Glu Pro Val
Gly Thr Gly 450 455 460
Asn Ser Ala Pro Thr Gln Thr Ser Tyr Gln Arg Arg Asp Thr Pro Thr 465
470 475 480 Leu Arg Ser Ala
Ala Lys Gln His Tyr Leu Lys His Ser Arg Tyr Leu 485
490 495 Pro Gly Ile Ser Ile Arg Asn Gly Ile
Leu Ser Asn Pro Gly Lys Glu 500 505
510 Phe Ile Pro Pro Asn Pro Trp Ser Ser Ser Gly Leu Ser Gly
Lys Ser 515 520 525
Ser Gly Thr Met Ser Val Ile Ser Lys Val Asn Ser Val Gly Ser Ser 530
535 540 Ser Thr Ser Ser Ser
Gly Leu Thr Gly Asn Tyr Val Pro Ser Phe Leu 545 550
555 560 Lys Lys Glu Ile Gly Ser Ala Met Gln Arg
Val His Leu Ala Pro Ile 565 570
575 Pro Asp Pro Ser Pro Gly Tyr Ser Ser Leu Lys Ala Met Arg Pro
His 580 585 590 Pro
Gly Arg Pro Phe Phe His Thr Gln Pro Arg Ser Thr Pro Gly Leu 595
600 605 Ile Pro Arg Pro Pro Ala
Ala Gln Pro Val His Gly Arg Thr Asp Trp 610 615
620 Ala Ser Lys Tyr Ala Ser Arg Arg 625
630 60455PRTHomo sapiens 60Met Ile Cys Cys Ser Ala Leu
Ser Pro Arg Ile His Leu Ser Phe His 1 5
10 15 Arg Ser Leu Thr Gly Ile Val Leu Ala Asn Ser
Ser Leu Asp Ile Val 20 25
30 Leu His Asp Thr Tyr Tyr Val Val Ala His Cys Gly Gly Asn Val
Arg 35 40 45 Arg
Leu His Cys Gly Gly Pro Ala Ser Arg Glu Arg Thr Ala Met Gln 50
55 60 Ala Leu Asn Ile Thr Pro
Glu Gln Phe Ser Arg Leu Leu Arg Asp His 65 70
75 80 Asn Leu Thr Arg Glu Gln Phe Ile Ala Leu Tyr
Arg Leu Arg Pro Leu 85 90
95 Val Tyr Thr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala Leu Val Leu
100 105 110 Thr Gly
Val Leu Ile Phe Ala Leu Ala Leu Phe Gly Asn Ala Leu Val 115
120 125 Phe Tyr Val Val Thr Arg Ser
Lys Ala Met Arg Thr Val Thr Asn Ile 130 135
140 Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile
Thr Phe Phe Cys 145 150 155
160 Ile Pro Val Thr Met Leu Gln Asn Ile Ser Asp Asn Trp Leu Gly Gly
165 170 175 Ala Phe Ile
Cys Lys Met Val Pro Phe Val Gln Ser Thr Ala Val Val 180
185 190 Thr Glu Ile Leu Thr Met Thr Cys
Ile Ala Val Glu Arg His Gln Gly 195 200
205 Leu Val His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn
Arg Arg Ala 210 215 220
Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val Gly Ser 225
230 235 240 Pro Met Trp His
Val Gln Gln Leu Glu Ile Lys Tyr Asp Phe Leu Tyr 245
250 255 Glu Lys Glu His Ile Cys Cys Leu Glu
Glu Trp Thr Ser Pro Val His 260 265
270 Gln Lys Ile Tyr Thr Thr Phe Ile Leu Ser Ser Ser Ser Ser
Cys Leu 275 280 285
Leu Trp Lys Lys Lys Arg Ala Val Ile Met Met Val Thr Val Val Ala 290
295 300 Leu Phe Ala Val Cys
Trp Ala Pro Phe His Val Val His Met Met Ile 305 310
315 320 Glu Tyr Ser Asn Phe Glu Lys Glu Tyr Asp
Asp Val Thr Ile Lys Met 325 330
335 Ile Phe Ala Ile Val Gln Ile Ile Gly Phe Ser Asn Ser Ile Cys
Asn 340 345 350 Pro
Ile Val Tyr Ala Phe Met Asn Glu Asn Phe Lys Lys Asn Val Leu 355
360 365 Ser Ala Val Cys Tyr Cys
Ile Val Asn Lys Thr Phe Ser Pro Ala Gln 370 375
380 Arg His Gly Asn Ser Gly Ile Thr Met Met Arg
Lys Lys Ala Lys Phe 385 390 395
400 Ser Leu Arg Glu Asn Pro Val Glu Glu Thr Lys Gly Glu Ala Phe Ser
405 410 415 Asp Gly
Asn Ile Glu Val Lys Leu Cys Glu Gln Thr Glu Glu Lys Lys 420
425 430 Lys Leu Lys Arg His Leu Ala
Leu Phe Arg Ser Glu Leu Ala Glu Asn 435 440
445 Ser Pro Leu Asp Ser Gly His 450
455 61431PRTHomo sapiens 61Met Gln Ala Leu Asn Ile Thr Pro Glu Gln
Phe Ser Arg Leu Leu Arg 1 5 10
15 Asp His Asn Leu Thr Arg Glu Gln Phe Ile Ala Val His Arg Leu
Arg 20 25 30 Pro
Leu Val Tyr Thr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala Leu 35
40 45 Val Leu Thr Gly Val Leu
Ile Phe Ala Leu Ala Leu Phe Gly Asn Ala 50 55
60 Leu Val Phe Tyr Val Val Thr Arg Ser Lys Ala
Met Arg Thr Val Thr 65 70 75
80 Asn Ile Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile Thr Phe
85 90 95 Phe Cys
Ile Pro Val Thr Met Leu Gln Asn Ile Ser Asp Asn Trp Leu 100
105 110 Gly Gly Ala Phe Ile Cys Lys
Met Val Pro Phe Val Gln Ser Thr Ala 115 120
125 Val Val Thr Glu Ile Leu Thr Met Thr Cys Ile Ala
Val Glu Arg His 130 135 140
Gln Gly Leu Val His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn Arg 145
150 155 160 Arg Ala Phe
Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val 165
170 175 Gly Ser Pro Met Trp His Val Gln
Gln Leu Glu Ile Lys Tyr Asp Phe 180 185
190 Leu Tyr Glu Lys Glu His Ile Cys Cys Leu Glu Glu Trp
Thr Ser Pro 195 200 205
Val His Gln Lys Ile Tyr Thr Thr Phe Ile Leu Val Ile Leu Phe Leu 210
215 220 Leu Pro Leu Met
Val Met Leu Ile Leu Tyr Ser Lys Ile Gly Tyr Glu 225 230
235 240 Leu Trp Ile Lys Lys Arg Val Gly Asp
Gly Ser Val Leu Arg Thr Ile 245 250
255 His Gly Lys Glu Met Ser Lys Ile Ala Arg Lys Lys Lys Arg
Ala Val 260 265 270
Ile Met Met Val Thr Val Val Ala Leu Phe Ala Val Cys Trp Ala Pro
275 280 285 Phe His Val Val
His Met Met Ile Glu Tyr Ser Asn Phe Glu Lys Glu 290
295 300 Tyr Asp Asp Val Thr Ile Lys Met
Ile Phe Ala Ile Val Gln Ile Ile 305 310
315 320 Gly Phe Ser Asn Ser Ile Cys Asn Pro Ile Val Tyr
Ala Phe Met Asn 325 330
335 Glu Asn Phe Lys Lys Asn Val Leu Ser Ala Val Cys Tyr Cys Ile Val
340 345 350 Asn Lys Thr
Phe Ser Pro Ala Gln Arg His Gly Asn Ser Gly Ile Thr 355
360 365 Met Met Arg Lys Lys Ala Lys Phe
Ser Leu Arg Glu Asn Pro Val Glu 370 375
380 Glu Thr Lys Gly Glu Ala Phe Ser Asp Gly Asn Ile Glu
Val Lys Leu 385 390 395
400 Cys Glu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu Ala Leu
405 410 415 Phe Arg Ser Glu
Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His 420
425 430 62647PRTHomo sapiens 62Met Ala Glu Glu Glu
Ala Pro Lys Lys Ser Arg Ala Ala Gly Gly Gly 1 5
10 15 Ala Ser Trp Glu Leu Cys Ala Gly Ala Leu
Ser Ala Arg Leu Ala Glu 20 25
30 Glu Gly Ser Gly Asp Ala Gly Gly Arg Arg Arg Pro Pro Val Asp
Pro 35 40 45 Arg
Arg Leu Ala Arg Gln Leu Leu Leu Leu Leu Trp Leu Leu Glu Ala 50
55 60 Pro Leu Leu Leu Gly Val
Arg Ala Gln Ala Ala Gly Gln Gly Pro Gly 65 70
75 80 Gln Gly Pro Gly Pro Gly Gln Gln Pro Pro Pro
Pro Pro Gln Gln Gln 85 90
95 Gln Ser Gly Gln Gln Tyr Asn Gly Glu Arg Gly Ile Ser Val Pro Asp
100 105 110 His Gly
Tyr Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala 115
120 125 Tyr Asn Gln Thr Ile Met Pro
Asn Leu Leu Gly His Thr Asn Gln Glu 130 135
140 Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu
Val Lys Val Gln 145 150 155
160 Cys Ser Ala Glu Leu Lys Phe Phe Leu Cys Ser Met Tyr Ala Pro Val
165 170 175 Cys Thr Val
Leu Glu Gln Ala Leu Pro Pro Cys Arg Ser Leu Cys Glu 180
185 190 Arg Ala Arg Gln Gly Cys Glu Ala
Leu Met Asn Lys Phe Gly Phe Gln 195 200
205 Trp Pro Asp Thr Leu Lys Cys Glu Lys Phe Pro Val His
Gly Ala Gly 210 215 220
Glu Leu Cys Val Gly Gln Asn Thr Ser Asp Lys Gly Thr Pro Thr Pro 225
230 235 240 Ser Leu Leu Pro
Glu Phe Trp Thr Ser Asn Pro Gln His Gly Gly Gly 245
250 255 Gly His Arg Gly Gly Phe Pro Gly Gly
Ala Gly Ala Ser Glu Arg Gly 260 265
270 Lys Phe Ser Cys Pro Arg Ala Leu Lys Val Pro Ser Tyr Leu
Asn Tyr 275 280 285
His Phe Leu Gly Glu Lys Asp Cys Gly Ala Pro Cys Glu Pro Thr Lys 290
295 300 Val Tyr Gly Leu Met
Tyr Phe Gly Pro Glu Glu Leu Arg Phe Ser Arg 305 310
315 320 Thr Trp Ile Gly Ile Trp Ser Val Leu Cys
Cys Ala Ser Thr Leu Phe 325 330
335 Thr Val Leu Thr Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro
Glu 340 345 350 Arg
Pro Ile Ile Phe Leu Ser Gly Cys Tyr Thr Ala Val Ala Val Ala 355
360 365 Tyr Ile Ala Gly Phe Leu
Leu Glu Asp Arg Val Val Cys Asn Asp Lys 370 375
380 Phe Ala Glu Asp Gly Ala Arg Thr Val Ala Gln
Gly Thr Lys Lys Glu 385 390 395
400 Gly Cys Thr Ile Leu Phe Met Met Leu Tyr Phe Phe Ser Met Ala Ser
405 410 415 Ser Ile
Trp Trp Val Ile Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly 420
425 430 Met Lys Trp Gly His Glu Ala
Ile Glu Ala Asn Ser Gln Tyr Phe His 435 440
445 Leu Ala Ala Trp Ala Val Pro Ala Ile Lys Thr Ile
Thr Ile Leu Ala 450 455 460
Leu Gly Gln Val Asp Gly Asp Val Leu Ser Gly Val Cys Phe Val Gly 465
470 475 480 Leu Asn Asn
Val Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe 485
490 495 Val Tyr Leu Phe Ile Gly Thr Ser
Phe Leu Leu Ala Gly Phe Val Ser 500 505
510 Leu Phe Arg Ile Arg Thr Ile Met Lys His Asp Gly Thr
Lys Thr Glu 515 520 525
Lys Leu Glu Lys Leu Met Val Arg Ile Gly Val Phe Ser Val Leu Tyr 530
535 540 Thr Val Pro Ala
Thr Ile Val Ile Ala Cys Tyr Phe Tyr Glu Gln Ala 545 550
555 560 Phe Arg Asp Gln Trp Glu Arg Ser Trp
Val Ala Gln Ser Cys Lys Ser 565 570
575 Tyr Ala Ile Pro Cys Pro His Leu Gln Ala Gly Gly Gly Ala
Pro Pro 580 585 590
His Pro Pro Met Ser Pro Asp Phe Thr Val Phe Met Ile Lys Tyr Leu
595 600 605 Met Thr Leu Ile
Val Gly Ile Thr Ser Gly Phe Trp Ile Trp Ser Gly 610
615 620 Lys Thr Leu Asn Ser Trp Arg Lys
Phe Tyr Thr Arg Leu Thr Asn Ser 625 630
635 640 Lys Gln Gly Glu Thr Thr Val 645
63258PRTHomo sapiens 63Met Leu Val Leu Tyr Gly His Ser Thr Gln Asp
Leu Pro Glu Thr Asn 1 5 10
15 Ala Arg Val Val Gly Gly Thr Glu Ala Gly Arg Asn Ser Trp Pro Ser
20 25 30 Gln Ile
Ser Leu Gln Tyr Arg Ser Gly Gly Ser Arg Tyr His Thr Cys 35
40 45 Gly Gly Thr Leu Ile Arg Gln
Asn Trp Val Met Thr Ala Ala His Cys 50 55
60 Val Asp Tyr Gln Lys Thr Phe Arg Val Val Ala Gly
Asp His Asn Leu 65 70 75
80 Ser Gln Asn Asp Gly Thr Glu Gln Tyr Val Ser Val Gln Lys Ile Val
85 90 95 Val His Pro
Tyr Trp Asn Ser Asp Asn Val Ala Ala Gly Tyr Asp Ile 100
105 110 Ala Leu Leu Arg Leu Ala Gln Ser
Val Thr Leu Asn Ser Tyr Val Gln 115 120
125 Leu Gly Val Leu Pro Gln Glu Gly Ala Ile Leu Ala Asn
Asn Ser Pro 130 135 140
Cys Tyr Ile Thr Gly Trp Gly Lys Thr Lys Thr Asn Gly Gln Leu Ala 145
150 155 160 Gln Thr Leu Gln
Gln Ala Tyr Leu Pro Ser Val Asp Tyr Ala Ile Cys 165
170 175 Ser Ser Ser Ser Tyr Trp Gly Ser Thr
Val Lys Asn Thr Met Val Cys 180 185
190 Ala Gly Gly Asp Gly Val Arg Ser Gly Cys Gln Gly Asp Ser
Gly Gly 195 200 205
Pro Leu His Cys Leu Val Asn Gly Lys Tyr Ser Val His Gly Val Thr 210
215 220 Ser Phe Val Ser Ser
Arg Gly Cys Asn Val Ser Arg Lys Pro Thr Val 225 230
235 240 Phe Thr Gln Val Ser Ala Tyr Ile Ser Trp
Ile Asn Asn Val Ile Ala 245 250
255 Ser Asn 642555PRTHomo sapiens 64Met Thr Ala Ile Thr His
Gly Ser Pro Val Gly Gly Asn Asp Ser Gln 1 5
10 15 Gly Gln Val Leu Asp Gly Gln Ser Gln His Leu
Phe Gln Gln Asn Gln 20 25
30 Thr Ser Ser Pro Asp Ser Ser Asn Glu Asn Ser Val Ala Thr Pro
Pro 35 40 45 Pro
Glu Glu Gln Gly Gln Gly Asp Ala Pro Pro Gln His Glu Asp Glu 50
55 60 Glu Pro Ala Phe Pro His
Thr Glu Leu Ala Asn Leu Asp Asp Met Ile 65 70
75 80 Asn Arg Pro Arg Trp Val Val Pro Val Leu Pro
Lys Gly Glu Leu Glu 85 90
95 Val Leu Leu Glu Ala Ala Ile Asp Leu Ser Val Lys Gly Leu Asp Val
100 105 110 Lys Ser
Glu Ala Cys Gln Arg Phe Phe Arg Asp Gly Leu Thr Ile Ser 115
120 125 Phe Thr Lys Ile Leu Met Asp
Glu Ala Val Ser Gly Trp Lys Phe Glu 130 135
140 Ile His Arg Cys Ile Ile Asn Asn Thr His Arg Leu
Val Glu Leu Cys 145 150 155
160 Val Ala Lys Leu Ser Gln Asp Trp Phe Pro Leu Leu Glu Leu Leu Ala
165 170 175 Met Ala Leu
Asn Pro His Cys Lys Phe His Ile Tyr Asn Gly Thr Arg 180
185 190 Pro Cys Glu Leu Ile Ser Ser Asn
Ala Gln Leu Pro Glu Glu Glu Leu 195 200
205 Phe Ala Arg Ser Ser Asp Pro Arg Ser Pro Lys Gly Trp
Leu Val Asp 210 215 220
Leu Ile Asn Lys Phe Gly Thr Leu Asn Gly Phe Gln Ile Leu His Asp 225
230 235 240 Arg Phe Phe Asn
Gly Ser Ala Leu Asn Ile Gln Ile Ile Ala Ala Leu 245
250 255 Ile Lys Pro Phe Gly Gln Cys Tyr Glu
Phe Leu Ser Gln His Thr Leu 260 265
270 Lys Lys Tyr Phe Ile Pro Val Ile Glu Ile Val Pro His Leu
Leu Glu 275 280 285
Asn Leu Thr Asp Glu Glu Leu Lys Lys Glu Ala Lys Asn Glu Ala Lys 290
295 300 Asn Asp Ala Leu Ser
Met Ile Ile Lys Ser Leu Lys Asn Leu Ala Ser 305 310
315 320 Arg Ile Ser Gly Gln Asp Glu Thr Ile Lys
Asn Leu Glu Ile Phe Arg 325 330
335 Leu Lys Met Ile Leu Arg Leu Leu Gln Ile Ser Ser Phe Asn Gly
Lys 340 345 350 Met
Asn Ala Leu Asn Glu Ile Asn Lys Val Ile Ser Ser Val Ser Tyr 355
360 365 Tyr Thr His Arg His Ser
Asn Pro Glu Glu Glu Glu Trp Leu Thr Ala 370 375
380 Glu Arg Met Ala Glu Trp Ile Gln Gln Asn Asn
Ile Leu Ser Ile Val 385 390 395
400 Leu Gln Asp Ser Leu His Gln Pro Gln Tyr Val Glu Lys Leu Glu Lys
405 410 415 Ile Leu
Arg Phe Val Ile Lys Glu Lys Ala Leu Thr Leu Gln Asp Leu 420
425 430 Asp Asn Ile Trp Ala Ala Gln
Ala Gly Lys His Glu Ala Ile Val Lys 435 440
445 Asn Val His Asp Leu Leu Ala Lys Leu Ala Trp Asp
Phe Ser Pro Gly 450 455 460
Gln Leu Asp His Leu Phe Asp Cys Phe Lys Ala Ser Trp Thr Asn Ala 465
470 475 480 Ser Lys Lys
Gln Arg Glu Lys Leu Leu Glu Leu Ile Arg Arg Leu Ala 485
490 495 Glu Asp Asp Lys Asp Gly Val Met
Ala His Lys Val Leu Asn Leu Leu 500 505
510 Trp Asn Leu Ala Gln Ser Asp Asp Val Pro Val Asp Ile
Met Asp Leu 515 520 525
Ala Leu Ser Ala His Ile Lys Ile Leu Asp Tyr Ser Cys Ser Gln Asp 530
535 540 Arg Asp Ala Gln
Lys Ile Gln Trp Ile Asp His Phe Ile Glu Glu Leu 545 550
555 560 Arg Thr Asn Asp Lys Trp Val Ile Pro
Ala Leu Lys Gln Ile Arg Glu 565 570
575 Ile Cys Ser Leu Phe Gly Glu Ala Ser Gln Asn Leu Ser Gln
Thr Gln 580 585 590
Arg Ser Pro His Ile Phe Tyr Arg His Asp Leu Ile Asn Gln Leu Gln
595 600 605 Gln Asn His Ala
Leu Val Thr Leu Val Ala Glu Asn Leu Ala Thr Tyr 610
615 620 Met Asn Ser Ile Arg Leu Tyr Ala
Gly Asp His Glu Asp Tyr Asp Pro 625 630
635 640 Gln Thr Val Arg Leu Gly Ser Arg Tyr Ser His Val
Gln Glu Val Gln 645 650
655 Glu Arg Leu Asn Phe Leu Arg Phe Leu Leu Lys Asp Gly Gln Leu Trp
660 665 670 Leu Cys Ala
Pro Gln Ala Lys Gln Ile Trp Lys Cys Leu Ala Glu Asn 675
680 685 Ala Val Tyr Leu Cys Asp Arg Glu
Ala Cys Phe Lys Trp Tyr Ser Lys 690 695
700 Leu Met Gly Asp Glu Pro Asp Leu Asp Pro Asp Ile Asn
Lys Asp Phe 705 710 715
720 Phe Glu Ser Asn Val Leu Gln Leu Asp Pro Ser Leu Leu Thr Glu Asn
725 730 735 Gly Met Lys Cys
Phe Glu Arg Phe Phe Lys Ala Val Asn Cys Arg Glu 740
745 750 Arg Lys Leu Ile Ala Lys Arg Arg Ser
Tyr Met Met Asp Asp Leu Glu 755 760
765 Leu Ile Gly Leu Asp Tyr Leu Trp Arg Val Val Ile Gln Ser
Ser Asp 770 775 780
Glu Ile Ala Asn Arg Ala Ile Asp Leu Leu Lys Glu Ile Tyr Thr Asn 785
790 795 800 Leu Gly Pro Arg Leu
Lys Ala Asn Gln Val Val Ile His Glu Asp Phe 805
810 815 Ile Gln Ser Cys Phe Asp Arg Leu Lys Ala
Ser Tyr Asp Thr Leu Cys 820 825
830 Val Phe Asp Gly Asp Lys Asn Ser Ile Asn Cys Ala Arg Gln Glu
Ala 835 840 845 Ile
Arg Met Val Arg Val Leu Thr Val Ile Lys Glu Tyr Ile Asn Glu 850
855 860 Cys Asp Ser Asp Tyr His
Lys Glu Arg Met Ile Leu Pro Met Ser Arg 865 870
875 880 Ala Phe Cys Gly Lys His Leu Ser Leu Ile Val
Arg Phe Pro Asn Gln 885 890
895 Gly Arg Gln Val Asp Glu Leu Asp Ile Trp Phe His Thr Asn Asp Thr
900 905 910 Ile Gly
Ser Val Arg Arg Cys Ile Val Asn Arg Ile Lys Ala Asn Val 915
920 925 Ala His Lys Lys Ile Glu Leu
Phe Val Gly Gly Glu Leu Ile Asp Ser 930 935
940 Glu Asn Asp Arg Lys Leu Ile Gly Gln Leu Asn Leu
Lys Asp Lys Ser 945 950 955
960 Leu Ile Thr Ala Lys Leu Thr Gln Ile Asn Phe Asn Met Pro Ser Ser
965 970 975 Pro Asp Ser
Ser Ser Asp Ser Ser Thr Ala Ser Pro Gly Asn His Arg 980
985 990 Asn His Tyr Asn Asp Gly Pro Asn
Leu Lys Val Glu Ser Cys Leu Pro 995 1000
1005 Gly Val Ile Met Ser Val His Pro Lys Tyr Ile
Ser Phe Leu Trp 1010 1015 1020
Gln Phe Ala Asn Leu Gly Ser Asn Leu Asn Met Pro Pro Leu Lys
1025 1030 1035 Asn Gly Ala
Arg Val Leu Met Lys Leu Met Pro Pro Asp Arg Thr 1040
1045 1050 Ala Val Glu Lys Leu Arg Thr Val
Cys Leu Asp His Ala Asn Leu 1055 1060
1065 Gly Glu Gly Lys Leu Ser Pro Pro Leu Asp Ser Leu Phe
Phe Gly 1070 1075 1080
Pro Ser Ala Ser Gln Val Leu Tyr Leu Thr Glu Val Val Tyr Ala 1085
1090 1095 Leu Leu Met Pro Ala
Gly Val Pro Leu Thr Asp Gly Ser Ser Asp 1100 1105
1110 Phe Gln Val His Phe Leu Lys Ser Gly Gly
Leu Pro Leu Val Leu 1115 1120 1125
Ser Met Leu Ile Arg Asn Asn Phe Leu Pro Asn Thr Asp Met Glu
1130 1135 1140 Thr Arg
Arg Gly Ala Tyr Leu Asn Ala Leu Lys Ile Ala Lys Leu 1145
1150 1155 Leu Leu Thr Ala Ile Gly Tyr
Gly His Val Arg Ala Val Ala Glu 1160 1165
1170 Ala Cys Gln Pro Val Val Asp Gly Thr Asp Pro Ile
Thr Gln Ile 1175 1180 1185
Asn Gln Val Thr His Asp Gln Ala Val Val Leu Gln Ser Ala Leu 1190
1195 1200 Gln Ser Ile Pro Asn
Pro Ser Ser Glu Cys Val Leu Arg Asn Glu 1205 1210
1215 Ser Ile Leu Leu Ala Gln Glu Ile Ser Asn
Glu Ala Ser Arg Tyr 1220 1225 1230
Met Pro Asp Ile Cys Val Ile Arg Ala Ile Gln Lys Ile Ile Trp
1235 1240 1245 Ala Ser
Ala Cys Gly Ala Leu Gly Leu Phe Phe Ser Pro Asn Glu 1250
1255 1260 Glu Ile Thr Lys Ile Tyr Gln
Met Thr Thr Asn Gly Ser Asn Lys 1265 1270
1275 Leu Glu Val Glu Asp Glu Gln Val Cys Cys Glu Ala
Leu Glu Val 1280 1285 1290
Met Thr Leu Cys Phe Ala Leu Leu Pro Thr Ala Leu Asp Ala Leu 1295
1300 1305 Ser Lys Glu Lys Ala
Trp Gln Thr Phe Ile Ile Asp Leu Leu Leu 1310 1315
1320 His Cys Pro Ser Lys Thr Val Arg Gln Leu
Ala Gln Glu Gln Phe 1325 1330 1335
Phe Leu Met Cys Thr Arg Cys Cys Met Gly His Arg Pro Leu Leu
1340 1345 1350 Phe Phe
Ile Thr Leu Leu Phe Thr Ile Leu Gly Ser Thr Ala Arg 1355
1360 1365 Glu Lys Gly Lys Tyr Ser Gly
Asp Tyr Phe Thr Leu Leu Arg His 1370 1375
1380 Leu Leu Asn Tyr Ala Tyr Asn Gly Asn Ile Asn Ile
Pro Asn Ala 1385 1390 1395
Glu Val Leu Leu Val Ser Glu Ile Asp Trp Leu Lys Arg Ile Arg 1400
1405 1410 Asp Asn Val Lys Asn
Thr Gly Glu Thr Gly Val Glu Glu Pro Ile 1415 1420
1425 Leu Glu Gly His Leu Gly Val Thr Lys Glu
Leu Leu Ala Phe Gln 1430 1435 1440
Thr Ser Glu Lys Lys Tyr His Phe Gly Cys Glu Lys Gly Gly Ala
1445 1450 1455 Asn Leu
Ile Lys Glu Leu Ile Asp Asp Phe Ile Phe Pro Ala Ser 1460
1465 1470 Lys Val Tyr Leu Gln Tyr Leu
Arg Ser Gly Glu Leu Pro Ala Glu 1475 1480
1485 Gln Ala Ile Pro Val Cys Ser Ser Pro Val Thr Ile
Asn Ala Gly 1490 1495 1500
Phe Glu Leu Leu Val Ala Leu Ala Ile Gly Cys Val Arg Asn Leu 1505
1510 1515 Lys Gln Ile Val Asp
Cys Leu Thr Glu Met Tyr Tyr Met Gly Thr 1520 1525
1530 Ala Ile Thr Thr Cys Glu Ala Leu Thr Glu
Trp Glu Tyr Leu Pro 1535 1540 1545
Pro Val Gly Pro Arg Pro Pro Lys Gly Phe Val Gly Leu Lys Asn
1550 1555 1560 Ala Gly
Ala Thr Cys Tyr Met Asn Ser Val Ile Gln Gln Leu Tyr 1565
1570 1575 Met Ile Pro Ser Ile Arg Asn
Ser Ile Leu Ala Ile Glu Gly Thr 1580 1585
1590 Gly Ser Asp Leu His Asp Asp Met Phe Gly Asp Glu
Lys Gln Asp 1595 1600 1605
