Patent application title: ACACS As Modifiers of the IGF Pathway and Methods of Use
Inventors:
Mark E. Maxwell (Lansdale, PA, US)
Michael Martin Ollmann (Redwood City, CA, US)
Timothy S. Heuer (El Granada, CA, US)
Lynn Margaret Bjerke (Surrey, GB)
Assignees:
Exelixis, Inc.
IPC8 Class: AA61K39395FI
USPC Class:
4241721
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds eukaryotic cell or component thereof or substance produced by said eukaryotic cell (e.g., honey, etc.)
Publication date: 2009-02-26
Patent application number: 20090053239
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Patent application title: ACACS As Modifiers of the IGF Pathway and Methods of Use
Inventors:
Timothy S. Heuer
Lynn Margaret Bjerke
Michael Martin Ollmann
Mark E. Maxwell
Agents:
MCDONNELL BOEHNEN HULBERT @ BERGHOFF LLP
Assignees:
EXELIXIS, INC.
Origin: CHICAGO, IL US
IPC8 Class: AA61K39395FI
USPC Class:
4241721
Abstract:
Human MPTEN genes are identified as modulators of the PTEN pathway, and
thus are therapeutic targets for disorders associated with defective PTEN
function. Methods for identifying modulators of PTEN, comprising
screening for agents that modulate the activity of MPTEN are provided.Claims:
1. A method of identifying a candidate IGF pathway modulating agent, said
method comprising the steps of:(a) providing an assay system comprising
an ACAC polypeptide or nucleic acid;(b) contacting the assay system with
a test agent under conditions whereby, but for the presence of the test
agent, the system provides a reference activity; and(c) detecting a test
agent-biased activity of the assay system, wherein a difference between
the test agent-biased activity and the reference activity identifies the
test agent as a candidate IGF pathway modulating agent.
2. The method of claim 1 wherein the assay system comprises cultured cells that express the ACAC polypeptide.
3. The method of claim 2 wherein the cultured cells additionally have defective IGF function.
4. The method of claim 1 wherein the assay system includes a screening assay comprising an ACAC polypeptide, and the candidate test agent is a small molecule modulator.
5. The method of claim 4 wherein the assay is a carboxylase assay.
6. The method of claim 1 wherein the assay system is selected from the group consisting of an apoptosis assay system, a cell proliferation assay system, an angiogenesis assay system, and a hypoxic induction assay system.
7. The method of claim 1 wherein the assay system includes a binding assay comprising an ACAC polypeptide and the candidate test agent is an antibody.
8. The method of claim 1 wherein the assay system includes an expression assay comprising an ACAC nucleic acid and the candidate test agent is a nucleic acid modulator.
9. The method of claim 8 wherein the nucleic acid modulator is an antisense oligomer.
10. The method of claim 8 wherein the nucleic acid modulator is a PMO.
11. The method of claim 1 additionally comprising:(d) administering the candidate IGF pathway modulating agent identified in (c) to a model system comprising cells defective in IGF function and, detecting a phenotypic change in the model system that indicates that the IGF function is restored.
12. The method of claim 11 wherein the model system is a mouse model with defective IGF function.
13. A method for modulating a IGF pathway of a cell comprising contacting a cell defective in IGF function with a candidate modulator that specifically binds to an ACAC polypeptide, whereby IGF function is restored.
14. The method of claim 13 wherein the candidate modulator is administered to a vertebrate animal predetermined to have a disease or disorder resulting from a defect in IGF function.
15. The method of claim 13 wherein the candidate modulator is selected from the group consisting of an antibody and a small molecule.
16. The method of claim 1, comprising the additional steps of:(d) providing a secondary assay system comprising cultured cells or a non-human animal expressing ACAC,(e) contacting the secondary assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and(f) detecting an agent-biased activity of the second assay system,wherein a difference between the agent-biased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate IGF pathway modulating agent,and wherein the second assay detects an agent-biased change in the IGF pathway.
17. The method of claim 16 wherein the secondary assay system comprises cultured cells.
18. The method of claim 16 wherein the secondary assay system comprises a non-human animal.
19. The method of claim 18 wherein the non-human animal mis-expresses a IGF pathway gene.
20. A method of modulating IGF pathway in a mammalian cell comprising contacting the cell with an agent that specifically binds an ACAC polypeptide or nucleic acid.
21. The method of claim 20 wherein the agent is administered to a mammalian animal predetermined to have a pathology associated with the IGF pathway.
22. The method of claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody.
23. A method for diagnosing a disease in a patient comprising:obtaining a biological sample from the patient;contacting the sample with a probe for ACAC expression;comparing results from step (b) with a control;determining whether step (c) indicates a likelihood of disease.
24. The method of claim 23 wherein said disease is cancer.
25. The method according to claim 24, wherein said cancer is a cancer as shown in Table 1 as having >25% expression level.
Description:
REFERENCE TO RELATED APPLICATIONS
[0001]This application claims priority to U.S. provisional patent application 60/581,689 filed Jun. 21, 2004. The contents of the prior application are hereby incorporated in their entirety.
BACKGROUND OF THE INVENTION
[0002]Somatic mutations in the PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) gene are known to cause tumors in a variety of human tissues. In addition, germline mutations in PTEN are the cause of human diseases (Cowden disease and Bannayan-Zonana syndrome) associated with increased risk of breast and thyroid cancer (Nelen M R et al. (1997) Hum Mol Genet, 8:1383-1387; Liaw D et al. (1997) Nat Genet, 1:64-67; Marsh D J et al. (1998) Hum Mol Genet, 3:507-515). PTEN is thought to act as a tumor suppressor by regulating several signaling pathways through the second messenger phosphatidylinositol 3,4,5 triphosphate (PIP3). PTEN dephosphorylates the D3 position of PIP3 and downregulates signaling events dependent on PIP3 levels (Maehama T and Dixon J E (1998) J Biol Chem, 22, 13375-8). In particular, pro-survival pathways downstream of the insulin-like growth factor (IGF) pathway are regulated by PTEN activity. Stimulation of the IGF pathway, or loss of PTEN function, elevates PIP3 levels and activates pro-survival pathways associated with tumorigenesis (Stambolic V et al. (1998) Cell, 95:29-39). Consistent with this model, elevated levels of insulin-like growth factors I and II correlate with increased risk of cancer (Yu H et al (1999) J Natl Cancer Inst 91:151-156) and poor prognosis (Takanami I et al, 1996, J Surg Oncol 61(3):205-8).
[0003]PTEN sequence is conserved in evolution, and exists in mouse (Hansen GM and Justice M J (1998) Mamm Genome, 9(1):88-90), Drosophila (Goberdhan D C et al (1999) Genes and Dev, 24:3244-58; Huang H et al (1999) Development 23:5365-72), and C. elegans (Ogg S and Ruvkun G, (1998) Mol Cell, (6):887-93). Studies in these model organisms have helped to elucidate the role of PTEN in processes relevant to tumorigenesis. In Drosophila, the PTEN homolog (dPTEN) has been shown to regulate cell size, survival, and proliferation (Huang et al, supra; Goberdhan et al, supra; Gao X et al, 2000, 221:404-418). In mice, loss of PTEN function increases cancer susceptibility (Di Cristofano A et al (1998) Nature Genetics, 19:348-355; Suzuki A et al (1998) Curr. Biol., 8:1169-78).
[0004]In addition, a member of the IGF/insulin receptor family exists in Drosophila and has been shown to respond to insulin stimulation (Fernandez-Almonacid R, and Rozen O M (1987) Mol Cell Bio, (8):2718-27). Similar to PTEN, studies in Drosophila (Brogiolo W et al (2001) Curr Biol, 11(4):213-21) and mouse (Moorehead R A et al (2003) Oncogene, 22(6):853-857) establish a conserved role for the IGF/insulin pathway in growth control.
[0005]Acetyl-CoA carboxylase (ACAC), 1 of 4 human biotin-dependent enzymes, is a pivotal enzyme in the synthesis of fatty acids. The other 3 enzymes are pyruvate carboxylase, which catalyzes the initial committed step in gluconeogenesis; propionyl-CoA carboxylase, which catabolizes the branched-chain amino acids valine, isoleucine, methionine, and threonine, as well as the odd-chain fatty acids and the side chain of cholesterol; and beta-methylcrotonyl CoA carboxylase, which catalyzes the catabolism of leucine. ACAC is a biotin-containing enzyme which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. There are two ACAC forms, alpha and beta, encoded by two different genes. ACAC-alpha (ACACA) is highly enriched in lipogenic tissues. The enzyme is under long term control at the transcriptional and translational levels and under short term regulation by the phosphorylation/dephosphorylation of targeted serine residues and by allosteric transformation by citrate or palmitoyl-CoA. Multiple alternatively spliced transcript variants divergent in the 5' sequence and encoding distinct isoforms have been found for this gene. ACAC-beta (ACACB) is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine-palmitoyl-CoA transferase I, the rate-limiting step in fatty acid uptake and oxidation by mitochondria. ACACB may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. There is evidence for the presence of two ACACB isoforms.
[0006]The ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Mechler B M et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M. 1996 Cell 86:521-529; Wassannan D A, et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth DR. 1999 Cancer Metastasis Rev. 18: 261-284). For example, a genetic screen can be carried out in an invertebrate model organism or cell having underexpression (e.g. knockout) or overexpression of a gene (referred to as a "genetic entry point") that yields a visible phenotype, such as altered cell growth. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a "modifier" involved in the same or overlapping pathway as the genetic entry point. When inactivation of either gene is not lethal, but inactivation of both genes results in reduced viability or death of the cell, tissue, or organism, the interaction is defined as "synthetic lethal" (Bender, A and Pringle J, (1991) Mol Cell Biol, 11: 1295-1305; Hartman J et al, (2001) Science 291:1001-1004; U.S. Pat. No. 6,489,127). In a synthetic lethal interaction, the modifier may also be identified as an "interactor". When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as the IGF pathway, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
[0007]All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.
SUMMARY OF THE INVENTION
[0008]We have discovered genes that modify the IGF pathway in Drosophila cells, and identified their human orthologs, hereinafter referred to as Acetyl-CoA carboxylase (ACAC). The invention provides methods for utilizing these IGF modifier genes and polypeptides to identify ACAC-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired IGF function and/or ACAC function. Preferred ACAC-modulating agents specifically bind to ACAC polypeptides and restore IGF function. Other preferred ACAC-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress ACAC gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
[0009]ACAC modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an ACAC polypeptide or nucleic acid. In one embodiment, candidate ACAC modulating agents are tested with an assay system comprising an ACAC polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate IGF modulating agents. The assay system may be cell-based or cell-free. ACAC-modulating agents include ACAC related proteins (e.g. dominant negative mutants, and biotherapeutics); ACAC-specific antibodies; ACAC-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with ACAC or compete with ACAC binding partner (e.g. by binding to an ACAC binding partner). In one specific embodiment, a small molecule modulator is identified using a carboxylase assay. In specific embodiments, the screening assay system is selected from a binding assay, an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.
[0010]In another embodiment, candidate IGF pathway modulating agents are further tested using a second assay system that detects changes in the IGF pathway, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the IGF pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).
[0011]The invention further provides methods for modulating the ACAC function and/or the IGF pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds an ACAC polypeptide or nucleic acid. The agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated with the IGF pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0012]The PTEN co-RNAi plus insulin synthetic interaction screen was designed to identify modifier genes that are lethal or reduce proliferation in cells with a hyperstimulated IGF/insulin pathway, but not in normal cells. We refer to these genes as "synthetic lethal" genes in the context of this screen. To identify these genes, we created cells with a hyperstimulated IGF/insulin pathway by treatment with insulin and RNAi-mediated inactivation of dPTEN, the Drosophila homologue of the human tumor suppressor PTEN. In addition to identifying genes with synthetic lethal interactions in insulin-treated, PTEN-deficient cells, this screen identified genes that, when inactivated, preferentially suppressed multiple readouts known to be regulated by IGF signaling. For our screen, these readouts included an expression assay for an IGF/insulin reporter gene and quantitative Western blot readouts for several nodes in the IGF/insulin pathway (phospho-4E-BP, phospho-MAPK, phospho-S6K, and total RpS6).
[0013]In a preferred embodiment, the Drosophila IGF modifier screen identified genes that, when inactivated, preferentially suppressed insulin-induced Lactate Dehydrogenase (LDH) expression and hence may be key mediators of IGF/PTEN signaling. Lactate Dehydrogenase (LDH) is a well-validated target of the Drosophila Insulin/IGF pathway. We confirmed this finding by analyzing gene expression in insulin-stimulated Drosophila S2 cells by microarray expression analysis (Affymetrix), which showed significant increases in expression of the LDH gene. This result was confirmed by Quantitative PCR (Taqman®) assay that detected a 12-fold increase in LDH expression in cells treated with either 1 μM insulin or dsRNA specific to the dPTEN gene. The use of LDH as a reporter gene has also been validated by RNAi of known positive mediators of IGF signaling such as InR, IRS, Tor, and Rheb, which results in substantially decreased LDH expression in the assay. In contrast, RNAi of known negative regulators of IGF signaling (TSC1 and TSC2) results in an increase in LDH expression. To further confirm that modifiers that decrease insulin-induced expression of LDH have relevance to IGF/PTEN signaling, we performed Quantitative Western Blots to determine whether RNAi of each modifier decreased phosphorylation of 4E-BP (a downstream gene that is phosphorylated by the Tor kinase) or affected S6K(Thr389) phosphorylation, MAPK phosphorylation, or total RPS6 protein levels.]
[0014]The CG 11198 gene was identified as having a synthetic interaction with the IGF/PTEN pathway. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, ACAC genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective IGF signaling pathway, such as cancer.
[0015]In vitro and in vivo methods of assessing ACAC function are provided herein. Modulation of the ACAC or their respective binding partners is useful for understanding the association of the IGF pathway and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for IGF related pathologies. ACAC-modulating agents that act by inhibiting or enhancing ACAC expression, directly or indirectly, for example, by affecting an ACAC function such as enzymatic (e.g., catalytic) or binding activity, can be identified using methods provided herein. ACAC modulating agents are useful in diagnosis, therapy and pharmaceutical development.
[0016]Nucleic Acids and Polypeptides of the Invention
[0017]Sequences related to ACAC nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) number) as GI #s 38679979 (SEQ ID NO:1), 38679959 (SEQ ID NO:2), 38679970 (SEQ ID NO:3), 38679973 (SEQ ID NO:4), 38679976 (SEQ ID NO:5), 33112864 (SEQ ID NO:6), 33112865 (SEQ ID NO:7), 33112866 (SEQ ID NO:8), 846082 (SEQ ID NO:9), 33112884 (SEQ ID NO:10), 61743949 (SEQ ID NO:11), 4501854 (SEQ ID NO:12), 1399289 (SEQ ID NO:13), 40019047 (SEQ ID NO:14), and 62087799 (SEQ ID NO:15) for nucleic acid, and GI #s 38679974 (SEQ ID NO:16), 4501855 (SEQ ID NO:17), and 61743950 (SEQ ID NO:18) for polypeptide sequences.
[0018]The term "ACAC polypeptide" refers to a full-length ACAC protein or a functionally active fragment or derivative thereof. A "functionally active" ACAC fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type ACAC protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind natural cellular substrates, etc. The functional activity of ACAC proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as further discussed below. In one embodiment, a functionally active ACAC polypeptide is an ACAC derivative capable of rescuing defective endogenous ACAC activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. For purposes herein, functionally active fragments also include, those fragments that comprise one or more structural domains of an ACAC, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2). For example, the carboxyl transferase domain (PFAM 01039) of ACAC from GI #s 38679974 and 61743950 (SEQ ID NOs:16 and 18, respectively) is located at approximately amino acid residues 1586-2145, and 1775-2334, respectively. Methods for obtaining ACAC polypeptides are also further described below. In some embodiments, preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of an ACAC. In further preferred embodiments, the fragment comprises the entire functionally active domain.