Ser Glu Ser Asn Val Asp Pro Arg Asp Asp Val Phe Gly Tyr Pro 1610
1615 1620 His Gln Phe Glu Asp
Lys Pro Ala Leu Ser Lys Thr Glu Asp Arg 1625 1630
1635 Lys Glu Tyr Asn Ile Gly Val Leu Arg His
Leu Gln Val Ile Phe 1640 1645 1650
Gly His Leu Ala Ala Ser Gln Leu Gln Tyr Tyr Val Pro Arg Gly
1655 1660 1665 Phe Trp
Lys Gln Phe Arg Leu Trp Gly Glu Pro Val Asn Leu Arg 1670
1675 1680 Glu Gln His Asp Ala Leu Glu
Phe Phe Asn Ser Leu Val Asp Ser 1685 1690
1695 Leu Asp Glu Ala Leu Lys Ala Leu Gly His Pro Ala
Ile Leu Ser 1700 1705 1710
Lys Val Leu Gly Gly Ser Phe Ala Asp Gln Lys Ile Cys Gln Gly 1715
1720 1725 Cys Pro His Arg Phe
Glu Cys Glu Glu Ser Phe Thr Thr Leu Asn 1730 1735
1740 Val Asp Ile Arg Asn His Gln Asn Leu Leu
Asp Ser Leu Glu Gln 1745 1750 1755
Tyr Ile Lys Gly Asp Leu Leu Glu Gly Ala Asn Ala Tyr His Cys
1760 1765 1770 Glu Lys
Cys Asp Lys Lys Val Asp Thr Val Lys Arg Leu Leu Ile 1775
1780 1785 Lys Lys Leu Pro Arg Val Leu
Ala Ile Gln Leu Lys Arg Phe Asp 1790 1795
1800 Tyr Asp Trp Glu Arg Glu Cys Ala Ile Lys Phe Asn
Asp Tyr Phe 1805 1810 1815
Glu Phe Pro Arg Glu Leu Asp Met Gly Pro Tyr Thr Val Ala Gly 1820
1825 1830 Val Ala Asn Leu Glu
Arg Asp Asn Val Asn Ser Glu Asn Glu Leu 1835 1840
1845 Ile Glu Gln Lys Glu Gln Ser Asp Asn Glu
Thr Ala Gly Gly Thr 1850 1855 1860
Lys Tyr Arg Leu Val Gly Val Leu Val His Ser Gly Gln Ala Ser
1865 1870 1875 Gly Gly
His Tyr Tyr Ser Tyr Ile Ile Gln Arg Asn Gly Lys Asp 1880
1885 1890 Asp Gln Thr Asp His Trp Tyr
Lys Phe Asp Asp Gly Asp Val Thr 1895 1900
1905 Glu Cys Lys Met Asp Asp Asp Glu Glu Met Lys Asn
Gln Cys Phe 1910 1915 1920
Gly Gly Glu Tyr Met Gly Glu Val Phe Asp His Met Met Lys Arg 1925
1930 1935 Met Ser Tyr Arg Arg
Gln Lys Arg Trp Trp Asn Ala Tyr Ile Pro 1940 1945
1950 Phe Tyr Glu Gln Met Asp Met Ile Asp Glu
Asp Asp Glu Met Ile 1955 1960 1965
Arg Tyr Ile Ser Glu Leu Thr Ile Ala Arg Pro His Gln Ile Ile
1970 1975 1980 Met Ser
Pro Ala Ile Glu Arg Ser Val Arg Lys Gln Asn Val Lys 1985
1990 1995 Phe Met His Asn Arg Leu Gln
Tyr Ser Leu Glu Tyr Phe Gln Phe 2000 2005
2010 Val Lys Lys Leu Leu Thr Cys Asn Gly Val Tyr Leu
Asn Pro Ala 2015 2020 2025
Pro Gly Gln Asp Tyr Leu Leu Pro Glu Ala Glu Glu Ile Thr Met 2030
2035 2040 Ile Ser Ile Gln Leu
Ala Ala Arg Phe Leu Phe Thr Thr Gly Phe 2045 2050
2055 His Thr Lys Lys Ile Val Arg Gly Pro Ala
Ser Asp Trp Tyr Asp 2060 2065 2070
Ala Leu Cys Val Leu Leu Arg His Ser Lys Asn Val Arg Phe Trp
2075 2080 2085 Phe Thr
His Asn Val Leu Phe Asn Val Ser Asn Arg Phe Ser Glu 2090
2095 2100 Tyr Leu Leu Glu Cys Pro Ser
Ala Glu Val Arg Gly Ala Phe Ala 2105 2110
2115 Lys Leu Ile Val Phe Ile Ala His Phe Ser Leu Gln
Asp Gly Ser 2120 2125 2130
Cys Pro Ser Pro Phe Ala Ser Pro Gly Pro Ser Ser Gln Ala Cys 2135
2140 2145 Asp Asn Leu Ser Leu
Ser Asp His Leu Leu Arg Ala Thr Leu Asn 2150 2155
2160 Leu Leu Arg Arg Glu Val Ser Glu His Gly
His His Leu Gln Gln 2165 2170 2175
Tyr Phe Asn Leu Phe Val Met Tyr Ala Asn Leu Gly Val Ala Glu
2180 2185 2190 Lys Thr
Gln Leu Leu Lys Leu Asn Val Pro Ala Thr Phe Met Leu 2195
2200 2205 Val Ser Leu Asp Glu Gly Pro
Gly Pro Pro Ile Lys Tyr Gln Tyr 2210 2215
2220 Ala Glu Leu Gly Lys Leu Tyr Ser Val Val Ser Gln
Leu Ile Arg 2225 2230 2235
Cys Cys Asn Val Ser Ser Thr Met Gln Ser Ser Ile Asn Gly Asn 2240
2245 2250 Pro Pro Leu Pro Asn
Pro Phe Gly Asp Leu Asn Leu Ser Gln Pro 2255 2260
2265 Ile Met Pro Ile Gln Gln Asn Val Leu Asp
Ile Leu Phe Val Arg 2270 2275 2280
Thr Ser Tyr Val Lys Lys Ile Ile Glu Asp Cys Ser Asn Ser Glu
2285 2290 2295 Asp Thr
Ile Lys Leu Leu Arg Phe Cys Ser Trp Glu Asn Pro Gln 2300
2305 2310 Phe Ser Ser Thr Val Leu Ser
Glu Leu Leu Trp Gln Val Ala Tyr 2315 2320
2325 Ser Tyr Thr Tyr Glu Leu Arg Pro Tyr Leu Asp Leu
Leu Phe Gln 2330 2335 2340
Ile Leu Leu Ile Glu Asp Ser Trp Gln Thr His Arg Ile His Asn 2345
2350 2355 Ala Leu Lys Gly Ile
Pro Asp Asp Arg Asp Gly Leu Phe Asp Thr 2360 2365
2370 Ile Gln Arg Ser Lys Asn His Tyr Gln Lys
Arg Ala Tyr Gln Cys 2375 2380 2385
Ile Lys Cys Met Val Ala Leu Phe Ser Ser Cys Pro Val Ala Tyr
2390 2395 2400 Gln Ile
Leu Gln Gly Asn Gly Asp Leu Lys Arg Lys Trp Thr Trp 2405
2410 2415 Ala Val Glu Trp Leu Gly Asp
Glu Leu Glu Arg Arg Pro Tyr Thr 2420 2425
2430 Gly Asn Pro Gln Tyr Ser Tyr Asn Asn Trp Ser Pro
Pro Val Gln 2435 2440 2445
Ser Asn Glu Thr Ala Asn Gly Tyr Phe Leu Glu Arg Ser His Ser 2450
2455 2460 Ala Arg Met Thr Leu
Ala Lys Ala Cys Glu Leu Cys Pro Glu Glu 2465 2470
2475 Glu Pro Asp Asp Gln Asp Ala Pro Asp Glu
His Glu Pro Ser Pro 2480 2485 2490
Ser Glu Asp Ala Pro Leu Tyr Pro His Ser Pro Ala Ser Gln Tyr
2495 2500 2505 Gln Gln
Asn Asn His Val His Gly Gln Pro Tyr Thr Gly Pro Ala 2510
2515 2520 Ala His His Leu Asn Asn Pro
Gln Lys Thr Gly Gln Arg Thr Gln 2525 2530
2535 Glu Asn Tyr Glu Gly Asn Glu Glu Val Ser Ser Pro
Gln Met Lys 2540 2545 2550
Asp Gln 2555 65330PRTHomo sapiens 65Met Ser Gln Trp His His Pro Arg
Ser Gly Trp Gly Arg Arg Arg Asp 1 5 10
15 Phe Ser Gly Arg Ser Ser Ala Lys Lys Lys Gly Gly Asn
His Ile Pro 20 25 30
Glu Arg Trp Lys Asp Tyr Leu Pro Val Gly Gln Arg Met Pro Gly Thr
35 40 45 Arg Phe Ile Ala
Phe Lys Val Pro Leu Gln Lys Ser Phe Glu Lys Lys 50
55 60 Leu Ala Pro Glu Glu Cys Phe Ser
Pro Leu Asp Leu Phe Asn Lys Ile 65 70
75 80 Arg Glu Gln Asn Glu Glu Leu Gly Leu Ile Ile Asp
Leu Thr Tyr Thr 85 90
95 Gln Arg Tyr Tyr Lys Pro Glu Asp Leu Pro Glu Thr Val Pro Tyr Leu
100 105 110 Lys Ile Phe
Thr Val Gly His Gln Val Pro Asp Asp Glu Thr Ile Phe 115
120 125 Lys Phe Lys His Ala Val Asn Gly
Phe Leu Lys Glu Asn Lys Asp Asn 130 135
140 Asp Lys Leu Ile Gly Val His Cys Thr His Gly Leu Asn
Arg Thr Gly 145 150 155
160 Tyr Leu Ile Cys Arg Tyr Leu Ile Asp Val Glu Gly Val Arg Pro Asp
165 170 175 Asp Ala Ile Glu
Leu Phe Asn Arg Cys Arg Gly His Cys Leu Glu Arg 180
185 190 Gln Asn Tyr Ile Glu Asp Leu Gln Asn
Gly Pro Ile Arg Lys Asn Trp 195 200
205 Asn Ser Ser Val Pro Arg Ser Ser Asp Phe Glu Asp Ser Ala
His Leu 210 215 220
Met Gln Pro Val His Asn Lys Pro Val Lys Gln Gly Pro Arg Tyr Asn 225
230 235 240 Leu His Gln Ile Gln
Gly His Ser Ala Pro Arg His Phe His Thr Gln 245
250 255 Thr Gln Ser Leu Gln Gln Ser Val Arg Lys
Phe Ser Glu Asn Pro His 260 265
270 Val Tyr Gln Arg His His Leu Pro Pro Pro Gly Pro Pro Gly Glu
Asp 275 280 285 Tyr
Ser His Arg Arg Tyr Ser Trp Asn Val Lys Pro Asn Ala Ser Arg 290
295 300 Ala Ala Gln Asp Arg Arg
Arg Trp Tyr Pro Tyr Asn Tyr Ser Arg Leu 305 310
315 320 Ser Tyr Pro Ala Cys Trp Glu Trp Thr Gln
325 330 66185PRTHomo sapiens 66Met Ser Gly
Ser Phe Glu Leu Ser Val Gln Asp Leu Asn Asp Leu Leu 1 5
10 15 Ser Asp Gly Ser Gly Cys Tyr Ser
Leu Pro Ser Gln Pro Cys Asn Glu 20 25
30 Val Thr Pro Arg Ile Tyr Val Gly Asn Ala Ser Val Ala
Gln Asp Ile 35 40 45
Pro Lys Leu Gln Lys Leu Gly Ile Thr His Val Leu Asn Ala Ala Glu 50
55 60 Gly Arg Ser Phe
Met His Val Asn Thr Asn Ala Asn Phe Tyr Lys Asp 65 70
75 80 Ser Gly Ile Thr Tyr Leu Gly Ile Lys
Ala Asn Asp Thr Gln Glu Phe 85 90
95 Asn Leu Ser Ala Tyr Phe Glu Arg Ala Ala Asp Phe Ile Asp
Gln Ala 100 105 110
Leu Ala Gln Lys Asn Gly Arg Val Leu Val His Cys Arg Glu Gly Tyr
115 120 125 Ser Arg Ser Pro
Thr Leu Val Ile Ala Tyr Leu Met Met Arg Gln Lys 130
135 140 Met Asp Val Lys Ser Ala Leu Ser
Ile Val Arg Gln Asn Arg Glu Ile 145 150
155 160 Gly Pro Asn Asp Gly Phe Leu Ala Gln Leu Cys Gln
Leu Asn Asp Arg 165 170
175 Leu Ala Lys Glu Gly Lys Leu Lys Pro 180
185 671154PRTHomo sapiens 67Met Gln Tyr Leu Asn Ile Lys Glu Asp Cys Asn
Ala Met Ala Phe Cys 1 5 10
15 Ala Lys Met Arg Ser Ser Lys Lys Thr Glu Val Asn Leu Glu Ala Pro
20 25 30 Glu Pro
Gly Val Glu Val Ile Phe Tyr Leu Ser Asp Arg Glu Pro Leu 35
40 45 Arg Leu Gly Ser Gly Glu Tyr
Thr Ala Glu Glu Leu Cys Ile Arg Ala 50 55
60 Ala Gln Ala Cys Arg Ile Ser Pro Leu Cys His Asn
Leu Phe Ala Leu 65 70 75
80 Tyr Asp Glu Asn Thr Lys Leu Trp Tyr Ala Pro Asn Arg Thr Ile Thr
85 90 95 Val Asp Asp
Lys Met Ser Leu Arg Leu His Tyr Arg Met Arg Phe Tyr 100
105 110 Phe Thr Asn Trp His Gly Thr Asn
Asp Asn Glu Gln Ser Val Trp Arg 115 120
125 His Ser Pro Lys Lys Gln Lys Asn Gly Tyr Glu Lys Lys
Lys Ile Pro 130 135 140
Asp Ala Thr Pro Leu Leu Asp Ala Ser Ser Leu Glu Tyr Leu Phe Ala 145
150 155 160 Gln Gly Gln Tyr
Asp Leu Val Lys Cys Leu Ala Pro Ile Arg Asp Pro 165
170 175 Lys Thr Glu Gln Asp Gly His Asp Ile
Glu Asn Glu Cys Leu Gly Met 180 185
190 Ala Val Leu Ala Ile Ser His Tyr Ala Met Met Lys Lys Met
Gln Leu 195 200 205
Pro Glu Leu Pro Lys Asp Ile Ser Tyr Lys Arg Tyr Ile Pro Glu Thr 210
215 220 Leu Asn Lys Ser Ile
Arg Gln Arg Asn Leu Leu Thr Arg Met Arg Ile 225 230
235 240 Asn Asn Val Phe Lys Asp Phe Leu Lys Glu
Phe Asn Asn Lys Thr Ile 245 250
255 Cys Asp Ser Ser Val Ser Thr His Asp Leu Lys Val Lys Tyr Leu
Ala 260 265 270 Thr
Leu Glu Thr Leu Thr Lys His Tyr Gly Ala Glu Ile Phe Glu Thr 275
280 285 Ser Met Leu Leu Ile Ser
Ser Glu Asn Glu Met Asn Trp Phe His Ser 290 295
300 Asn Asp Gly Gly Asn Val Leu Tyr Tyr Glu Val
Met Val Thr Gly Asn 305 310 315
320 Leu Gly Ile Gln Trp Arg His Lys Pro Asn Val Val Ser Val Glu Lys
325 330 335 Glu Lys
Asn Lys Leu Lys Arg Lys Lys Leu Glu Asn Lys Asp Lys Lys 340
345 350 Asp Glu Glu Lys Asn Lys Ile
Arg Glu Glu Trp Asn Asn Phe Ser Phe 355 360
365 Phe Pro Glu Ile Thr His Ile Val Ile Lys Glu Ser
Val Val Ser Ile 370 375 380
Asn Lys Gln Asp Asn Lys Lys Met Glu Leu Lys Leu Ser Ser His Glu 385
390 395 400 Glu Ala Leu
Ser Phe Val Ser Leu Val Asp Gly Tyr Phe Arg Leu Thr 405
410 415 Ala Asp Ala His His Tyr Leu Cys
Thr Asp Val Ala Pro Pro Leu Ile 420 425
430 Val His Asn Ile Gln Asn Gly Cys His Gly Pro Ile Cys
Thr Glu Tyr 435 440 445
Ala Ile Asn Lys Leu Arg Gln Glu Gly Ser Glu Glu Gly Met Tyr Val 450
455 460 Leu Arg Trp Ser
Cys Thr Asp Phe Asp Asn Ile Leu Met Thr Val Thr 465 470
475 480 Cys Phe Glu Lys Ser Glu Gln Val Gln
Gly Ala Gln Lys Gln Phe Lys 485 490
495 Asn Phe Gln Ile Glu Val Gln Lys Gly Arg Tyr Ser Leu His
Gly Ser 500 505 510
Asp Arg Ser Phe Pro Ser Leu Gly Asp Leu Met Ser His Leu Lys Lys
515 520 525 Gln Ile Leu Arg
Thr Asp Asn Ile Ser Phe Met Leu Lys Arg Cys Cys 530
535 540 Gln Pro Lys Pro Arg Glu Ile Ser
Asn Leu Leu Val Ala Thr Lys Lys 545 550
555 560 Ala Gln Glu Trp Gln Pro Val Tyr Pro Met Ser Gln
Leu Ser Phe Asp 565 570
575 Arg Ile Leu Lys Lys Asp Leu Val Gln Gly Glu His Leu Gly Arg Gly
580 585 590 Thr Arg Thr
His Ile Tyr Ser Gly Thr Leu Met Asp Tyr Lys Asp Asp 595
600 605 Glu Gly Thr Ser Glu Glu Lys Lys
Ile Lys Val Ile Leu Lys Val Leu 610 615
620 Asp Pro Ser His Arg Asp Ile Ser Leu Ala Phe Phe Glu
Ala Ala Ser 625 630 635
640 Met Met Arg Gln Val Ser His Lys His Ile Val Tyr Leu Tyr Gly Val
645 650 655 Cys Val Arg Asp
Val Glu Asn Ile Met Val Glu Glu Phe Val Glu Gly 660
665 670 Gly Pro Leu Asp Leu Phe Met His Arg
Lys Ser Asp Val Leu Thr Thr 675 680
685 Pro Trp Lys Phe Lys Val Ala Lys Gln Leu Ala Ser Ala Leu
Ser Tyr 690 695 700
Leu Glu Asp Lys Asp Leu Val His Gly Asn Val Cys Thr Lys Asn Leu 705
710 715 720 Leu Leu Ala Arg Glu
Gly Ile Asp Ser Glu Cys Gly Pro Phe Ile Lys 725
730 735 Leu Ser Asp Pro Gly Ile Pro Ile Thr Val
Leu Ser Arg Gln Glu Cys 740 745
750 Ile Glu Arg Ile Pro Trp Ile Ala Pro Glu Cys Val Glu Asp Ser
Lys 755 760 765 Asn
Leu Ser Val Ala Ala Asp Lys Trp Ser Phe Gly Thr Thr Leu Trp 770
775 780 Glu Ile Cys Tyr Asn Gly
Glu Ile Pro Leu Lys Asp Lys Thr Leu Ile 785 790
795 800 Glu Lys Glu Arg Phe Tyr Glu Ser Arg Cys Arg
Pro Val Thr Pro Ser 805 810
815 Cys Lys Glu Leu Ala Asp Leu Met Thr Arg Cys Met Asn Tyr Asp Pro
820 825 830 Asn Gln
Arg Pro Phe Phe Arg Ala Ile Met Arg Asp Ile Asn Lys Leu 835
840 845 Glu Glu Gln Asn Pro Asp Ile
Val Ser Arg Lys Lys Asn Gln Pro Thr 850 855
860 Glu Val Asp Pro Thr His Phe Glu Lys Arg Phe Leu
Lys Arg Ile Arg 865 870 875
880 Asp Leu Gly Glu Gly His Phe Gly Lys Val Glu Leu Cys Arg Tyr Asp
885 890 895 Pro Glu Asp
Asn Thr Gly Glu Gln Val Ala Val Lys Ser Leu Lys Pro 900
905 910 Glu Ser Gly Gly Asn His Ile Ala
Asp Leu Lys Lys Glu Ile Glu Ile 915 920
925 Leu Arg Asn Leu Tyr His Glu Asn Ile Val Lys Tyr Lys
Gly Ile Cys 930 935 940
Thr Glu Asp Gly Gly Asn Gly Ile Lys Leu Ile Met Glu Phe Leu Pro 945
950 955 960 Ser Gly Ser Leu
Lys Glu Tyr Leu Pro Lys Asn Lys Asn Lys Ile Asn 965
970 975 Leu Lys Gln Gln Leu Lys Tyr Ala Val
Gln Ile Cys Lys Gly Met Asp 980 985
990 Tyr Leu Gly Ser Arg Gln Tyr Val His Arg Asp Leu Ala
Ala Arg Asn 995 1000 1005
Val Leu Val Glu Ser Glu His Gln Val Lys Ile Gly Asp Phe Gly
1010 1015 1020 Leu Thr Lys
Ala Ile Glu Thr Asp Lys Glu Tyr Tyr Thr Val Lys 1025
1030 1035 Asp Asp Arg Asp Ser Pro Val Phe
Trp Tyr Ala Pro Glu Cys Leu 1040 1045
1050 Met Gln Ser Lys Phe Tyr Ile Ala Ser Asp Val Trp Ser
Phe Gly 1055 1060 1065
Val Thr Leu His Glu Leu Leu Thr Tyr Cys Asp Ser Asp Ser Ser 1070
1075 1080 Pro Met Ala Leu Phe
Leu Lys Met Ile Gly Pro Thr His Gly Gln 1085 1090
1095 Met Thr Val Thr Arg Leu Val Asn Thr Leu
Lys Glu Gly Lys Arg 1100 1105 1110
Leu Pro Cys Pro Pro Asn Cys Pro Asp Glu Val Tyr Gln Leu Met
1115 1120 1125 Arg Lys
Cys Trp Glu Phe Gln Pro Ser Asn Arg Thr Ser Phe Gln 1130
1135 1140 Asn Leu Ile Glu Gly Phe Glu
Ala Leu Leu Lys 1145 1150
681141PRTHomo sapiens 68Met Ala Phe Cys Ala Lys Met Arg Ser Ser Lys Lys
Thr Glu Val Asn 1 5 10
15 Leu Glu Ala Pro Glu Pro Gly Val Glu Val Ile Phe Tyr Leu Ser Asp
20 25 30 Arg Glu Pro
Leu Arg Leu Gly Ser Gly Glu Tyr Thr Ala Glu Glu Leu 35
40 45 Cys Ile Arg Ala Ala Gln Ala
Cys Arg Ile Ser Pro Leu Cys His Asn 50 55
60 Leu Phe Ala Leu Tyr Asp Glu Asn Thr Lys Leu Trp
Tyr Ala Pro Asn 65 70 75
80 Arg Thr Ile Thr Val Asp Asp Lys Met Ser Leu Arg Leu His Tyr Arg
85 90 95 Met Arg Phe
Tyr Phe Thr Asn Trp His Gly Thr Asn Asp Asn Glu Gln 100
105 110 Ser Val Trp Arg His Ser Pro Lys
Lys Gln Lys Asn Gly Tyr Glu Lys 115 120
125 Lys Lys Ile Pro Asp Ala Thr Pro Leu Leu Asp Ala Ser
Ser Leu Glu 130 135 140
Tyr Leu Phe Ala Gln Gly Gln Tyr Asp Leu Val Lys Cys Leu Ala Pro 145
150 155 160 Ile Arg Asp Pro
Lys Thr Glu Gln Asp Gly His Asp Ile Glu Asn Glu 165
170 175 Cys Leu Gly Met Ala Val Leu Ala Ile
Ser His Tyr Ala Met Met Lys 180 185
190 Lys Met Gln Leu Pro Glu Leu Pro Lys Asp Ile Ser Tyr Lys
Arg Tyr 195 200 205
Ile Pro Glu Thr Leu Asn Lys Ser Ile Arg Gln Arg Asn Leu Leu Thr 210
215 220 Arg Met Arg Ile Asn
Asn Val Phe Lys Asp Phe Leu Lys Glu Phe Asn 225 230
235 240 Asn Lys Thr Ile Cys Asp Ser Ser Val Ser
Thr His Asp Leu Lys Val 245 250
255 Lys Tyr Leu Ala Thr Leu Glu Thr Leu Thr Lys His Tyr Gly Ala
Glu 260 265 270 Ile
Phe Glu Thr Ser Met Leu Leu Ile Ser Ser Glu Asn Glu Met Asn 275
280 285 Trp Phe His Ser Asn Asp
Gly Gly Asn Val Leu Tyr Tyr Glu Val Met 290 295
300 Val Thr Gly Asn Leu Gly Ile Gln Trp Arg His
Lys Pro Asn Val Val 305 310 315
320 Ser Val Glu Lys Glu Lys Asn Lys Leu Lys Arg Lys Lys Leu Glu Asn
325 330 335 Lys Asp
Lys Lys Asp Glu Glu Lys Asn Lys Ile Arg Glu Glu Trp Asn 340
345 350 Asn Phe Ser Phe Phe Pro Glu
Ile Thr His Ile Val Ile Lys Glu Ser 355 360
365 Val Val Ser Ile Asn Lys Gln Asp Asn Lys Lys Met
Glu Leu Lys Leu 370 375 380
Ser Ser His Glu Glu Ala Leu Ser Phe Val Ser Leu Val Asp Gly Tyr 385
390 395 400 Phe Arg Leu
Thr Ala Asp Ala His His Tyr Leu Cys Thr Asp Val Ala 405
410 415 Pro Pro Leu Ile Val His Asn Ile
Gln Asn Gly Cys His Gly Pro Ile 420 425
430 Cys Thr Glu Tyr Ala Ile Asn Lys Leu Arg Gln Glu Gly
Ser Glu Glu 435 440 445
Gly Met Tyr Val Leu Arg Trp Ser Cys Thr Asp Phe Asp Asn Ile Leu 450
455 460 Met Thr Val Thr
Cys Phe Glu Lys Ser Glu Gln Val Gln Gly Ala Gln 465 470
475 480 Lys Gln Phe Lys Asn Phe Gln Ile Glu
Val Gln Lys Gly Arg Tyr Ser 485 490
495 Leu His Gly Ser Asp Arg Ser Phe Pro Ser Leu Gly Asp Leu
Met Ser 500 505 510
His Leu Lys Lys Gln Ile Leu Arg Thr Asp Asn Ile Ser Phe Met Leu
515 520 525 Lys Arg Cys Cys
Gln Pro Lys Pro Arg Glu Ile Ser Asn Leu Leu Val 530
535 540 Ala Thr Lys Lys Ala Gln Glu Trp
Gln Pro Val Tyr Pro Met Ser Gln 545 550
555 560 Leu Ser Phe Asp Arg Ile Leu Lys Lys Asp Leu Val
Gln Gly Glu His 565 570
575 Leu Gly Arg Gly Thr Arg Thr His Ile Tyr Ser Gly Thr Leu Met Asp
580 585 590 Tyr Lys Asp
Asp Glu Gly Thr Ser Glu Glu Lys Lys Ile Lys Val Ile 595
600 605 Leu Lys Val Leu Asp Pro Ser His
Arg Asp Ile Ser Leu Ala Phe Phe 610 615
620 Glu Ala Ala Ser Met Met Arg Gln Val Ser His Lys His
Ile Val Tyr 625 630 635
640 Leu Tyr Gly Val Cys Val Arg Asp Val Glu Asn Ile Met Val Glu Glu
645 650 655 Phe Val Glu Gly
Gly Pro Leu Asp Leu Phe Met His Arg Lys Ser Asp 660
665 670 Val Leu Thr Thr Pro Trp Lys Phe Lys
Val Ala Lys Gln Leu Ala Ser 675 680
685 Ala Leu Ser Tyr Leu Glu Asp Lys Asp Leu Val His Gly Asn
Val Cys 690 695 700
Thr Lys Asn Leu Leu Leu Ala Arg Glu Gly Ile Asp Ser Glu Cys Gly 705
710 715 720 Pro Phe Ile Lys Leu
Ser Asp Pro Gly Ile Pro Ile Thr Val Leu Ser 725
730 735 Arg Gln Glu Cys Ile Glu Arg Ile Pro Trp
Ile Ala Pro Glu Cys Val 740 745
750 Glu Asp Ser Lys Asn Leu Ser Val Ala Ala Asp Lys Trp Ser Phe
Gly 755 760 765 Thr
Thr Leu Trp Glu Ile Cys Tyr Asn Gly Glu Ile Pro Leu Lys Asp 770
775 780 Lys Thr Leu Ile Glu Lys
Glu Arg Phe Tyr Glu Ser Arg Cys Arg Pro 785 790
795 800 Val Thr Pro Ser Cys Lys Glu Leu Ala Asp Leu
Met Thr Arg Cys Met 805 810
815 Asn Tyr Asp Pro Asn Gln Arg Pro Phe Phe Arg Ala Ile Met Arg Asp
820 825 830 Ile Asn
Lys Leu Glu Glu Gln Asn Pro Asp Ile Val Ser Arg Lys Lys 835
840 845 Asn Gln Pro Thr Glu Val Asp
Pro Thr His Phe Glu Lys Arg Phe Leu 850 855
860 Lys Arg Ile Arg Asp Leu Gly Glu Gly His Phe Gly
Lys Val Glu Leu 865 870 875
880 Cys Arg Tyr Asp Pro Glu Asp Asn Thr Gly Glu Gln Val Ala Val Lys
885 890 895 Ser Leu Lys
Pro Glu Ser Gly Gly Asn His Ile Ala Asp Leu Lys Lys 900
905 910 Glu Ile Glu Ile Leu Arg Asn Leu
Tyr His Glu Asn Ile Val Lys Tyr 915 920
925 Lys Gly Ile Cys Thr Glu Asp Gly Gly Asn Gly Ile Lys
Leu Ile Met 930 935 940
Glu Phe Leu Pro Ser Gly Ser Leu Lys Glu Tyr Leu Pro Lys Asn Lys 945
950 955 960 Asn Lys Ile Asn
Leu Lys Gln Gln Leu Lys Tyr Ala Val Gln Ile Cys 965