[0019]The term "ACAC nucleic acid" refers to a DNA or RNA molecule that encodes an ACAC polypeptide. Preferably, the ACAC polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with human ACAC. Methods of identifying orthlogs are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A et al., Genome Research (2000) 10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. In evolution, when a gene duplication event follows specification, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human. As used herein, the term "orthologs" encompasses paralogs. As used herein, "percent (%) sequence identity" with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410) with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity" is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
[0020]A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
[0021]Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Molec. Biol., 147:195-197; Nicholas et al., 1998, "A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www.psc.edu) and references cited therein; W. R. Pearson, 1991, Genomics 11:635-650). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated, the "Match" value reflects "sequence identity."
[0022]Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of an ACAC. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of an ACAC under high stringency hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h in a solution containing 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate).
[0023]In other embodiments, moderately stringent hybridization conditions are used that, are: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C. in a solution containing 2×SSC and 0.1% SDS.
[0024]Alternatively, low stringency conditions can be used that are: incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1×SSC at about 37° C. for 1 hour.
[0025]Isolation, Production, Expression, and Mis-Expression of ACAC Nucleic Acids and Polypeptides
[0026]ACAC nucleic acids and polypeptides are useful for identifying and testing agents that modulate ACAC function and for other applications related to the involvement of ACAC in the IGF pathway. ACAC nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art. In general, the particular use for the protein will dictate the particulars of expression, production, and purification methods. For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes. Expression of proteins to be purified for screening or antibody production may require the addition of specific tags (e.g., generation of fusion proteins). Overexpression of an ACAC protein for assays used to assess ACAC function, such as involvement in cell cycle regulation or hypoxic response, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefore may be used (e.g., Higgins S J and Hames B D (eds.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury P F et al., Principles of Fermentation Technology, 2nd edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan J E et al, Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York). In particular embodiments, recombinant ACAC is expressed in a cell line known to have defective IGF function. The recombinant cells are used in cell-based screening assay systems of the invention, as described further below.
[0027]The nucleotide sequence encoding an ACAC polypeptide can be inserted into any appropriate expression vector. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native ACAC gene and/or its flanking regions or can be heterologous. A variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA. An isolated host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.
[0028]To detect expression of the ACAC gene product, the expression vector can comprise a promoter operably linked to an ACAC gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the ACAC gene product based on the physical or functional properties of the ACAC protein in in vitro assay systems (e.g. immunoassays).
[0029]The ACAC protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection. A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).
[0030]Once a recombinant cell that expresses the ACAC gene sequence is identified, the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). Alternatively, native ACAC proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.
[0031]The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of ACAC or other genes associated with the IGF pathway. As used herein, mis-expression encompasses ectopic expression, over-expression, under-expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur).
[0032]Genetically Modified Animals
[0033]Animal models that have been genetically modified to alter ACAC expression may be used in in vivo assays to test for activity of a candidate IGF modulating agent, or to further assess the role of ACAC in a IGF pathway process such as apoptosis or cell proliferation. Preferably, the altered ACAC expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal ACAC expression. The genetically modified animal may additionally have altered IGF expression (e.g. IGF knockout). Preferred genetically modified animals are mammals such as primates, rodents (preferably mice or rats), among others. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i.e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763) or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.
[0034]Methods of making transgenic animals are well-known in the art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No. 4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin and Spradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see Berghammer A. J. et al., A Universal Marker for Transgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafish see Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000); 136:375-3830); for microinjection procedures for fish, amphibian eggs and birds see Houdebine and Chourrout, Experientia (1991) 47:897-905; for transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e.g., Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press (1987)). Clones of the nonhuman transgenic animals can be produced according to available methods (see Wilmut, I. et al. (1997) Nature 385:810-813; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).
[0035]In one embodiment, the transgenic animal is a "knock-out" animal having a heterozygous or homozygous alteration in the sequence of an endogenous ACAC gene that results in a decrease of ACAC function, preferably such that ACAC expression is undetectable or insignificant. Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it. The transgene can be a human gene (e.g., from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species. For example, a mouse ACAC gene is used to construct a homologous recombination vector suitable for altering an endogenous ACAC gene in the mouse genome. Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244:1288-1292; Joyner et al., Nature (1989) 338:153-156). Procedures for the production of non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra; Pursel et al., Science (1989)244:1281-1288; Simms et al., Bio/Technology (1988)6:179-183). In a preferred embodiment, knock-out animals, such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson M H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995) J Biol Chem. 270:8397-400).
[0036]In another embodiment, the transgenic animal is a "knock-in" animal having an alteration in its genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of the ACAC gene, e.g., by introduction of additional copies of ACAC, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the ACAC gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. The knock-in can be homozygous or heterozygous.
[0037]Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene. One example of such a system that may be produced is the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a preferred embodiment, both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25:83-6).
[0038]The genetically modified animals can be used in genetic studies to further elucidate the IGF pathway, as animal models of disease and disorders implicating defective IGF function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below. The candidate therapeutic agents are administered to a genetically modified animal having altered ACAC function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered ACAC expression that receive candidate therapeutic agent.
[0039]In addition to the above-described genetically modified animals having altered ACAC function, animal models having defective IGF function (and otherwise normal ACAC function), can be used in the methods of the present invention. For example, a mouse with defective PTEN function can be used to assess, in vivo, the activity of a candidate PTEN modulating agent identified in one of the in vitro assays described below. Transgenic mice with defective PTEN function have been described in literature (Di Cristofano et al, supra). Preferably, the candidate IGF modulating agent when administered to a model system with cells defective in IGF function, produces a detectable phenotypic change in the model system indicating that the IGF function is restored, i.e., the cells exhibit normal cell cycle progression.
[0040]Modulating Agents
[0041]The invention provides methods to identify agents that interact with and/or modulate the function of ACAC and/or the IGF pathway. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the IGF pathway, as well as in further analysis of the ACAC protein and its contribution to the IGF pathway. Accordingly, the invention also provides methods for modulating the IGF pathway comprising the step of specifically modulating ACAC activity by administering an ACAC-interacting or -modulating agent.
[0042]As used herein, an "ACAC-modulating agent" is any agent that modulates ACAC function, for example, an agent that interacts with ACAC to inhibit or enhance ACAC activity or otherwise affect normal ACAC function. ACAC function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the ACAC-modulating agent specifically modulates the function of the ACAC. The phrases "specific modulating agent", "specifically modulates", etc., are used herein to refer to modulating agents that directly bind to the ACAC polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the ACAC. These phrases also encompass modulating agents that alter the interaction of the ACAC with a binding partner, substrate, or cofactor (e.g. by binding to a binding partner of an ACAC, or to a protein/binding partner complex, and altering ACAC function). In a further preferred embodiment, the ACAC-modulating agent is a modulator of the IGF pathway (e.g. it restores and/or upregulates IGF function) and thus is also a IGF-modulating agent.
[0043]Preferred ACAC-modulating agents include small molecule compounds; ACAC-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors. The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, Pa., 19th edition.
[0044]Small Molecule Modulators
[0045]Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains. Chemical agents, referred to in the art as "small molecule" compounds are typically organic, non-peptide molecules, having a molecular weight up to 10,000, preferably up to 5,000, more preferably up to 1,000, and most preferably up to 500 daltons. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the ACAC protein or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for ACAC-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber S L, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).
[0046]Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the IGF pathway. The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
[0047]Protein Modulators
[0048]Specific ACAC-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the IGF pathway and related disorders, as well as in validation assays for other ACAC-modulating agents. In a preferred embodiment, ACAC-interacting proteins affect normal ACAC function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, ACAC-interacting proteins are useful in detecting and providing information about the function of ACAC proteins, as is relevant to IGF related disorders, such as cancer (e.g., for diagnostic means).
[0049]An ACAC-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an ACAC, such as a member of the ACAC pathway that modulates ACAC expression, localization, and/or activity. ACAC-modulators include dominant negative forms of ACAC-interacting proteins and of ACAC proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous ACAC-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema S F et al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R 3rd, Trends Genet (2000) 16:5-8).
[0050]An ACAC-interacting protein may be an exogenous protein, such as an ACAC-specific antibody or a T-cell antigen receptor (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). ACAC antibodies are further discussed below.
[0051]In preferred embodiments, an ACAC-interacting protein specifically binds an ACAC protein. In alternative preferred embodiments, an ACAC-modulating agent binds an ACAC substrate, binding partner, or cofactor.
[0052]Antibodies
[0053]In another embodiment, the protein modulator is an ACAC specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify ACAC modulators. The antibodies can also be used in dissecting the portions of the ACAC pathway responsible for various cellular responses and in the general processing and maturation of the ACAC.
[0054]Antibodies that specifically bind ACAC polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of ACAC polypeptide, and more preferably, to human ACAC. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Epitopes of ACAC which are particularly antigenic can be selected, for example, by routine screening of ACAC polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89; Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence of an ACAC. Monoclonal antibodies with affinities of 108 M-1 preferably 109 M-1 to 1010 M-1, or stronger can be made by standard procedures as described (Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577). Antibodies may be generated against crude cell extracts of ACAC or substantially purified fragments thereof. If ACAC fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an ACAC protein. In a particular embodiment, ACAC-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response. For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.
[0055]The presence of ACAC-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding ACAC polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.
[0056]Chimeric antibodies specific to ACAC polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323: 323-327). Humanized antibodies contain ˜10% murine sequences and ˜90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co M S, and Queen C. 1991 Nature 351: 501-501; Morrison S L. 1992 Ann. Rev. Immun. 10:239-265). Humanized antibodies and methods of their production are well-known in the art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).
[0057]ACAC-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced by methods known in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989) 334:544-546).
[0058]Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-1281). As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
[0059]The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers (1989) 4:131-134). A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also, recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. No. 6,086,900).
[0060]When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously. The therapeutically effective dose and dosage regimen is determined by clinical studies. Typically, the amount of antibody administered is in the range of about 0.1 mg/kg-to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further described in the literature (U.S. Pat. No. 5,859,206; WO0073469).
[0061]Nucleic Acid Modulators
[0062]Other preferred ACAC-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit ACAC activity. Preferred nucleic acid modulators interfere with the function of the ACAC nucleic acid such as DNA replication, transcription, translocation of the ACAC RNA to the site of protein translation, translation of protein from the ACAC RNA, splicing of the ACAC RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the ACAC RNA.
[0063]In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to an ACAC mRNA to bind to and prevent translation, preferably by binding to the 5' untranslated region. ACAC-specific antisense oligonucleotides, preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.
[0064]In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.: 7:187-95; U.S. Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).
[0065]Alternative preferred ACAC nucleic acid modulators are double-stranded RNA species mediating RNA interference (RNAi). RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619; Elbashir S M, et al., 2001 Nature 411:494-498; Novina C D and Sharp P. 2004 Nature 430:161-164; Soutschek J et al 2004 Nature 432:173-178; Hsieh A C et al. (2004) NAR 32(3):893-901).
[0066]Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used to elucidate the function of particular genes (see, for example, U.S. Pat. No. 6,165,790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway. For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan J F, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the invention, an ACAC-specific nucleic acid modulator is used in an assay to further elucidate the role of the ACAC in the IGF pathway, and/or its relationship to other members of the pathway. In another aspect of the invention, an ACAC-specific antisense oligomer is used as a therapeutic agent for treatment of IGF-related disease states.
[0067]Assay Systems
[0068]The invention provides assay systems and screening methods for identifying specific modulators of ACAC activity. As used herein, an "assay system" encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the ACAC nucleic acid or protein. In general, secondary assays further assess the activity of an ACAC modulating agent identified by a primary assay and may confirm that the modulating agent affects ACAC in a manner relevant to the IGF pathway. In some cases, ACAC modulators will be directly tested in a secondary assay.
[0069]In a preferred embodiment, the screening method comprises contacting a suitable assay system comprising an ACAC polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. carboxylase activity), which is based on the particular molecular event the screening method detects. A statistically significant difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates ACAC activity, and hence the IGF pathway. The ACAC polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.
[0070]Primary Assays
[0071]The type of modulator tested generally determines the type of primary assay.
[0072]Primary Assays for Small Molecule Modulators
[0073]For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying references). As used herein the term "cell-based" refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term "cell free" encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts. Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicity and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, calorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.
[0074]Cell-based screening assays usually require systems for recombinant expression of ACAC and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications, when ACAC-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the ACAC protein may be assayed by various known methods such as substrate processing (e.g. ability of the candidate ACAC-specific binding agents to function as negative effectors in ACAC-expressing cells), binding equilibrium constants (usually at least about 107 M-1, preferably at least about 108 M-1, more preferably at least about 109 M31 1), and immunogenicity (e.g. ability to elicit ACAC specific antibody in a heterologous host such as a mouse, rat, goat or rabbit). For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.
[0075]The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of an ACAC polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The ACAC polypeptide can be full length or a fragment thereof that retains functional ACAC activity. The ACAC polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The ACAC polypeptide is preferably human ACAC, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of ACAC interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has ACAC-specific binding activity, and can be used to assess normal ACAC gene function.
[0076]Suitable assay formats that may be adapted to screen for ACAC modulators are known in the art. Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In one preferred embodiment, screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4; Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000) 4:445-451).
[0077]A variety of suitable assay systems may be used to identify candidate ACAC and IGF pathway modulators (e.g. U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosis assays); and U.S. Pat. Nos. 5,976,782, 6,225,118 and 6,444,434 (angiogenesis assays), among others). Specific preferred assays are described in more detail below.
[0078]High throughput assays for measuring Acetyl COA carboxylase activity based on radiochemical methods have been described (Harwood et al (2003) J. Biol. Chem., 278: 37099-37111).
[0079]Apoptosis assays. Apoptosis or programmed cell death is a suicide program is activated within the cell, leading to fragmentation of DNA, shrinkage of the cytoplasm, membrane changes and cell death. Apoptosis is mediated by proteolytic enzymes of the caspase family. Many of the altering parameters of a cell are measurable during apoptosis. Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature 371, 346), by following the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747). Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41). Other cell-based apoptosis assays include the caspase-3/7 assay and the cell death nucleosome ELISA assay. The caspase 3/7 assay is based on the activation of the caspase cleavage activity as part of a cascade of events that occur during programmed cell death in many apoptotic pathways. In the caspase 3/7 assay (commercially available Apo-ONE® Homogeneous Caspase-3/7 assay from Promega, cat #67790), lysis buffer and caspase substrate are mixed and added to cells. The caspase substrate becomes fluorescent when cleaved by active caspase 3/7. The nucleosome ELISA assay is a general cell death assay known to those skilled in the art, and available commercially (Roche, Cat #1774425). This assay is a quantitative sandwich-enzyme-immunoassay which uses monoclonal antibodies directed against DNA and histones respectively, thus specifically determining amount of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Mono and oligonucleosomes are enriched in the cytoplasm during apoptosis due to the fact that DNA fragmentation occurs several hours before the plasma membrane breaks down, allowing for accumulation in the cytoplasm. Nucleosomes are not present in the cytoplasmic fraction of cells that are not undergoing apoptosis. The Phospho-histone H2B assay is another apoptosis assay, based on phosphorylation of histone H2B as a result of apoptosis. Fluorescent dyes that are associated with phosphohistone H2B may be used to measure the increase of phosphohistone H2B as a result of apoptosis. Apoptosis assays that simultaneously measure multiple parameters associated with apoptosis have also been developed. In such assays, various cellular parameters that can be associated with antibodies or fluorescent dyes, and that mark various stages of apoptosis are labeled, and the results are measured using instruments such as Cellomics® ArrayScan® HCS System. The measurable parameters and their markers include anti-active caspase-3 antibody which marks intermediate stage apoptosis, anti-PARP-p85 antibody (cleaved PARP) which marks late stage apoptosis, Hoechst labels which label the nucleus and are used to measure nuclear swelling as a measure of early apoptosis and nuclear condensation as a measure of late apoptosis, TOTO-3 fluorescent dye which labels DNA of dead cells with high cell membrane permeability, and anti-alpha-tubulin or F-actin labels, which assess cytoskeletal changes in cells and correlate well with TOTO-3 label.