970 975 Lys Gly Met Asp Tyr Leu Gly Ser Arg
Gln Tyr Val His Arg Asp Leu 980 985
990 Ala Ala Arg Asn Val Leu Val Glu Ser Glu His Gln Val
Lys Ile Asp 995 1000 1005
Phe Gly Leu Thr Lys Ala Ile Glu Thr Asp Lys Glu Tyr Tyr Thr
1010 1015 1020 Val Lys Asp
Asp Arg Asp Ser Pro Val Phe Trp Tyr Ala Pro Glu 1025
1030 1035 Cys Leu Met Gln Ser Lys Phe Tyr
Ile Ala Ser Asp Val Trp Ser 1040 1045
1050 Phe Gly Val Thr Leu His Glu Leu Leu Thr Tyr Cys Asp
Ser Asp 1055 1060 1065
Ser Ser Pro Met Ala Leu Phe Leu Lys Met Ile Gly Pro Thr His 1070
1075 1080 Gly Gln Met Thr Val
Thr Arg Leu Val Asn Thr Leu Lys Glu Gly 1085 1090
1095 Lys Arg Leu Pro Cys Pro Pro Asn Cys Pro
Asp Glu Val Tyr Gln 1100 1105 1110
Leu Met Arg Lys Cys Trp Glu Phe Gln Pro Ser Asn Arg Thr Ser
1115 1120 1125 Phe Gln
Asn Leu Ile Glu Gly Phe Glu Ala Leu Leu Lys 1130
1135 1140 69666PRTHomo sapiens 69Met Ala Cys Leu Met
Ala Ala Phe Ser Val Gly Thr Ala Met Asn Ala 1 5
10 15 Ser Ser Tyr Ser Ala Glu Met Thr Glu Pro
Lys Ser Val Cys Val Ser 20 25
30 Val Asp Glu Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr Asp
Leu 35 40 45 Leu
Asn Gly His Leu Lys Lys Val Asp Asn Asn Leu Thr Glu Ala Gln 50
55 60 Arg Phe Ser Ser Leu Pro
Arg Arg Ala Ala Val Asn Ile Glu Phe Arg 65 70
75 80 Asp Leu Ser Tyr Ser Val Pro Glu Gly Pro Trp
Trp Arg Lys Lys Gly 85 90
95 Tyr Lys Thr Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu
100 105 110 Leu Val
Ala Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr Leu Met 115
120 125 Asn Ile Leu Ala Gly Tyr Arg
Glu Thr Gly Met Lys Gly Ala Val Leu 130 135
140 Ile Asn Gly Leu Pro Arg Asp Leu Arg Cys Phe Arg
Lys Val Ser Cys 145 150 155
160 Tyr Ile Met Gln Asp Asp Met Leu Leu Pro His Leu Thr Val Gln Glu
165 170 175 Ala Met Met
Val Ser Ala His Leu Lys Leu Gln Glu Lys Asp Glu Gly 180
185 190 Arg Arg Glu Met Val Lys Glu Ile
Leu Thr Ala Leu Gly Leu Leu Ser 195 200
205 Cys Ala Asn Thr Arg Thr Gly Ser Leu Ser Gly Gly Gln
Arg Lys Arg 210 215 220
Leu Ala Ile Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met Phe Phe 225
230 235 240 Asp Glu Pro Thr
Ser Gly Leu Asp Ser Ala Ser Cys Phe Gln Val Val 245
250 255 Ser Leu Met Lys Gly Leu Ala Gln Gly
Gly Arg Ser Ile Ile Cys Thr 260 265
270 Ile His Gln Pro Ser Ala Lys Leu Phe Glu Leu Phe Asp Gln
Leu Tyr 275 280 285
Val Leu Ser Gln Gly Gln Cys Val Tyr Arg Gly Lys Val Cys Asn Leu 290
295 300 Val Pro Tyr Leu Arg
Asp Leu Gly Leu Asn Cys Pro Thr Tyr His Asn 305 310
315 320 Pro Ala Asp Phe Val Met Glu Val Ala Ser
Gly Glu Tyr Gly Asp Gln 325 330
335 Asn Ser Arg Leu Val Arg Ala Val Arg Glu Gly Met Cys Asp Ser
Asp 340 345 350 His
Lys Arg Asp Leu Gly Gly Asp Ala Glu Val Asn Pro Phe Leu Trp 355
360 365 His Arg Pro Ser Glu Glu
Asp Ser Ser Ser Met Glu Gly Cys His Ser 370 375
380 Phe Ser Ala Ser Cys Leu Thr Gln Phe Cys Ile
Leu Phe Lys Arg Thr 385 390 395
400 Phe Leu Ser Ile Met Arg Asp Ser Val Leu Thr His Leu Arg Ile Thr
405 410 415 Ser His
Ile Gly Ile Gly Leu Leu Ile Gly Leu Leu Tyr Leu Gly Ile 420
425 430 Gly Asn Glu Ala Lys Lys Val
Leu Ser Asn Ser Gly Phe Leu Phe Phe 435 440
445 Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met Pro
Thr Val Leu Thr 450 455 460
Phe Pro Leu Glu Met Gly Val Phe Leu Arg Glu His Leu Asn Tyr Trp 465
470 475 480 Tyr Ser Leu
Lys Ala Tyr Tyr Leu Ala Lys Thr Met Ala Asp Val Pro 485
490 495 Phe Gln Ile Met Phe Pro Val Ala
Tyr Cys Ser Ile Val Tyr Trp Met 500 505
510 Thr Ser Gln Pro Ser Asp Ala Val Arg Phe Val Leu Phe
Ala Ala Leu 515 520 525
Gly Thr Met Thr Ser Leu Val Ala Gln Ser Leu Gly Leu Leu Ile Gly 530
535 540 Ala Ala Ser Thr
Ser Leu Gln Val Ala Thr Phe Val Gly Pro Val Thr 545 550
555 560 Ala Ile Pro Val Leu Leu Phe Ser Gly
Phe Phe Val Ser Phe Asp Thr 565 570
575 Ile Pro Thr Tyr Leu Gln Trp Met Ser Tyr Ile Ser Tyr Val
Arg Tyr 580 585 590
Gly Phe Glu Gly Val Ile Leu Ser Ile Tyr Gly Leu Asp Arg Glu Asp
595 600 605 Leu His Cys Asp
Ile Asp Glu Thr Cys His Phe Gln Lys Ser Glu Ala 610
615 620 Ile Leu Arg Glu Leu Asp Val Glu
Asn Ala Lys Leu Tyr Leu Asp Phe 625 630
635 640 Ile Val Leu Gly Ile Phe Phe Ile Ser Leu Arg Leu
Ile Ala Tyr Phe 645 650
655 Val Leu Arg Tyr Lys Ile Arg Ala Glu Arg 660
665 70678PRTHomo sapiens 70Met Ala Cys Leu Met Ala Ala Phe Ser
Val Gly Thr Ala Met Asn Ala 1 5 10
15 Ser Ser Tyr Ser Ala Glu Met Thr Glu Pro Lys Ser Val Cys
Val Ser 20 25 30
Val Asp Glu Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr Asp Leu
35 40 45 Leu Asn Gly His
Leu Lys Lys Val Asp Asn Asn Leu Thr Glu Ala Gln 50
55 60 Arg Phe Ser Ser Leu Pro Arg Arg
Ala Ala Val Asn Ile Glu Phe Arg 65 70
75 80 Asp Leu Ser Tyr Ser Val Pro Glu Gly Pro Trp Trp
Arg Lys Lys Gly 85 90
95 Tyr Lys Thr Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu
100 105 110 Leu Val Ala
Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr Leu Met 115
120 125 Asn Ile Leu Ala Gly Tyr Arg Glu
Thr Gly Met Lys Gly Ala Val Leu 130 135
140 Ile Asn Gly Leu Pro Arg Asp Leu Arg Cys Phe Arg Lys
Val Ser Cys 145 150 155
160 Tyr Ile Met Gln Asp Asp Met Leu Leu Pro His Leu Thr Val Gln Glu
165 170 175 Ala Met Met Val
Ser Ala His Leu Lys Leu Gln Glu Lys Asp Glu Gly 180
185 190 Arg Arg Glu Met Val Lys Glu Ile Leu
Thr Ala Leu Gly Leu Leu Ser 195 200
205 Cys Ala Asn Thr Arg Thr Gly Ser Leu Ser Gly Gly Gln Arg
Lys Arg 210 215 220
Leu Ala Ile Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met Phe Phe 225
230 235 240 Asp Glu Pro Thr Ser
Gly Leu Asp Ser Ala Ser Cys Phe Gln Val Val 245
250 255 Ser Leu Met Lys Gly Leu Ala Gln Gly Gly
Arg Ser Ile Ile Cys Thr 260 265
270 Ile His Gln Pro Ser Ala Lys Leu Phe Glu Leu Phe Asp Gln Leu
Tyr 275 280 285 Val
Leu Ser Gln Gly Gln Cys Val Tyr Arg Gly Lys Val Cys Asn Leu 290
295 300 Val Pro Tyr Leu Arg Asp
Leu Gly Leu Asn Cys Pro Thr Tyr His Asn 305 310
315 320 Pro Ala Asp Phe Val Met Glu Val Ala Ser Gly
Glu Tyr Gly Asp Gln 325 330
335 Asn Ser Arg Leu Val Arg Ala Val Arg Glu Gly Met Cys Asp Ser Asp
340 345 350 His Lys
Arg Asp Leu Gly Gly Asp Ala Glu Val Asn Pro Phe Leu Trp 355
360 365 His Arg Pro Ser Glu Glu Val
Lys Gln Thr Lys Arg Leu Lys Gly Leu 370 375
380 Arg Lys Asp Ser Ser Ser Met Glu Gly Cys His Ser
Phe Ser Ala Ser 385 390 395
400 Cys Leu Thr Gln Phe Cys Ile Leu Phe Lys Arg Thr Phe Leu Ser Ile
405 410 415 Met Arg Asp
Ser Val Leu Thr His Leu Arg Ile Thr Ser His Ile Gly 420
425 430 Ile Gly Leu Leu Ile Gly Leu Leu
Tyr Leu Gly Ile Gly Asn Glu Ala 435 440
445 Lys Lys Val Leu Ser Asn Ser Gly Phe Leu Phe Phe Ser
Met Leu Phe 450 455 460
Leu Met Phe Ala Ala Leu Met Pro Thr Val Leu Thr Phe Pro Leu Glu 465
470 475 480 Met Gly Val Phe
Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu Lys 485
490 495 Ala Tyr Tyr Leu Ala Lys Thr Met Ala
Asp Val Pro Phe Gln Ile Met 500 505
510 Phe Pro Val Ala Tyr Cys Ser Ile Val Tyr Trp Met Thr Ser
Gln Pro 515 520 525
Ser Asp Ala Val Arg Phe Val Leu Phe Ala Ala Leu Gly Thr Met Thr 530
535 540 Ser Leu Val Ala Gln
Ser Leu Gly Leu Leu Ile Gly Ala Ala Ser Thr 545 550
555 560 Ser Leu Gln Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val 565 570
575 Leu Leu Phe Ser Gly Phe Phe Val Ser Phe Asp Thr Ile Pro Thr
Tyr 580 585 590 Leu
Gln Trp Met Ser Tyr Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly 595
600 605 Val Ile Leu Ser Ile Tyr
Gly Leu Asp Arg Glu Asp Leu His Cys Asp 610 615
620 Ile Asp Glu Thr Cys His Phe Gln Lys Ser Glu
Ala Ile Leu Arg Glu 625 630 635
640 Leu Asp Val Glu Asn Ala Lys Leu Tyr Leu Asp Phe Ile Val Leu Gly
645 650 655 Ile Phe
Phe Ile Ser Leu Arg Leu Ile Ala Tyr Phe Val Leu Arg Tyr 660
665 670 Lys Ile Arg Ala Glu Arg
675 71677PRTHomo sapiens 71Met Arg Ile Ser Leu Pro Arg
Ala Pro Glu Arg Asp Gly Gly Val Ser 1 5
10 15 Ala Ser Ser Leu Leu Asp Thr Val Thr Asn Ala
Ser Ser Tyr Ser Ala 20 25
30 Glu Met Thr Glu Pro Lys Ser Val Cys Val Ser Val Asp Glu Val
Val 35 40 45 Ser
Ser Asn Met Glu Ala Thr Glu Thr Asp Leu Leu Asn Gly His Leu 50
55 60 Lys Lys Val Asp Asn Asn
Leu Thr Glu Ala Gln Arg Phe Ser Ser Leu 65 70
75 80 Pro Arg Arg Ala Ala Val Asn Ile Glu Phe Arg
Asp Leu Ser Tyr Ser 85 90
95 Val Pro Glu Gly Pro Trp Trp Arg Lys Lys Gly Tyr Lys Thr Leu Leu
100 105 110 Lys Gly
Ile Ser Gly Lys Phe Asn Ser Gly Glu Leu Val Ala Ile Met 115
120 125 Gly Pro Ser Gly Ala Gly Lys
Ser Thr Leu Met Asn Ile Leu Ala Gly 130 135
140 Tyr Arg Glu Thr Gly Met Lys Gly Ala Val Leu Ile
Asn Gly Leu Pro 145 150 155
160 Arg Asp Leu Arg Cys Phe Arg Lys Val Ser Cys Tyr Ile Met Gln Asp
165 170 175 Asp Met Leu
Leu Pro His Leu Thr Val Gln Glu Ala Met Met Val Ser 180
185 190 Ala His Leu Lys Leu Gln Glu Lys
Asp Glu Gly Arg Arg Glu Met Val 195 200
205 Lys Glu Ile Leu Thr Ala Leu Gly Leu Leu Ser Cys Ala
Asn Thr Arg 210 215 220
Thr Gly Ser Leu Ser Gly Gly Gln Arg Lys Arg Leu Ala Ile Ala Leu 225
230 235 240 Glu Leu Val Asn
Asn Pro Pro Val Met Phe Phe Asp Glu Pro Thr Ser 245
250 255 Gly Leu Asp Ser Ala Ser Cys Phe Gln
Val Val Ser Leu Met Lys Gly 260 265
270 Leu Ala Gln Gly Gly Arg Ser Ile Ile Cys Thr Ile His Gln
Pro Ser 275 280 285
Ala Lys Leu Phe Glu Leu Phe Asp Gln Leu Tyr Val Leu Ser Gln Gly 290
295 300 Gln Cys Val Tyr Arg
Gly Lys Val Cys Asn Leu Val Pro Tyr Leu Arg 305 310
315 320 Asp Leu Gly Leu Asn Cys Pro Thr Tyr His
Asn Pro Ala Asp Phe Val 325 330
335 Met Glu Val Ala Ser Gly Glu Tyr Gly Asp Gln Asn Ser Arg Leu
Val 340 345 350 Arg
Ala Val Arg Glu Gly Met Cys Asp Ser Asp His Lys Arg Asp Leu 355
360 365 Gly Gly Asp Ala Glu Val
Asn Pro Phe Leu Trp His Arg Pro Ser Glu 370 375
380 Glu Asp Ser Ser Ser Met Glu Gly Cys His Ser
Phe Ser Ala Ser Cys 385 390 395
400 Leu Thr Gln Phe Cys Ile Leu Phe Lys Arg Thr Phe Leu Ser Ile Met
405 410 415 Arg Asp
Ser Val Leu Thr His Leu Arg Ile Thr Ser His Ile Gly Ile 420
425 430 Gly Leu Leu Ile Gly Leu Leu
Tyr Leu Gly Ile Gly Asn Glu Ala Lys 435 440
445 Lys Val Leu Ser Asn Ser Gly Phe Leu Phe Phe Ser
Met Leu Phe Leu 450 455 460
Met Phe Ala Ala Leu Met Pro Thr Val Leu Thr Phe Pro Leu Glu Met 465
470 475 480 Gly Val Phe
Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu Lys Ala 485
490 495 Tyr Tyr Leu Ala Lys Thr Met Ala
Asp Val Pro Phe Gln Ile Met Phe 500 505
510 Pro Val Ala Tyr Cys Ser Ile Val Tyr Trp Met Thr Ser
Gln Pro Ser 515 520 525
Asp Ala Val Arg Phe Val Leu Phe Ala Ala Leu Gly Thr Met Thr Ser 530
535 540 Leu Val Ala Gln
Ser Leu Gly Leu Leu Ile Gly Ala Ala Ser Thr Ser 545 550
555 560 Leu Gln Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu 565 570
575 Leu Phe Ser Gly Phe Phe Val Ser Phe Asp Thr Ile Pro Thr
Tyr Leu 580 585 590
Gln Trp Met Ser Tyr Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val
595 600 605 Ile Leu Ser Ile
Tyr Gly Leu Asp Arg Glu Asp Leu His Cys Asp Ile 610
615 620 Asp Glu Thr Cys His Phe Gln Lys
Ser Glu Ala Ile Leu Arg Glu Leu 625 630
635 640 Asp Val Glu Asn Ala Lys Leu Tyr Leu Asp Phe Ile
Val Leu Gly Ile 645 650
655 Phe Phe Ile Ser Leu Arg Leu Ile Ala Tyr Phe Val Leu Arg Tyr Lys
660 665 670 Ile Arg Ala
Glu Arg 675 72668PRTHomo sapiens 72Met Leu Gly Thr Gln
Gly Trp Thr Lys Gln Arg Lys Pro Cys Pro Gln 1 5
10 15 Asn Ala Ser Ser Tyr Ser Ala Glu Met Thr
Glu Pro Lys Ser Val Cys 20 25
30 Val Ser Val Asp Glu Val Val Ser Ser Asn Met Glu Ala Thr Glu
Thr 35 40 45 Asp
Leu Leu Asn Gly His Leu Lys Lys Val Asp Asn Asn Leu Thr Glu 50
55 60 Ala Gln Arg Phe Ser Ser
Leu Pro Arg Arg Ala Ala Val Asn Ile Glu 65 70
75 80 Phe Arg Asp Leu Ser Tyr Ser Val Pro Glu Gly
Pro Trp Trp Arg Lys 85 90
95 Lys Gly Tyr Lys Thr Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser
100 105 110 Gly Glu
Leu Val Ala Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr 115
120 125 Leu Met Asn Ile Leu Ala Gly
Tyr Arg Glu Thr Gly Met Lys Gly Ala 130 135
140 Val Leu Ile Asn Gly Leu Pro Arg Asp Leu Arg Cys
Phe Arg Lys Val 145 150 155
160 Ser Cys Tyr Ile Met Gln Asp Asp Met Leu Leu Pro His Leu Thr Val
165 170 175 Gln Glu Ala
Met Met Val Ser Ala His Leu Lys Leu Gln Glu Lys Asp 180
185 190 Glu Gly Arg Arg Glu Met Val Lys
Glu Ile Leu Thr Ala Leu Gly Leu 195 200
205 Leu Ser Cys Ala Asn Thr Arg Thr Gly Ser Leu Ser Gly
Gly Gln Arg 210 215 220
Lys Arg Leu Ala Ile Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met 225
230 235 240 Phe Phe Asp Glu
Pro Thr Ser Gly Leu Asp Ser Ala Ser Cys Phe Gln 245
250 255 Val Val Ser Leu Met Lys Gly Leu Ala
Gln Gly Gly Arg Ser Ile Ile 260 265
270 Cys Thr Ile His Gln Pro Ser Ala Lys Leu Phe Glu Leu Phe
Asp Gln 275 280 285
Leu Tyr Val Leu Ser Gln Gly Gln Cys Val Tyr Arg Gly Lys Val Cys 290
295 300 Asn Leu Val Pro Tyr
Leu Arg Asp Leu Gly Leu Asn Cys Pro Thr Tyr 305 310
315 320 His Asn Pro Ala Asp Phe Val Met Glu Val
Ala Ser Gly Glu Tyr Gly 325 330
335 Asp Gln Asn Ser Arg Leu Val Arg Ala Val Arg Glu Gly Met Cys
Asp 340 345 350 Ser
Asp His Lys Arg Asp Leu Gly Gly Asp Ala Glu Val Asn Pro Phe 355
360 365 Leu Trp His Arg Pro Ser
Glu Glu Asp Ser Ser Ser Met Glu Gly Cys 370 375
380 His Ser Phe Ser Ala Ser Cys Leu Thr Gln Phe
Cys Ile Leu Phe Lys 385 390 395
400 Arg Thr Phe Leu Ser Ile Met Arg Asp Ser Val Leu Thr His Leu Arg
405 410 415 Ile Thr
Ser His Ile Gly Ile Gly Leu Leu Ile Gly Leu Leu Tyr Leu 420
425 430 Gly Ile Gly Asn Glu Ala Lys
Lys Val Leu Ser Asn Ser Gly Phe Leu 435 440
445 Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu
Met Pro Thr Val 450 455 460
Leu Thr Phe Pro Leu Glu Met Gly Val Phe Leu Arg Glu His Leu Asn 465
470 475 480 Tyr Trp Tyr
Ser Leu Lys Ala Tyr Tyr Leu Ala Lys Thr Met Ala Asp 485
490 495 Val Pro Phe Gln Ile Met Phe Pro
Val Ala Tyr Cys Ser Ile Val Tyr 500 505
510 Trp Met Thr Ser Gln Pro Ser Asp Ala Val Arg Phe Val
Leu Phe Ala 515 520 525
Ala Leu Gly Thr Met Thr Ser Leu Val Ala Gln Ser Leu Gly Leu Leu 530
535 540 Ile Gly Ala Ala
Ser Thr Ser Leu Gln Val Ala Thr Phe Val Gly Pro 545 550
555 560 Val Thr Ala Ile Pro Val Leu Leu Phe
Ser Gly Phe Phe Val Ser Phe 565 570
575 Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met Ser Tyr Ile Ser
Tyr Val 580 585 590
Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile Tyr Gly Leu Asp Arg
595 600 605 Glu Asp Leu His
Cys Asp Ile Asp Glu Thr Cys His Phe Gln Lys Ser 610
615 620 Glu Ala Ile Leu Arg Glu Leu Asp
Val Glu Asn Ala Lys Leu Tyr Leu 625 630
635 640 Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg Leu Ile Ala 645 650
655 Tyr Phe Val Leu Arg Tyr Lys Ile Arg Ala Glu Arg 660
665 73644PRTHomo sapiens 73Met Thr Glu Pro Lys
Ser Val Cys Val Ser Val Asp Glu Val Val Ser 1 5
10 15 Ser Asn Met Glu Ala Thr Glu Thr Asp Leu
Leu Asn Gly His Leu Lys 20 25
30 Lys Val Asp Asn Asn Leu Thr Glu Ala Gln Arg Phe Ser Ser Leu
Pro 35 40 45 Arg
Arg Ala Ala Val Asn Ile Glu Phe Arg Asp Leu Ser Tyr Ser Val 50
55 60 Pro Glu Gly Pro Trp Trp
Arg Lys Lys Gly Tyr Lys Thr Leu Leu Lys 65 70
75 80 Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu Leu
Val Ala Ile Met Gly 85 90
95 Pro Ser Gly Ala Gly Lys Ser Thr Leu Met Asn Ile Leu Ala Gly Tyr
100 105 110 Arg Glu
Thr Gly Met Lys Gly Ala Val Leu Ile Asn Gly Leu Pro Arg 115
120 125 Asp Leu Arg Cys Phe Arg Lys
Val Ser Cys Tyr Ile Met Gln Asp Asp 130 135
140 Met Leu Leu Pro His Leu Thr Val Gln Glu Ala Met
Met Val Ser Ala 145 150 155
160 His Leu Lys Leu Gln Glu Lys Asp Glu Gly Arg Arg Glu Met Val Lys
165 170 175 Glu Ile Leu
Thr Ala Leu Gly Leu Leu Ser Cys Ala Asn Thr Arg Thr 180
185 190 Gly Ser Leu Ser Gly Gly Gln Arg
Lys Arg Leu Ala Ile Ala Leu Glu 195 200
205 Leu Val Asn Asn Pro Pro Val Met Phe Phe Asp Glu Pro
Thr Ser Gly 210 215 220
Leu Asp Ser Ala Ser Cys Phe Gln Val Val Ser Leu Met Lys Gly Leu 225
230 235 240 Ala Gln Gly Gly
Arg Ser Ile Ile Cys Thr Ile His Gln Pro Ser Ala 245
250 255 Lys Leu Phe Glu Leu Phe Asp Gln Leu
Tyr Val Leu Ser Gln Gly Gln 260 265
270 Cys Val Tyr Arg Gly Lys Val Cys Asn Leu Val Pro Tyr Leu
Arg Asp 275 280 285
Leu Gly Leu Asn Cys Pro Thr Tyr His Asn Pro Ala Asp Phe Val Met 290
295 300 Glu Val Ala Ser Gly
Glu Tyr Gly Asp Gln Asn Ser Arg Leu Val Arg 305 310
315 320 Ala Val Arg Glu Gly Met Cys Asp Ser Asp
His Lys Arg Asp Leu Gly 325 330
335 Gly Asp Ala Glu Val Asn Pro Phe Leu Trp His Arg Pro Ser Glu
Glu 340 345 350 Asp
Ser Ser Ser Met Glu Gly Cys His Ser Phe Ser Ala Ser Cys Leu 355
360 365 Thr Gln Phe Cys Ile Leu
Phe Lys Arg Thr Phe Leu Ser Ile Met Arg 370 375
380 Asp Ser Val Leu Thr His Leu Arg Ile Thr Ser
His Ile Gly Ile Gly 385 390 395
400 Leu Leu Ile Gly Leu Leu Tyr Leu Gly Ile Gly Asn Glu Ala Lys Lys
405 410 415 Val Leu
Ser Asn Ser Gly Phe Leu Phe Phe Ser Met Leu Phe Leu Met 420
425 430 Phe Ala Ala Leu Met Pro Thr
Val Leu Thr Phe Pro Leu Glu Met Gly 435 440
445 Val Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser
Leu Lys Ala Tyr 450 455 460
Tyr Leu Ala Lys Thr Met Ala Asp Val Pro Phe Gln Ile Met Phe Pro 465
470 475 480 Val Ala Tyr
Cys Ser Ile Val Tyr Trp Met Thr Ser Gln Pro Ser Asp 485
490 495 Ala Val Arg Phe Val Leu Phe Ala
Ala Leu Gly Thr Met Thr Ser Leu 500 505
510 Val Ala Gln Ser Leu Gly Leu Leu Ile Gly Ala Ala Ser
Thr Ser Leu 515 520 525
Gln Val Ala Thr Phe Val Gly Pro Val Thr Ala Ile Pro Val Leu Leu 530
535 540 Phe Ser Gly Phe
Phe Val Ser Phe Asp Thr Ile Pro Thr Tyr Leu Gln 545 550
555 560 Trp Met Ser Tyr Ile Ser Tyr Val Arg
Tyr Gly Phe Glu Gly Val Ile 565 570
575 Leu Ser Ile Tyr Gly Leu Asp Arg Glu Asp Leu His Cys Asp
Ile Asp 580 585 590
Glu Thr Cys His Phe Gln Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp
595 600 605 Val Glu Asn Ala
Lys Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe 610
615 620 Phe Ile Ser Leu Arg Leu Ile Ala
Tyr Phe Val Leu Arg Tyr Lys Ile 625 630
635 640 Arg Ala Glu Arg 74663PRTHomo sapiens 74Met Ile
Met Arg Leu Pro Gln Pro His Gly Thr Asn Ala Ser Ser Tyr 1 5
10 15 Ser Ala Glu Met Thr Glu Pro
Lys Ser Val Cys Val Ser Val Asp Glu 20 25
30 Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr Asp
Leu Leu Asn Gly 35 40 45
His Leu Lys Lys Val Asp Asn Asn Leu Thr Glu Ala Gln Arg Phe Ser
50 55 60 Ser Leu Pro
Arg Arg Ala Ala Val Asn Ile Glu Phe Arg Asp Leu Ser 65
70 75 80 Tyr Ser Val Pro Glu Gly Pro
Trp Trp Arg Lys Lys Gly Tyr Lys Thr 85
90 95 Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser
Gly Glu Leu Val Ala 100 105
110 Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr Leu Met Asn Ile
Leu 115 120 125 Ala
Gly Tyr Arg Glu Thr Gly Met Lys Gly Ala Val Leu Ile Asn Gly 130
135 140 Leu Pro Arg Asp Leu Arg
Cys Phe Arg Lys Val Ser Cys Tyr Ile Met 145 150
155 160 Gln Asp Asp Met Leu Leu Pro His Leu Thr Val
Gln Glu Ala Met Met 165 170
175 Val Ser Ala His Leu Lys Leu Gln Glu Lys Asp Glu Gly Arg Arg Glu
180 185 190 Met Val
Lys Glu Ile Leu Thr Ala Leu Gly Leu Leu Ser Cys Ala Asn 195
200 205 Thr Arg Thr Gly Ser Leu Ser
Gly Gly Gln Arg Lys Arg Leu Ala Ile 210 215
220 Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met Phe
Phe Asp Glu Pro 225 230 235
240 Thr Ser Gly Leu Asp Ser Ala Ser Cys Phe Gln Val Val Ser Leu Met
245 250 255 Lys Gly Leu
Ala Gln Gly Gly Arg Ser Ile Ile Cys Thr Ile His Gln 260
265 270 Pro Ser Ala Lys Leu Phe Glu Leu
Phe Asp Gln Leu Tyr Val Leu Ser 275 280
285 Gln Gly Gln Cys Val Tyr Arg Gly Lys Val Cys Asn Leu
Val Pro Tyr 290 295 300
Leu Arg Asp Leu Gly Leu Asn Cys Pro Thr Tyr His Asn Pro Ala Asp 305
310 315 320 Phe Val Met Glu
Val Ala Ser Gly Glu Tyr Gly Asp Gln Asn Ser Arg 325
330 335 Leu Val Arg Ala Val Arg Glu Gly Met
Cys Asp Ser Asp His Lys Arg 340 345
350 Asp Leu Gly Gly Asp Ala Glu Val Asn Pro Phe Leu Trp His
Arg Pro 355 360 365
Ser Glu Glu Asp Ser Ser Ser Met Glu Gly Cys His Ser Phe Ser Ala 370
375 380 Ser Cys Leu Thr Gln
Phe Cys Ile Leu Phe Lys Arg Thr Phe Leu Ser 385 390
395 400 Ile Met Arg Asp Ser Val Leu Thr His Leu
Arg Ile Thr Ser His Ile 405 410
415 Gly Ile Gly Leu Leu Ile Gly Leu Leu Tyr Leu Gly Ile Gly Asn
Glu 420 425 430 Ala
Lys Lys Val Leu Ser Asn Ser Gly Phe Leu Phe Phe Ser Met Leu 435
440 445 Phe Leu Met Phe Ala Ala
Leu Met Pro Thr Val Leu Thr Phe Pro Leu 450 455
460 Glu Met Gly Val Phe Leu Arg Glu His Leu Asn
Tyr Trp Tyr Ser Leu 465 470 475
480 Lys Ala Tyr Tyr Leu Ala Lys Thr Met Ala Asp Val Pro Phe Gln Ile
485 490 495 Met Phe
Pro Val Ala Tyr Cys Ser Ile Val Tyr Trp Met Thr Ser Gln 500
505 510 Pro Ser Asp Ala Val Arg Phe
Val Leu Phe Ala Ala Leu Gly Thr Met 515 520
525 Thr Ser Leu Val Ala Gln Ser Leu Gly Leu Leu Ile
Gly Ala Ala Ser 530 535 540
Thr Ser Leu Gln Val Ala Thr Phe Val Gly Pro Val Thr Ala Ile Pro 545
550 555 560 Val Leu Leu
Phe Ser Gly Phe Phe Val Ser Phe Asp Thr Ile Pro Thr 565
570 575 Tyr Leu Gln Trp Met Ser Tyr Ile
Ser Tyr Val Arg Tyr Gly Phe Glu 580 585
590 Gly Val Ile Leu Ser Ile Tyr Gly Leu Asp Arg Glu Asp
Leu His Cys 595 600 605
Asp Ile Asp Glu Thr Cys His Phe Gln Lys Ser Glu Ala Ile Leu Arg 610
615 620 Glu Leu Asp Val
Glu Asn Ala Lys Leu Tyr Leu Asp Phe Ile Val Leu 625 630
635 640 Gly Ile Phe Phe Ile Ser Leu Arg Leu
Ile Ala Tyr Phe Val Leu Arg 645 650
655 Tyr Lys Ile Arg Ala Glu Arg 660
75824PRTHomo sapiens 75Met Leu Ala Val Gln Gln Thr Glu His Leu Pro Ala
Cys Pro Pro Ala 1 5 10
15 Arg Arg Trp Ser Ser Asn Phe Cys Pro Glu Ser Thr Glu Gly Gly Pro
20 25 30 Ser Leu Leu
Gly Leu Arg Asp Met Val Arg Arg Gly Trp Ser Val Cys 35
40 45 Thr Ala Ile Leu Leu Ala Arg Leu
Trp Cys Leu Val Pro Thr His Thr 50 55
60 Phe Leu Ser Glu Tyr Pro Glu Ala Ala Glu Tyr Pro His
Pro Gly Trp 65 70 75
80 Val Tyr Trp Leu Gln Met Ala Val Ala Pro Gly His Leu Arg Ala Trp
85 90 95 Val Met Arg Asn
Asn Val Thr Thr Asn Ile Pro Ser Ala Phe Ser Gly 100
105 110 Thr Leu Thr His Glu Glu Lys Ala Val
Leu Thr Val Phe Thr Gly Thr 115 120
125 Ala Thr Ala Val His Val Gln Val Ala Ala Leu Ala Ser Ala
Lys Leu 130 135 140
Glu Ser Ser Val Phe Val Thr Asp Cys Val Ser Cys Lys Ile Glu Asn 145
150 155 160 Val Cys Asp Ser Ala
Leu Gln Gly Lys Arg Val Pro Met Ser Gly Leu 165
170 175 Gln Gly Ser Ser Ile Val Ile Met Pro Pro
Ser Asn Arg Pro Leu Ala 180 185
190 Ser Ala Ala Ser Cys Thr Trp Ser Val Gln Val Gln Gly Gly Pro
His 195 200 205 His
Leu Gly Val Val Ala Ile Ser Gly Lys Val Leu Ser Ala Ala His 210
215 220 Gly Ala Gly Arg Ala Tyr
Gly Trp Gly Phe Pro Gly Asp Pro Met Glu 225 230
235 240 Glu Gly Tyr Lys Thr Leu Leu Lys Gly Ile Ser
Gly Lys Phe Asn Ser 245 250
255 Gly Glu Leu Val Ala Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr
260 265 270 Leu Met
Asn Ile Leu Ala Gly Tyr Arg Glu Thr Gly Met Lys Gly Ala 275
280 285 Val Leu Ile Asn Gly Leu Pro
Arg Asp Leu Arg Cys Phe Arg Lys Val 290 295
300 Ser Cys Tyr Ile Met Gln Asp Asp Met Leu Leu Pro
His Leu Thr Val 305 310 315
320 Gln Glu Ala Met Met Val Ser Ala His Leu Lys Leu Gln Glu Lys Asp
325 330 335 Glu Gly Arg
Arg Glu Met Val Lys Glu Ile Leu Thr Ala Leu Gly Leu 340
345 350 Leu Ser Cys Ala Asn Thr Arg Thr
Gly Ser Leu Ser Gly Gly Gln Arg 355 360
365 Lys Arg Leu Ala Ile Ala Leu Glu Leu Val Asn Asn Pro
Pro Val Met 370 375 380
Phe Phe Asp Glu Pro Thr Ser Gly Leu Asp Ser Ala Ser Cys Phe Gln 385
390 395 400 Val Val Ser Leu
Met Lys Gly Leu Ala Gln Gly Gly Arg Ser Ile Ile 405
410 415 Cys Thr Ile His Gln Pro Ser Ala Lys
Leu Phe Glu Leu Phe Asp Gln 420 425
430 Leu Tyr Val Leu Ser Gln Gly Gln Cys Val Tyr Arg Gly Lys
Val Cys 435 440 445
Asn Leu Val Pro Tyr Leu Arg Asp Leu Gly Leu Asn Cys Pro Thr Tyr 450
455 460 His Asn Pro Ala Asp
Phe Val Met Glu Val Ala Ser Gly Glu Tyr Gly 465 470
475 480 Asp Gln Asn Ser Arg Leu Val Arg Ala Val
Arg Glu Gly Met Cys Asp 485 490
495 Ser Asp His Lys Arg Asp Leu Gly Gly Asp Ala Glu Val Asn Pro
Phe 500 505 510 Leu
Trp His Arg Pro Ser Glu Glu Val Lys Gln Thr Lys Arg Leu Lys 515
520 525 Gly Leu Arg Lys Asp Ser
Ser Ser Met Glu Gly Cys His Ser Phe Ser 530 535
540 Ala Ser Cys Leu Thr Gln Phe Cys Ile Leu Phe
Lys Arg Thr Phe Leu 545 550 555
560 Ser Ile Met Arg Asp Ser Val Leu Thr His Leu Arg Ile Thr Ser His
565 570 575 Ile Gly
Ile Gly Leu Leu Ile Gly Leu Leu Tyr Leu Gly Ile Gly Asn 580
585 590 Glu Ala Lys Lys Val Leu Ser
Asn Ser Gly Phe Leu Phe Phe Ser Met 595 600
605 Leu Phe Leu Met Phe Ala Ala Leu Met Pro Thr Val
Leu Thr Phe Pro 610 615 620
Leu Glu Met Gly Val Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser 625
630 635 640 Leu Lys Ala
Tyr Tyr Leu Ala Lys Thr Met Ala Asp Val Pro Phe Gln 645
650 655 Ile Met Phe Pro Val Ala Tyr Cys
Ser Ile Val Tyr Trp Met Thr Ser 660 665
670 Gln Pro Ser Asp Ala Val Arg Phe Val Leu Phe Ala Ala
Leu Gly Thr 675 680 685
Met Thr Ser Leu Val Ala Gln Ser Leu Gly Leu Leu Ile Gly Ala Ala 690
695 700 Ser Thr Ser Leu
Gln Val Ala Thr Phe Val Gly Pro Val Thr Ala Ile 705 710
715 720 Pro Val Leu Leu Phe Ser Gly Phe Phe
Val Ser Phe Asp Thr Ile Pro 725 730
735 Thr Tyr Leu Gln Trp Met Ser Tyr Ile Ser Tyr Val Arg Tyr
Gly Phe 740 745 750
Glu Gly Val Ile Leu Ser Ile Tyr Gly Leu Asp Arg Glu Asp Leu His
755 760 765 Cys Asp Ile Asp
Glu Thr Cys His Phe Gln Lys Ser Glu Ala Ile Leu 770
775 780 Arg Glu Leu Asp Val Glu Asn Ala
Lys Leu Tyr Leu Asp Phe Ile Val 785 790
795 800 Leu Gly Ile Phe Phe Ile Ser Leu Arg Leu Ile Ala
Tyr Phe Val Leu 805 810
815 Arg Tyr Lys Ile Arg Ala Glu Arg 820
76970PRTHomo sapiens 76Met Ala Gln Leu Glu Arg Ser Ala Ile Ser Gly Phe
Ser Ser Lys Ser 1 5 10
15 Arg Arg Asn Ser Phe Ala Tyr Asp Val Lys Arg Glu Val Tyr Asn Glu
20 25 30 Glu Thr Phe
Gln Gln Glu His Lys Arg Lys Ala Ser Ser Ser Gly Asn 35
40 45 Met Asn Ile Asn Ile Thr Thr Phe
Arg His His Val Gln Cys Arg Cys 50 55
60 Ser Trp His Arg Phe Leu Arg Cys Val Leu Thr Ile Phe
Pro Phe Leu 65 70 75
80 Glu Trp Met Cys Met Tyr Arg Leu Lys Asp Trp Leu Leu Gly Asp Leu
85 90 95 Leu Ala Gly Ile
Ser Val Gly Leu Val Gln Val Pro Gln Gly Leu Thr 100
105 110 Leu Ser Leu Leu Ala Arg Gln Leu Ile
Pro Pro Leu Asn Ile Ala Tyr 115 120
125 Ala Ala Phe Cys Ser Ser Val Ile Tyr Val Ile Phe Gly Ser
Cys His 130 135 140
Gln Met Ser Ile Gly Ser Phe Phe Leu Val Ser Ala Leu Leu Ile Asn 145
150 155 160 Val Leu Lys Val Ser
Pro Phe Asn Asn Gly Gln Leu Val Met Gly Ser 165
170 175 Phe Val Lys Asn Glu Phe Ser Ala Pro Ser
Tyr Leu Met Gly Tyr Asn 180 185
190 Lys Ser Leu Ser Val Val Ala Thr Thr Thr Phe Leu Thr Gly Ile
Ile 195 200 205 Gln
Leu Ile Met Gly Val Leu Gly Leu Gly Phe Ile Ala Thr Tyr Leu 210
215 220 Pro Glu Ser Ala Met Ser
Ala Tyr Leu Ala Ala Val Ala Leu His Ile 225 230
235 240 Met Leu Ser Gln Leu Thr Phe Ile Phe Gly Ile
Met Ile Ser Phe His 245 250
255 Ala Gly Pro Ile Ser Phe Phe Tyr Asp Ile Ile Asn Tyr Cys Val Ala
260 265 270 Leu Pro
Lys Ala Asn Ser Thr Ser Ile Leu Val Phe Leu Thr Val Val 275
280 285 Val Ala Leu Arg Ile Asn Lys
Cys Ile Arg Ile Ser Phe Asn Gln Tyr 290 295
300 Pro Ile Glu Phe Pro Met Glu Leu Phe Leu Ile Ile
Gly Phe Thr Val 305 310 315
320 Ile Ala Asn Lys Ile Ser Met Ala Thr Glu Thr Ser Gln Thr Leu Ile
325 330 335 Asp Met Ile
Pro Tyr Ser Phe Leu Leu Pro Val Thr Pro Asp Phe Ser 340
345 350 Leu Leu Pro Lys Ile Ile Leu Gln
Ala Phe Ser Leu Ser Leu Val Ser 355 360
365 Ser Phe Leu Leu Ile Phe Leu Gly Lys Lys Ile Ala Ser
Leu His Asn 370 375 380
Tyr Ser Val Asn Ser Asn Gln Asp Leu Ile Ala Ile Gly Leu Cys Asn 385
390 395 400 Val Val Ser Ser
Phe Phe Arg Ser Cys Val Phe Thr Gly Ala Ile Ala 405
410 415 Arg Thr Ile Ile Gln Asp Lys Ser Gly
Gly Arg Gln Gln Phe Ala Ser 420 425
430 Leu Val Gly Ala Gly Val Met Leu Leu Leu Met Val Lys Met
Gly His 435 440 445
Phe Phe Tyr Thr Leu Pro Asn Ala Val Leu Ala Gly Ile Ile Leu Ser 450
455 460 Asn Val Ile Pro Tyr
Leu Glu Thr Ile Ser Asn Leu Pro Ser Leu Trp 465 470
475 480 Arg Gln Asp Gln Tyr Asp Cys Ala Leu Trp
Met Met Thr Phe Ser Ser 485 490
495 Ser Ile Phe Leu Gly Leu Asp Ile Gly Leu Ile Ile Ser Val Val
Ser 500 505 510 Ala
Phe Phe Ile Thr Thr Val Arg Ser His Arg Ala Lys Ile Leu Leu 515
520 525 Leu Gly Gln Ile Pro Asn
Thr Asn Ile Tyr Arg Ser Ile Asn Asp Tyr 530 535
540 Arg Glu Ile Ile Thr Ile Pro Gly Val Lys Ile
Phe Gln Cys Cys Ser 545 550 555
560 Ser Ile Thr Phe Val Asn Val Tyr Tyr Leu Lys His Lys Leu Leu Lys
565 570 575 Glu Val
Asp Met Val Lys Val Pro Leu Lys Glu Glu Glu Ile Phe Ser 580
585 590 Leu Phe Asn Ser Ser Asp Thr
Asn Leu Gln Gly Gly Lys Ile Cys Arg 595 600
605 Cys Phe Cys Asn Cys Asp Asp Leu Glu Pro Leu Pro
Arg Ile Leu Tyr 610 615 620
Thr Glu Arg Phe Glu Asn Lys Leu Asp Pro Glu Ala Ser Ser Ile Asn 625
630 635 640 Leu Ile His
Cys Ser His Phe Glu Ser Met Asn Thr Ser Gln Thr Ala 645
650 655 Ser Glu Asp Gln Val Pro Tyr Thr
Val Ser Ser Val Ser Gln Lys Asn 660 665
670 Gln Gly Gln Gln Tyr Glu Glu Val Glu Glu Val Trp Leu
Pro Asn Asn 675 680 685
Ser Ser Arg Asn Ser Ser Pro Gly Leu Pro Asp Val Ala Glu Ser Gln 690
695 700 Gly Arg Arg Ser
Leu Ile Pro Tyr Ser Asp Ala Ser Leu Leu Pro Ser 705 710
715 720 Val His Thr Ile Ile Leu Asp Phe Ser
Met Val His Tyr Val Asp Ser 725 730
735 Arg Gly Leu Val Val Leu Arg Gln Ile Cys Asn Ala Phe Gln
Asn Ala 740 745 750
Asn Ile Leu Ile Leu Ile Ala Gly Cys His Ser Ser Ile Val Arg Ala
755 760 765 Phe Glu Arg Asn
Asp Phe Phe Asp Ala Gly Ile Thr Lys Thr Gln Leu 770
775 780 Phe Leu Ser Val His Asp Ala Val
Leu Phe Ala Leu Ser Arg Lys Val 785 790
795 800 Ile Gly Ser Ser Glu Leu Ser Ile Asp Glu Ser Glu
Thr Val Ile Arg 805 810
815 Glu Thr Tyr Ser Glu Thr Asp Lys Asn Asp Asn Ser Arg Tyr Lys Met
820 825 830 Ser Ser Ser
Phe Leu Gly Ser Gln Lys Asn Val Ser Pro Gly Phe Ile 835
840 845 Lys Ile Gln Gln Pro Val Glu Glu
Glu Ser Glu Leu Asp Leu Glu Leu 850 855
860 Glu Ser Glu Gln Glu Ala Gly Leu Gly Leu Asp Leu Asp
Leu Asp Arg 865 870 875
880 Glu Leu Glu Pro Glu Met Glu Pro Lys Ala Glu Thr Glu Thr Lys Thr
885 890 895 Gln Thr Glu Met
Glu Pro Gln Pro Glu Thr Glu Pro Glu Met Glu Pro 900
905 910 Asn Pro Lys Ser Arg Pro Arg Ala His
Thr Phe Pro Gln Gln Arg Tyr 915 920
925 Trp Pro Met Tyr His Pro Ser Met Ala Ser Thr Gln Ser Gln
Thr Gln 930 935 940
Thr Arg Thr Trp Ser Val Glu Arg Arg Arg His Pro Met Asp Ser Tyr 945
950 955 960 Ser Pro Glu Gly Asn
Ser Asn Glu Asp Val 965 970 77225PRTHomo
sapiens 77Met Thr Ser Glu Phe Phe Ser Ala Gln Leu Arg Ala Gln Ile Ser Asp
1 5 10 15 Asp Thr
Thr His Pro Ile Ser Tyr Tyr Lys Pro Glu Phe Tyr Met Pro 20
25 30 Asp Asp Gly Gly Thr Ala His
Leu Ser Val Val Ala Glu Asp Gly Ser 35 40
45 Ala Val Ser Ala Thr Ser Thr Ile Asn Leu Tyr Phe
Gly Ser Lys Val 50 55 60
Arg Ser Pro Val Ser Gly Ile Leu Leu Asn Asn Glu Met Asp Asp Phe 65
70 75 80 Ser Ser Thr
Ser Ile Thr Asn Glu Phe Gly Val Pro Pro Ser Pro Ala 85
90 95 Asn Phe Ile Gln Pro Gly Lys Gln
Pro Leu Ser Ser Met Cys Pro Thr 100 105
110 Ile Met Val Gly Gln Asp Gly Gln Val Arg Met Val Val
Gly Ala Ala 115 120 125
Gly Gly Thr Gln Ile Thr Met Ala Thr Ala Leu Ala Ile Ile Tyr Asn 130
135 140 Leu Trp Phe Gly
Tyr Asp Val Lys Trp Ala Val Glu Glu Pro Arg Leu 145 150
155 160 His Asn Gln Leu Leu Pro Asn Val Thr
Thr Val Glu Arg Asn Ile Asp 165 170
175 Gln Glu Val Thr Ala Ala Leu Glu Thr Arg His His His Thr
Gln Ile 180 185 190
Thr Ser Thr Phe Ile Ala Val Val Gln Ala Ile Val Arg Met Ala Gly
195 200 205 Gly Trp Ala Ala
Ala Ser Asp Ser Arg Lys Gly Gly Glu Pro Ala Gly 210
215 220 Tyr 225 78225PRTHomo sapiens
78Met Thr Ser Glu Phe Phe Ser Ala Gln Leu Arg Ala Gln Ile Ser Asp 1
5 10 15 Asp Thr Thr His
Pro Ile Ser Tyr Tyr Lys Pro Glu Phe Tyr Met Pro 20
25 30 Asp Asp Gly Gly Thr Ala His Leu Ser
Val Val Ala Glu Asp Gly Ser 35 40
45 Ala Val Ser Ala Thr Ser Thr Ile Asn Leu Tyr Phe Gly Ser
Lys Val 50 55 60
Arg Ser Pro Val Ser Gly Ile Leu Leu Asn Asn Glu Met Asp Asp Phe 65
70 75 80 Ser Ser Thr Ser Ile
Thr Asn Glu Phe Gly Val Pro Pro Ser Pro Ala 85
90 95 Asn Phe Ile Gln Pro Gly Lys Gln Pro Leu
Ser Ser Met Cys Pro Thr 100 105
110 Ile Met Val Gly Gln Asp Gly Gln Val Arg Met Val Val Gly Ala
Ala 115 120 125 Gly
Gly Thr Gln Ile Thr Met Ala Thr Ala Leu Ala Ile Ile Tyr Asn 130
135 140 Leu Trp Phe Gly Tyr Asp
Val Lys Trp Ala Val Glu Glu Pro Arg Leu 145 150
155 160 His Asn Gln Leu Leu Pro Asn Val Thr Thr Val
Glu Arg Asn Ile Asp 165 170
175 Gln Glu Val Thr Ala Ala Leu Glu Thr Arg His His His Thr Gln Ile
180 185 190 Thr Ser
Thr Phe Ile Ala Val Val Gln Ala Ile Val Arg Met Ala Gly 195
200 205 Gly Trp Ala Ala Ala Ser Asp
Ser Arg Lys Gly Gly Glu Pro Ala Gly 210 215
220 Tyr 225 79225PRTHomo sapiens 79Met Thr Ser Glu
Phe Phe Ser Ala Gln Leu Arg Ala Gln Ile Ser Asp 1 5
10 15 Asp Thr Thr His Pro Ile Ser Tyr Tyr
Lys Pro Glu Phe Tyr Met Pro 20 25
30 Asp Asp Gly Gly Thr Ala His Leu Ser Val Val Ala Glu Asp
Gly Ser 35 40 45
Ala Val Ser Ala Thr Ser Thr Ile Asn Leu Tyr Phe Gly Ser Lys Val 50
55 60 Arg Ser Pro Val Ser
Gly Ile Leu Leu Asn Asn Glu Met Asp Asp Phe 65 70
75 80 Ser Ser Thr Ser Ile Thr Asn Glu Phe Gly
Val Pro Pro Ser Pro Ala 85 90
95 Asn Phe Ile Gln Pro Gly Lys Gln Pro Leu Ser Ser Met Cys Pro
Thr 100 105 110 Ile
Met Val Gly Gln Asp Gly Gln Val Arg Met Val Val Gly Ala Ala 115
120 125 Gly Gly Thr Gln Ile Thr
Met Ala Thr Ala Leu Ala Ile Ile Tyr Asn 130 135
140 Leu Trp Phe Gly Tyr Asp Val Lys Trp Ala Val
Glu Glu Pro Arg Leu 145 150 155
160 His Asn Gln Leu Leu Pro Asn Val Thr Thr Val Glu Arg Asn Ile Asp
165 170 175 Gln Glu
Val Thr Ala Ala Leu Glu Thr Arg His His His Thr Gln Ile 180
185 190 Thr Ser Thr Phe Ile Ala Val
Val Gln Ala Ile Val Arg Met Ala Gly 195 200
205 Gly Trp Ala Ala Ala Ser Asp Ser Arg Lys Gly Gly
Glu Pro Ala Gly 210 215 220
Tyr 225 80545PRTHomo sapiens 80Met Gly Ser Ser Ala Thr Glu Ile Glu
Glu Leu Glu Asn Thr Thr Phe 1 5 10
15 Lys Tyr Leu Thr Gly Glu Gln Thr Glu Lys Met Trp Gln Arg
Leu Lys 20 25 30
Gly Ile Leu Arg Cys Leu Val Lys Gln Leu Glu Arg Gly Asp Val Asn
35 40 45 Val Val Asp Leu
Lys Lys Asn Ile Glu Tyr Ala Ala Ser Val Leu Glu 50
55 60 Ala Val Tyr Ile Asp Glu Thr Arg
Arg Leu Leu Asp Thr Glu Asp Glu 65 70
75 80 Leu Ser Asp Ile Gln Thr Asp Ser Val Pro Ser Glu
Val Arg Asp Trp 85 90
95 Leu Ala Ser Thr Phe Thr Arg Lys Met Gly Met Thr Lys Lys Lys Pro
100 105 110 Glu Glu Lys
Pro Lys Phe Arg Ser Ile Val His Ala Val Gln Ala Gly 115
120 125 Ile Phe Val Glu Arg Met Tyr Arg
Lys Thr Tyr His Met Val Gly Leu 130 135
140 Ala Tyr Pro Ala Ala Val Ile Val Thr Leu Lys Asp Val
Asp Lys Trp 145 150 155
160 Ser Phe Asp Val Phe Ala Leu Asn Glu Ala Ser Gly Glu His Ser Leu
165 170 175 Lys Phe Met Ile
Tyr Glu Leu Phe Thr Arg Tyr Asp Leu Ile Asn Arg 180
185 190 Phe Lys Ile Pro Val Ser Cys Leu Ile
Thr Phe Ala Glu Ala Leu Glu 195 200
205 Val Gly Tyr Ser Lys Tyr Lys Asn Pro Tyr His Asn Leu Ile
His Ala 210 215 220
Ala Asp Val Thr Gln Thr Val His Tyr Ile Met Leu His Thr Gly Ile 225
230 235 240 Met His Trp Leu Thr
Glu Leu Glu Ile Leu Ala Met Val Phe Ala Ala 245
250 255 Ala Ile His Asp Tyr Glu His Thr Gly Thr
Thr Asn Asn Phe His Ile 260 265
270 Gln Thr Arg Ser Asp Val Ala Ile Leu Tyr Asn Asp Arg Ser Val
Leu 275 280 285 Glu
Asn His His Val Ser Ala Ala Tyr Arg Leu Met Gln Glu Glu Glu 290
295 300 Met Asn Ile Leu Ile Asn
Leu Ser Lys Asp Asp Trp Arg Asp Leu Arg 305 310
315 320 Asn Leu Val Ile Glu Met Val Leu Ser Thr Asp
Met Ser Gly His Phe 325 330
335 Gln Gln Ile Lys Asn Ile Arg Asn Ser Leu Gln Gln Pro Glu Gly Ile
340 345 350 Asp Arg
Ala Lys Thr Met Ser Leu Ile Leu His Ala Ala Asp Ile Ser 355
360 365 His Pro Ala Lys Ser Trp Lys
Leu His Tyr Arg Trp Thr Met Ala Leu 370 375
380 Met Glu Glu Phe Phe Leu Gln Gly Asp Lys Glu Ala
Glu Leu Gly Leu 385 390 395
400 Pro Phe Ser Pro Leu Cys Asp Arg Lys Ser Thr Met Val Ala Gln Ser
405 410 415 Gln Ile Gly
Phe Ile Asp Phe Ile Val Glu Pro Thr Phe Ser Leu Leu 420
425 430 Thr Asp Ser Thr Glu Lys Ile Val
Ile Pro Leu Ile Glu Glu Ala Ser 435 440
445 Lys Ala Glu Thr Ser Ser Tyr Val Ala Ser Ser Ser Thr
Thr Ile Val 450 455 460
Gly Leu His Ile Ala Asp Ala Leu Arg Arg Ser Asn Thr Lys Gly Ser 465
470 475 480 Met Ser Asp Gly
Ser Tyr Ser Pro Asp Tyr Ser Leu Ala Ala Val Asp 485
490 495 Leu Lys Ser Phe Lys Asn Asn Leu Val
Asp Ile Ile Gln Gln Asn Lys 500 505
510 Glu Arg Trp Lys Glu Leu Ala Ala Gln Gly Glu Ser Asp Leu
His Lys 515 520 525
Asn Ser Glu Asp Leu Val Asn Ala Glu Glu Lys His Asp Glu Thr His 530
535 540 Ser 545
81535PRTHomo sapiens 81Met Gly Ser Ser Ala Thr Glu Ile Glu Glu Leu Glu