[0080]An apoptosis assay system may comprise a cell that expresses an ACAC, and that optionally has defective IGF function (e.g. IGF is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate IGF modulating agents. In some embodiments of the invention, an apoptosis assay may be used as a secondary assay to test a candidate IGF modulating agents that is initially identified using a cell-free assay system. An apoptosis assay may also be used to test whether ACAC function plays a direct role in apoptosis. For example, an apoptosis assay may be performed on cells that over- or under-express ACAC relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the ACAC plays a direct role in the apoptotic response. Apoptosis assays are described further in U.S. Pat. No. 6,133,437.
[0081]Cell proliferation and cell cycle assays. Cell proliferation may be assayed via bromodeoxyuridine (BRDU) incorporation. This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA. Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or by other means.
[0082]Cell proliferation is also assayed via phospho-histone H3 staining, which identifies a cell population undergoing mitosis by phosphorylation of histone H3. Phosphorylation of histone H3 at serine 10 is detected using an antibody specific to the phosphorylated form of the serine 10 residue of histone H3. (Chadlee, D. N. 1995, J. Biol. Chem 270:20098-105). Cell Proliferation may also be examined using [3H]-thymidine incorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This assay allows for quantitative characterization of S-phase DNA syntheses. In this assay, cells synthesizing DNA will incorporate [3H]-thymidine into newly synthesized DNA. Incorporation can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e.g., Beckman LS 3800 Liquid Scintillation Counter). Another proliferation assay uses the dye Alamar Blue (available from Biosource International), which fluoresces when reduced in living cells and provides an indirect measurement of cell number (Voytik-Harbin S L et al., 1998, In Vitro Cell Dev Biol Anim 34:239-46). Yet another proliferation assay, the MTS assay, is based on in vitro cytotoxicity assessment of industrial chemicals, and uses the soluble tetrazolium salt, MTS. MTS assays are commercially available, for example, the Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Cat. #G5421).
[0083]Cell proliferation may also be assayed by colony formation in soft agar, or clonogenic survival assay (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). For example, cells transformed with ACAC are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.
[0084]Cell proliferation may also be assayed by measuring ATP levels as indicator of metabolically active cells. Such assays are commercially available, for example Cell Titer-Glo®, which is a luminescent homogeneous assay available from Promega.
[0085]Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55). Cells transfected with an ACAC may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson), which indicates accumulation of cells in different stages of the cell cycle.
[0086]Involvement of a gene in cell cycle may also be assayed by FOXO nuclear translocation assays. The FOXO family of transcription factors are mediators of various cellular functions including cell cycle progression and cell death, and are negatively regulated by activation of the PI3 kinase pathway. Akt phosphorylation of FOXO family members leads to FOXO sequestration in the cytoplasm and transcriptional inactivation (Medema, R. H et al (2000) Nature 404: 782-787). PTEN is a negative regulator of PI3 kinase pathway. Activation of PTEN, or loss of PI3 kinase or AKT, prevents phosphorylation of FOXO, leading to accumulation of FOXO in the nucleus, transcriptional activation of FOXO regulated genes, and apoptosis. Alternatively, loss of PTEN leads to pathway activation and cell survival (Nakamura, N. et al (2000) Mol Cell Biol 20: 8969-8982). FOXO translocation into the cytoplasm is used in assays and screens to identify members and/or modulators of the PTEN pathway. FOXO translocation assays using GFP or luciferase as detection reagents are known in the art (e.g., Zhang X et al (2002) J Biol Chem 277:45276-45284; and Li et al (2003) Mol Cell Biol 23:104-118).
[0087]Accordingly, a cell proliferation or cell cycle assay system may comprise a cell that expresses an ACAC, and that optionally has defective IGF function (e.g. IGF is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the assay system and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate IGF modulating agents. In some embodiments of the invention, the cell proliferation or cell cycle assay may be used as a secondary assay to test a candidate IGF modulating agents that is initially identified using another assay system such as a cell-free assay system. A cell proliferation assay may also be used to test whether ACAC function plays a direct role in cell proliferation or cell cycle. For example, a cell proliferation or cell cycle assay may be performed on cells that over- or under-express ACAC relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the ACAC plays a direct role in cell proliferation or cell cycle.
[0088]Angiogenesis. Angiogenesis may be assayed using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on Matrigel® (Becton Dickinson). Accordingly, an angiogenesis assay system may comprise a cell that expresses an ACAC, and that optionally has defective IGF function (e.g. IGF is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate IGF modulating agents. In some embodiments of the invention, the angiogenesis assay may be used as a secondary assay to test a candidate IGF modulating agents that is initially identified using another assay system. An angiogenesis assay may also be used to test whether ACAC function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over- or under-express ACAC relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the ACAC plays a direct role in angiogenesis. U.S. Pat. Nos. 5,976,782, 6,225,118 and 6,444,434, among others, describe various angiogenesis assays.
[0089]Hypoxic induction. The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glycolytic enzymes and VEGF. Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with ACAC in hypoxic conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001 incubator (Precision Scientific)) and normoxic conditions, followed by assessment of gene activity or expression by Taqman®. For example, a hypoxic induction assay system may comprise a cell that expresses an ACAC, and that optionally has defective IGF function (e.g. IGF is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate IGF modulating agents. In some embodiments of the invention, the hypoxic induction assay may be used as a secondary assay to test a candidate IGF modulating agents that is initially identified using another assay system. A hypoxic induction assay may also be used to test whether ACAC function plays a direct role in the hypoxic response. For example, a hypoxic induction assay may be performed on cells that over- or under-express ACAC relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the ACAC plays a direct role in hypoxic induction.
[0090]Cell adhesion. Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents. Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2× final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.
[0091]Cell-cell adhesion assays measure the ability of agents to modulate binding of cell adhesion proteins with their native ligands. These assays use cells that naturally or recombinantly express the adhesion protein of choice. In an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate. Cells expressing the ligand are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate agents. Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader.
[0092]High-throughput cell adhesion assays have also been described. In one such assay, small molecule ligands and peptides are bound to the surface of microscope slides using a microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off. In this assay, not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey J R et al., Bioconjug Chem. 2001 May-June; 12(3):346-53).
Primary Assays for Antibody Modulators
[0093]For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the ACAC protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting ACAC-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
[0094]In some cases, screening assays described for small molecule modulators may also be used to test antibody modulators.
Primary Assays for Nucleic Acid Modulators
[0095]For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance ACAC gene expression, preferably mRNA expression. In general, expression analysis comprises comparing ACAC expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express ACAC) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), or microarray analysis may be used to confirm that ACAC mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the ACAC protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra). In some cases, screening assays described for small molecule modulators, particularly in assay systems that involve ACAC mRNA expression, may also be used to test nucleic acid modulators.
Secondary Assays
[0096]Secondary assays may be used to further assess the activity of ACAC-modulating agent identified by any of the above methods to confirm that the modulating agent affects ACAC in a manner relevant to the IGF pathway. As used herein, ACAC-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with ACAC.
[0097]Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express ACAC) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate ACAC-modulating agent results in changes in the IGF pathway in comparison to untreated (or mock- or placebo-treated) cells or animals. Certain assays use "sensitized genetic backgrounds", which, as used herein, describe cells or animals engineered for altered expression of genes in the IGF or interacting pathways.
Cell-Based Assays
[0098]Cell based assays may detect endogenous IGF pathway activity or may rely on recombinant expression of IGF pathway components. Any of the aforementioned assays may be used in this cell-based format. Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
Animal Assays
[0099]A variety of non-human animal models of normal or defective IGF pathway may be used to test candidate ACAC modulators. Models for defective IGF pathway typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in the IGF pathway. Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.
[0100]In a preferred embodiment, IGF pathway activity is assessed by monitoring neovascularization and angiogenesis. Animal models with defective and normal IGF are used to test the candidate modulator's affect on ACAC in Matrigel® assays. Matrigel® is an extract of basement membrane proteins, and is composed primarily of laminin, collagen IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at 4° C., but rapidly forms a solid gel at 37° C. Liquid Matrigel® is mixed with various angiogenic agents, such as bFGF and VEGF, or with human tumor cells which over-express the ACAC. The mixture is then injected subcutaneously (SC) into female athymic nude mice (Taconic, Germantown, N.Y.) to support an intense vascular response. Mice with Matrigel® pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IV) routes with the candidate modulator. Mice are euthanized 5-12 days post-injection, and the Matrigel® pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.
[0101]In another preferred embodiment, the effect of the candidate modulator on ACAC is assessed via tumorigenicity assays. Tumor xenograft assays are known in the art (see, e.g., Ogawa K et al., 2000, Oncogene 19:6043-6052). Xenografts are typically implanted SC into female athymic mice, 6-7 week old, as single cell suspensions either from a pre-existing tumor or from in vitro culture. The tumors which express the ACAC endogenously are injected in the flank, 1×105 to 1×107 cells per mouse in a volume of 100 μL using a 27 gauge needle. Mice are then ear tagged and tumors are measured twice weekly. Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus administration. Depending upon the pharmacokinetics of each unique candidate modulator, dosing can be performed multiple times per day. The tumor weight is assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions. At the end of the experiment, the excised tumors may be utilized for biomarker identification or further analyses. For immunohistochemistry staining, xenograft tumors are fixed in 4% paraformaldehyde, 0.1M phosphate, pH 7.2, for 6 hours at 4° C., immersed in 30% sucrose in PBS, and rapidly frozen in isopentane cooled with liquid nitrogen.
[0102]In another preferred embodiment, tumorigenicity is monitored using a hollow fiber assay, which is described in U.S. Pat. No. 5,698,413. Briefly, the method comprises implanting into a laboratory animal a biocompatible, semi-permeable encapsulation device containing target cells, treating the laboratory animal with a candidate modulating agent, and evaluating the target cells for reaction to the candidate modulator. Implanted cells are generally human cells from a pre-existing tumor or a tumor cell line. After an appropriate period of time, generally around six days, the implanted samples are harvested for evaluation of the candidate modulator. Tumorigenicity and modulator efficacy may be evaluated by assaying the quantity of viable cells present in the macrocapsule, which can be determined by tests known in the art, for example, MTT dye conversion assay, neutral red dye uptake, trypan blue staining, viable cell counts, the number of colonies formed in soft agar, the capacity of the cells to recover and replicate in vitro, etc.
[0103]In another preferred embodiment, a tumorigenicity assay use a transgenic animal, usually a mouse, carrying a dominant oncogene or tumor suppressor gene knockout under the control of tissue specific regulatory sequences; these assays are generally referred to as transgenic tumor assays. In a preferred application, tumor development in the transgenic model is well characterized or is controlled. In an exemplary model, the "RIP1-Tag2" transgene, comprising the SV40 large T-antigen oncogene under control of the insulin gene regulatory regions is expressed in pancreatic beta cells and results in islet cell carcinomas (Hanahan D, 1985, Nature 315:115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA 93: 2002-2007; Bergers G et al, 1999, Science 284:808-812). An "angiogenic switch," occurs at approximately five weeks, as normally quiescent capillaries in a subset of hyperproliferative islets become angiogenic. The RIP1-TAG2 mice die by age 14 weeks. Candidate modulators may be administered at a variety of stages, including just prior to the angiogenic switch (e.g., for a model of tumor prevention), during the growth of small tumors (e.g., for a model of intervention), or during the growth of large and/or invasive tumors (e.g., for a model of regression). Tumorigenicity and modulator efficacy can be evaluating life-span extension and/or tumor characteristics, including number of tumors, tumor size, tumor morphology, vessel density, apoptotic index, etc.
Diagnostic and Therapeutic Uses
[0104]Specific ACAC-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the IGF pathway, such as angiogenic, apoptotic, or cell proliferation disorders. Accordingly, the invention also provides methods for modulating the IGF pathway in a cell, preferably a cell pre-determined to have defective or impaired IGF function (e.g. due to overexpression, underexpression, or misexpression of IGF, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates ACAC activity. Preferably, the modulating agent produces a detectable phenotypic change in the cell indicating that the IGF function is restored. The phrase "function is restored", and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored IGF function, cell proliferation and/or progression through cell cycle may normalize, or be brought closer to normal relative to untreated cells. The invention also provides methods for treating disorders or disease associated with impaired IGF function by administering a therapeutically effective amount of an ACAC-modulating agent that modulates the IGF pathway. The invention further provides methods for modulating ACAC function in a cell, preferably a cell pre-determined to have defective or impaired ACAC function, by administering an ACAC-modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired ACAC function by administering a therapeutically effective amount of an ACAC-modulating agent.
[0105]The discovery that ACAC is implicated in IGF pathway provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders involving defects in the IGF pathway and for the identification of subjects having a predisposition to such diseases and disorders.
[0106]Various expression analysis methods can be used to diagnose whether ACAC expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis. (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001, 12:41-47). Tissues having a disease or disorder implicating defective IGF signaling that express an ACAC, are identified as amenable to treatment with an ACAC modulating agent. In a preferred application, the IGF defective tissue overexpresses an ACAC relative to normal tissue. For example, a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial ACAC cDNA sequences as probes, can determine whether particular tumors express or overexpress ACAC. Alternatively, the TaqMan® is used for quantitative RT-PCR analysis of ACAC expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).
[0107]Various other diagnostic methods may be performed, for example, utilizing reagents such as the ACAC oligonucleotides, and antibodies directed against an ACAC, as described above for: (1) the detection of the presence of ACAC gene mutations, or the detection of either over- or under-expression of ACAC mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of ACAC gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by ACAC. Kits for detecting expression of ACAC in various samples, comprising at least one antibody specific to ACAC, all reagents and/or devices suitable for the detection of antibodies, the immobilization of antibodies, and the like, and instructions for using such kits in diagnosis or therapy are also provided.
[0108]Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in ACAC expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for ACAC expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder. Preferably, the disease is cancer, most preferably a cancer as shown in TABLE 1. The probe may be either DNA or protein, including an antibody.
EXAMPLES
[0109]The following experimental section and examples are offered by way of illustration and not by way of limitation.