Asn Thr Thr Phe 1 5 10
15 Lys Tyr Leu Thr Gly Glu Gln Thr Glu Lys Met Trp Gln Arg Leu Lys
20 25 30 Gly Ile Leu
Arg Cys Leu Val Lys Gln Leu Glu Arg Gly Asp Val Asn 35
40 45 Val Val Asp Leu Lys Lys Asn Ile
Glu Tyr Ala Ala Ser Val Leu Glu 50 55
60 Ala Val Tyr Ile Asp Glu Thr Arg Arg Leu Leu Asp Thr
Glu Asp Glu 65 70 75
80 Leu Ser Asp Ile Gln Thr Asp Ser Val Pro Ser Glu Val Arg Asp Trp
85 90 95 Leu Ala Ser Thr
Phe Thr Arg Lys Met Gly Met Thr Lys Lys Lys Pro 100
105 110 Glu Glu Lys Pro Lys Phe Arg Ser Ile
Val His Ala Val Gln Ala Gly 115 120
125 Ile Phe Val Glu Arg Met Tyr Arg Lys Thr Tyr His Met Val
Gly Leu 130 135 140
Ala Tyr Pro Ala Ala Val Ile Val Thr Leu Lys Asp Val Asp Lys Trp 145
150 155 160 Ser Phe Asp Val Phe
Ala Leu Asn Glu Ala Ser Gly Glu His Ser Leu 165
170 175 Lys Phe Met Ile Tyr Glu Leu Phe Thr Arg
Tyr Asp Leu Ile Asn Arg 180 185
190 Phe Lys Ile Pro Val Ser Cys Leu Ile Thr Phe Ala Glu Ala Leu
Glu 195 200 205 Val
Gly Tyr Ser Lys Tyr Lys Asn Pro Tyr His Asn Leu Ile His Ala 210
215 220 Ala Asp Val Thr Gln Thr
Val His Tyr Ile Met Leu His Thr Gly Ile 225 230
235 240 Met His Trp Leu Thr Glu Leu Glu Ile Leu Ala
Met Val Phe Ala Ala 245 250
255 Ala Ile His Asp Tyr Glu His Thr Gly Thr Thr Asn Asn Phe His Ile
260 265 270 Gln Thr
Arg Ser Asp Val Ala Ile Leu Tyr Asn Asp Arg Ser Val Leu 275
280 285 Glu Asn His His Val Ser Ala
Ala Tyr Arg Leu Met Gln Glu Glu Glu 290 295
300 Met Asn Ile Leu Ile Asn Leu Ser Lys Asp Asp Trp
Arg Asp Leu Arg 305 310 315
320 Asn Leu Val Ile Glu Met Val Leu Ser Thr Asp Met Ser Gly His Phe
325 330 335 Gln Gln Ile
Lys Asn Ile Arg Asn Ser Leu Gln Gln Pro Glu Gly Ile 340
345 350 Asp Arg Ala Lys Thr Met Ser Leu
Ile Leu His Ala Ala Asp Ile Ser 355 360
365 His Pro Ala Lys Ser Trp Lys Leu His Tyr Arg Trp Thr
Met Ala Leu 370 375 380
Met Glu Glu Phe Phe Leu Gln Gly Asp Lys Glu Ala Glu Leu Gly Leu 385
390 395 400 Pro Phe Ser Pro
Leu Cys Asp Arg Lys Ser Thr Met Val Ala Gln Ser 405
410 415 Gln Ile Gly Phe Ile Asp Phe Ile Val
Glu Pro Thr Phe Ser Leu Leu 420 425
430 Thr Asp Ser Thr Glu Lys Ile Val Ile Pro Leu Ile Glu Glu
Ala Ser 435 440 445
Lys Ala Glu Thr Ser Ser Tyr Val Ala Ser Ser Ser Thr Thr Ile Val 450
455 460 Gly Leu His Ile Ala
Asp Ala Leu Arg Arg Ser Asn Thr Lys Gly Ser 465 470
475 480 Met Ser Asp Gly Ser Tyr Ser Pro Asp Tyr
Ser Leu Ala Ala Val Asp 485 490
495 Leu Lys Ser Phe Lys Asn Asn Leu Val Asp Ile Ile Gln Gln Asn
Lys 500 505 510 Glu
Arg Trp Lys Glu Leu Ala Ala Gln Glu Ala Arg Thr Ser Ser Gln 515
520 525 Lys Cys Glu Phe Ile His
Gln 530 535 82729PRTHomo sapiens 82Met Asn Pro Phe
Gln Lys Asn Glu Ser Lys Glu Thr Leu Phe Ser Pro 1 5
10 15 Val Ser Ile Glu Glu Val Pro Pro Arg
Pro Pro Ser Pro Pro Lys Lys 20 25
30 Pro Ser Pro Thr Ile Cys Gly Ser Asn Tyr Pro Leu Ser Ile
Ala Phe 35 40 45
Ile Val Val Asn Glu Phe Cys Glu Arg Phe Ser Tyr Tyr Gly Met Lys 50
55 60 Ala Val Leu Ile Leu
Tyr Phe Leu Tyr Phe Leu His Trp Asn Glu Asp 65 70
75 80 Thr Ser Thr Ser Ile Tyr His Ala Phe Ser
Ser Leu Cys Tyr Phe Thr 85 90
95 Pro Ile Leu Gly Ala Ala Ile Ala Asp Ser Trp Leu Gly Lys Phe
Lys 100 105 110 Thr
Ile Ile Tyr Leu Ser Leu Val Tyr Val Leu Gly His Val Ile Lys 115
120 125 Ser Leu Gly Ala Leu Pro
Ile Leu Gly Gly Gln Val Val His Thr Val 130 135
140 Leu Ser Leu Ile Gly Leu Ser Leu Ile Ala Leu
Gly Thr Gly Gly Ile 145 150 155
160 Lys Pro Cys Val Ala Ala Phe Gly Gly Asp Gln Phe Glu Glu Lys His
165 170 175 Ala Glu
Glu Arg Thr Arg Tyr Phe Ser Val Phe Tyr Leu Ser Ile Asn 180
185 190 Ala Gly Ser Leu Ile Ser Thr
Phe Ile Thr Pro Met Leu Arg Gly Asp 195 200
205 Val Gln Cys Phe Gly Glu Asp Cys Tyr Ala Leu Ala
Phe Gly Val Pro 210 215 220
Gly Leu Leu Met Val Ile Ala Leu Val Val Phe Ala Met Gly Ser Lys 225
230 235 240 Ile Tyr Asn
Lys Pro Pro Pro Glu Gly Asn Ile Val Ala Gln Val Phe 245
250 255 Lys Cys Ile Trp Phe Ala Ile Ser
Asn Arg Phe Lys Asn Arg Ser Gly 260 265
270 Asp Ile Pro Lys Arg Gln His Trp Leu Asp Trp Ala Ala
Glu Lys Tyr 275 280 285
Pro Lys Gln Leu Ile Met Asp Val Lys Ala Leu Thr Arg Val Leu Phe 290
295 300 Leu Tyr Ile Pro
Leu Pro Met Phe Trp Ala Leu Leu Asp Gln Gln Gly 305 310
315 320 Ser Arg Trp Thr Leu Gln Ala Ile Arg
Met Asn Arg Asn Leu Gly Phe 325 330
335 Phe Val Leu Gln Pro Asp Gln Met Gln Val Leu Asn Pro Leu
Leu Val 340 345 350
Leu Ile Phe Ile Pro Leu Phe Asp Phe Val Ile Tyr Arg Leu Val Ser
355 360 365 Lys Cys Gly Ile
Asn Phe Ser Ser Leu Arg Lys Met Ala Val Gly Met 370
375 380 Ile Leu Ala Cys Leu Ala Phe Ala
Val Ala Ala Ala Val Glu Ile Lys 385 390
395 400 Ile Asn Glu Met Ala Pro Ala Gln Pro Gly Pro Gln
Glu Val Phe Leu 405 410
415 Gln Val Leu Asn Leu Ala Asp Asp Glu Val Lys Val Thr Val Val Gly
420 425 430 Asn Glu Asn
Asn Ser Leu Leu Ile Glu Ser Ile Lys Ser Phe Gln Lys 435
440 445 Thr Pro His Tyr Ser Lys Leu His
Leu Lys Thr Lys Ser Gln Asp Phe 450 455
460 His Phe His Leu Lys Tyr His Asn Leu Ser Leu Tyr Thr
Glu His Ser 465 470 475
480 Val Gln Glu Lys Asn Trp Tyr Ser Leu Val Ile Arg Glu Asp Gly Asn
485 490 495 Ser Ile Ser Ser
Met Met Val Lys Asp Thr Glu Ser Arg Thr Thr Asn 500
505 510 Gly Met Thr Thr Val Arg Phe Val Asn
Thr Leu His Lys Asp Val Asn 515 520
525 Ile Ser Leu Ser Thr Asp Thr Ser Leu Asn Val Gly Glu Asp
Tyr Gly 530 535 540
Val Ser Ala Tyr Arg Thr Val Gln Arg Gly Glu Tyr Pro Ala Val His 545
550 555 560 Cys Arg Thr Glu Asp
Lys Asn Phe Ser Leu Asn Leu Gly Leu Leu Asp 565
570 575 Phe Gly Ala Ala Tyr Leu Phe Val Ile Thr
Asn Asn Thr Asn Gln Gly 580 585
590 Leu Gln Ala Trp Lys Ile Glu Asp Ile Pro Ala Asn Lys Met Ser
Ile 595 600 605 Ala
Trp Gln Leu Pro Gln Tyr Ala Leu Val Thr Ala Gly Glu Val Met 610
615 620 Phe Ser Val Thr Gly Leu
Glu Phe Ser Tyr Ser Gln Ala Pro Ser Gly 625 630
635 640 Met Lys Ser Val Leu Gln Ala Ala Trp Leu Leu
Thr Ile Ala Val Gly 645 650
655 Asn Ile Ile Val Leu Val Val Ala Gln Phe Ser Gly Leu Val Gln Trp
660 665 670 Ala Glu
Phe Ile Leu Phe Ser Cys Leu Leu Leu Val Ile Cys Leu Ile 675
680 685 Phe Ser Ile Met Gly Tyr Tyr
Tyr Val Pro Val Lys Thr Glu Asp Met 690 695
700 Arg Gly Pro Ala Asp Lys His Ile Pro His Ile Gln
Gly Asn Met Ile 705 710 715
720 Lys Leu Glu Thr Lys Lys Thr Lys Leu 725
838RNAArtificial SequenceSynthetic oligonucleotide 83uugcuaua
88419DNAArtificial
SequenceSynthetic oligonucleotide 84ttccatggta atggtgtgc
198519DNAArtificial SequenceSynthetic
oligonucleotide 85ccgaagcaag gaataatcc
198619DNAArtificial SequenceSynthetic oligonucleotide
86tatgtttcgc ctttatgac
198719DNAArtificial SequenceSynthetic oligonucleotide 87ggattcaagg
aggaatgac
198819DNAArtificial SequenceSynthetic oligonucleotide 88ctccctcttg
catcaagac
198919DNAArtificial SequenceSynthetic oligonucleotide 89aaatcctcgt
caggtttac
199019DNAArtificial SequenceSynthetic oligonucleotide 90agtgccttac
agtatcatc
199119DNAArtificial SequenceSynthetic oligonucleotide 91cctgaatgtg
actgtggac
199219DNAArtificial SequenceSynthetic oligonucleotide 92gactgactgg
cctgaaggc
199319DNAArtificial SequenceSynthetic oligonucleotide 93gcagcactat
ttgaagcac
199419DNAArtificial SequenceSynthetic oligonucleotide 94gcctgagaac
ctcctctgc
199519DNAArtificial SequenceSynthetic oligonucleotide 95acagctagtc
aggcacttc
199619DNAArtificial SequenceSynthetic oligonucleotide 96tataagaaat
ggcatactc
199719DNAArtificial SequenceSynthetic oligonucleotide 97ctaatccatg
gtctagttc
199819DNAArtificial SequenceSynthetic oligonucleotide 98gtctgctata
aggaatatc
199919DNAArtificial SequenceSynthetic oligonucleotide 99aagtactcct
gaggtctac
1910019DNAArtificial SequenceSynthetic oligonucleotide 100aggcaccagg
gacttgtgc
1910119DNAArtificial SequenceSynthetic oligonucleotide 101gatctacacc
accttcatc
1910219DNAArtificial SequenceSynthetic oligonucleotide 102tggtgttcta
cgtggtgac
1910319DNAArtificial SequenceSynthetic oligonucleotide 103tgttaggcgc
ctgcattgc
1910419DNAArtificial SequenceSynthetic oligonucleotide 104caacatcttt
atctgctcc
1910519DNAArtificial SequenceSynthetic oligonucleotide 105cgaagggctt
tcacaatgc
1910619DNAArtificial SequenceSynthetic oligonucleotide 106gtactacgtt
gtagcccac
1910719DNAArtificial SequenceSynthetic oligonucleotide 107tgcaggcgct
taacattac
1910819DNAArtificial SequenceSynthetic oligonucleotide 108ggtgtatggg
ctcatgtac
1910919DNAArtificial SequenceSynthetic oligonucleotide 109catcgtcatc
gcctgctac
1911019DNAArtificial SequenceSynthetic oligonucleotide 110gctcatggtg
cgcattggc
1911119DNAArtificial SequenceSynthetic oligonucleotide 111gtaccttatg
acgctgatc
1911219DNAArtificial SequenceSynthetic oligonucleotide 112acctggtatg
ggtttggcc
1911319DNAArtificial SequenceSynthetic oligonucleotide 113atgtgtgcag
gtctactgc
1911419DNAArtificial SequenceSynthetic oligonucleotide 114ttatttaggg
cggtttaac
1911519DNAArtificial SequenceSynthetic oligonucleotide 115taatgtcatc
gcctccaac
1911619DNAArtificial SequenceSynthetic oligonucleotide 116agaactgggt
gatgacagc
1911719DNAArtificial SequenceSynthetic oligonucleotide 117caacagtccc
tgctacatc
1911819DNAArtificial SequenceSynthetic oligonucleotide 118gatcgtggtg
catccatac
1911919DNAArtificial SequenceSynthetic oligonucleotide 119gcgtggatta
ccagaagac
1912019DNAArtificial SequenceSynthetic oligonucleotide 120taacaacagt
ccctgctac
1912119DNAArtificial SequenceSynthetic oligonucleotide 121ccattgcttg
gtgaatggc
1912219DNAArtificial SequenceSynthetic oligonucleotide 122attctctcca
tggagtgac
1912319DNAArtificial SequenceSynthetic oligonucleotide 123atgaactctg
tgatccagc
1912419DNAArtificial SequenceSynthetic oligonucleotide 124aggttggcta
gtggatctc
1912519DNAArtificial SequenceSynthetic oligonucleotide 125aagtgggtaa
ttcctgctc
1912619DNAArtificial SequenceSynthetic oligonucleotide 126cgaatggcag
aatggatac
1912719DNAArtificial SequenceSynthetic oligonucleotide 127tctggcaggt
tgcatattc
1912819DNAArtificial SequenceSynthetic oligonucleotide 128ctgcaagttt
catatctac
1912919DNAArtificial SequenceSynthetic oligonucleotide 129atagcatcag
attgtatgc
1913019DNAArtificial SequenceSynthetic oligonucleotide 130tttacacgat
gatatgttc
1913119DNAArtificial SequenceSynthetic oligonucleotide 131cagaggattt
gccagaaac
1913219DNAArtificial SequenceSynthetic oligonucleotide 132ttggaattcc
agtgtaccc
1913319DNAArtificial SequenceSynthetic oligonucleotide 133cagagacacc
atctccctc
1913419DNAArtificial SequenceSynthetic oligonucleotide 134acccagaccc
aaagtttgc
1913519DNAArtificial SequenceSynthetic oligonucleotide 135aaggtggaaa
gactatctc
1913619DNAArtificial SequenceSynthetic oligonucleotide 136tataaaccag
aggatttgc
1913719DNAArtificial SequenceSynthetic oligonucleotide 137gtataatcta
catcagatc
1913819DNAArtificial SequenceSynthetic oligonucleotide 138ccacatgttt
accagagac
1913919DNAArtificial SequenceSynthetic oligonucleotide 139gctagttatc
gcctacctc
1914019DNAArtificial SequenceSynthetic oligonucleotide 140gacacacagg
agttcaacc
1914119DNAArtificial SequenceSynthetic oligonucleotide 141gctgcagaaa
ctaggcatc
1914219DNAArtificial SequenceSynthetic oligonucleotide 142tgccaacttc
tacaaggac
1914319DNAArtificial SequenceSynthetic oligonucleotide 143cgacacacag
gagttcaac
1914419DNAArtificial SequenceSynthetic oligonucleotide 144gatggacgtc
aagtctgcc
1914519DNAArtificial SequenceSynthetic oligonucleotide 145acaggagttc
aacctcagc
1914619DNAArtificial SequenceSynthetic oligonucleotide 146ttggcatgga
accaacgac
1914719DNAArtificial SequenceSynthetic oligonucleotide 147cctctttgcc
ctgtatgac
1914819DNAArtificial SequenceSynthetic oligonucleotide 148agattccaga
tgcaacccc
1914919DNAArtificial SequenceSynthetic oligonucleotide 149catgagccag
ctgagtttc
1915019DNAArtificial SequenceSynthetic oligonucleotide 150gtggaagtga
tcttctatc
1915119DNAArtificial SequenceSynthetic oligonucleotide 151tggctgtcat
ggtccaatc
1915219DNAArtificial SequenceSynthetic oligonucleotide 152ccgctgcatg
aactatgac
1915319DNAArtificial SequenceSynthetic oligonucleotide 153ttggagactt
cggtttaac
1915419DNAArtificial SequenceSynthetic oligonucleotide 154tgtgattcag
attctagtc
1915519DNAArtificial SequenceSynthetic oligonucleotide 155agtggatgtc
ctacatctc
1915619DNAArtificial SequenceSynthetic oligonucleotide 156atcatgcagg
atgacatgc
1915719DNAArtificial SequenceSynthetic oligonucleotide 157ctgaactact
ggtacagcc
1915819DNAArtificial SequenceSynthetic oligonucleotide 158cagctttacg
tcctgagtc
1915919DNAArtificial SequenceSynthetic oligonucleotide 159tcagaccaca
agagagacc
1916019DNAArtificial SequenceSynthetic oligonucleotide 160gtacctacag
tggatgtcc
1916119DNAArtificial SequenceSynthetic oligonucleotide 161tggtcaagga
gatactgac
1916219DNAArtificial SequenceSynthetic oligonucleotide 162ccctccagtc
atgttcttc
1916319DNAArtificial SequenceSynthetic oligonucleotide 163ttctgcaact
gtgatgatc
1916419DNAArtificial SequenceSynthetic oligonucleotide 164gtacactacg
tggattcac
1916519DNAArtificial SequenceSynthetic oligonucleotide 165gcgaattcca
ccagcattc
1916619DNAArtificial SequenceSynthetic oligonucleotide 166tcttccagtg
ctgcagctc
1916719DNAArtificial SequenceSynthetic oligonucleotide 167tcagaacaag
aggctgggc
1916819DNAArtificial SequenceSynthetic oligonucleotide 168gaagattgcc
agtcttcac
1916919DNAArtificial SequenceSynthetic oligonucleotide 169gattcctcct
ctcaacatc
1917019DNAArtificial SequenceSynthetic oligonucleotide 170gcattctagt
atttctaac
1917119DNAArtificial SequenceSynthetic oligonucleotide 171ttacagtgtc
aattccaac
1917219DNAArtificial SequenceSynthetic oligonucleotide 172tgattatcgg
gagatcatc
1917319DNAArtificial SequenceSynthetic oligonucleotide 173gaatggatgt
gtatgtatc
1917419DNAArtificial SequenceSynthetic oligonucleotide 174tgacatgatt
ccttatagc
1917519DNAArtificial SequenceSynthetic oligonucleotide 175tctacacact
gccaaatgc
1917619DNAArtificial SequenceSynthetic oligonucleotide 176actggccatc
atctacaac
1917719DNAArtificial SequenceSynthetic oligonucleotide 177tgctcacctg
tctgtggtc
1917819DNAArtificial SequenceSynthetic oligonucleotide 178acattgacca
ggaagtgac
1917919DNAArtificial SequenceSynthetic oligonucleotide 179tggatgactt
cagctctac
1918019DNAArtificial SequenceSynthetic oligonucleotide 180ctacaacctc
tggttcggc
1918119DNAArtificial SequenceSynthetic oligonucleotide 181cacgacagtg
gagagaaac
1918219DNAArtificial SequenceSynthetic oligonucleotide 182gttctacatg
ccggatgac
1918319DNAArtificial SequenceSynthetic oligonucleotide 183aggtatcatg
cactggctc
1918419DNAArtificial SequenceSynthetic oligonucleotide 184gaagctgaat
tagggcttc
1918519DNAArtificial SequenceSynthetic oligonucleotide 185ctggtggaca
tcattcagc
1918619DNAArtificial SequenceSynthetic oligonucleotide 186tttgtgatcg
gaagtcaac
1918719DNAArtificial SequenceSynthetic oligonucleotide 187attgctgatg
cactaagac
1918819DNAArtificial SequenceSynthetic oligonucleotide 188cagatatgat
cttatcaac
1918919DNAArtificial SequenceSynthetic oligonucleotide 189actgtgcatt
acataatgc
1919019DNAArtificial SequenceSynthetic oligonucleotide 190cacgtgagtg
cagcttatc
1919119DNAArtificial SequenceSynthetic oligonucleotide 191agtcctatca
ttgatcggc
1919219DNAArtificial SequenceSynthetic oligonucleotide 192gaagccatct
ccgacaatc
1919319DNAArtificial SequenceSynthetic oligonucleotide 193atggctgttg
gtatgatcc
1919419DNAArtificial SequenceSynthetic oligonucleotide 194ccgtgaggtt
tgttaacac
1919519DNAArtificial SequenceSynthetic oligonucleotide 195ttgggtgcct
taccaatac
1919619DNAArtificial SequenceSynthetic oligonucleotide 196ctccaagtgt
ggaattaac
1919719DNAArtificial SequenceSynthetic oligonucleotide 197gcatgatggt
aaaggatac
19198109PRTHomo sapiens 198Met Ile Cys Cys Ser Ala Leu Ser Pro Arg Ile
His Leu Ser Phe His 1 5 10
15 Arg Ser Leu Thr Gly Ile Val Leu Ala Asn Ser Ser Leu Asp Ile Val
20 25 30 Leu His
Asp Thr Tyr Tyr Val Val Ala His Cys Gly Gly Asn Val Arg 35
40 45 Arg Leu His Cys Gly Gly Pro
Ala Ser Arg Glu Arg Thr Ala Met Gln 50 55
60 Ala Leu Asn Ile Thr Pro Glu Gln Phe Ser Arg Leu
Leu Arg Asp His 65 70 75
80 Asn Leu Thr Arg Glu Gln Phe Ile Ala Leu Tyr Arg Leu Arg Pro Leu
85 90 95 Val Tyr Thr
Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala 100
105 19923PRTHomo sapiens 199Leu Val Leu Thr Gly Val Leu
Ile Phe Ala Leu Ala Leu Phe Gly Asn 1 5
10 15 Ala Leu Val Phe Tyr Val Val 20
20011PRTHomo sapiens 200Thr Arg Ser Lys Ala Met Arg Thr Val Thr
Asn 1 5 10 20123PRTHomo sapiens
201Ile Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile Thr Phe Phe 1
5 10 15 Cys Ile Pro Val
Thr Met Leu 20 20214PRTHomo sapiens 202Gln Asn
Ile Ser Asp Asn Trp Leu Gly Gly Ala Phe Ile Cys 1 5
10 20323PRTHomo sapiens 203Lys Met Val Pro Phe
Val Gln Ser Thr Ala Val Val Thr Glu Ile Leu 1 5
10 15 Thr Met Thr Cys Ile Ala Val
20 20420PRTHomo sapiens 204Glu Arg His Gln Gly Leu Val His
Pro Phe Lys Met Lys Trp Gln Tyr 1 5 10
15 Thr Asn Arg Arg 20 20523PRTHomo
sapiens 205Ala Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val
Gly 1 5 10 15 Ser
Pro Met Trp His Val Gln 20 20648PRTHomo sapiens
206Gln Leu Glu Ile Lys Tyr Asp Phe Leu Tyr Glu Lys Glu His Ile Cys 1
5 10 15 Cys Leu Glu Glu
Trp Thr Ser Pro Val His Gln Lys Ile Tyr Thr Thr 20
25 30 Phe Ile Leu Ser Ser Ser Ser Ser Cys
Leu Leu Trp Lys Lys Lys Arg 35 40
45 20723PRTHomo sapiens 207Ala Val Ile Met Met Val Thr Val
Val Ala Leu Phe Ala Val Cys Trp 1 5 10
15 Ala Pro Phe His Val Val His 20
20819PRTHomo sapiens 208Met Met Ile Glu Tyr Ser Asn Phe Glu Lys Glu
Tyr Asp Asp Val Thr 1 5 10
15 Ile Lys Met 20923PRTHomo sapiens 209Ile Phe Ala Ile Val Gln Ile
Ile Gly Phe Ser Asn Ser Ile Cys Asn 1 5
10 15 Pro Ile Val Tyr Ala Phe Met 20
21096PRTHomo sapiens 210Asn Glu Asn Phe Lys Lys Asn Val Leu Ser
Ala Val Cys Tyr Cys Ile 1 5 10
15 Val Asn Lys Thr Phe Ser Pro Ala Gln Arg His Gly Asn Ser Gly
Ile 20 25 30 Thr
Met Met Arg Lys Lys Ala Lys Phe Ser Leu Arg Glu Asn Pro Val 35
40 45 Glu Glu Thr Lys Gly Glu
Ala Phe Ser Asp Gly Asn Ile Glu Val Lys 50 55
60 Leu Cys Glu Gln Thr Glu Glu Lys Lys Lys Leu
Lys Arg His Leu Ala 65 70 75
80 Leu Phe Arg Ser Glu Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His
85 90 95
21147PRTHomo sapiens 211Met Gln Ala Leu Asn Ile Thr Pro Glu Gln Phe Ser
Arg Leu Leu Arg 1 5 10
15 Asp His Asn Leu Thr Arg Glu Gln Phe Ile Ala Val His Arg Leu Arg
20 25 30 Pro Leu Val
Tyr Thr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala 35
40 45 21223PRTHomo sapiens 212Leu Val Leu
Thr Gly Val Leu Ile Phe Ala Leu Ala Leu Phe Gly Asn 1 5
10 15 Ala Leu Val Phe Tyr Val Val
20 21311PRTHomo sapiens 213Thr Arg Ser Lys Ala Met
Arg Thr Val Thr Asn 1 5 10
21423PRTHomo sapiens 214Ile Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu
Ile Thr Phe Phe 1 5 10
15 Cys Ile Pro Val Thr Met Leu 20
21514PRTHomo sapiens 215Gln Asn Ile Ser Asp Asn Trp Leu Gly Gly Ala Phe
Ile Cys 1 5 10
21623PRTHomo sapiens 216Lys Met Val Pro Phe Val Gln Ser Thr Ala Val Val
Thr Glu Ile Leu 1 5 10
15 Thr Met Thr Cys Ile Ala Val 20
21720PRTHomo sapiens 217Glu Arg His Gln Gly Leu Val His Pro Phe Lys Met
Lys Trp Gln Tyr 1 5 10
15 Thr Asn Arg Arg 20 21823PRTHomo sapiens 218Ala Phe
Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val Gly 1 5
10 15 Ser Pro Met Trp His Val Gln
20 21928PRTHomo sapiens 219Gln Leu Glu Ile Lys
Tyr Asp Phe Leu Tyr Glu Lys Glu His Ile Cys 1 5
10 15 Cys Leu Glu Glu Trp Thr Ser Pro Val His
Gln Lys 20 25 22023PRTHomo
sapiens 220Ile Tyr Thr Thr Phe Ile Leu Val Ile Leu Phe Leu Leu Pro Leu
Met 1 5 10 15 Val
Met Leu Ile Leu Tyr Ser 20 22134PRTHomo sapiens
221Lys Ile Gly Tyr Glu Leu Trp Ile Lys Lys Arg Val Gly Asp Gly Ser 1
5 10 15 Val Leu Arg Thr
Ile His Gly Lys Glu Met Ser Lys Ile Ala Arg Lys 20
25 30 Lys Lys 22223PRTHomo sapiens 222Arg
Ala Val Ile Met Met Val Thr Val Val Ala Leu Phe Ala Val Cys 1
5 10 15 Trp Ala Pro Phe His Val
Val 20 22319PRTHomo sapiens 223His Met Met Ile
Glu Tyr Ser Asn Phe Glu Lys Glu Tyr Asp Asp Val 1 5
10 15 Thr Ile Lys 22423PRTHomo sapiens
224Met Ile Phe Ala Ile Val Gln Ile Ile Gly Phe Ser Asn Ser Ile Cys 1
5 10 15 Asn Pro Ile Val
Tyr Ala Phe 20 22597PRTHomo sapiens 225Met Asn
Glu Asn Phe Lys Lys Asn Val Leu Ser Ala Val Cys Tyr Cys 1 5
10 15 Ile Val Asn Lys Thr Phe Ser
Pro Ala Gln Arg His Gly Asn Ser Gly 20 25
30 Ile Thr Met Met Arg Lys Lys Ala Lys Phe Ser Leu
Arg Glu Asn Pro 35 40 45
Val Glu Glu Thr Lys Gly Glu Ala Phe Ser Asp Gly Asn Ile Glu Val
50 55 60 Lys Leu Cys
Glu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu 65
70 75 80 Ala Leu Phe Arg Ser Glu Leu
Ala Glu Asn Ser Pro Leu Asp Ser Gly 85
90 95 His 226320PRTHomo sapiens 226Met Ala Glu Glu
Glu Ala Pro Lys Lys Ser Arg Ala Ala Gly Gly Gly 1 5
10 15 Ala Ser Trp Glu Leu Cys Ala Gly Ala
Leu Ser Ala Arg Leu Ala Glu 20 25
30 Glu Gly Ser Gly Asp Ala Gly Gly Arg Arg Arg Pro Pro Val
Asp Pro 35 40 45
Arg Arg Leu Ala Arg Gln Leu Leu Leu Leu Leu Trp Leu Leu Glu Ala 50
55 60 Pro Leu Leu Leu Gly
Val Arg Ala Gln Ala Ala Gly Gln Gly Pro Gly 65 70
75 80 Gln Gly Pro Gly Pro Gly Gln Gln Pro Pro
Pro Pro Pro Gln Gln Gln 85 90
95 Gln Ser Gly Gln Gln Tyr Asn Gly Glu Arg Gly Ile Ser Val Pro
Asp 100 105 110 His
Gly Tyr Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala 115
120 125 Tyr Asn Gln Thr Ile Met
Pro Asn Leu Leu Gly His Thr Asn Gln Glu 130 135
140 Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro
Leu Val Lys Val Gln 145 150 155
160 Cys Ser Ala Glu Leu Lys Phe Phe Leu Cys Ser Met Tyr Ala Pro Val
165 170 175 Cys Thr
Val Leu Glu Gln Ala Leu Pro Pro Cys Arg Ser Leu Cys Glu 180
185 190 Arg Ala Arg Gln Gly Cys Glu
Ala Leu Met Asn Lys Phe Gly Phe Gln 195 200
205 Trp Pro Asp Thr Leu Lys Cys Glu Lys Phe Pro Val
His Gly Ala Gly 210 215 220
Glu Leu Cys Val Gly Gln Asn Thr Ser Asp Lys Gly Thr Pro Thr Pro 225
230 235 240 Ser Leu Leu
Pro Glu Phe Trp Thr Ser Asn Pro Gln His Gly Gly Gly 245
250 255 Gly His Arg Gly Gly Phe Pro Gly
Gly Ala Gly Ala Ser Glu Arg Gly 260 265
270 Lys Phe Ser Cys Pro Arg Ala Leu Lys Val Pro Ser Tyr
Leu Asn Tyr 275 280 285
His Phe Leu Gly Glu Lys Asp Cys Gly Ala Pro Cys Glu Pro Thr Lys 290
295 300 Val Tyr Gly Leu
Met Tyr Phe Gly Pro Glu Glu Leu Arg Phe Ser Arg 305 310
315 320 22723PRTHomo sapiens 227Thr Trp Ile
Gly Ile Trp Ser Val Leu Cys Cys Ala Ser Thr Leu Phe 1 5
10 15 Thr Val Leu Thr Tyr Leu Val
20 22812PRTHomo sapiens 228Asp Met Arg Arg Phe Ser
Tyr Pro Glu Arg Pro Ile 1 5 10
22920PRTHomo sapiens 229Ile Phe Leu Ser Gly Cys Tyr Thr Ala Val Ala Val
Ala Tyr Ile Ala 1 5 10
15 Gly Phe Leu Leu 20 23027PRTHomo sapiens 230Glu Asp
Arg Val Val Cys Asn Asp Lys Phe Ala Glu Asp Gly Ala Arg 1 5
10 15 Thr Val Ala Gln Gly Thr Lys
Lys Glu Gly Cys 20 25 23123PRTHomo
sapiens 231Thr Ile Leu Phe Met Met Leu Tyr Phe Phe Ser Met Ala Ser Ser
Ile 1 5 10 15 Trp
Trp Val Ile Leu Ser Leu 20 23220PRTHomo sapiens
232Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly His Glu Ala Ile Glu 1
5 10 15 Ala Asn Ser Gln
20 23323PRTHomo sapiens 233Tyr Phe His Leu Ala Ala Trp Ala
Val Pro Ala Ile Lys Thr Ile Thr 1 5 10
15 Ile Leu Ala Leu Gly Gln Val 20
23423PRTHomo sapiens 234Asp Gly Asp Val Leu Ser Gly Val Cys Phe Val
Gly Leu Asn Asn Val 1 5 10
15 Asp Ala Leu Arg Gly Phe Val 20
23523PRTHomo sapiens 235Leu Ala Pro Leu Phe Val Tyr Leu Phe Ile Gly Thr
Ser Phe Leu Leu 1 5 10
15 Ala Gly Phe Val Ser Leu Phe 20
23620PRTHomo sapiens 236Arg Ile Arg Thr Ile Met Lys His Asp Gly Thr Lys
Thr Glu Lys Leu 1 5 10
15 Glu Lys Leu Met 20 23723PRTHomo sapiens 237Val Arg
Ile Gly Val Phe Ser Val Leu Tyr Thr Val Pro Ala Thr Ile 1 5
10 15 Val Ile Ala Cys Tyr Phe Tyr
20 23842PRTHomo sapiens 238Glu Gln Ala Phe Arg
Asp Gln Trp Glu Arg Ser Trp Val Ala Gln Ser 1 5
10 15 Cys Lys Ser Tyr Ala Ile Pro Cys Pro His
Leu Gln Ala Gly Gly Gly 20 25
30 Ala Pro Pro His Pro Pro Met Ser Pro Asp 35
40 23923PRTHomo sapiens 239Phe Thr Val Phe Met Ile Lys
Tyr Leu Met Thr Leu Ile Val Gly Ile 1 5
10 15 Thr Ser Gly Phe Trp Ile Trp 20
24025PRTHomo sapiens 240Ser Gly Lys Thr Leu Asn Ser Trp Arg Lys
Phe Tyr Thr Arg Leu Thr 1 5 10
15 Asn Ser Lys Gln Gly Glu Thr Thr Val 20
25 241409PRTHomo sapiens 241Met Ala Cys Leu Met Ala Ala Phe Ser
Val Gly Thr Ala Met Asn Ala 1 5 10
15 Ser Ser Tyr Ser Ala Glu Met Thr Glu Pro Lys Ser Val Cys
Val Ser 20 25 30
Val Asp Glu Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr Asp Leu
35 40 45 Leu Asn Gly His
Leu Lys Lys Val Asp Asn Asn Leu Thr Glu Ala Gln 50
55 60 Arg Phe Ser Ser Leu Pro Arg Arg
Ala Ala Val Asn Ile Glu Phe Arg 65 70
75 80 Asp Leu Ser Tyr Ser Val Pro Glu Gly Pro Trp Trp
Arg Lys Lys Gly 85 90
95 Tyr Lys Thr Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu
100 105 110 Leu Val Ala
Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr Leu Met 115
120 125 Asn Ile Leu Ala Gly Tyr Arg Glu
Thr Gly Met Lys Gly Ala Val Leu 130 135
140 Ile Asn Gly Leu Pro Arg Asp Leu Arg Cys Phe Arg Lys
Val Ser Cys 145 150 155
160 Tyr Ile Met Gln Asp Asp Met Leu Leu Pro His Leu Thr Val Gln Glu
165 170 175 Ala Met Met Val
Ser Ala His Leu Lys Leu Gln Glu Lys Asp Glu Gly 180
185 190 Arg Arg Glu Met Val Lys Glu Ile Leu
Thr Ala Leu Gly Leu Leu Ser 195 200
205 Cys Ala Asn Thr Arg Thr Gly Ser Leu Ser Gly Gly Gln Arg
Lys Arg 210 215 220
Leu Ala Ile Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met Phe Phe 225
230 235 240 Asp Glu Pro Thr Ser
Gly Leu Asp Ser Ala Ser Cys Phe Gln Val Val 245
250 255 Ser Leu Met Lys Gly Leu Ala Gln Gly Gly
Arg Ser Ile Ile Cys Thr 260 265
270 Ile His Gln Pro Ser Ala Lys Leu Phe Glu Leu Phe Asp Gln Leu
Tyr 275 280 285 Val
Leu Ser Gln Gly Gln Cys Val Tyr Arg Gly Lys Val Cys Asn Leu 290
295 300 Val Pro Tyr Leu Arg Asp
Leu Gly Leu Asn Cys Pro Thr Tyr His Asn 305 310
315 320 Pro Ala Asp Phe Val Met Glu Val Ala Ser Gly
Glu Tyr Gly Asp Gln 325 330
335 Asn Ser Arg Leu Val Arg Ala Val Arg Glu Gly Met Cys Asp Ser Asp
340 345 350 His Lys
Arg Asp Leu Gly Gly Asp Ala Glu Val Asn Pro Phe Leu Trp 355
360 365 His Arg Pro Ser Glu Glu Asp
Ser Ser Ser Met Glu Gly Cys His Ser 370 375
380 Phe Ser Ala Ser Cys Leu Thr Gln Phe Cys Ile Leu
Phe Lys Arg Thr 385 390 395
400 Phe Leu Ser Ile Met Arg Asp Ser Val 405
24223PRTHomo sapiens 242Leu Thr His Leu Arg Ile Thr Ser His Ile Gly
Ile Gly Leu Leu Ile 1 5 10
15 Gly Leu Leu Tyr Leu Gly Ile 20
24312PRTHomo sapiens 243Gly Asn Glu Ala Lys Lys Val Leu Ser Asn Ser Gly 1
5 10 24423PRTHomo sapiens 244Phe
Leu Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met Pro 1
5 10 15 Thr Val Leu Thr Phe Pro
Leu 20 24523PRTHomo sapiens 245Glu Met Gly Val
Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu 1 5
10 15 Lys Ala Tyr Tyr Leu Ala Lys
20 24623PRTHomo sapiens 246Thr Met Ala Asp Val Pro Phe
Gln Ile Met Phe Pro Val Ala Tyr Cys 1 5
10 15 Ser Ile Val Tyr Trp Met Thr 20
2476PRTHomo sapiens 247Ser Gln Pro Ser Asp Ala 1
5 24823PRTHomo sapiens 248Val Arg Phe Val Leu Phe Ala Ala Leu Gly
Thr Met Thr Ser Leu Val 1 5 10
15 Ala Gln Ser Leu Gly Leu Leu 20
2499PRTHomo sapiens 249Ile Gly Ala Ala Ser Thr Ser Leu Gln 1
5 25023PRTHomo sapiens 250Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe 20
25112PRTHomo sapiens 251Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met
Ser Tyr 1 5 10 25218PRTHomo
sapiens 252Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile
Tyr 1 5 10 15 Gly
Leu 25331PRTHomo sapiens 253Asp Arg Glu Asp Leu His Cys Asp Ile Asp Glu
Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn Ala Lys
20 25 30 25423PRTHomo
sapiens 254Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg 1 5 10 15 Leu
Ile Ala Tyr Phe Val Leu 20 2558PRTHomo sapiens
255Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
256421PRTHomo sapiens 256Met Ala Cys Leu Met Ala Ala Phe Ser Val Gly Thr
Ala Met Asn Ala 1 5 10
15 Ser Ser Tyr Ser Ala Glu Met Thr Glu Pro Lys Ser Val Cys Val Ser
20 25 30 Val Asp Glu
Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr Asp Leu 35
40 45 Leu Asn Gly His Leu Lys Lys Val
Asp Asn Asn Leu Thr Glu Ala Gln 50 55
60 Arg Phe Ser Ser Leu Pro Arg Arg Ala Ala Val Asn Ile
Glu Phe Arg 65 70 75
80 Asp Leu Ser Tyr Ser Val Pro Glu Gly Pro Trp Trp Arg Lys Lys Gly
85 90 95 Tyr Lys Thr Leu
Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu 100
105 110 Leu Val Ala Ile Met Gly Pro Ser Gly
Ala Gly Lys Ser Thr Leu Met 115 120
125 Asn Ile Leu Ala Gly Tyr Arg Glu Thr Gly Met Lys Gly Ala
Val Leu 130 135 140
Ile Asn Gly Leu Pro Arg Asp Leu Arg Cys Phe Arg Lys Val Ser Cys 145
150 155 160 Tyr Ile Met Gln Asp
Asp Met Leu Leu Pro His Leu Thr Val Gln Glu 165
170 175 Ala Met Met Val Ser Ala His Leu Lys Leu
Gln Glu Lys Asp Glu Gly 180 185
190 Arg Arg Glu Met Val Lys Glu Ile Leu Thr Ala Leu Gly Leu Leu
Ser 195 200 205 Cys
Ala Asn Thr Arg Thr Gly Ser Leu Ser Gly Gly Gln Arg Lys Arg 210
215 220 Leu Ala Ile Ala Leu Glu
Leu Val Asn Asn Pro Pro Val Met Phe Phe 225 230
235 240 Asp Glu Pro Thr Ser Gly Leu Asp Ser Ala Ser
Cys Phe Gln Val Val 245 250
255 Ser Leu Met Lys Gly Leu Ala Gln Gly Gly Arg Ser Ile Ile Cys Thr
260 265 270 Ile His
Gln Pro Ser Ala Lys Leu Phe Glu Leu Phe Asp Gln Leu Tyr 275
280 285 Val Leu Ser Gln Gly Gln Cys
Val Tyr Arg Gly Lys Val Cys Asn Leu 290 295
300 Val Pro Tyr Leu Arg Asp Leu Gly Leu Asn Cys Pro
Thr Tyr His Asn 305 310 315
320 Pro Ala Asp Phe Val Met Glu Val Ala Ser Gly Glu Tyr Gly Asp Gln
325 330 335 Asn Ser Arg
Leu Val Arg Ala Val Arg Glu Gly Met Cys Asp Ser Asp 340
345 350 His Lys Arg Asp Leu Gly Gly Asp
Ala Glu Val Asn Pro Phe Leu Trp 355 360
365 His Arg Pro Ser Glu Glu Val Lys Gln Thr Lys Arg Leu
Lys Gly Leu 370 375 380
Arg Lys Asp Ser Ser Ser Met Glu Gly Cys His Ser Phe Ser Ala Ser 385
390 395 400 Cys Leu Thr Gln
Phe Cys Ile Leu Phe Lys Arg Thr Phe Leu Ser Ile 405
410 415 Met Arg Asp Ser Val 420
25723PRTHomo sapiens 257Leu Thr His Leu Arg Ile Thr Ser His Ile Gly
Ile Gly Leu Leu Ile 1 5 10
15 Gly Leu Leu Tyr Leu Gly Ile 20
25812PRTHomo sapiens 258Gly Asn Glu Ala Lys Lys Val Leu Ser Asn Ser Gly 1
5 10 25923PRTHomo sapiens 259Phe
Leu Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met Pro 1
5 10 15 Thr Val Leu Thr Phe Pro
Leu 20 26023PRTHomo sapiens 260Glu Met Gly Val
Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu 1 5
10 15 Lys Ala Tyr Tyr Leu Ala Lys
20 26123PRTHomo sapiens 261Thr Met Ala Asp Val Pro Phe
Gln Ile Met Phe Pro Val Ala Tyr Cys 1 5
10 15 Ser Ile Val Tyr Trp Met Thr 20
2626PRTHomo sapiens 262Ser Gln Pro Ser Asp Ala 1
5 26323PRTHomo sapiens 263Val Arg Phe Val Leu Phe Ala Ala Leu Gly
Thr Met Thr Ser Leu Val 1 5 10
15 Ala Gln Ser Leu Gly Leu Leu 20
2649PRTHomo sapiens 264Ile Gly Ala Ala Ser Thr Ser Leu Gln 1
5 26523PRTHomo sapiens 265Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe 20
26612PRTHomo sapiens 266Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met
Ser Tyr 1 5 10 26718PRTHomo
sapiens 267Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile
Tyr 1 5 10 15 Gly
Leu 26831PRTHomo sapiens 268Asp Arg Glu Asp Leu His Cys Asp Ile Asp Glu
Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn Ala Lys
20 25 30 26923PRTHomo
sapiens 269Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg 1 5 10 15 Leu
Ile Ala Tyr Phe Val Leu 20 2708PRTHomo sapiens
270Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
271420PRTHomo sapiens 271Met Arg Ile Ser Leu Pro Arg Ala Pro Glu Arg Asp
Gly Gly Val Ser 1 5 10
15 Ala Ser Ser Leu Leu Asp Thr Val Thr Asn Ala Ser Ser Tyr Ser Ala
20 25 30 Glu Met Thr
Glu Pro Lys Ser Val Cys Val Ser Val Asp Glu Val Val 35
40 45 Ser Ser Asn Met Glu Ala Thr Glu
Thr Asp Leu Leu Asn Gly His Leu 50 55
60 Lys Lys Val Asp Asn Asn Leu Thr Glu Ala Gln Arg Phe
Ser Ser Leu 65 70 75
80 Pro Arg Arg Ala Ala Val Asn Ile Glu Phe Arg Asp Leu Ser Tyr Ser
85 90 95 Val Pro Glu Gly
Pro Trp Trp Arg Lys Lys Gly Tyr Lys Thr Leu Leu 100
105 110 Lys Gly Ile Ser Gly Lys Phe Asn Ser
Gly Glu Leu Val Ala Ile Met 115 120
125 Gly Pro Ser Gly Ala Gly Lys Ser Thr Leu Met Asn Ile Leu
Ala Gly 130 135 140
Tyr Arg Glu Thr Gly Met Lys Gly Ala Val Leu Ile Asn Gly Leu Pro 145
150 155 160 Arg Asp Leu Arg Cys
Phe Arg Lys Val Ser Cys Tyr Ile Met Gln Asp 165
170 175 Asp Met Leu Leu Pro His Leu Thr Val Gln
Glu Ala Met Met Val Ser 180 185
190 Ala His Leu Lys Leu Gln Glu Lys Asp Glu Gly Arg Arg Glu Met
Val 195 200 205 Lys
Glu Ile Leu Thr Ala Leu Gly Leu Leu Ser Cys Ala Asn Thr Arg 210
215 220 Thr Gly Ser Leu Ser Gly
Gly Gln Arg Lys Arg Leu Ala Ile Ala Leu 225 230
235 240 Glu Leu Val Asn Asn Pro Pro Val Met Phe Phe
Asp Glu Pro Thr Ser 245 250
255 Gly Leu Asp Ser Ala Ser Cys Phe Gln Val Val Ser Leu Met Lys Gly
260 265 270 Leu Ala
Gln Gly Gly Arg Ser Ile Ile Cys Thr Ile His Gln Pro Ser 275
280 285 Ala Lys Leu Phe Glu Leu Phe
Asp Gln Leu Tyr Val Leu Ser Gln Gly 290 295
300 Gln Cys Val Tyr Arg Gly Lys Val Cys Asn Leu Val
Pro Tyr Leu Arg 305 310 315
320 Asp Leu Gly Leu Asn Cys Pro Thr Tyr His Asn Pro Ala Asp Phe Val
325 330 335 Met Glu Val
Ala Ser Gly Glu Tyr Gly Asp Gln Asn Ser Arg Leu Val 340
345 350 Arg Ala Val Arg Glu Gly Met Cys
Asp Ser Asp His Lys Arg Asp Leu 355 360
365 Gly Gly Asp Ala Glu Val Asn Pro Phe Leu Trp His Arg
Pro Ser Glu 370 375 380
Glu Asp Ser Ser Ser Met Glu Gly Cys His Ser Phe Ser Ala Ser Cys 385
390 395 400 Leu Thr Gln Phe
Cys Ile Leu Phe Lys Arg Thr Phe Leu Ser Ile Met 405
410 415 Arg Asp Ser Val 420
27223PRTHomo sapiens 272Leu Thr His Leu Arg Ile Thr Ser His Ile Gly Ile
Gly Leu Leu Ile 1 5 10
15 Gly Leu Leu Tyr Leu Gly Ile 20
27312PRTHomo sapiens 273Gly Asn Glu Ala Lys Lys Val Leu Ser Asn Ser Gly 1
5 10 27423PRTHomo sapiens 274Phe
Leu Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met Pro 1
5 10 15 Thr Val Leu Thr Phe Pro
Leu 20 27523PRTHomo sapiens 275Glu Met Gly Val
Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu 1 5
10 15 Lys Ala Tyr Tyr Leu Ala Lys
20 27623PRTHomo sapiens 276Thr Met Ala Asp Val Pro Phe
Gln Ile Met Phe Pro Val Ala Tyr Cys 1 5
10 15 Ser Ile Val Tyr Trp Met Thr 20
2776PRTHomo sapiens 277Ser Gln Pro Ser Asp Ala 1
5 27823PRTHomo sapiens 278Val Arg Phe Val Leu Phe Ala Ala Leu Gly
Thr Met Thr Ser Leu Val 1 5 10
15 Ala Gln Ser Leu Gly Leu Leu 20
2799PRTHomo sapiens 279Ile Gly Ala Ala Ser Thr Ser Leu Gln 1
5 28023PRTHomo sapiens 280Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe 20
28112PRTHomo sapiens 281Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met
Ser Tyr 1 5 10 28218PRTHomo
sapiens 282Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile
Tyr 1 5 10 15 Gly
Leu 28331PRTHomo sapiens 283Asp Arg Glu Asp Leu His Cys Asp Ile Asp Glu
Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn Ala Lys
20 25 30 28423PRTHomo
sapiens 284Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg 1 5 10 15 Leu
Ile Ala Tyr Phe Val Leu 20 2858PRTHomo sapiens
285Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
286411PRTHomo sapiens 286Met Leu Gly Thr Gln Gly Trp Thr Lys Gln Arg Lys
Pro Cys Pro Gln 1 5 10
15 Asn Ala Ser Ser Tyr Ser Ala Glu Met Thr Glu Pro Lys Ser Val Cys
20 25 30 Val Ser Val
Asp Glu Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr 35
40 45 Asp Leu Leu Asn Gly His Leu Lys
Lys Val Asp Asn Asn Leu Thr Glu 50 55
60 Ala Gln Arg Phe Ser Ser Leu Pro Arg Arg Ala Ala Val
Asn Ile Glu 65 70 75
80 Phe Arg Asp Leu Ser Tyr Ser Val Pro Glu Gly Pro Trp Trp Arg Lys
85 90 95 Lys Gly Tyr Lys
Thr Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser 100
105 110 Gly Glu Leu Val Ala Ile Met Gly Pro
Ser Gly Ala Gly Lys Ser Thr 115 120
125 Leu Met Asn Ile Leu Ala Gly Tyr Arg Glu Thr Gly Met Lys
Gly Ala 130 135 140
Val Leu Ile Asn Gly Leu Pro Arg Asp Leu Arg Cys Phe Arg Lys Val 145
150 155 160 Ser Cys Tyr Ile Met
Gln Asp Asp Met Leu Leu Pro His Leu Thr Val 165
170 175 Gln Glu Ala Met Met Val Ser Ala His Leu
Lys Leu Gln Glu Lys Asp 180 185
190 Glu Gly Arg Arg Glu Met Val Lys Glu Ile Leu Thr Ala Leu Gly
Leu 195 200 205 Leu
Ser Cys Ala Asn Thr Arg Thr Gly Ser Leu Ser Gly Gly Gln Arg 210
215 220 Lys Arg Leu Ala Ile Ala
Leu Glu Leu Val Asn Asn Pro Pro Val Met 225 230