I. Drosophila Cell IGF Screen
[0110]RNA interference (RNAi) was used to create dPTEN-deficient cultured Drosophila cells (Schneider S2 cells (Schneider, I. (1972) J. Embryol. Exp. Morph. 27, 363), adapted to serum-free media, from Invitrogen Corp., Carlsbad, Calif.). Cells were treated for 3 days with dPTEN double stranded RNA (dsRNA) or a control dsRNA representing sequences from a Renilla luciferase cDNA. After a 3 day dsRNA pretreatment, 1 μM bovine insulin was added to cells treated with dPTEN dsRNA to provide additional stimulation of the IGF/insulin pathway. PTEN-deficient, insulin-stimulated cells and control cells were plated in 384-well format and dsRNA representing approximately 10,000 different Drosophila genes were added to individual wells. A cell proliferation assay (AqueousOne® assay-Promega Corp, Madison, Wis.) was used to quantify cell viability after 96-hours incubation. For each of the greater than 6000 dsRNA sequences tested in this manner, cell viability data was obtained on dPTEN-deficient, insulin-stimulated cells (insulin and dPTEN dsRNA-treated) and control cells (Renilla luciferase dsRNA-treated). Comparison of this data for each dsRNA identified dsRNA sequences that preferentially reduced the viability of insulin and dPTEN dsRNA treated cells. Additionally, the screen identified sequences that when inactivated, preferentially suppressed insulin induced LDH (lactate dehydrogenase) expression relative to normal cells. The LDH expression levels were detected by TaqMan® analysis.
[0111]Dmel cells were treated with 1 μM bovine insulin to stimulate the IGF/insulin pathway. The insulin-stimulated cells were plated into 384-well plates and dsRNA representing approximately 10,000 different Drosophila genes were added to individual wells. After a 96-hour incubation cells were lysed using Cells-to-cDNAII cell lysis buffer (Ambion) and a 384 format, multiplexed, RT-PCR TaqMan® assay was run on the lysates. The TaqMan® assay identifies changes in expression of lactate dehydrogenase, a IGF reporter gene, and Rp49, an internal standard to normalize values for cell number and RT-PCR efficiency. For each of the greater than 13,000 dsRNA sequences tested in this manner, effects on LDH and rp49 expression were analyzed. Selections of genes with the greatest reduction in LDH expression were further analyzed in a multiplexed Western Blot assay that examines phosphorylation and overall levels of several proteins simultaneously. The multiplexed assay measured changes in phosphorylation of 4E-BP1 (Thr37/46), MAPK1(Thr202/Tyr204) and either S6K(Thr389) phosphorylation or total RPS6 protein levels. The multiplexed Western assay was done on lysates from cells treated with 1 μM insulin plated in 96 format and treated with target dsRNA for 96 hrs. Each lysate was tested for its differences in phosphorylation of 4E-BP 1 (Thr37/46), MAPK1 (Thr202/Tyr204), and either S6K(Thr389) phosphorylation or total RPS6 protein relative to negative control dsRNA (luciferase dsRNA). The quantitative Western blot assay, like the LDH reporter assay, was validated as a readout for IGF signaling by RNAi of known pathway components. CG11198 was identified as having synthetic lethal interaction from the screen. Orthologs of CG11198 are referred to herein as ACAC.
[0112]BLAST analysis (Altschul et al., supra) was employed to identify orthologs of Drosophila modifiers. For example, representative sequences from ACAC, GI #38679974 (SEQ ID NO:16), and GI #61743950 (SEQ ID NO:18) share 61% and 60% amino acid identity, respectively, with the Drosophila CG11198.
[0113]Various domains, signals, and functional subunits in proteins were analyzed using the PSORT (Nakai K., and Horton P., Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting signals and prediction of subcellular localization, Adv. Protein Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2), SMART (Ponting C P, et al., SMART: identification and annotation of domains from signaling and extracellular protein sequences. Nucleic Acids Res. 1999 Jan. 1; 27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998), and clust (Remm M, and Sonnhammer E. Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs. Genome Res. 2000 November; 10(11): 1679-89) programs. For example, the carboxyl transferase domain (PFAM 01039) of ACAC from GI #s 38679974 and 61743950 (SEQ ID NOs:16 and 18, respectively) is located at approximately amino acid residues 1586-2145, and 1775-2334, respectively.
II. High-Throughput In Vitro Fluorescence Polarization Assay
[0114]Fluorescently-labeled ACAC peptide/substrate are added to each well of a 96-well microtiter plate, along with a test agent in a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes in fluorescence polarization, determined by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc), relative to control values indicates the test compound is a candidate modifier of ACAC activity.
III. High-Throughput In Vitro Binding Assay.
33P-labeled ACAC peptide is added in an assay buffer (100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1% glycerol, 0.5% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a test agent to the wells of a Neutralite-avidin coated assay plate and incubated at 25° C. for 1 hour. Biotinylated substrate is then added to each well and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter. Test agents that cause a difference in activity relative to control without test agent are identified as candidate IGF modulating agents.
IV. Immunoprecipitations and Immunoblotting
[0116]For coprecipitation of transfected proteins, 3×106 appropriate recombinant cells containing the ACAC proteins are plated on 10-cm dishes and transfected on the following day with expression constructs. The total amount of DNA is kept constant in each transfection by adding empty vector. After 24 h, cells are collected, washed once with phosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysis buffer containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, protease inhibitors (complete, Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removed by centrifugation twice at 15,000×g for 15 min. The cell lysate is incubated with 25 μl of M2 beads (Sigma) for 2 h at 4° C. with gentle rocking.
[0117]After extensive washing with lysis buffer, proteins bound to the beads are solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane and blotted with the indicated antibodies. The reactive bands are visualized with horseradish peroxidase coupled to the appropriate secondary antibodies and the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).
V. Expression Analysis
[0118]All cell lines used in the following experiments are NCI (National Cancer Institute) lines, and are available from ATCC (American Type Culture Collection, Manassas, Va. 20110-2209). Normal and tumor tissues were obtained from Impath, UC Davis, Clontech, Stratagene, Ardais, Genome Collaborative, and Ambion.
[0119]TaqMan® analysis was used to assess expression levels of the disclosed genes in various samples.
[0120]RNA was extracted from each tissue sample using Qiagen (Valencia, Calif.) RNeasy kits, following manufacturer's protocols, to a final concentration of 50 ng/μl. Single stranded cDNA was then synthesized by reverse transcribing the RNA samples using random hexamers and 500 ng of total RNA per reaction, following protocol 430-4965 of Applied Biosystems (Foster City, Calif.).
[0121]Primers for expression analysis using TaqMan® assay (Applied Biosystems, Foster City, Calif.) were prepared according to the TaqMan® protocols, and the following criteria: a) primer pairs were designed to span introns to eliminate genomic contamination, and b) each primer pair produced only one product. Expression analysis was performed using a 7900HT instrument.
[0122]TaqMan® reactions were carried out following manufacturer's protocols, in 25 μl total volume for 96-well plates and 10 μl total volume for 384-well plates, using 300 nM primer and 250 nM probe, and approximately 25 ng of cDNA. The standard curve for result analysis was prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be present in appreciable amounts is good. The raw data were normalized using 18S rRNA (universally expressed in all tissues and cells).
[0123]For each expression analysis, tumor tissue samples were compared with matched normal tissues from the same patient. A gene was considered overexpressed in a tumor when the level of expression of the gene was 2 fold or higher in the tumor compared with its matched normal sample. In cases where normal tissue was not available, a universal pool of cDNA samples was used instead. In these cases, a gene was considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type was greater than 2 times the standard deviation of all normal samples (i.e., Tumor-average (all normal samples)>2×STDEV (all normal samples)).
[0124]Results are shown in Table 1. Number of pairs of tumor samples and matched normal tissue from the same patient are shown for each tumor type. Percentage of the samples with at least two-fold overexpression for each tumor type is provided. A modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator. Prior to treating a patient with the modulator, the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator. The expression data for the gene(s) can also be used as a diagnostic marker for disease progression. The assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.
TABLE-US-00001 TABLE 1 Gene Name ACACA ACACB SEQ ID NO 5 11 Breast 36% 0% # of Pairs 36 36 Colon 22% 5% # of Pairs 40 40 Head And 46% 8% Neck # of Pairs 13 13 Liver 56% 0% # of Pairs 9 9 Lung 5% 0% # of Pairs 40 40 Lymphoma 0% 0% # of Pairs 4 4 Ovary 32% 0% # of Pairs 19 19 Pancreas 64% 42% # of Pairs 11 12 Prostate 17% 4% # of Pairs 24 24 Skin 14% 0% # of Pairs 7 7 Stomach 9% 9% # of Pairs 11 11 Testis 0% 0% # of Pairs 8 8 Thyroid 21% 0% Gland # of Pairs 14 14 Uterus 26% 0% # of Pairs 23 23
VI. ACAC Functional Assays
[0125]RNAi experiments were carried out to knock down expression of ACAC (SEQ ID NO:5) in various cell lines using small interfering RNAs (siRNA, Elbashir et al, supra).
[0126]Effect of ACAC RNAi on cell proliferation and growth. BrdU and Cell Titer-Glo® assays, as described above, were employed to study the effects of decreased ACAC expression on cell proliferation. The results of these experiments indicated that RNAi of ACAC decreased proliferation in 231T breast cancer cells, A549 lung cancer cells, and U87MG glioblastoma cells.
[0127][3H]-thymidine incorporation assay, as described above, was also employed to study the effects of decreased ACAC expression on cell proliferation. The results of this experiment indicated that RNAi of ACAC decreased proliferation in A549 lung cancer cells and RD1 rhabdomyosarcoma cells.
[0128]Effect of ACAC RNAi on apoptosis. The Phospho-histone H2B assay, as described above, was employed to study the effects of decreased ACAC expression on apoptosis. The results of this experiment indicated that RNAi of ACAC increased apoptosis in 231t Breast cancer cells and A549 lung cancer cells.
[0129]Multiple parameter apoptosis assay, as described above, was also used to study the effects of decreased ACAC expression on apoptosis. The results of this experiment indicated that RNAi of ACAC increased apoptosis in A2780 ovarian cancer and A549 lung cancer cells. Further, RNAi of ACAC also caused reduced cell count in 231T breast cancer cells.
[0130]Involvement in PTEN/IGF pathway: ACAC FOXO nuclear translocation assays. FOXO nuclear translocation assays, as described above, were employed to assess involvement of ACAC in the PTEN/IGF pathway. In these experiments, cells with reduced expression of ACAC by RNAi were transiently transfected with a plasmid expressing GFP-tagged FOXO. Automated imaging of cellular components, such as nucleus and cytoplasm were then carried out to assess translocation of FOXO. Alternatively, cells were co-transfected with siRNA directed to ACAC along with a plasmid containing FOXO, and a cassette containing a promoter, a FOXO response element, and luciferase. Cells were then analyzed for luciferase activity and compared with cells with no siRNA. Results indicated that reduced expression of ACAC led to retention of FOXO in the nucleus in PC3 prostate cancer cells, similar to a reduced AKT effect. These results suggest involvement of ACAC in the PTEN/IGF pathway.
[0131]Pan-AKT assays. This assay was developed to detect involvement of ACAC in the PTEN/IGF pathway. The assay detects changes in phosphorylation for several substrates of AKT, such as PRAS40, BAD, 4EBP1, and RPS6. For this experiment, antibodies were raised against phosphorylated AKT substrates, including the consensus phosphorylated AKT substrate sequence RxRxxS/T. Expression levels of phosphorylated substrates were then quantitated at normal levels, in presence of a negative control, a positive control (AKT), and then with ACAC knockout. For example, when AKT levels were reduced, expression of all its substrates was also reduced. Results indicated that reduced expression of ACAC was similar to reduced AKT levels in 231T breast cancer cells.
[0132]We used 4EBP1 as the substrate for a subset of the experiments. For this substrate, AKT pathway inhibition causes decreased cytoplasmic staining and unchanged or increased nuclear staining. Cells were plated in 96 well plates, transfected with RNAi for ACAC, fixed, permeabilized and stained with anti-phospho-4EBP1 antibody. Measurements were based on the percentage of the population of cells with a decreased Cytoplasmic/Nuclear staining ratio compared with negative or positive control cells. Results of this experiment showed that reduced expression of ACAC caused a reduction in the level of phospho-4EBP1 protein in the cytoplasm in 231T breast cancer cells, thus suggesting an involvement in the IGF pathway.
[0133]We used PRAS40 as the substrate for another subset of experiments. For this substrate, pathway inhibition causes decreased cytoplasmic staining and increased nuclear and perinuclear staining. Cells were plated in 96 well plates, transfected with RNAi for ACAC, fixed, treated with PRAS40 antibody, and stained. Measurements were based on percentage of population of cells with increased or decreased nuclear/cytoplasmic staining ratio compared with negative or positive control cells. Results of this experiment showed that reduced expression of ACAC altered the level of phospho PRAS40 protein in 231 T breast cancer cells and A549 lung cancer cells, again suggesting an involvement in the IGF pathway.
[0134]We used BAD as the substrate for another subset of the experiments. For this substrate, AKT pathway inhibition causes decreased cytoplasmic staining and unchanged or increased nuclear staining. Cells were plated in 96 well plates, transfected with RNAi for ACAC, fixed, permeabilized and stained with anti-phospho-BAD antibody. Measurements were based on the percentage of the population of cells with a decreased Cytoplasmic/Nuclear staining ratio compared with negative or positive control cells. Results of this experiment showed that reduced expression of ACAC caused a reduction in the level of phospho-BAD protein in the cytoplasm in 231T breast cancer and PC3 prostate cancer cells, again suggesting an involvement in the IGF pathway. Taken together, the results of the pan-AKT assay suggest involvement of ACAC in the PTEN/IGF pathway.