235 240 Phe Phe Asp Glu Pro Thr Ser Gly Leu Asp Ser
Ala Ser Cys Phe Gln 245 250
255 Val Val Ser Leu Met Lys Gly Leu Ala Gln Gly Gly Arg Ser Ile Ile
260 265 270 Cys Thr
Ile His Gln Pro Ser Ala Lys Leu Phe Glu Leu Phe Asp Gln 275
280 285 Leu Tyr Val Leu Ser Gln Gly
Gln Cys Val Tyr Arg Gly Lys Val Cys 290 295
300 Asn Leu Val Pro Tyr Leu Arg Asp Leu Gly Leu Asn
Cys Pro Thr Tyr 305 310 315
320 His Asn Pro Ala Asp Phe Val Met Glu Val Ala Ser Gly Glu Tyr Gly
325 330 335 Asp Gln Asn
Ser Arg Leu Val Arg Ala Val Arg Glu Gly Met Cys Asp 340
345 350 Ser Asp His Lys Arg Asp Leu Gly
Gly Asp Ala Glu Val Asn Pro Phe 355 360
365 Leu Trp His Arg Pro Ser Glu Glu Asp Ser Ser Ser Met
Glu Gly Cys 370 375 380
His Ser Phe Ser Ala Ser Cys Leu Thr Gln Phe Cys Ile Leu Phe Lys 385
390 395 400 Arg Thr Phe Leu
Ser Ile Met Arg Asp Ser Val 405 410
28723PRTHomo sapiens 287Leu Thr His Leu Arg Ile Thr Ser His Ile Gly Ile
Gly Leu Leu Ile 1 5 10
15 Gly Leu Leu Tyr Leu Gly Ile 20
28812PRTHomo sapiens 288Gly Asn Glu Ala Lys Lys Val Leu Ser Asn Ser Gly 1
5 10 28923PRTHomo sapiens 289Phe
Leu Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met Pro 1
5 10 15 Thr Val Leu Thr Phe Pro
Leu 20 29023PRTHomo sapiens 290Glu Met Gly Val
Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu 1 5
10 15 Lys Ala Tyr Tyr Leu Ala Lys
20 29123PRTHomo sapiens 291Thr Met Ala Asp Val Pro Phe
Gln Ile Met Phe Pro Val Ala Tyr Cys 1 5
10 15 Ser Ile Val Tyr Trp Met Thr 20
2926PRTHomo sapiens 292Ser Gln Pro Ser Asp Ala 1
5 29323PRTHomo sapiens 293Val Arg Phe Val Leu Phe Ala Ala Leu Gly
Thr Met Thr Ser Leu Val 1 5 10
15 Ala Gln Ser Leu Gly Leu Leu 20
2949PRTHomo sapiens 294Ile Gly Ala Ala Ser Thr Ser Leu Gln 1
5 29523PRTHomo sapiens 295Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe 20
29612PRTHomo sapiens 296Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met
Ser Tyr 1 5 10 29718PRTHomo
sapiens 297Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile
Tyr 1 5 10 15 Gly
Leu 29831PRTHomo sapiens 298Asp Arg Glu Asp Leu His Cys Asp Ile Asp Glu
Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn Ala Lys
20 25 30 29923PRTHomo
sapiens 299Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg 1 5 10 15 Leu
Ile Ala Tyr Phe Val Leu 20 3008PRTHomo sapiens
300Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
301387PRTHomo sapiens 301Met Thr Glu Pro Lys Ser Val Cys Val Ser Val Asp
Glu Val Val Ser 1 5 10
15 Ser Asn Met Glu Ala Thr Glu Thr Asp Leu Leu Asn Gly His Leu Lys
20 25 30 Lys Val Asp
Asn Asn Leu Thr Glu Ala Gln Arg Phe Ser Ser Leu Pro 35
40 45 Arg Arg Ala Ala Val Asn Ile Glu
Phe Arg Asp Leu Ser Tyr Ser Val 50 55
60 Pro Glu Gly Pro Trp Trp Arg Lys Lys Gly Tyr Lys Thr
Leu Leu Lys 65 70 75
80 Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu Leu Val Ala Ile Met Gly
85 90 95 Pro Ser Gly Ala
Gly Lys Ser Thr Leu Met Asn Ile Leu Ala Gly Tyr 100
105 110 Arg Glu Thr Gly Met Lys Gly Ala Val
Leu Ile Asn Gly Leu Pro Arg 115 120
125 Asp Leu Arg Cys Phe Arg Lys Val Ser Cys Tyr Ile Met Gln
Asp Asp 130 135 140
Met Leu Leu Pro His Leu Thr Val Gln Glu Ala Met Met Val Ser Ala 145
150 155 160 His Leu Lys Leu Gln
Glu Lys Asp Glu Gly Arg Arg Glu Met Val Lys 165
170 175 Glu Ile Leu Thr Ala Leu Gly Leu Leu Ser
Cys Ala Asn Thr Arg Thr 180 185
190 Gly Ser Leu Ser Gly Gly Gln Arg Lys Arg Leu Ala Ile Ala Leu
Glu 195 200 205 Leu
Val Asn Asn Pro Pro Val Met Phe Phe Asp Glu Pro Thr Ser Gly 210
215 220 Leu Asp Ser Ala Ser Cys
Phe Gln Val Val Ser Leu Met Lys Gly Leu 225 230
235 240 Ala Gln Gly Gly Arg Ser Ile Ile Cys Thr Ile
His Gln Pro Ser Ala 245 250
255 Lys Leu Phe Glu Leu Phe Asp Gln Leu Tyr Val Leu Ser Gln Gly Gln
260 265 270 Cys Val
Tyr Arg Gly Lys Val Cys Asn Leu Val Pro Tyr Leu Arg Asp 275
280 285 Leu Gly Leu Asn Cys Pro Thr
Tyr His Asn Pro Ala Asp Phe Val Met 290 295
300 Glu Val Ala Ser Gly Glu Tyr Gly Asp Gln Asn Ser
Arg Leu Val Arg 305 310 315
320 Ala Val Arg Glu Gly Met Cys Asp Ser Asp His Lys Arg Asp Leu Gly
325 330 335 Gly Asp Ala
Glu Val Asn Pro Phe Leu Trp His Arg Pro Ser Glu Glu 340
345 350 Asp Ser Ser Ser Met Glu Gly Cys
His Ser Phe Ser Ala Ser Cys Leu 355 360
365 Thr Gln Phe Cys Ile Leu Phe Lys Arg Thr Phe Leu Ser
Ile Met Arg 370 375 380
Asp Ser Val 385 30223PRTHomo sapiens 302Leu Thr His Leu Arg Ile
Thr Ser His Ile Gly Ile Gly Leu Leu Ile 1 5
10 15 Gly Leu Leu Tyr Leu Gly Ile 20
30312PRTHomo sapiens 303Gly Asn Glu Ala Lys Lys Val Leu Ser
Asn Ser Gly 1 5 10 30423PRTHomo
sapiens 304Phe Leu Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met
Pro 1 5 10 15 Thr
Val Leu Thr Phe Pro Leu 20 30523PRTHomo sapiens
305Glu Met Gly Val Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu 1
5 10 15 Lys Ala Tyr Tyr
Leu Ala Lys 20 30623PRTHomo sapiens 306Thr Met
Ala Asp Val Pro Phe Gln Ile Met Phe Pro Val Ala Tyr Cys 1 5
10 15 Ser Ile Val Tyr Trp Met Thr
20 3076PRTHomo sapiens 307Ser Gln Pro Ser Asp
Ala 1 5 30823PRTHomo sapiens 308Val Arg Phe Val Leu
Phe Ala Ala Leu Gly Thr Met Thr Ser Leu Val 1 5
10 15 Ala Gln Ser Leu Gly Leu Leu
20 3099PRTHomo sapiens 309Ile Gly Ala Ala Ser Thr Ser Leu
Gln 1 5 31023PRTHomo sapiens 310Val Ala
Thr Phe Val Gly Pro Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe
20 31112PRTHomo sapiens 311Asp Thr Ile Pro Thr
Tyr Leu Gln Trp Met Ser Tyr 1 5 10
31218PRTHomo sapiens 312Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile
Leu Ser Ile Tyr 1 5 10
15 Gly Leu 31331PRTHomo sapiens 313Asp Arg Glu Asp Leu His Cys Asp
Ile Asp Glu Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn
Ala Lys 20 25 30
31423PRTHomo sapiens 314Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe
Ile Ser Leu Arg 1 5 10
15 Leu Ile Ala Tyr Phe Val Leu 20
3158PRTHomo sapiens 315Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
316406PRTHomo sapiens 316Met Ile Met Arg Leu Pro Gln Pro His
Gly Thr Asn Ala Ser Ser Tyr 1 5 10
15 Ser Ala Glu Met Thr Glu Pro Lys Ser Val Cys Val Ser Val
Asp Glu 20 25 30
Val Val Ser Ser Asn Met Glu Ala Thr Glu Thr Asp Leu Leu Asn Gly
35 40 45 His Leu Lys Lys
Val Asp Asn Asn Leu Thr Glu Ala Gln Arg Phe Ser 50
55 60 Ser Leu Pro Arg Arg Ala Ala Val
Asn Ile Glu Phe Arg Asp Leu Ser 65 70
75 80 Tyr Ser Val Pro Glu Gly Pro Trp Trp Arg Lys Lys
Gly Tyr Lys Thr 85 90
95 Leu Leu Lys Gly Ile Ser Gly Lys Phe Asn Ser Gly Glu Leu Val Ala
100 105 110 Ile Met Gly
Pro Ser Gly Ala Gly Lys Ser Thr Leu Met Asn Ile Leu 115
120 125 Ala Gly Tyr Arg Glu Thr Gly Met
Lys Gly Ala Val Leu Ile Asn Gly 130 135
140 Leu Pro Arg Asp Leu Arg Cys Phe Arg Lys Val Ser Cys
Tyr Ile Met 145 150 155
160 Gln Asp Asp Met Leu Leu Pro His Leu Thr Val Gln Glu Ala Met Met
165 170 175 Val Ser Ala His
Leu Lys Leu Gln Glu Lys Asp Glu Gly Arg Arg Glu 180
185 190 Met Val Lys Glu Ile Leu Thr Ala Leu
Gly Leu Leu Ser Cys Ala Asn 195 200
205 Thr Arg Thr Gly Ser Leu Ser Gly Gly Gln Arg Lys Arg Leu
Ala Ile 210 215 220
Ala Leu Glu Leu Val Asn Asn Pro Pro Val Met Phe Phe Asp Glu Pro 225
230 235 240 Thr Ser Gly Leu Asp
Ser Ala Ser Cys Phe Gln Val Val Ser Leu Met 245
250 255 Lys Gly Leu Ala Gln Gly Gly Arg Ser Ile
Ile Cys Thr Ile His Gln 260 265
270 Pro Ser Ala Lys Leu Phe Glu Leu Phe Asp Gln Leu Tyr Val Leu
Ser 275 280 285 Gln
Gly Gln Cys Val Tyr Arg Gly Lys Val Cys Asn Leu Val Pro Tyr 290
295 300 Leu Arg Asp Leu Gly Leu
Asn Cys Pro Thr Tyr His Asn Pro Ala Asp 305 310
315 320 Phe Val Met Glu Val Ala Ser Gly Glu Tyr Gly
Asp Gln Asn Ser Arg 325 330
335 Leu Val Arg Ala Val Arg Glu Gly Met Cys Asp Ser Asp His Lys Arg
340 345 350 Asp Leu
Gly Gly Asp Ala Glu Val Asn Pro Phe Leu Trp His Arg Pro 355
360 365 Ser Glu Glu Asp Ser Ser Ser
Met Glu Gly Cys His Ser Phe Ser Ala 370 375
380 Ser Cys Leu Thr Gln Phe Cys Ile Leu Phe Lys Arg
Thr Phe Leu Ser 385 390 395
400 Ile Met Arg Asp Ser Val 405 31723PRTHomo
sapiens 317Leu Thr His Leu Arg Ile Thr Ser His Ile Gly Ile Gly Leu Leu
Ile 1 5 10 15 Gly
Leu Leu Tyr Leu Gly Ile 20 31812PRTHomo sapiens
318Gly Asn Glu Ala Lys Lys Val Leu Ser Asn Ser Gly 1 5
10 31923PRTHomo sapiens 319Phe Leu Phe Phe Ser Met
Leu Phe Leu Met Phe Ala Ala Leu Met Pro 1 5
10 15 Thr Val Leu Thr Phe Pro Leu 20
32023PRTHomo sapiens 320Glu Met Gly Val Phe Leu Arg Glu His
Leu Asn Tyr Trp Tyr Ser Leu 1 5 10
15 Lys Ala Tyr Tyr Leu Ala Lys 20
32123PRTHomo sapiens 321Thr Met Ala Asp Val Pro Phe Gln Ile Met Phe Pro
Val Ala Tyr Cys 1 5 10
15 Ser Ile Val Tyr Trp Met Thr 20
3226PRTHomo sapiens 322Ser Gln Pro Ser Asp Ala 1 5
32323PRTHomo sapiens 323Val Arg Phe Val Leu Phe Ala Ala Leu Gly Thr Met
Thr Ser Leu Val 1 5 10
15 Ala Gln Ser Leu Gly Leu Leu 20
3249PRTHomo sapiens 324Ile Gly Ala Ala Ser Thr Ser Leu Gln 1
5 32523PRTHomo sapiens 325Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe 20
32612PRTHomo sapiens 326Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met
Ser Tyr 1 5 10 32718PRTHomo
sapiens 327Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile
Tyr 1 5 10 15 Gly
Leu 32831PRTHomo sapiens 328Asp Arg Glu Asp Leu His Cys Asp Ile Asp Glu
Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn Ala Lys
20 25 30 32923PRTHomo
sapiens 329Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg 1 5 10 15 Leu
Ile Ala Tyr Phe Val Leu 20 3308PRTHomo sapiens
330Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
331567PRTHomo sapiens 331Met Leu Ala Val Gln Gln Thr Glu His Leu Pro Ala
Cys Pro Pro Ala 1 5 10
15 Arg Arg Trp Ser Ser Asn Phe Cys Pro Glu Ser Thr Glu Gly Gly Pro
20 25 30 Ser Leu Leu
Gly Leu Arg Asp Met Val Arg Arg Gly Trp Ser Val Cys 35
40 45 Thr Ala Ile Leu Leu Ala Arg Leu
Trp Cys Leu Val Pro Thr His Thr 50 55
60 Phe Leu Ser Glu Tyr Pro Glu Ala Ala Glu Tyr Pro His
Pro Gly Trp 65 70 75
80 Val Tyr Trp Leu Gln Met Ala Val Ala Pro Gly His Leu Arg Ala Trp
85 90 95 Val Met Arg Asn
Asn Val Thr Thr Asn Ile Pro Ser Ala Phe Ser Gly 100
105 110 Thr Leu Thr His Glu Glu Lys Ala Val
Leu Thr Val Phe Thr Gly Thr 115 120
125 Ala Thr Ala Val His Val Gln Val Ala Ala Leu Ala Ser Ala
Lys Leu 130 135 140
Glu Ser Ser Val Phe Val Thr Asp Cys Val Ser Cys Lys Ile Glu Asn 145
150 155 160 Val Cys Asp Ser Ala
Leu Gln Gly Lys Arg Val Pro Met Ser Gly Leu 165
170 175 Gln Gly Ser Ser Ile Val Ile Met Pro Pro
Ser Asn Arg Pro Leu Ala 180 185
190 Ser Ala Ala Ser Cys Thr Trp Ser Val Gln Val Gln Gly Gly Pro
His 195 200 205 His
Leu Gly Val Val Ala Ile Ser Gly Lys Val Leu Ser Ala Ala His 210
215 220 Gly Ala Gly Arg Ala Tyr
Gly Trp Gly Phe Pro Gly Asp Pro Met Glu 225 230
235 240 Glu Gly Tyr Lys Thr Leu Leu Lys Gly Ile Ser
Gly Lys Phe Asn Ser 245 250
255 Gly Glu Leu Val Ala Ile Met Gly Pro Ser Gly Ala Gly Lys Ser Thr
260 265 270 Leu Met
Asn Ile Leu Ala Gly Tyr Arg Glu Thr Gly Met Lys Gly Ala 275
280 285 Val Leu Ile Asn Gly Leu Pro
Arg Asp Leu Arg Cys Phe Arg Lys Val 290 295
300 Ser Cys Tyr Ile Met Gln Asp Asp Met Leu Leu Pro
His Leu Thr Val 305 310 315
320 Gln Glu Ala Met Met Val Ser Ala His Leu Lys Leu Gln Glu Lys Asp
325 330 335 Glu Gly Arg
Arg Glu Met Val Lys Glu Ile Leu Thr Ala Leu Gly Leu 340
345 350 Leu Ser Cys Ala Asn Thr Arg Thr
Gly Ser Leu Ser Gly Gly Gln Arg 355 360
365 Lys Arg Leu Ala Ile Ala Leu Glu Leu Val Asn Asn Pro
Pro Val Met 370 375 380
Phe Phe Asp Glu Pro Thr Ser Gly Leu Asp Ser Ala Ser Cys Phe Gln 385
390 395 400 Val Val Ser Leu
Met Lys Gly Leu Ala Gln Gly Gly Arg Ser Ile Ile 405
410 415 Cys Thr Ile His Gln Pro Ser Ala Lys
Leu Phe Glu Leu Phe Asp Gln 420 425
430 Leu Tyr Val Leu Ser Gln Gly Gln Cys Val Tyr Arg Gly Lys
Val Cys 435 440 445
Asn Leu Val Pro Tyr Leu Arg Asp Leu Gly Leu Asn Cys Pro Thr Tyr 450
455 460 His Asn Pro Ala Asp
Phe Val Met Glu Val Ala Ser Gly Glu Tyr Gly 465 470
475 480 Asp Gln Asn Ser Arg Leu Val Arg Ala Val
Arg Glu Gly Met Cys Asp 485 490
495 Ser Asp His Lys Arg Asp Leu Gly Gly Asp Ala Glu Val Asn Pro
Phe 500 505 510 Leu
Trp His Arg Pro Ser Glu Glu Val Lys Gln Thr Lys Arg Leu Lys 515
520 525 Gly Leu Arg Lys Asp Ser
Ser Ser Met Glu Gly Cys His Ser Phe Ser 530 535
540 Ala Ser Cys Leu Thr Gln Phe Cys Ile Leu Phe
Lys Arg Thr Phe Leu 545 550 555
560 Ser Ile Met Arg Asp Ser Val 565
33223PRTHomo sapiens 332Leu Thr His Leu Arg Ile Thr Ser His Ile Gly Ile
Gly Leu Leu Ile 1 5 10
15 Gly Leu Leu Tyr Leu Gly Ile 20
33312PRTHomo sapiens 333Gly Asn Glu Ala Lys Lys Val Leu Ser Asn Ser Gly 1
5 10 33423PRTHomo sapiens 334Phe
Leu Phe Phe Ser Met Leu Phe Leu Met Phe Ala Ala Leu Met Pro 1
5 10 15 Thr Val Leu Thr Phe Pro
Leu 20 33523PRTHomo sapiens 335Glu Met Gly Val
Phe Leu Arg Glu His Leu Asn Tyr Trp Tyr Ser Leu 1 5
10 15 Lys Ala Tyr Tyr Leu Ala Lys
20 33623PRTHomo sapiens 336Thr Met Ala Asp Val Pro Phe
Gln Ile Met Phe Pro Val Ala Tyr Cys 1 5
10 15 Ser Ile Val Tyr Trp Met Thr 20
3376PRTHomo sapiens 337Ser Gln Pro Ser Asp Ala 1
5 33823PRTHomo sapiens 338Val Arg Phe Val Leu Phe Ala Ala Leu Gly
Thr Met Thr Ser Leu Val 1 5 10
15 Ala Gln Ser Leu Gly Leu Leu 20
3399PRTHomo sapiens 339Ile Gly Ala Ala Ser Thr Ser Leu Gln 1
5 34023PRTHomo sapiens 340Val Ala Thr Phe Val Gly Pro
Val Thr Ala Ile Pro Val Leu Leu Phe 1 5
10 15 Ser Gly Phe Phe Val Ser Phe 20
34112PRTHomo sapiens 341Asp Thr Ile Pro Thr Tyr Leu Gln Trp Met
Ser Tyr 1 5 10 34218PRTHomo
sapiens 342Ile Ser Tyr Val Arg Tyr Gly Phe Glu Gly Val Ile Leu Ser Ile
Tyr 1 5 10 15 Gly
Leu 34331PRTHomo sapiens 343Asp Arg Glu Asp Leu His Cys Asp Ile Asp Glu
Thr Cys His Phe Gln 1 5 10
15 Lys Ser Glu Ala Ile Leu Arg Glu Leu Asp Val Glu Asn Ala Lys
20 25 30 34423PRTHomo
sapiens 344Leu Tyr Leu Asp Phe Ile Val Leu Gly Ile Phe Phe Ile Ser Leu
Arg 1 5 10 15 Leu
Ile Ala Tyr Phe Val Leu 20 3458PRTHomo sapiens
345Arg Tyr Lys Ile Arg Ala Glu Arg 1 5
34690PRTHomo sapiens 346Met Ala Gln Leu Glu Arg Ser Ala Ile Ser Gly Phe
Ser Ser Lys Ser 1 5 10
15 Arg Arg Asn Ser Phe Ala Tyr Asp Val Lys Arg Glu Val Tyr Asn Glu
20 25 30 Glu Thr Phe
Gln Gln Glu His Lys Arg Lys Ala Ser Ser Ser Gly Asn 35
40 45 Met Asn Ile Asn Ile Thr Thr Phe
Arg His His Val Gln Cys Arg Cys 50 55
60 Ser Trp His Arg Phe Leu Arg Cys Val Leu Thr Ile Phe
Pro Phe Leu 65 70 75
80 Glu Trp Met Cys Met Tyr Arg Leu Lys Asp 85
90 34723PRTHomo sapiens 347Trp Leu Leu Gly Asp Leu Leu Ala Gly Ile
Ser Val Gly Leu Val Gln 1 5 10
15 Val Pro Gln Gly Leu Thr Leu 20
3485PRTHomo sapiens 348Ser Leu Leu Ala Arg 1 5
34923PRTHomo sapiens 349Gln Leu Ile Pro Pro Leu Asn Ile Ala Tyr Ala Ala
Phe Cys Ser Ser 1 5 10
15 Val Ile Tyr Val Ile Phe Gly 20
3506PRTHomo sapiens 350Ser Cys His Gln Met Ser 1 5
35120PRTHomo sapiens 351Ile Gly Ser Phe Phe Leu Val Ser Ala Leu Leu Ile
Asn Val Leu Lys 1 5 10
15 Val Ser Pro Phe 20 35229PRTHomo sapiens 352Asn Asn
Gly Gln Leu Val Met Gly Ser Phe Val Lys Asn Glu Phe Ser 1 5
10 15 Ala Pro Ser Tyr Leu Met Gly
Tyr Asn Lys Ser Leu Ser 20 25
35323PRTHomo sapiens 353Val Val Ala Thr Thr Thr Phe Leu Thr Gly Ile Ile
Gln Leu Ile Met 1 5 10
15 Gly Val Leu Gly Leu Gly Phe 20
35412PRTHomo sapiens 354Ile Ala Thr Tyr Leu Pro Glu Ser Ala Met Ser Ala 1
5 10 35523PRTHomo sapiens 355Tyr
Leu Ala Ala Val Ala Leu His Ile Met Leu Ser Gln Leu Thr Phe 1
5 10 15 Ile Phe Gly Ile Met Ile
Ser 20 35614PRTHomo sapiens 356Phe His Ala Gly
Pro Ile Ser Phe Phe Tyr Asp Ile Ile Asn 1 5
10 35723PRTHomo sapiens 357Tyr Cys Val Ala Leu Pro Lys
Ala Asn Ser Thr Ser Ile Leu Val Phe 1 5
10 15 Leu Thr Val Val Val Ala Leu 20
35820PRTHomo sapiens 358Arg Ile Asn Lys Cys Ile Arg Ile Ser Phe
Asn Gln Tyr Pro Ile Glu 1 5 10
15 Phe Pro Met Glu 20 35918PRTHomo sapiens 359Leu
Phe Leu Ile Ile Gly Phe Thr Val Ile Ala Asn Lys Ile Ser Met 1
5 10 15 Ala Thr 36027PRTHomo
sapiens 360Glu Thr Ser Gln Thr Leu Ile Asp Met Ile Pro Tyr Ser Phe Leu
Leu 1 5 10 15 Pro
Val Thr Pro Asp Phe Ser Leu Leu Pro Lys 20
25 36120PRTHomo sapiens 361Ile Ile Leu Gln Ala Phe Ser Leu Ser
Leu Val Ser Ser Phe Leu Leu 1 5 10
15 Ile Phe Leu Gly 20 36253PRTHomo sapiens
362Lys Lys Ile Ala Ser Leu His Asn Tyr Ser Val Asn Ser Asn Gln Asp 1
5 10 15 Leu Ile Ala Ile
Gly Leu Cys Asn Val Val Ser Ser Phe Phe Arg Ser 20
25 30 Cys Val Phe Thr Gly Ala Ile Ala Arg
Thr Ile Ile Gln Asp Lys Ser 35 40
45 Gly Gly Arg Gln Gln 50 36323PRTHomo
sapiens 363Phe Ala Ser Leu Val Gly Ala Gly Val Met Leu Leu Leu Met Val
Lys 1 5 10 15 Met
Gly His Phe Phe Tyr Thr 20 3644PRTHomo sapiens
364Leu Pro Asn Ala 1 36520PRTHomo sapiens 365Val Leu Ala
Gly Ile Ile Leu Ser Asn Val Ile Pro Tyr Leu Glu Thr 1 5
10 15 Ile Ser Asn Leu 20
36620PRTHomo sapiens 366Pro Ser Leu Trp Arg Gln Asp Gln Tyr Asp Cys Ala
Leu Trp Met Met 1 5 10
15 Thr Phe Ser Ser 20 36723PRTHomo sapiens 367Ser Ile
Phe Leu Gly Leu Asp Ile Gly Leu Ile Ile Ser Val Val Ser 1 5
10 15 Ala Phe Phe Ile Thr Thr Val
20 368451PRTHomo sapiens 368Arg Ser His Arg Ala
Lys Ile Leu Leu Leu Gly Gln Ile Pro Asn Thr 1 5
10 15 Asn Ile Tyr Arg Ser Ile Asn Asp Tyr Arg
Glu Ile Ile Thr Ile Pro 20 25
30 Gly Val Lys Ile Phe Gln Cys Cys Ser Ser Ile Thr Phe Val Asn
Val 35 40 45 Tyr
Tyr Leu Lys His Lys Leu Leu Lys Glu Val Asp Met Val Lys Val 50
55 60 Pro Leu Lys Glu Glu Glu
Ile Phe Ser Leu Phe Asn Ser Ser Asp Thr 65 70
75 80 Asn Leu Gln Gly Gly Lys Ile Cys Arg Cys Phe
Cys Asn Cys Asp Asp 85 90
95 Leu Glu Pro Leu Pro Arg Ile Leu Tyr Thr Glu Arg Phe Glu Asn Lys
100 105 110 Leu Asp
Pro Glu Ala Ser Ser Ile Asn Leu Ile His Cys Ser His Phe 115
120 125 Glu Ser Met Asn Thr Ser Gln
Thr Ala Ser Glu Asp Gln Val Pro Tyr 130 135
140 Thr Val Ser Ser Val Ser Gln Lys Asn Gln Gly Gln
Gln Tyr Glu Glu 145 150 155
160 Val Glu Glu Val Trp Leu Pro Asn Asn Ser Ser Arg Asn Ser Ser Pro
165 170 175 Gly Leu Pro
Asp Val Ala Glu Ser Gln Gly Arg Arg Ser Leu Ile Pro 180
185 190 Tyr Ser Asp Ala Ser Leu Leu Pro
Ser Val His Thr Ile Ile Leu Asp 195 200
205 Phe Ser Met Val His Tyr Val Asp Ser Arg Gly Leu Val
Val Leu Arg 210 215 220
Gln Ile Cys Asn Ala Phe Gln Asn Ala Asn Ile Leu Ile Leu Ile Ala 225
230 235 240 Gly Cys His Ser
Ser Ile Val Arg Ala Phe Glu Arg Asn Asp Phe Phe 245