Sequence CWU
1
18110128DNAHomo sapiens 1ggcccccgcc gggtgctgag cgcccgccac cgccctcttg
gccttttccc ggtccccgcc 60gcgcgcctgc tgctgtcccc gtgcccgagg actgggagga
tggggctgag ccgcttgcct 120gacttttgat ccgaccagta atcactttgc ccgtgtggcg
cgtggcccgt tgcctgaggc 180ttcctggcgc gtggggctgt ttggtcccct ccggagtgcg
gtgaggcggg ccgagccggg 240actgcctggg tttggggata acgttcccat ctccacccct
gttgcagcaa gggaaattga 300ggctgaggga actgggccca gggacggcga gccgtggctg
cctctccagt ccgggccccg 360aagggctgct cgtggatgaa ccagactgat tttaaggggt
gaagagggtg cgtttcaatc 420agatgcttct ggaacgtcga aattgtcttc tttggaaaga
accatcccct ctttgggctt 480cagaggccca aattgaggcg caatgatgag aggatgtggt
ggtctactct gatgtcaatc 540ttgagggcta ggtctttctg gaagtggata tctactcaga
cagtaagaat tataagagct 600gtaagagctc attttggagg aataatggat gaaccatctc
ccttggccca acctctggag 660ctgaaccagc actctcgatt cataataggt tctgtgtctg
aagataactc agaggatgag 720atcagcaacc tggtgaagtt ggacctactg gaggagaagg
agggctcctt gtcacctgct 780tctgttggct cagatacact ctctgatttg gggatctcta
gcctacagga tggcttggcc 840ttgcacataa gactttgaaa tactcgtggg atatgaagtg
gaggtgccta gagggttgaa 900gataatgagg acagatgaag aaatgtttga ttgtgccata
cttacagttg ctgattttca 960aggtccagca tgtctggctt gcacctagta aagcagggcc
gagacagaaa gaaaatagat 1020tctcaacgag atttcactgt ggcttctcca gcagaatttg
ttactcgctt tgggggaaat 1080aaagtgattg agaaggttct tattgctaac aatggcattg
cagcagtgaa atgcatgcgg 1140tctatccgta ggtggtctta tgaaatgttt cgaaatgaac
gtgcaattag attcgttgtc 1200atggtcacac ctgaagacct taaagccaat gcagaataca
ttaagatggc agatcactat 1260gtgccagtgc ctggaggacc aaacaacaac aactatgcaa
atgtggaatt aattcttgat 1320attgctaaaa ggatcccagt acaagcagtg tgggctggct
ggggtcatgc ttctgagaat 1380cccaaactac cggaacttct cttgaaaaat ggcattgcct
tcatgggtcc tccaagccag 1440gccatgtggg ctttagggga taagattgca tcttccatag
tggctcaaac tgcaggtatc 1500ccaactcttc cctggagcgg cagtggtctt cgtgtggact
ggcaggaaaa tgatttttca 1560aaacgtatct taaatgttcc ccaggagcta tatgaaaaag
gttatgtgaa agatgtggat 1620gatgggctac aggcagctga ggaagttgga tatccagtaa
tgatcaaggc ctcagaggga 1680ggaggaggga agggaattag aaaagtcaac aatgcagatg
acttccctaa tctcttcaga 1740caggttcaag ctgaagttcc tggatctccc atatttgtga
tgagactagc caaacaatct 1800cgtcatctgg aggtgcagat cttagcggac caatatggca
atgctatctc tttgtttggt 1860cgtgattgct ctgtacaacg caggcatcag aagattattg
aagaagcacc tgctactatt 1920gctactccag cagtatttga acacatggaa cagtgtgcgg
tgaaacttgc caaaatggtg 1980ggttatgtga gtgctgggac tgtggaatac ctgtacagcc
aggatggcag cttctacttt 2040ctggaattga atcctcggct gcaggtagag cacccttgta
cagagatggt ggctgatgtc 2100aatctccctg cagcacagct ccagattgcc atggggattc
ctctatatag aatcaaggat 2160atccgtatga tgtatggggt atctccctgg ggtgattctc
ccattgattt tgaagattct 2220gcacacgttc cttgtccaag gggccatgtt attgctgctc
ggatcactag tgaaaatcca 2280gatgagggtt ttaagcccag ctcaggaaca gttcaggagc
taaatttccg cagcaataag 2340aatgtttggg gatatttcag tgttgctgct gcagggggac
ttcatgaatt tgctgattct 2400cagtttggtc actgcttttc ttggggagaa aacagagaag
aggcaatttc aaacatggtg 2460gtggctttga aggagctgtc tattcggggt gactttcgaa
ctacagttga atacctgatc 2520aaattgttag agactgaaag ctttcagatg aacagaattg
atactggctg gctggacaga 2580ctgatagcag aaaaagtaca ggctgagcga cctgacacca
tgttgggggt tgtgtgtggt 2640gccctccacg tggcagatgt gagcctgcgg aatagcgtct
ctaacttcct tcactcctta 2700gaaaggggtc aagtccttcc tgctcataca cttctgaata
cagtagatgt tgaacttatc 2760tatgagggag tcaagtatgt acttaaggtg actcgacagt
cccccaactc ctatgtggtg 2820atcatgaatg gctcatgtgt agaagtagat gtacatcggc
tgagtgacgg tggactgctc 2880ttgtcctatg atggcagcag ttatactacg tatatgaaag
aggaagtgga tagatatcgc 2940atcacaattg gcaataaaac ctgtgtgttt gagaaggaaa
atgacccatc ggtgatgcgc 3000tcaccttctg ctgggaagtt aatccagtac attgtagaag
atggaggtca tgtgtttgcc 3060ggccagtgct atgctgagat tgaggtaatg aagatggtaa
tgaccttaac agctgtggag 3120tctggctgta tccattacgt caagcgacct ggagcagctc
ttgaccctgg ctgtgtacta 3180gccaaaatgc aactggacaa ccccagcaag gttcagcagg
ctgaacttca cacaggtagt 3240ctgccacgga tccagagcac ggcactcaga ggcgagaaac
tccatcgagt gttccattat 3300gtcctggata atctggtcaa tgtaatgaat ggatactgcc
ttccagatcc tttctttagc 3360agcaaggtaa aagactgggt agagcgattg atgaaaaccc
tcagagatcc ctccctgcct 3420ctcctagaat tgcaagatat tatgaccagt gtgtctggcc
gcattccccc caatgtggag 3480aagtctatca agaaggaaat ggctcagtat gctagcaaca
tcacatcagt cctctgtcag 3540tttcccagcc agcagattgc aaacatccta gatagccatg
cagctacatt gaaccggaaa 3600tctgaacggg aagtcttctt tatgaatact cagagcattg
ttcagctggt acagaggtac 3660cgaagtggca tccgaggcca catgaaggct gtggtgatgg
atctgctccg gcagtacctg 3720cgagtagaga cacaattcca gaatggtcac tatgacaaat
gtgtattcgc cctccgagaa 3780gagaataaaa gtgacatgaa cactgtactg aactacatct
tctctcacgc tcaagtcacc 3840aagaagaatc ttctggtcac aatgcttatt gatcagttgt
gtggccggga ccctactctc 3900actgatgagc tgctgaatat tctcacagag ctaactcaac
tcagtaagac caccaatgcc 3960aaagtagcac ttcgagcacg ccaggttctt attgcctccc
atttgccatc atatgagctt 4020cgccataacc aagtagagtc tatcttccta tcagctattg
acatgtatgg acatcaattt 4080tgcattgaga acctgcagaa actcatccta tcagaaacat
ctatttttga tgtcctacca 4140aacttcttct atcacagcaa ccaagtagtg aggatggcag
ctctggaggt gtatgttcga 4200agggcttata ttgcctatga acttaacagc gtacaacacc
gccagcttaa ggacaacacc 4260tgtgtggtgg aattccagtt catgctgccc acatctcatc
caaacagagg gaacatccct 4320acgctaaaca gaatgtcctt ctcctccaac ctcaaccact
atggcatgac ccatgtagct 4380agtgtcagcg atgtactgtt ggacaactca ttcactccac
cttgtcagcg gatgggcgga 4440atggtctctt ttcggacttt tgaagatttt gtcaggatct
ttgatgaagt gatgggctgc 4500ttctctgact ccccacccca gagtcccaca ttccctgagg
caggtcacac gtctctttat 4560gatgaggata aggttcccag ggatgaacca attcacattc
tcaatgtggc tatcaagact 4620gactgtgata ttgaggatga caggctggca gctatgttca
gagaatttac ccagcaaaat 4680aaagctaccc tggttgacca tgggatccgg cgccttactt
tcctggttgc acaaaaggat 4740ttcagaaagc aggtcaacta tgaggtggat cggagatttc
atagagaatt ccctaaattt 4800tttacattcc gagcaaggga taagtttgag gaggatcgta
tctatcgtca tctggagcct 4860gctctggctt tccagttaga gctgaaccgg atgagaaatt
ttgacctcac tgccattcca 4920tgtgctaatc acaagatgca cctgtatctc ggggcagcca
aggtggaagt gggcacagaa 4980gtgacagact acaggttctt tgttcgtgca atcatcaggc
attctgatct ggtcaccaag 5040gaagcttctt ttgaatatct gcaaaatgaa ggggagcggc
tactcctgga agccatggat 5100gagttggaag ttgcttttaa caatacaaat gtccgcactg
actgtaacca catcttcctc 5160aactttgtgc ccacggttat catggaccca tcaaagattg
aggaatccgt gcggagcatg 5220gtaatgcggt atggaagtcg cctgtggaaa ttgcgcgtcc
tccaggcaga actgaaaatc 5280aacattcgcc tgacgccaac tggaaaagca attcccatcc
gcctcttcct gacaaacgag 5340tctggctatt acttggatat cagcctatac aaggaagtga
ctgactccag gacagcacag 5400atcatgtttc aggcatatgg agacaaacag ggaccactgc
atggaatgtt aatcaatact 5460ccatatgtga ccaaagacct gctgcaatca aagaggttcc
aggcacaatc cttagggaca 5520acatacatat atgatatccc agagatgttt cggcagtccc
tgatcaaact ctgggagtct 5580atgtccactc aagcatttct tccatctccc cctctgcctt
ctgacatgct gacttacact 5640gaactggtac tggatgatca aggtcagctg gtccacatga
acaggcttcc aggaggaaat 5700gagattggca tggtagcttg gaaaatgacc tttaaaagtc
ctgaatatcc agaaggccga 5760gatatcattg ttattggcaa tgacatcaca taccgaattg
ggtcctttgg gcctcaagag 5820gatttgttat ttctcagagc ttccgaactt gctagggcag
aaggtattcc acgcatctat 5880gtatcagcca acagtggagc aagaatcgga ctggcagaag
aaattcgcca tatgtttcat 5940gtggcctggg tagatcctga ggatccttac aagggataca
ggtatttata tctgactcct 6000caagattata agagagtcag tgctctcaac tctgtccatt
gtgaacacgt ggaagatgaa 6060ggagaatcca ggtacaagat aacagatatt attgggaaag
aagagggaat tggacccgag 6120aaccttcgag gttctggaat gattgctgga gaatcctcat
tggcctataa tgagatcatt 6180accatcagcc tggtgacgtg ccgggccatt gggattgggg
cttaccttgt ccggctggga 6240cagagaacca tccaggttga gaattctcac ttaattctaa
caggagctgg agccctcaac 6300aaagtcctcg ggcgggaagt gtacacctcc aataaccagc
tggggggcat ccagattatg 6360cacaacaatg gggtgaccca ctgcactgtg tgtgatgact
ttgaaggggt tttcactgtc 6420ctgcactggc tgtcttacat gcccaagagc gtgcacagtt
cagttcctct tctgaactca 6480aaggatccta tagacagaat catcgagttt gttcccacaa
agaccccata cgatcctcga 6540tggatgctag caggccgtcc tcacccaacc caaaaaggtc
agtggttgag tggctttttt 6600gactatggat ctttctcaga gattatgcag ccctgggcac
agactgtggt ggttggtaga 6660gccaggctag gaggaatacc tgtgggagtt gttgctgtag
aaacccgaac agtagaacta 6720agtatcccag ctgatccagc aaacctggat tctgaagcca
agataatcca gcaggctggc 6780caggtttggt tcccagattc tgcgtttaag acgtatcagg
ccatcaagga cttcaaccgg 6840gaagggctgc ctctgatggt ctttgccaac tggagaggct
tctctggtgg aatgaaagat 6900atgtacgacc aagtgctgaa gtttggtgct tacattgtgg
atggcttgag ggagtgctgc 6960cagcctgtgc tggtttacat tcctccccag gctgagctgc
ggggtggctc ctgggtggtg 7020attgactcct ccatcaaccc ccggcacatg gagatgtatg
ctgaccgaga aagcagggga 7080tctgttctgg agccagaagg gacagtagaa atcaaattcc
gcagaaagga tctggtgaaa 7140accatgcgtc gggtggaccc agtctacatc cacttggctg
agcgattggg gaccccagag 7200ctaagcacag ctgagcggaa ggagttggag aacaagttga
aggagcggga ggaattccta 7260attcccattt accatcaggt agccgtgcag tttgctgact
tgcacgacac accaggccgg 7320atgcaggaga agggtgttat tagcgatatc ctggattgga
aaacatcccg taccttcttc 7380tactggcggc tgaggcgtct tctgctggag gacctggtca
agaagaaaat ccacaatgcc 7440aaccctgagc tgactgatgg ccagattcaa gccatgttaa
ggcgctggtt tgtggaagtg 7500gaaggaacag tgaaggctta tgtttgggac aataataagg
atctggcgga gtggctagag 7560aaacagctga cagaggagga tggtgttcac tcggtaatag
aggaaaacat caaatgcatc 7620agcagagact acgtcctcaa gcaaatccgc agcttggtcc
aggccaatcc agaggttgcc 7680atggattcca tcatccatat gacgcagcac atatcaccca
ctcagcgagc agaagtcata 7740cggatcctct ccacaatgga ttccccttcc acgtaggaag
agcttcctgc ctgtccctgc 7800cctgtctctg gagaaaaggg ctagagctgc cttttacaac
tgtaaccact gtaatgagaa 7860ggcacaggag acccagcact ggagtcaaat ggcattttac
ttcctctcgt ttcaggttat 7920gcatgacatc ctgggatgta agatcacaga atccccctcc
agcccaccag tcacacctac 7980cccattcagt atttattacc ctggccaggc ctagtcctcc
actccctgca caggactgag 8040aaggcaatga aaggtacaaa catgtaccat gaggtcttac
taaccaaagt agggctgccc 8100ctcctgtcct gacagcccct tggcctccca gcatggggaa
gcgtgaggag ttgcccagca 8160gtgagcagcc cccctcactc ctggccccat gagccgcagc
cacaggcagc agaggagggc 8220taaggagagg aggaagcctc aagtccattg tttattaccc
cgactcttag cccagcacac 8280agtaggcact ggagaggaat gattcccagt ttaaccacac
tacggtacct tttatgaaga 8340aaaattagag cataaaatct actacaagct ccataggaac
tcaaagatga gggcaaaact 8400gtgagccaag aagcagagaa agaaaataga accagttatt
cttgatttag gggacctcaa 8460ccttgggttc agtctctgag gacaaaggga aaggtagttg
ttggcctgcc tctcgcctgc 8520acgtcactgc tggactagct gtcgcatgtg gctgggagct
gcaaggccag tgcttgaggg 8580gccccagcag ttccacaggt ggtgaagcct gagttggcag
aggaggagcc agaagagaac 8640tgccctttct gcactggtgg aaactagtta tttatgccat
gtggagagcc agtgagatag 8700atagatagtc tgtttgtttt gaggacttgg aaagttgttc
ctatgaagcc tggagcttgg 8760atggttttga gaggttaatg gtgcctccac actcactctt
ccctagttcc aggattactg 8820tcctagcagc taacggttct actctcttcc ccagagtgta
gacaggcagc aggtctcccc 8880acagctctga aaggaccctg gtgacagcta caccctcagc
accaggagct ggccttcctg 8940atgagggagg cttccaggaa acacagaatc cacatgacct
taagattatt tacaactcag 9000tcatggtgct gctgtcctcc aggcttactg gcccctcctg
actggcatca ggggcttcct 9060caggtggtgg agagagttta ctttcaacaa ctagtttatt
caagaaaaga acttactgat 9120tcctctgttc ctaaagcaag agtggcaggt gatcagggct
ggtgtagcat ccggttcctt 9180tagtgcagct aactgcattt gtcactgatg accaaggagg
aaatcactaa gacatttgag 9240aagcagtggt atgaacgttc ttggacaagc cacagttctg
agccttaacc ctgtagtttg 9300cacacaagaa cgagctccac ctccccttct tcaggaggaa
tctgtgcgga tagattggct 9360ggacttttca atggttctgg gttgcaggtg ggcactgtat
ggctgggtat ggagcggaca 9420gcccccagga gtcagagcct cagcccggct gccctggtgg
aaggtacagg tgttcagcac 9480cttcagaaaa gggcataaag tggtggggga caattctcag
tccaggaaaa tgcattgacc 9540attgctggct atttgcttac ctagtaagaa ttggattcat
ttttgaccag attattcttc 9600tatgcttttt tgcaataaat caaatcccac atatctacaa
gtggtatgaa gtcctgcacc 9660ccccaggagg cctgtccagg catgtcttca gaggcagggt
gggttacact catttacctc 9720ccctctcccc accaaattat gacacaaacg agtatgtttc
ctctctagaa ccctgtaatg 9780cctcctcccc catccccaga gctccttact gtaggtctta
ccctggacaa ggattttttc 9840aagttggagg cacagaacat gagcaatctg acattcccac
agcccctcaa acatgcaagg 9900ctactaaggc aggaggagta taaatgatgg atattgacca
agacctgctt ggacggagac 9960cgccatatta tctgttctct tcgttcacaa aacagccttc
acttgtctca gaatttgatg 10020gacacatact gtgatgagca ggagcttcag atgcactctt
tacacatttt gttgaaataa 10080acctctacat ttgtagaaga aaaaaaaaaa aaaaaaaaaa
aaaaaaaa 10128210018DNAHomo sapiens 2ggcccccgcc gggtgctgag
cgcccgccac cgccctcttg gccttttccc ggtccccgcc 60gcgcgcctgc tgctgtcccc
gtgcccgagg actgggagga tggggctgag ccgcttgcct 120gacttttgat ccgaccagta
atcactttgc ccgtgtggcg cgtggcccgt tgcctgaggc 180ttcctggcgc gtggggctgt
ttggtcccct ccggagtgcg gtgaggcggg ccgagccggg 240actgcctggg tttggggata
acgttcccat ctccacccct gttgcagcaa gggaaattga 300ggctgaggga actgggccca
gggacggcga gccgtggctg cctctccagt ccgggccccg 360aagggctgct cgtggatgaa
ccagactgat tttaaggggt gaagagggtg cgtttcaatc 420agatgcttct ggaacgtcga
aattgtcttc tttggaaaga accatcccct ctttgggctt 480cagaggccca aattgaggcg
caatgatgag aggatgtggt ggtctactct gatgtcaatc 540ttgagggcta ggtctttctg
gaagtggata tctactcaga cagtaagaat tataagagct 600gtaagagctc attttggagg
aataatggat gaaccatctc ccttggccca acctctggag 660ctgaaccagc actctcgatt
cataataggt tctgtgtctg aagataactc agaggatgag 720atcagcaacc tggtgaagtt
ggacctactg gaggagaagg agggctcctt gtcacctgct 780tctgttggct cagatacact
ctctgatttg gggatctcta gcctacagga tggcttggcc 840ttgcacataa ggtccagcat
gtctggcttg cacctagtaa agcagggccg agacagaaag 900aaaatagatt ctcaacgaga
tttcactgtg gcttctccag cagaatttgt tactcgcttt 960gggggaaata aagtgattga
gaaggttctt attgctaaca atggcattgc agcagtgaaa 1020tgcatgcggt ctatccgtag
gtggtcttat gaaatgtttc gaaatgaacg tgcaattaga 1080ttcgttgtca tggtcacacc
tgaagacctt aaagccaatg cagaatacat taagatggca 1140gatcactatg tgccagtgcc
tggaggacca aacaacaaca actatgcaaa tgtggaatta 1200attcttgata ttgctaaaag
gatcccagta caagcagtgt gggctggctg gggtcatgct 1260tctgagaatc ccaaactacc
ggaacttctc ttgaaaaatg gcattgcctt catgggtcct 1320ccaagccagg ccatgtgggc
tttaggggat aagattgcat cttccatagt ggctcaaact 1380gcaggtatcc caactcttcc
ctggagcggc agtggtcttc gtgtggactg gcaggaaaat 1440gatttttcaa aacgtatctt
aaatgttccc caggagctat atgaaaaagg ttatgtgaaa 1500gatgtggatg atgggctaca
ggcagctgag gaagttggat atccagtaat gatcaaggcc 1560tcagagggag gaggagggaa
gggaattaga aaagtcaaca atgcagatga cttccctaat 1620ctcttcagac aggttcaagc
tgaagttcct ggatctccca tatttgtgat gagactagcc 1680aaacaatctc gtcatctgga
ggtgcagatc ttagcggacc aatatggcaa tgctatctct 1740ttgtttggtc gtgattgctc
tgtacaacgc aggcatcaga agattattga agaagcacct 1800gctactattg ctactccagc
agtatttgaa cacatggaac agtgtgcggt gaaacttgcc 1860aaaatggtgg gttatgtgag
tgctgggact gtggaatacc tgtacagcca ggatggcagc 1920ttctactttc tggaattgaa
tcctcggctg caggtagagc acccttgtac agagatggtg 1980gctgatgtca atctccctgc
agcacagctc cagattgcca tggggattcc tctatataga 2040atcaaggata tccgtatgat
gtatggggta tctccctggg gtgattctcc cattgatttt 2100gaagattctg cacacgttcc
ttgtccaagg ggccatgtta ttgctgctcg gatcactagt 2160gaaaatccag atgagggttt
taagcccagc tcaggaacag ttcaggagct aaatttccgc 2220agcaataaga atgtttgggg
atatttcagt gttgctgctg cagggggact tcatgaattt 2280gctgattctc agtttggtca
ctgcttttct tggggagaaa acagagaaga ggcaatttca 2340aacatggtgg tggctttgaa
ggagctgtct attcggggtg actttcgaac tacagttgaa 2400tacctgatca aattgttaga
gactgaaagc tttcagatga acagaattga tactggctgg 2460ctggacagac tgatagcaga
aaaagtacag gctgagcgac ctgacaccat gttgggggtt 2520gtgtgtggtg ccctccacgt
ggcagatgtg agcctgcgga atagcgtctc taacttcctt 2580cactccttag aaaggggtca
agtccttcct gctcatacac ttctgaatac agtagatgtt 2640gaacttatct atgagggagt
caagtatgta cttaaggtga ctcgacagtc ccccaactcc 2700tatgtggtga tcatgaatgg
ctcatgtgta gaagtagatg tacatcggct gagtgacggt 2760ggactgctct tgtcctatga
tggcagcagt tatactacgt atatgaaaga ggaagtggat 2820agatatcgca tcacaattgg
caataaaacc tgtgtgtttg agaaggaaaa tgacccatcg 2880gtgatgcgct caccttctgc
tgggaagtta atccagtaca ttgtagaaga tggaggtcat 2940gtgtttgccg gccagtgcta
tgctgagatt gaggtaatga agatggtaat gaccttaaca 3000gctgtggagt ctggctgtat
ccattacgtc aagcgacctg gagcagctct tgaccctggc 3060tgtgtactag ccaaaatgca
actggacaac cccagcaagg ttcagcaggc tgaacttcac 3120acaggtagtc tgccacggat
ccagagcacg gcactcagag gcgagaaact ccatcgagtg 3180ttccattatg tcctggataa
tctggtcaat gtaatgaatg gatactgcct tccagatcct 3240ttctttagca gcaaggtaaa
agactgggta gagcgattga tgaaaaccct cagagatccc 3300tccctgcctc tcctagaatt
gcaagatatt atgaccagtg tgtctggccg cattcccccc 3360aatgtggaga agtctatcaa
gaaggaaatg gctcagtatg ctagcaacat cacatcagtc 3420ctctgtcagt ttcccagcca
gcagattgca aacatcctag atagccatgc agctacattg 3480aaccggaaat ctgaacggga
agtcttcttt atgaatactc agagcattgt tcagctggta 3540cagaggtacc gaagtggcat
ccgaggccac atgaaggctg tggtgatgga tctgctccgg 3600cagtacctgc gagtagagac
acaattccag aatggtcact atgacaaatg tgtattcgcc 3660ctccgagaag agaataaaag
tgacatgaac actgtactga actacatctt ctctcacgct 3720caagtcacca agaagaatct
tctggtcaca atgcttattg atcagttgtg tggccgggac 3780cctactctca ctgatgagct
gctgaatatt ctcacagagc taactcaact cagtaagacc 3840accaatgcca aagtagcact
tcgagcacgc caggttctta ttgcctccca tttgccatca 3900tatgagcttc gccataacca
agtagagtct atcttcctat cagctattga catgtatgga 3960catcaatttt gcattgagaa
cctgcagaaa ctcatcctat cagaaacatc tatttttgat 4020gtcctaccaa acttcttcta
tcacagcaac caagtagtga ggatggcagc tctggaggtg 4080tatgttcgaa gggcttatat
tgcctatgaa cttaacagcg tacaacaccg ccagcttaag 4140gacaacacct gtgtggtgga
attccagttc atgctgccca catctcatcc aaacagaggg 4200aacatcccta cgctaaacag
aatgtccttc tcctccaacc tcaaccacta tggcatgacc 4260catgtagcta gtgtcagcga
tgtactgttg gacaactcat tcactccacc ttgtcagcgg 4320atgggcggaa tggtctcttt
tcggactttt gaagattttg tcaggatctt tgatgaagtg 4380atgggctgct tctctgactc
cccaccccag agtcccacat tccctgaggc aggtcacacg 4440tctctttatg atgaggataa
ggttcccagg gatgaaccaa ttcacattct caatgtggct 4500atcaagactg actgtgatat
tgaggatgac aggctggcag ctatgttcag agaatttacc 4560cagcaaaata aagctaccct
ggttgaccat gggatccggc gccttacttt cctggttgca 4620caaaaggatt tcagaaagca
ggtcaactat gaggtggatc ggagatttca tagagaattc 4680cctaaatttt ttacattccg
agcaagggat aagtttgagg aggatcgtat ctatcgtcat 4740ctggagcctg ctctggcttt
ccagttagag ctgaaccgga tgagaaattt tgacctcact 4800gccattccat gtgctaatca
caagatgcac ctgtatctcg gggcagccaa ggtggaagtg 4860ggcacagaag tgacagacta
caggttcttt gttcgtgcaa tcatcaggca ttctgatctg 4920gtcaccaagg aagcttcttt
tgaatatctg caaaatgaag gggagcggct actcctggaa 4980gccatggatg agttggaagt
tgcttttaac aatacaaatg tccgcactga ctgtaaccac 5040atcttcctca actttgtgcc
cacggttatc atggacccat caaagattga ggaatccgtg 5100cggagcatgg taatgcggta
tggaagtcgc ctgtggaaat tgcgcgtcct ccaggcagaa 5160ctgaaaatca acattcgcct
gacgccaact ggaaaagcaa ttcccatccg cctcttcctg 5220acaaacgagt ctggctatta
cttggatatc agcctataca aggaagtgac tgactccagg 5280acagcacaga tcatgtttca
ggcatatgga gacaaacagg gaccactgca tggaatgtta 5340atcaatactc catatgtgac
caaagacctg ctgcaatcaa agaggttcca ggcacaatcc 5400ttagggacaa catacatata
tgatatccca gagatgtttc ggcagtccct gatcaaactc 5460tgggagtcta tgtccactca
agcatttctt ccatctcccc ctctgccttc tgacatgctg 5520acttacactg aactggtact
ggatgatcaa ggtcagctgg tccacatgaa caggcttcca 5580ggaggaaatg agattggcat
ggtagcttgg aaaatgacct ttaaaagtcc tgaatatcca 5640gaaggccgag atatcattgt
tattggcaat gacatcacat accgaattgg gtcctttggg 5700cctcaagagg atttgttatt
tctcagagct tccgaacttg ctagggcaga aggtattcca 5760cgcatctatg tatcagccaa
cagtggagca agaatcggac tggcagaaga aattcgccat 5820atgtttcatg tggcctgggt
agatcctgag gatccttaca agggatacag gtatttatat 5880ctgactcctc aagattataa
gagagtcagt gctctcaact ctgtccattg tgaacacgtg 5940gaagatgaag gagaatccag
gtacaagata acagatatta ttgggaaaga agagggaatt 6000ggacccgaga accttcgagg
ttctggaatg attgctggag aatcctcatt ggcctataat 6060gagatcatta ccatcagcct
ggtgacgtgc cgggccattg ggattggggc ttaccttgtc 6120cggctgggac agagaaccat
ccaggttgag aattctcact taattctaac aggagctgga 6180gccctcaaca aagtcctcgg
gcgggaagtg tacacctcca ataaccagct ggggggcatc 6240cagattatgc acaacaatgg
ggtgacccac tgcactgtgt gtgatgactt tgaaggggtt 6300ttcactgtcc tgcactggct
gtcttacatg cccaagagcg tgcacagttc agttcctctt 6360ctgaactcaa aggatcctat
agacagaatc atcgagtttg ttcccacaaa gaccccatac 6420gatcctcgat ggatgctagc
aggccgtcct cacccaaccc aaaaaggtca gtggttgagt 6480ggcttttttg actatggatc
tttctcagag attatgcagc cctgggcaca gactgtggtg 6540gttggtagag ccaggctagg
aggaatacct gtgggagttg ttgctgtaga aacccgaaca 6600gtagaactaa gtatcccagc
tgatccagca aacctggatt ctgaagccaa gataatccag 6660caggctggcc aggtttggtt
cccagattct gcgtttaaga cgtatcaggc catcaaggac 6720ttcaaccggg aagggctgcc
tctgatggtc tttgccaact ggagaggctt ctctggtgga 6780atgaaagata tgtacgacca
agtgctgaag tttggtgctt acattgtgga tggcttgagg 6840gagtgctgcc agcctgtgct
ggtttacatt cctccccagg ctgagctgcg gggtggctcc 6900tgggtggtga ttgactcctc
catcaacccc cggcacatgg agatgtatgc tgaccgagaa 6960agcaggggat ctgttctgga
gccagaaggg acagtagaaa tcaaattccg cagaaaggat 7020ctggtgaaaa ccatgcgtcg
ggtggaccca gtctacatcc acttggctga gcgattgggg 7080accccagagc taagcacagc
tgagcggaag gagttggaga acaagttgaa ggagcgggag 7140gaattcctaa ttcccattta
ccatcaggta gccgtgcagt ttgctgactt gcacgacaca 7200ccaggccgga tgcaggagaa
gggtgttatt agcgatatcc tggattggaa aacatcccgt 7260accttcttct actggcggct
gaggcgtctt ctgctggagg acctggtcaa gaagaaaatc 7320cacaatgcca accctgagct
gactgatggc cagattcaag ccatgttaag gcgctggttt 7380gtggaagtgg aaggaacagt
gaaggcttat gtttgggaca ataataagga tctggcggag 7440tggctagaga aacagctgac
agaggaggat ggtgttcact cggtaataga ggaaaacatc 7500aaatgcatca gcagagacta
cgtcctcaag caaatccgca gcttggtcca ggccaatcca 7560gaggttgcca tggattccat
catccatatg acgcagcaca tatcacccac tcagcgagca 7620gaagtcatac ggatcctctc
cacaatggat tccccttcca cgtaggaaga gcttcctgcc 7680tgtccctgcc ctgtctctgg
agaaaagggc tagagctgcc ttttacaact gtaaccactg 7740taatgagaag gcacaggaga
cccagcactg gagtcaaatg gcattttact tcctctcgtt 7800tcaggttatg catgacatcc
tgggatgtaa gatcacagaa tccccctcca gcccaccagt 7860cacacctacc ccattcagta
tttattaccc tggccaggcc tagtcctcca ctccctgcac 7920aggactgaga aggcaatgaa