250 255 Asp Ala Gly Ile Thr Lys Thr Gln Leu
Phe Leu Ser Val His Asp Ala 260 265
270 Val Leu Phe Ala Leu Ser Arg Lys Val Ile Gly Ser Ser Glu
Leu Ser 275 280 285
Ile Asp Glu Ser Glu Thr Val Ile Arg Glu Thr Tyr Ser Glu Thr Asp 290
295 300 Lys Asn Asp Asn Ser
Arg Tyr Lys Met Ser Ser Ser Phe Leu Gly Ser 305 310
315 320 Gln Lys Asn Val Ser Pro Gly Phe Ile Lys
Ile Gln Gln Pro Val Glu 325 330
335 Glu Glu Ser Glu Leu Asp Leu Glu Leu Glu Ser Glu Gln Glu Ala
Gly 340 345 350 Leu
Gly Leu Asp Leu Asp Leu Asp Arg Glu Leu Glu Pro Glu Met Glu 355
360 365 Pro Lys Ala Glu Thr Glu
Thr Lys Thr Gln Thr Glu Met Glu Pro Gln 370 375
380 Pro Glu Thr Glu Pro Glu Met Glu Pro Asn Pro
Lys Ser Arg Pro Arg 385 390 395
400 Ala His Thr Phe Pro Gln Gln Arg Tyr Trp Pro Met Tyr His Pro Ser
405 410 415 Met Ala
Ser Thr Gln Ser Gln Thr Gln Thr Arg Thr Trp Ser Val Glu 420
425 430 Arg Arg Arg His Pro Met Asp
Ser Tyr Ser Pro Glu Gly Asn Ser Asn 435 440
445 Glu Asp Val 450 36957PRTHomo sapiens
369Met Asn Pro Phe Gln Lys Asn Glu Ser Lys Glu Thr Leu Phe Ser Pro 1
5 10 15 Val Ser Ile Glu
Glu Val Pro Pro Arg Pro Pro Ser Pro Pro Lys Lys 20
25 30 Pro Ser Pro Thr Ile Cys Gly Ser Asn
Tyr Pro Leu Ser Ile Ala Phe 35 40
45 Ile Val Val Asn Glu Phe Cys Glu Arg 50
55 37018PRTHomo sapiens 370Phe Ser Tyr Tyr Gly Met Lys Ala
Val Leu Ile Leu Tyr Phe Leu Tyr 1 5 10
15 Phe Leu 3719PRTHomo sapiens 371His Trp Asn Glu Asp
Thr Ser Thr Ser 1 5 37223PRTHomo sapiens
372Ile Tyr His Ala Phe Ser Ser Leu Cys Tyr Phe Thr Pro Ile Leu Gly 1
5 10 15 Ala Ala Ile Ala
Asp Ser Trp 20 3736PRTHomo sapiens 373Leu Gly
Lys Phe Lys Thr 1 5 37423PRTHomo sapiens 374Ile Ile
Tyr Leu Ser Leu Val Tyr Val Leu Gly His Val Ile Lys Ser 1 5
10 15 Leu Gly Ala Leu Pro Ile Leu
20 3753PRTHomo sapiens 375Gly Gly Gln 1
37623PRTHomo sapiens 376Val Val His Thr Val Leu Ser Leu Ile Gly Leu Ser
Leu Ile Ala Leu 1 5 10
15 Gly Thr Gly Gly Ile Lys Pro 20
37720PRTHomo sapiens 377Cys Val Ala Ala Phe Gly Gly Asp Gln Phe Glu Glu
Lys His Ala Glu 1 5 10
15 Glu Arg Thr Arg 20 37820PRTHomo sapiens 378Tyr Phe
Ser Val Phe Tyr Leu Ser Ile Asn Ala Gly Ser Leu Ile Ser 1 5
10 15 Thr Phe Ile Thr
20 37914PRTHomo sapiens 379Pro Met Leu Arg Gly Asp Val Gln Cys Phe Gly
Glu Asp Cys 1 5 10
38020PRTHomo sapiens 380Tyr Ala Leu Ala Phe Gly Val Pro Gly Leu Leu Met
Val Ile Ala Leu 1 5 10
15 Val Val Phe Ala 20 38161PRTHomo sapiens 381Met Gly
Ser Lys Ile Tyr Asn Lys Pro Pro Pro Glu Gly Asn Ile Val 1 5
10 15 Ala Gln Val Phe Lys Cys Ile
Trp Phe Ala Ile Ser Asn Arg Phe Lys 20 25
30 Asn Arg Ser Gly Asp Ile Pro Lys Arg Gln His Trp
Leu Asp Trp Ala 35 40 45
Ala Glu Lys Tyr Pro Lys Gln Leu Ile Met Asp Val Lys 50
55 60 38219PRTHomo sapiens 382Ala Leu Thr
Arg Val Leu Phe Leu Tyr Ile Pro Leu Pro Met Phe Trp 1 5
10 15 Ala Leu Leu 38329PRTHomo
sapiens 383Asp Gln Gln Gly Ser Arg Trp Thr Leu Gln Ala Ile Arg Met Asn
Arg 1 5 10 15 Asn
Leu Gly Phe Phe Val Leu Gln Pro Asp Gln Met Gln 20
25 38423PRTHomo sapiens 384Val Leu Asn Pro Leu Leu
Val Leu Ile Phe Ile Pro Leu Phe Asp Phe 1 5
10 15 Val Ile Tyr Arg Leu Val Ser 20
38511PRTHomo sapiens 385Lys Cys Gly Ile Asn Phe Ser Ser Leu
Arg Lys 1 5 10 38618PRTHomo sapiens
386Met Ala Val Gly Met Ile Leu Ala Cys Leu Ala Phe Ala Val Ala Ala 1
5 10 15 Ala Val
387250PRTHomo sapiens 387Glu Ile Lys Ile Asn Glu Met Ala Pro Ala Gln Pro
Gly Pro Gln Glu 1 5 10
15 Val Phe Leu Gln Val Leu Asn Leu Ala Asp Asp Glu Val Lys Val Thr
20 25 30 Val Val Gly
Asn Glu Asn Asn Ser Leu Leu Ile Glu Ser Ile Lys Ser 35
40 45 Phe Gln Lys Thr Pro His Tyr Ser
Lys Leu His Leu Lys Thr Lys Ser 50 55
60 Gln Asp Phe His Phe His Leu Lys Tyr His Asn Leu Ser
Leu Tyr Thr 65 70 75
80 Glu His Ser Val Gln Glu Lys Asn Trp Tyr Ser Leu Val Ile Arg Glu
85 90 95 Asp Gly Asn Ser
Ile Ser Ser Met Met Val Lys Asp Thr Glu Ser Arg 100
105 110 Thr Thr Asn Gly Met Thr Thr Val Arg
Phe Val Asn Thr Leu His Lys 115 120
125 Asp Val Asn Ile Ser Leu Ser Thr Asp Thr Ser Leu Asn Val
Gly Glu 130 135 140
Asp Tyr Gly Val Ser Ala Tyr Arg Thr Val Gln Arg Gly Glu Tyr Pro 145
150 155 160 Ala Val His Cys Arg
Thr Glu Asp Lys Asn Phe Ser Leu Asn Leu Gly 165
170 175 Leu Leu Asp Phe Gly Ala Ala Tyr Leu Phe
Val Ile Thr Asn Asn Thr 180 185
190 Asn Gln Gly Leu Gln Ala Trp Lys Ile Glu Asp Ile Pro Ala Asn
Lys 195 200 205 Met
Ser Ile Ala Trp Gln Leu Pro Gln Tyr Ala Leu Val Thr Ala Gly 210
215 220 Glu Val Met Phe Ser Val
Thr Gly Leu Glu Phe Ser Tyr Ser Gln Ala 225 230
235 240 Pro Ser Gly Met Lys Ser Val Leu Gln Ala
245 250 38823PRTHomo sapiens 388Ala Trp Leu
Leu Thr Ile Ala Val Gly Asn Ile Ile Val Leu Val Val 1 5
10 15 Ala Gln Phe Ser Gly Leu Val
20 3894PRTHomo sapiens 389Gln Trp Ala Glu 1
39023PRTHomo sapiens 390Phe Ile Leu Phe Ser Cys Leu Leu Leu Val Ile
Cys Leu Ile Phe Ser 1 5 10
15 Ile Met Gly Tyr Tyr Tyr Val 20
39132PRTHomo sapiens 391Pro Val Lys Thr Glu Asp Met Arg Gly Pro Ala Asp
Lys His Ile Pro 1 5 10
15 His Ile Gln Gly Asn Met Ile Lys Leu Glu Thr Lys Lys Thr Lys Leu
20 25 30
39219DNAArtificial SequenceSynthetic oligonucleotide 392ataagcggtt
atcactgcc
1939319DNAArtificial SequenceSynthetic oligonucleotide 393gctgggattc
caagtggac
1939419DNAArtificial SequenceSynthetic oligonucleotide 394aactgtgcag
ggcctctcc
1939519DNAArtificial SequenceSynthetic oligonucleotide 395gctgctggat
gtcattcac
1939619DNAArtificial SequenceSynthetic oligonucleotide 396agagacacag
tgcccatcc
1939719DNAArtificial SequenceSynthetic oligonucleotide 397actgaacctc
cgaaatgcc
1939819DNAArtificial SequenceSynthetic oligonucleotide 398gtgctggagt
gcttccatc
1939919DNAArtificial SequenceSynthetic oligonucleotide 399ttcagaccta
ccttcagtc
1940019DNAArtificial SequenceSynthetic oligonucleotide 400gagtcacaca
gagatgagc
1940119DNAArtificial SequenceSynthetic oligonucleotide 401cgatgtgcct
tcaagattc
1940219DNAArtificial SequenceSynthetic oligonucleotide 402cagtggtttg
ggaatctgc
1940319DNAArtificial SequenceSynthetic oligonucleotide 403gtgactacac
aaggactcc
1940419DNAArtificial SequenceSynthetic oligonucleotide 404gactgattcg
ctctttgcc
1940519DNAArtificial SequenceSynthetic oligonucleotide 405agtgcagcct
tgtgggttc
1940619DNAArtificial SequenceSynthetic oligonucleotide 406taacactcac
tgcacctgc
1940719DNAArtificial SequenceSynthetic oligonucleotide 407taactgaaac
tcagctagc
1940819DNAArtificial SequenceSynthetic oligonucleotide 408actgaagtag
ccctccttc
1940919DNAArtificial SequenceSynthetic oligonucleotide 409agtgcagtac
agcgatgac
1941019DNAArtificial SequenceSynthetic oligonucleotide 410ttcacatcgc
tgagcaccc
1941119DNAArtificial SequenceSynthetic oligonucleotide 411agccagcaac
gacatgtac
1941219DNAArtificial SequenceSynthetic oligonucleotide 412gctgctgggc
atgtccttc
1941319DNAArtificial SequenceSynthetic oligonucleotide 413tgtgatcgtc
atcacagtc
1941419DNAArtificial SequenceSynthetic oligonucleotide 414aacatgatat
gtgctggac
1941519DNAArtificial SequenceSynthetic oligonucleotide 415ctgagaaggc
ttccactgc
1941619DNAArtificial SequenceSynthetic oligonucleotide 416tgatacgtgg
atccaggcc
1941719DNAArtificial SequenceSynthetic oligonucleotide 417ctacagtgac
aaggctaac
1941819DNAArtificial SequenceSynthetic oligonucleotide 418gaactggata
gccctcatc
1941919DNAArtificial SequenceSynthetic oligonucleotide 419ccctggtaaa
gctgcattc
1942019DNAArtificial SequenceSynthetic oligonucleotide 420gatgaaggct
tcgggcttc
1942119DNAArtificial SequenceSynthetic oligonucleotide 421tgtaaagctg
gaaagggac
1942219DNAArtificial SequenceSynthetic oligonucleotide 422ctgaagaagc
tggagttgc
1942319DNAArtificial SequenceSynthetic oligonucleotide 423tccttgcagc
aggcacatc
1942419DNAArtificial SequenceSynthetic oligonucleotide 424tctgtgcgtg
gactggaac
1942519DNAArtificial SequenceSynthetic oligonucleotide 425ctttgctcgg
aagacgttc
1942619DNAArtificial SequenceSynthetic oligonucleotide 426gaaggctttg
gaaagtgtc
1942719DNAArtificial SequenceSynthetic oligonucleotide 427gtgaactctg
ctgcgactc
1942819DNAArtificial SequenceSynthetic oligonucleotide 428gacaaggcta
tgatgctgc
1942919DNAArtificial SequenceSynthetic oligonucleotide 429ggatgtgtgg
tgctgtcac
1943019DNAArtificial SequenceSynthetic oligonucleotide 430ctctgtgttc
cacttcggc
1943119DNAArtificial SequenceSynthetic oligonucleotide 431cagcaatgca
gagtgtgac
1943219DNAArtificial SequenceSynthetic oligonucleotide 432caaagctggc
tactactac
1943319DNAArtificial SequenceSynthetic oligonucleotide 433cagtgcaaag
agcccaaac
1943419DNAArtificial SequenceSynthetic oligonucleotide 434gtattctgta
caccctggc
1943519DNAArtificial SequenceSynthetic oligonucleotide 435gtgatcgaca
ggattgctc
1943619DNAArtificial SequenceSynthetic oligonucleotide 436cacagtgaaa
ccttcctgc
1943719DNAArtificial SequenceSynthetic oligonucleotide 437atctgtgaca
ctggatcgc
1943819DNAArtificial SequenceSynthetic oligonucleotide 438agagactgga
gttgtcagc
1943919DNAArtificial SequenceSynthetic oligonucleotide 439cctgagttga
atgtcatac
1944019DNAArtificial SequenceSynthetic oligonucleotide 440ctgaactagt
gactatccc
1944119DNAArtificial SequenceSynthetic oligonucleotide 441ataagcaccg
tgagcgacc
1944219DNAArtificial SequenceSynthetic oligonucleotide 442cattgggcca
cagacctac
1944319DNAArtificial SequenceSynthetic oligonucleotide 443gatgaagaca
gcaaccaac
1944419DNAArtificial SequenceSynthetic oligonucleotide 444agcatatgat
gaccttggc
1944519DNAArtificial SequenceSynthetic oligonucleotide 445attccactac
tacagctgc
1944619DNAArtificial SequenceSynthetic oligonucleotide 446gaaactgtgg
caggctaac
1944719DNAArtificial SequenceSynthetic oligonucleotide 447ctgatgaagg
ccttcgacc
1944819DNAArtificial SequenceSynthetic oligonucleotide 448ttgaaacaag
aggaagtcc
1944919DNAArtificial SequenceSynthetic oligonucleotide 449tgaacttgct
ctgagctgc
1945019DNAArtificial SequenceSynthetic oligonucleotide 450atctgtaacc
tcagcacac
1945119DNAArtificial SequenceSynthetic oligonucleotide 451gaagctaagc
ctcggttac
1945219DNAArtificial SequenceSynthetic oligonucleotide 452taaccgtggc
atctacctc
1945319DNAArtificial SequenceSynthetic oligonucleotide 453tgaccacctg
gagtatcac
1945419DNAArtificial SequenceSynthetic oligonucleotide 454gtggacatct
ttgagcttc
1945519DNAArtificial SequenceSynthetic oligonucleotide 455gctgagaagt
acttccacc
1945619DNAArtificial SequenceSynthetic oligonucleotide 456agactactgc
aagggcggc
1945719DNAArtificial SequenceSynthetic oligonucleotide 457gagtatttgc
tggcattcc
1945819DNAArtificial SequenceSynthetic oligonucleotide 458ggagacacgg
aataaactc
1945919DNAArtificial SequenceSynthetic oligonucleotide 459ccgagaccac
ctcaatgtc
1946019DNAArtificial SequenceSynthetic oligonucleotide 460atggacatct
ccacgggac
1946119DNAArtificial SequenceSynthetic oligonucleotide 461tatcctgacc
ttcctgcgc
1946219DNAArtificial SequenceSynthetic oligonucleotide 462cacatgatca
agctaggtc
1946319DNAArtificial SequenceSynthetic oligonucleotide 463gaagccaggc
atcttcatc
1946419DNAArtificial SequenceSynthetic oligonucleotide 464gctgaagtta
tccagtctc
1946519DNAArtificial SequenceSynthetic oligonucleotide 465agcattggac
cagttgatc
1946619DNAArtificial SequenceSynthetic oligonucleotide 466gtgatctacg
tgaactggc
1946719DNAArtificial SequenceSynthetic oligonucleotide 467gccgacagtg
gtgcactac
1946819DNAArtificial SequenceSynthetic oligonucleotide 468aacatgatgg
ctcagaacc
1946919DNAArtificial SequenceSynthetic oligonucleotide 469tacagtgatg
gatcatagc
1947019DNAArtificial SequenceSynthetic oligonucleotide 470accaatatgc
ctaccttcc
1947119DNAArtificial SequenceSynthetic oligonucleotide 471actgtatccc
agcagtccc
1947219DNAArtificial SequenceSynthetic oligonucleotide 472aagctgaaca
taaccttgc
1947319DNAArtificial SequenceSynthetic oligonucleotide 473ttgaatagct
cggtgtccc
1947419DNAArtificial SequenceSynthetic oligonucleotide 474gtggaaggca
agatcttcc
1947519DNAArtificial SequenceSynthetic oligonucleotide 475tgtatggctg
gtcgatcac
1947619DNAArtificial SequenceSynthetic oligonucleotide 476gctgcgacaa
cttctgttc
1947719DNAArtificial SequenceSynthetic oligonucleotide 477gcccacggtc
ttccactac
1947819DNAArtificial SequenceSynthetic oligonucleotide 478gactgaatca
ggccttccc
194792196DNAhomo sapiens 479ccggcatttg cgtttggggc gccctccctg cgccgggggc
gggagcccag cgagcgcaga 60gccccggccc cgcgcggccc gagtgccaca tcactgcgct
ggccgtccaa ggtccgccgc 120cccaccatgc cgcccccgcc gccgctgctg ctccttacag
tcctggtcgt cgccgctgcc 180cggccggggt gcgagtttga gcggaacccc gccgccacct
gcgtggacct gcagctcagg 240acctgcagcg atgccgccta caaccacacc accttcccca
acctgcttca gcaccggtcg 300tgggaggtgg tggaggccag ctccgagtac atcctgctga
gcgttctaca ccagctcctg 360gaaggccagt gcaacccgga cctgcggctg ctgggctgtg
ctgtgctggc cccccggtgt 420gagggcggct gggtgcgcag accctgccgg cacatctgcg
agggcctgcg ggaggtctgc 480cagcccgcct tcgacgccat tgacatggcc tggccctact
tccttgactg ccaccgctac 540ttcacgagag aggacgaggg ctgctatgac ccgctggaga
agcttcgggg aggcctggag 600gctgacgagg cactgccctc agggctgccg cccaccttca
tccgcttcag ccaccactcc 660tacgcccaga tggtgcgtgt gctgaggcgg acggcctccc
gctgcgccca cgtggccagg 720acctacagca tcgggcgcag cttcgacggc agggagctgc
tggtcatcga gttctccagc 780cgccccggcc agcacgagct gatggagccc gaggtgaagc
tcatcggcaa cattcatggc 840aacgaggtgg cgggccggga gatgctcatc tacctagccc
agtacctgtg ctctgagtac 900ctgcttggta acccccgcat ccagcgcctg ctcaacacca
cccgcatcca cctgctgccc 960tccatgaacc ctgacggcta tgaggtggca gctgccgagg
gtgccggcta caacgggtgg 1020acgagcggga ggcagaacgc gcagaacctg gatctgaacc
gaaatttccc ggacctgacg 1080tccgagtact accggctggc ggagacccgc ggcgcacgca
gcgaccacat ccccatcccc 1140cagcactact ggtggggtaa ggtggccccg gagacaaagg
caatcatgaa gtggatgcag 1200accataccct ttgtgctctc agccagcctt catgggggcg
acctggtggt gtcctacccc 1260ttcgacttct ccaagcaccc ccaggaggag aagatgtttt
ctcccacgcc cgacgagaag 1320atgttcaagc tgctgtccag agcctacgct gacgtccacc
ccatgatgat ggacaggtcg 1380gagaataggt gtggaggcaa tttcctgaag agggggagca
tcatcaacgg ggcggactgg 1440tacagcttca cgggaggcat gtccgatttc aactacctgc
acaccaactg ctttgagatc 1500acggtagagc tgggctgtgt gaagttcccc cccgaggagg
ccctgtacac actctggcag 1560cacaacaagg agtcactcct gaatttcgtg gagacggtgc
accggggcat caaaggtgtg 1620gtgacagata aattcggcaa gccagtcaaa aacgcccgga
tctcagtcaa aggcattcgc 1680cacgacatca ccacagcccc agatggtgac tactggagac
tgctgccccc aggtatccac 1740attgtcattg cccaagcccc tggctacgcc aaagtcatca
agaaagtcat catccccgcc 1800cggatgaaga gggctggccg tgtggacttc attctgcaac
ctctggggat gggacccaag 1860aactttattc atgggctgcg gaggactggg ccccacgacc
cgctgggagg tgccagctct 1920ttgggggagg ccacggagcc cgacccgctc cgggcgcgca
ggcagccctc ggccgacggg 1980agtaagccct ggtggtggtc ctacttcaca tcgctgagca
cccacaggcc acgctggctg 2040ctcaagtact agccccggcc ccagcacccg ccaggatgtg
gagaccgagg cccatctccg 2100catcccgggc tcctggctct tgattttgtc tgccacagac
atcccacaaa gccgctgcca 2160ttttattaaa gtgttttgat ccactttcca ctggaa
2196480641PRThomo sapiens 480Met Pro Pro Pro Pro
Pro Leu Leu Leu Leu Thr Val Leu Val Val Ala 1 5
10 15 Ala Ala Arg Pro Gly Cys Glu Phe Glu Arg
Asn Pro Ala Ala Thr Cys 20 25
30 Val Asp Leu Gln Leu Arg Thr Cys Ser Asp Ala Ala Tyr Asn His
Thr 35 40 45 Thr
Phe Pro Asn Leu Leu Gln His Arg Ser Trp Glu Val Val Glu Ala 50
55 60 Ser Ser Glu Tyr Ile Leu
Leu Ser Val Leu His Gln Leu Leu Glu Gly 65 70
75 80 Gln Cys Asn Pro Asp Leu Arg Leu Leu Gly Cys
Ala Val Leu Ala Pro 85 90
95 Arg Cys Glu Gly Gly Trp Val Arg Arg Pro Cys Arg His Ile Cys Glu
100 105 110 Gly Leu
Arg Glu Val Cys Gln Pro Ala Phe Asp Ala Ile Asp Met Ala 115
120 125 Trp Pro Tyr Phe Leu Asp Cys
His Arg Tyr Phe Thr Arg Glu Asp Glu 130 135
140 Gly Cys Tyr Asp Pro Leu Glu Lys Leu Arg Gly Gly
Leu Glu Ala Asp 145 150 155
160 Glu Ala Leu Pro Ser Gly Leu Pro Pro Thr Phe Ile Arg Phe Ser His
165 170 175 His Ser Tyr
Ala Gln Met Val Arg Val Leu Arg Arg Thr Ala Ser Arg 180
185 190 Cys Ala His Val Ala Arg Thr Tyr
Ser Ile Gly Arg Ser Phe Asp Gly 195 200
205 Arg Glu Leu Leu Val Ile Glu Phe Ser Ser Arg Pro Gly
Gln His Glu 210 215 220
Leu Met Glu Pro Glu Val Lys Leu Ile Gly Asn Ile His Gly Asn Glu 225
230 235 240 Val Ala Gly Arg
Glu Met Leu Ile Tyr Leu Ala Gln Tyr Leu Cys Ser 245
250 255 Glu Tyr Leu Leu Gly Asn Pro Arg Ile
Gln Arg Leu Leu Asn Thr Thr 260 265
270 Arg Ile His Leu Leu Pro Ser Met Asn Pro Asp Gly Tyr Glu
Val Ala 275 280 285
Ala Ala Glu Gly Ala Gly Tyr Asn Gly Trp Thr Ser Gly Arg Gln Asn 290
295 300 Ala Gln Asn Leu Asp
Leu Asn Arg Asn Phe Pro Asp Leu Thr Ser Glu 305 310
315 320 Tyr Tyr Arg Leu Ala Glu Thr Arg Gly Ala
Arg Ser Asp His Ile Pro 325 330
335 Ile Pro Gln His Tyr Trp Trp Gly Lys Val Ala Pro Glu Thr Lys
Ala 340 345 350 Ile
Met Lys Trp Met Gln Thr Ile Pro Phe Val Leu Ser Ala Ser Leu 355
360 365 His Gly Gly Asp Leu Val
Val Ser Tyr Pro Phe Asp Phe Ser Lys His 370 375
380 Pro Gln Glu Glu Lys Met Phe Ser Pro Thr Pro
Asp Glu Lys Met Phe 385 390 395
400 Lys Leu Leu Ser Arg Ala Tyr Ala Asp Val His Pro Met Met Met Asp
405 410 415 Arg Ser
Glu Asn Arg Cys Gly Gly Asn Phe Leu Lys Arg Gly Ser Ile 420
425 430 Ile Asn Gly Ala Asp Trp Tyr
Ser Phe Thr Gly Gly Met Ser Asp Phe 435 440
445 Asn Tyr Leu His Thr Asn Cys Phe Glu Ile Thr Val
Glu Leu Gly Cys 450 455 460
Val Lys Phe Pro Pro Glu Glu Ala Leu Tyr Thr Leu Trp Gln His Asn 465
470 475 480 Lys Glu Ser
Leu Leu Asn Phe Val Glu Thr Val His Arg Gly Ile Lys 485
490 495 Gly Val Val Thr Asp Lys Phe Gly
Lys Pro Val Lys Asn Ala Arg Ile 500 505
510 Ser Val Lys Gly Ile Arg His Asp Ile Thr Thr Ala Pro
Asp Gly Asp 515 520 525
Tyr Trp Arg Leu Leu Pro Pro Gly Ile His Ile Val Ile Ala Gln Ala 530
535 540 Pro Gly Tyr Ala
Lys Val Ile Lys Lys Val Ile Ile Pro Ala Arg Met 545 550
555 560 Lys Arg Ala Gly Arg Val Asp Phe Ile
Leu Gln Pro Leu Gly Met Gly 565 570
575 Pro Lys Asn Phe Ile His Gly Leu Arg Arg Thr Gly Pro His
Asp Pro 580 585 590
Leu Gly Gly Ala Ser Ser Leu Gly Glu Ala Thr Glu Pro Asp Pro Leu
595 600 605 Arg Ala Arg Arg
Gln Pro Ser Ala Asp Gly Ser Lys Pro Trp Trp Trp 610
615 620 Ser Tyr Phe Thr Ser Leu Ser Thr
His Arg Pro Arg Trp Leu Leu Lys 625 630
635 640 Tyr
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