aggtacaaac atgtaccatg aggtcttact aaccaaagta 7980gggctgcccc tcctgtcctg
acagcccctt ggcctcccag catggggaag cgtgaggagt 8040tgcccagcag tgagcagccc
ccctcactcc tggccccatg agccgcagcc acaggcagca 8100gaggagggct aaggagagga
ggaagcctca agtccattgt ttattacccc gactcttagc 8160ccagcacaca gtaggcactg
gagaggaatg attcccagtt taaccacact acggtacctt 8220ttatgaagaa aaattagagc
ataaaatcta ctacaagctc cataggaact caaagatgag 8280ggcaaaactg tgagccaaga
agcagagaaa gaaaatagaa ccagttattc ttgatttagg 8340ggacctcaac cttgggttca
gtctctgagg acaaagggaa aggtagttgt tggcctgcct 8400ctcgcctgca cgtcactgct
ggactagctg tcgcatgtgg ctgggagctg caaggccagt 8460gcttgagggg ccccagcagt
tccacaggtg gtgaagcctg agttggcaga ggaggagcca 8520gaagagaact gccctttctg
cactggtgga aactagttat ttatgccatg tggagagcca 8580gtgagataga tagatagtct
gtttgttttg aggacttgga aagttgttcc tatgaagcct 8640ggagcttgga tggttttgag
aggttaatgg tgcctccaca ctcactcttc cctagttcca 8700ggattactgt cctagcagct
aacggttcta ctctcttccc cagagtgtag acaggcagca 8760ggtctcccca cagctctgaa
aggaccctgg tgacagctac accctcagca ccaggagctg 8820gccttcctga tgagggaggc
ttccaggaaa cacagaatcc acatgacctt aagattattt 8880acaactcagt catggtgctg
ctgtcctcca ggcttactgg cccctcctga ctggcatcag 8940gggcttcctc aggtggtgga
gagagtttac tttcaacaac tagtttattc aagaaaagaa 9000cttactgatt cctctgttcc
taaagcaaga gtggcaggtg atcagggctg gtgtagcatc 9060cggttccttt agtgcagcta
actgcatttg tcactgatga ccaaggagga aatcactaag 9120acatttgaga agcagtggta
tgaacgttct tggacaagcc acagttctga gccttaaccc 9180tgtagtttgc acacaagaac
gagctccacc tccccttctt caggaggaat ctgtgcggat 9240agattggctg gacttttcaa
tggttctggg ttgcaggtgg gcactgtatg gctgggtatg 9300gagcggacag cccccaggag
tcagagcctc agcccggctg ccctggtgga aggtacaggt 9360gttcagcacc ttcagaaaag
ggcataaagt ggtgggggac aattctcagt ccaggaaaat 9420gcattgacca ttgctggcta
tttgcttacc tagtaagaat tggattcatt tttgaccaga 9480ttattcttct atgctttttt
gcaataaatc aaatcccaca tatctacaag tggtatgaag 9540tcctgcaccc cccaggaggc
ctgtccaggc atgtcttcag aggcagggtg ggttacactc 9600atttacctcc cctctcccca
ccaaattatg acacaaacga gtatgtttcc tctctagaac 9660cctgtaatgc ctcctccccc
atccccagag ctccttactg taggtcttac cctggacaag 9720gattttttca agttggaggc
acagaacatg agcaatctga cattcccaca gcccctcaaa 9780catgcaaggc tactaaggca
ggaggagtat aaatgatgga tattgaccaa gacctgcttg 9840gacggagacc gccatattat
ctgttctctt cgttcacaaa acagccttca cttgtctcag 9900aatttgatgg acacatactg
tgatgagcag gagcttcaga tgcactcttt acacattttg 9960ttgaaataaa cctctacatt
tgtagaagaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1001839655DNAHomo sapiens
3ggcagccagg agaggcagtg gagcttttct ccgttgactg tcagaagaaa gtgactgttg
60ttgaaaatgt ttgctgcaac taaaggagag ctgctaagat ctactttcta gttaaacaag
120cttttatatt tctccccggc tcctctcagt tcccttggga aatcaactaa ttgtgtgtgt
180aagctaagtg tgtggaggag agaagatgca aaaaggaaga gaaaggtggg gaaagaagga
240aattgggaac aattagacaa aaagactata gtttgaggcc atcaggagta ttctagtctg
300actgaagcca ggcagaaggc acagtatctc ttgtcctcct gggttttaag cagctgcaac
360aggaggagga actgcggctt gtgtttacaa ggccgaaatc tgaagaaaaa gcttccattc
420ttttcccatc ttgatatgga gggttctcca gaggaaaata aggaaatgag atattacatg
480cttcaaaggt ccagcatgtc tggcttgcac ctagtaaagc agggccgaga cagaaagaaa
540atagattctc aacgagattt cactgtggct tctccagcag aatttgttac tcgctttggg
600ggaaataaag tgattgagaa ggttcttatt gctaacaatg gcattgcagc agtgaaatgc
660atgcggtcta tccgtaggtg gtcttatgaa atgtttcgaa atgaacgtgc aattagattc
720gttgtcatgg tcacacctga agaccttaaa gccaatgcag aatacattaa gatggcagat
780cactatgtgc cagtgcctgg aggaccaaac aacaacaact atgcaaatgt ggaattaatt
840cttgatattg ctaaaaggat cccagtacaa gcagtgtggg ctggctgggg tcatgcttct
900gagaatccca aactaccgga acttctcttg aaaaatggca ttgccttcat gggtcctcca
960agccaggcca tgtgggcttt aggggataag attgcatctt ccatagtggc tcaaactgca
1020ggtatcccaa ctcttccctg gagcggcagt ggtcttcgtg tggactggca ggaaaatgat
1080ttttcaaaac gtatcttaaa tgttccccag gagctatatg aaaaaggtta tgtgaaagat
1140gtggatgatg ggctacaggc agctgaggaa gttggatatc cagtaatgat caaggcctca
1200gagggaggag gagggaaggg aattagaaaa gtcaacaatg cagatgactt ccctaatctc
1260ttcagacagg ttcaagctga agttcctgga tctcccatat ttgtgatgag actagccaaa
1320caatctcgtc atctggaggt gcagatctta gcggaccaat atggcaatgc tatctctttg
1380tttggtcgtg attgctctgt acaacgcagg catcagaaga ttattgaaga agcacctgct
1440actattgcta ctccagcagt atttgaacac atggaacagt gtgcggtgaa acttgccaaa
1500atggtgggtt atgtgagtgc tgggactgtg gaatacctgt acagccagga tggcagcttc
1560tactttctgg aattgaatcc tcggctgcag gtagagcacc cttgtacaga gatggtggct
1620gatgtcaatc tccctgcagc acagctccag attgccatgg ggattcctct atatagaatc
1680aaggatatcc gtatgatgta tggggtatct ccctggggtg attctcccat tgattttgaa
1740gattctgcac acgttccttg tccaaggggc catgttattg ctgctcggat cactagtgaa
1800aatccagatg agggttttaa gcccagctca ggaacagttc aggagctaaa tttccgcagc
1860aataagaatg tttggggata tttcagtgtt gctgctgcag ggggacttca tgaatttgct
1920gattctcagt ttggtcactg cttttcttgg ggagaaaaca gagaagaggc aatttcaaac
1980atggtggtgg ctttgaagga gctgtctatt cggggtgact ttcgaactac agttgaatac
2040ctgatcaaat tgttagagac tgaaagcttt cagatgaaca gaattgatac tggctggctg
2100gacagactga tagcagaaaa agtacaggct gagcgacctg acaccatgtt gggggttgtg
2160tgtggtgccc tccacgtggc agatgtgagc ctgcggaata gcgtctctaa cttccttcac
2220tccttagaaa ggggtcaagt ccttcctgct catacacttc tgaatacagt agatgttgaa
2280cttatctatg agggagtcaa gtatgtactt aaggtgactc gacagtcccc caactcctat
2340gtggtgatca tgaatggctc atgtgtagaa gtagatgtac atcggctgag tgacggtgga
2400ctgctcttgt cctatgatgg cagcagttat actacgtata tgaaagagga agtggataga
2460tatcgcatca caattggcaa taaaacctgt gtgtttgaga aggaaaatga cccatcggtg
2520atgcgctcac cttctgctgg gaagttaatc cagtacattg tagaagatgg aggtcatgtg
2580tttgccggcc agtgctatgc tgagattgag gtaatgaaga tggtaatgac cttaacagct
2640gtggagtctg gctgtatcca ttacgtcaag cgacctggag cagctcttga ccctggctgt
2700gtactagcca aaatgcaact ggacaacccc agcaaggttc agcaggctga acttcacaca
2760ggtagtctgc cacggatcca gagcacggca ctcagaggcg agaaactcca tcgagtgttc
2820cattatgtcc tggataatct ggtcaatgta atgaatggat actgccttcc agatcctttc
2880tttagcagca aggtaaaaga ctgggtagag cgattgatga aaaccctcag agatccctcc
2940ctgcctctcc tagaattgca agatattatg accagtgtgt ctggccgcat tccccccaat
3000gtggagaagt ctatcaagaa ggaaatggct cagtatgcta gcaacatcac atcagtcctc
3060tgtcagtttc ccagccagca gattgcaaac atcctagata gccatgcagc tacattgaac
3120cggaaatctg aacgggaagt cttctttatg aatactcaga gcattgttca gctggtacag
3180aggtaccgaa gtggcatccg aggccacatg aaggctgtgg tgatggatct gctccggcag
3240tacctgcgag tagagacaca attccagaat ggtcactatg acaaatgtgt attcgccctc
3300cgagaagaga ataaaagtga catgaacact gtactgaact acatcttctc tcacgctcaa
3360gtcaccaaga agaatcttct ggtcacaatg cttattgatc agttgtgtgg ccgggaccct
3420actctcactg atgagctgct gaatattctc acagagctaa ctcaactcag taagaccacc
3480aatgccaaag tagcacttcg agcacgccag gttcttattg cctcccattt gccatcatat
3540gagcttcgcc ataaccaagt agagtctatc ttcctatcag ctattgacat gtatggacat
3600caattttgca ttgagaacct gcagaaactc atcctatcag aaacatctat ttttgatgtc
3660ctaccaaact tcttctatca cagcaaccaa gtagtgagga tggcagctct ggaggtgtat
3720gttcgaaggg cttatattgc ctatgaactt aacagcgtac aacaccgcca gcttaaggac
3780aacacctgtg tggtggaatt ccagttcatg ctgcccacat ctcatccaaa cagagggaac
3840atccctacgc taaacagaat gtccttctcc tccaacctca accactatgg catgacccat
3900gtagctagtg tcagcgatgt actgttggac aactcattca ctccaccttg tcagcggatg
3960ggcggaatgg tctcttttcg gacttttgaa gattttgtca ggatctttga tgaagtgatg
4020ggctgcttct ctgactcccc accccagagt cccacattcc ctgaggcagg tcacacgtct
4080ctttatgatg aggataaggt tcccagggat gaaccaattc acattctcaa tgtggctatc
4140aagactgact gtgatattga ggatgacagg ctggcagcta tgttcagaga atttacccag
4200caaaataaag ctaccctggt tgaccatggg atccggcgcc ttactttcct ggttgcacaa
4260aaggatttca gaaagcaggt caactatgag gtggatcgga gatttcatag agaattccct
4320aaatttttta cattccgagc aagggataag tttgaggagg atcgtatcta tcgtcatctg
4380gagcctgctc tggctttcca gttagagctg aaccggatga gaaattttga cctcactgcc
4440attccatgtg ctaatcacaa gatgcacctg tatctcgggg cagccaaggt ggaagtgggc
4500acagaagtga cagactacag gttctttgtt cgtgcaatca tcaggcattc tgatctggtc
4560accaaggaag cttcttttga atatctgcaa aatgaagggg agcggctact cctggaagcc
4620atggatgagt tggaagttgc ttttaacaat acaaatgtcc gcactgactg taaccacatc
4680ttcctcaact ttgtgcccac ggttatcatg gacccatcaa agattgagga atccgtgcgg
4740agcatggtaa tgcggtatgg aagtcgcctg tggaaattgc gcgtcctcca ggcagaactg
4800aaaatcaaca ttcgcctgac gccaactgga aaagcaattc ccatccgcct cttcctgaca
4860aacgagtctg gctattactt ggatatcagc ctatacaagg aagtgactga ctccaggaca
4920gcacagatca tgtttcaggc atatggagac aaacagggac cactgcatgg aatgttaatc
4980aatactccat atgtgaccaa agacctgctg caatcaaaga ggttccaggc acaatcctta
5040gggacaacat acatatatga tatcccagag atgtttcggc agtccctgat caaactctgg
5100gagtctatgt ccactcaagc atttcttcca tctccccctc tgccttctga catgctgact
5160tacactgaac tggtactgga tgatcaaggt cagctggtcc acatgaacag gcttccagga
5220ggaaatgaga ttggcatggt agcttggaaa atgaccttta aaagtcctga atatccagaa
5280ggccgagata tcattgttat tggcaatgac atcacatacc gaattgggtc ctttgggcct
5340caagaggatt tgttatttct cagagcttcc gaacttgcta gggcagaagg tattccacgc
5400atctatgtat cagccaacag tggagcaaga atcggactgg cagaagaaat tcgccatatg
5460tttcatgtgg cctgggtaga tcctgaggat ccttacaagg gatacaggta tttatatctg
5520actcctcaag attataagag agtcagtgct ctcaactctg tccattgtga acacgtggaa
5580gatgaaggag aatccaggta caagataaca gatattattg ggaaagaaga gggaattgga
5640cccgagaacc ttcgaggttc tggaatgatt gctggagaat cctcattggc ctataatgag
5700atcattacca tcagcctggt gacgtgccgg gccattggga ttggggctta ccttgtccgg
5760ctgggacaga gaaccatcca ggttgagaat tctcacttaa ttctaacagg agctggagcc
5820ctcaacaaag tcctcgggcg ggaagtgtac acctccaata accagctggg gggcatccag
5880attatgcaca acaatggggt gacccactgc actgtgtgtg atgactttga aggggttttc
5940actgtcctgc actggctgtc ttacatgccc aagagcgtgc acagttcagt tcctcttctg
6000aactcaaagg atcctataga cagaatcatc gagtttgttc ccacaaagac cccatacgat
6060cctcgatgga tgctagcagg ccgtcctcac ccaacccaaa aaggtcagtg gttgagtggc
6120ttttttgact atggatcttt ctcagagatt atgcagccct gggcacagac tgtggtggtt
6180ggtagagcca ggctaggagg aatacctgtg ggagttgttg ctgtagaaac ccgaacagta
6240gaactaagta tcccagctga tccagcaaac ctggattctg aagccaagat aatccagcag
6300gctggccagg tttggttccc agattctgcg tttaagacgt atcaggccat caaggacttc
6360aaccgggaag ggctgcctct gatggtcttt gccaactgga gaggcttctc tggtggaatg
6420aaagatatgt acgaccaagt gctgaagttt ggtgcttaca ttgtggatgg cttgagggag
6480tgctgccagc ctgtgctggt ttacattcct ccccaggctg agctgcgggg tggctcctgg
6540gtggtgattg actcctccat caacccccgg cacatggaga tgtatgctga ccgagaaagc
6600aggggatctg ttctggagcc agaagggaca gtagaaatca aattccgcag aaaggatctg
6660gtgaaaacca tgcgtcgggt ggacccagtc tacatccact tggctgagcg attggggacc
6720ccagagctaa gcacagctga gcggaaggag ttggagaaca agttgaagga gcgggaggaa
6780ttcctaattc ccatttacca tcaggtagcc gtgcagtttg ctgacttgca cgacacacca
6840ggccggatgc aggagaaggg tgttattagc gatatcctgg attggaaaac atcccgtacc
6900ttcttctact ggcggctgag gcgtcttctg ctggaggacc tggtcaagaa gaaaatccac
6960aatgccaacc ctgagctgac tgatggccag attcaagcca tgttaaggcg ctggtttgtg
7020gaagtggaag gaacagtgaa ggcttatgtt tgggacaata ataaggatct ggcggagtgg
7080ctagagaaac agctgacaga ggaggatggt gttcactcgg taatagagga aaacatcaaa
7140tgcatcagca gagactacgt cctcaagcaa atccgcagct tggtccaggc caatccagag
7200gttgccatgg attccatcat ccatatgacg cagcacatat cacccactca gcgagcagaa
7260gtcatacgga tcctctccac aatggattcc ccttccacgt aggaagagct tcctgcctgt
7320ccctgccctg tctctggaga aaagggctag agctgccttt tacaactgta accactgtaa
7380tgagaaggca caggagaccc agcactggag tcaaatggca ttttacttcc tctcgtttca
7440ggttatgcat gacatcctgg gatgtaagat cacagaatcc ccctccagcc caccagtcac
7500acctacccca ttcagtattt attaccctgg ccaggcctag tcctccactc cctgcacagg
7560actgagaagg caatgaaagg tacaaacatg taccatgagg tcttactaac caaagtaggg
7620ctgcccctcc tgtcctgaca gccccttggc ctcccagcat ggggaagcgt gaggagttgc
7680ccagcagtga gcagcccccc tcactcctgg ccccatgagc cgcagccaca ggcagcagag
7740gagggctaag gagaggagga agcctcaagt ccattgttta ttaccccgac tcttagccca
7800gcacacagta ggcactggag aggaatgatt cccagtttaa ccacactacg gtacctttta
7860tgaagaaaaa ttagagcata aaatctacta caagctccat aggaactcaa agatgagggc
7920aaaactgtga gccaagaagc agagaaagaa aatagaacca gttattcttg atttagggga
7980cctcaacctt gggttcagtc tctgaggaca aagggaaagg tagttgttgg cctgcctctc
8040gcctgcacgt cactgctgga ctagctgtcg catgtggctg ggagctgcaa ggccagtgct
8100tgaggggccc cagcagttcc acaggtggtg aagcctgagt tggcagagga ggagccagaa
8160gagaactgcc ctttctgcac tggtggaaac tagttattta tgccatgtgg agagccagtg
8220agatagatag atagtctgtt tgttttgagg acttggaaag ttgttcctat gaagcctgga
8280gcttggatgg ttttgagagg ttaatggtgc ctccacactc actcttccct agttccagga
8340ttactgtcct agcagctaac ggttctactc tcttccccag agtgtagaca ggcagcaggt
8400ctccccacag ctctgaaagg accctggtga cagctacacc ctcagcacca ggagctggcc
8460ttcctgatga gggaggcttc caggaaacac agaatccaca tgaccttaag attatttaca
8520actcagtcat ggtgctgctg tcctccaggc ttactggccc ctcctgactg gcatcagggg
8580cttcctcagg tggtggagag agtttacttt caacaactag tttattcaag aaaagaactt
8640actgattcct ctgttcctaa agcaagagtg gcaggtgatc agggctggtg tagcatccgg
8700ttcctttagt gcagctaact gcatttgtca ctgatgacca aggaggaaat cactaagaca
8760tttgagaagc agtggtatga acgttcttgg acaagccaca gttctgagcc ttaaccctgt
8820agtttgcaca caagaacgag ctccacctcc ccttcttcag gaggaatctg tgcggataga
8880ttggctggac ttttcaatgg ttctgggttg caggtgggca ctgtatggct gggtatggag
8940cggacagccc ccaggagtca gagcctcagc ccggctgccc tggtggaagg tacaggtgtt
9000cagcaccttc agaaaagggc ataaagtggt gggggacaat tctcagtcca ggaaaatgca
9060ttgaccattg ctggctattt gcttacctag taagaattgg attcattttt gaccagatta
9120ttcttctatg cttttttgca ataaatcaaa tcccacatat ctacaagtgg tatgaagtcc
9180tgcacccccc aggaggcctg tccaggcatg tcttcagagg cagggtgggt tacactcatt
9240tacctcccct ctccccacca aattatgaca caaacgagta tgtttcctct ctagaaccct
9300gtaatgcctc ctcccccatc cccagagctc cttactgtag gtcttaccct ggacaaggat
9360tttttcaagt tggaggcaca gaacatgagc aatctgacat tcccacagcc cctcaaacat
9420gcaaggctac taaggcagga ggagtataaa tgatggatat tgaccaagac ctgcttggac
9480ggagaccgcc atattatctg ttctcttcgt tcacaaaaca gccttcactt gtctcagaat
9540ttgatggaca catactgtga tgagcaggag cttcagatgc actctttaca cattttgttg
9600aaataaacct ctacatttgt agaagaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa
965549766DNAHomo sapiens 4ggcagccagg agaggcagtg gagcttttct ccgttgactg
tcagaagaaa gtgactgttg 60ttgaaaatgt ttgctgcaac taaaggagag ctgctaagat
ctactttcta gttaaacaag 120cttttatatt tctccccggc tcctctcagt tcccttggga
aatcaactaa ttgtgtgtgt 180aagctaagtg tgtggaggag agaagatgca aaaaggaaga
gaaaggtggg gaaagaagga 240aattgggaac aattagacaa aaagactata gtttgaggcc
atcaggagta ttctagtctg 300actgaagcca ggcagaaggc acagtatctc ttgtcctcct
gggttttaag cagctgcaac 360aggaggagga actgcggctt gtgtttacaa ggccgaaatc
tgaagaaaaa gcttccattc 420ttttcccatc ttgatatgga gggttctcca gaggaaaata
aggaaatgag atattacatg 480cttcaaagac tttgaaatac tcgtgggata tgaagtggag
gtgcctagag ggttgaagat 540aatgaggaca gatgaagaaa tgtttgattg tgccatactt
acagttgctg attttcaagg 600tccagcatgt ctggcttgca cctagtaaag cagggccgag
acagaaagaa aatagattct 660caacgagatt tcactgtggc ttctccagca gaatttgtta
ctcgctttgg gggaaataaa 720gtgattgaga aggttcttat tgctaacaat ggcattgcag
cagtgaaatg catgcggtct 780atccgtaggt ggtcttatga aatgtttcga aatgaacgtg
caattagatt cgttgtcatg 840gtcacacctg aagaccttaa agccaatgca gaatacatta
agatggcaga tcactatgtg 900ccagtgcctg gaggaccaaa caacaacaac tatgcaaatg
tggaattaat tcttgatatt 960gctaaaagga tcccagtaca agcagtgtgg gctggctggg
gtcatgcttc tgagaatccc 1020aaactaccgg aacttctctt gaaaaatggc attgccttca
tgggtcctcc aagccaggcc 1080atgtgggctt taggggataa gattgcatct tccatagtgg
ctcaaactgc aggtatccca 1140actcttccct ggagcggcag tggtcttcgt gtggactggc
aggaaaatga tttttcaaaa 1200cgtatcttaa atgttcccca ggagctatat gaaaaaggtt
atgtgaaaga tgtggatgat 1260gggctacagg cagctgagga agttggatat ccagtaatga
tcaaggcctc agagggagga 1320ggagggaagg gaattagaaa agtcaacaat gcagatgact
tccctaatct cttcagacag 1380gttcaagctg aagttcctgg atctcccata tttgtgatga
gactagccaa acaatctcgt 1440catctggagg tgcagatctt agcggaccaa tatggcaatg
ctatctcttt gtttggtcgt 1500gattgctctg tacaacgcag gcatcagaag attattgaag
aagcacctgc tactattgct 1560actccagcag tatttgaaca catggaacag tgtgcggtga
aacttgccaa aatggtgggt 1620tatgtgagtg ctgggactgt ggaatacctg tacagccagg
atggcagctt ctactttctg 1680gaattgaatc ctcggctgca ggtagagcac ccttgtacag
agatggtggc tgatgtcaat 1740ctccctgcag cacagctcca gattgccatg gggattcctc
tatatagaat caaggatatc 1800cgtatgatgt atggggtatc tccctggggt gattctccca
ttgattttga agattctgca 1860cacgttcctt gtccaagggg ccatgttatt gctgctcgga
tcactagtga aaatccagat 1920gagggtttta agcccagctc aggaacagtt caggagctaa
atttccgcag caataagaat 1980gtttggggat atttcagtgt tgctgctgca gggggacttc
atgaatttgc tgattctcag 2040tttggtcact gcttttcttg gggagaaaac agagaagagg
caatttcaaa catggtggtg 2100gctttgaagg agctgtctat tcggggtgac tttcgaacta
cagttgaata cctgatcaaa 2160ttgttagaga ctgaaagctt tcagatgaac agaattgata
ctggctggct ggacagactg 2220atagcagaaa aagtacaggc tgagcgacct gacaccatgt
tgggggttgt gtgtggtgcc 2280ctccacgtgg cagatgtgag cctgcggaat agcgtctcta
acttccttca ctccttagaa 2340aggggtcaag tccttcctgc tcatacactt ctgaatacag
tagatgttga acttatctat 2400gagggagtca agtatgtact taaggtgact cgacagtccc
ccaactccta tgtggtgatc 2460atgaatggct catgtgtaga agtagatgta catcggctga
gtgacggtgg actgctcttg 2520tcctatgatg gcagcagtta tactacgtat atgaaagagg
aagtggatag atatcgcatc 2580acaattggca ataaaacctg tgtgtttgag aaggaaaatg
acccatcggt gatgcgctca 2640ccttctgctg ggaagttaat ccagtacatt gtagaagatg
gaggtcatgt gtttgccggc 2700cagtgctatg ctgagattga ggtaatgaag atggtaatga
ccttaacagc tgtggagtct 2760ggctgtatcc attacgtcaa gcgacctgga gcagctcttg
accctggctg tgtactagcc 2820aaaatgcaac tggacaaccc cagcaaggtt cagcaggctg
aacttcacac aggtagtctg 2880ccacggatcc agagcacggc actcagaggc gagaaactcc
atcgagtgtt ccattatgtc 2940ctggataatc tggtcaatgt aatgaatgga tactgccttc
cagatccttt ctttagcagc 3000aaggtaaaag actgggtaga gcgattgatg aaaaccctca
gagatccctc cctgcctctc 3060ctagaattgc aagatattat gaccagtgtg tctggccgca
ttccccccaa tgtggagaag 3120tctatcaaga aggaaatggc tcagtatgct agcaacatca
catcagtcct ctgtcagttt 3180cccagccagc agattgcaaa catcctagat agccatgcag
ctacattgaa ccggaaatct 3240gaacgggaag tcttctttat gaatactcag agcattgttc
agctggtaca gaggtaccga 3300agtggcatcc gaggccacat gaaggctgtg gtgatggatc
tgctccggca gtacctgcga 3360gtagagacac aattccagaa tggtcactat gacaaatgtg
tattcgccct ccgagaagag 3420aataaaagtg acatgaacac tgtactgaac tacatcttct
ctcacgctca agtcaccaag 3480aagaatcttc tggtcacaat gcttattgat cagttgtgtg
gccgggaccc tactctcact 3540gatgagctgc tgaatattct cacagagcta actcaactca
gtaagaccac caatgccaaa 3600gtagcacttc gagcacgcca ggttcttatt gcctcccatt
tgccatcata tgagcttcgc 3660cataaccaag tagagtctat cttcctatca gctattgaca
tgtatggaca tcaattttgc 3720attgagaacc tgcagaaact catcctatca gaaacatcta
tttttgatgt cctaccaaac 3780ttcttctatc acagcaacca agtagtgagg atggcagctc
tggaggtgta tgttcgaagg 3840gcttatattg cctatgaact taacagcgta caacaccgcc
agcttaagga caacacctgt 3900gtggtggaat tccagttcat gctgcccaca tctcatccaa
acagagggaa catccctacg 3960ctaaacagaa tgtccttctc ctccaacctc aaccactatg
gcatgaccca tgtagctagt 4020gtcagcgatg tactgttgga caactcattc actccacctt
gtcagcggat gggcggaatg 4080gtctcttttc ggacttttga agattttgtc aggatctttg
atgaagtgat gggctgcttc 4140tctgactccc caccccagag tcccacattc cctgaggcag
gtcacacgtc tctttatgat 4200gaggataagg ttcccaggga tgaaccaatt cacattctca
atgtggctat caagactgac 4260tgtgatattg aggatgacag gctggcagct atgttcagag
aatttaccca gcaaaataaa 4320gctaccctgg ttgaccatgg gatccggcgc cttactttcc
tggttgcaca aaaggatttc 4380agaaagcagg tcaactatga ggtggatcgg agatttcata
gagaattccc taaatttttt 4440acattccgag caagggataa gtttgaggag gatcgtatct
atcgtcatct ggagcctgct 4500ctggctttcc agttagagct gaaccggatg agaaattttg
acctcactgc cattccatgt 4560gctaatcaca agatgcacct gtatctcggg gcagccaagg
tggaagtggg cacagaagtg 4620acagactaca ggttctttgt tcgtgcaatc atcaggcatt
ctgatctggt caccaaggaa 4680gcttcttttg aatatctgca aaatgaaggg gagcggctac
tcctggaagc catggatgag 4740ttggaagttg cttttaacaa tacaaatgtc cgcactgact
gtaaccacat cttcctcaac 4800tttgtgccca cggttatcat ggacccatca aagattgagg
aatccgtgcg gagcatggta 4860atgcggtatg gaagtcgcct gtggaaattg cgcgtcctcc
aggcagaact gaaaatcaac 4920attcgcctga cgccaactgg aaaagcaatt cccatccgcc
tcttcctgac aaacgagtct 4980ggctattact tggatatcag cctatacaag gaagtgactg
actccaggac agcacagatc 5040atgtttcagg catatggaga caaacaggga ccactgcatg
gaatgttaat caatactcca 5100tatgtgacca aagacctgct gcaatcaaag aggttccagg
cacaatcctt agggacaaca 5160tacatatatg atatcccaga gatgtttcgg cagtccctga
tcaaactctg ggagtctatg 5220tccactcaag catttcttcc atctccccct ctgccttctg
acatgctgac ttacactgaa 5280ctggtactgg atgatcaagg tcagctggtc cacatgaaca
ggcttccagg aggaaatgag 5340attggcatgg tagcttggaa aatgaccttt aaaagtcctg
aatatccaga aggccgagat 5400atcattgtta ttggcaatga catcacatac cgaattgggt
cctttgggcc tcaagaggat 5460ttgttatttc tcagagcttc cgaacttgct agggcagaag
gtattccacg catctatgta 5520tcagccaaca gtggagcaag aatcggactg gcagaagaaa
ttcgccatat gtttcatgtg 5580gcctgggtag atcctgagga tccttacaag ggatacaggt
atttatatct gactcctcaa 5640gattataaga gagtcagtgc tctcaactct gtccattgtg
aacacgtgga agatgaagga 5700gaatccaggt acaagataac agatattatt gggaaagaag
agggaattgg acccgagaac 5760cttcgaggtt ctggaatgat tgctggagaa tcctcattgg
cctataatga gatcattacc 5820atcagcctgg tgacgtgccg ggccattggg attggggctt
accttgtccg gctgggacag 5880agaaccatcc aggttgagaa ttctcactta attctaacag
gagctggagc cctcaacaaa 5940gtcctcgggc gggaagtgta cacctccaat aaccagctgg
ggggcatcca gattatgcac 6000aacaatgggg tgacccactg cactgtgtgt gatgactttg
aaggggtttt cactgtcctg 6060cactggctgt cttacatgcc caagagcgtg cacagttcag
ttcctcttct gaactcaaag 6120gatcctatag acagaatcat cgagtttgtt cccacaaaga
ccccatacga tcctcgatgg 6180atgctagcag gccgtcctca cccaacccaa aaaggtcagt
ggttgagtgg cttttttgac 6240tatggatctt tctcagagat tatgcagccc tgggcacaga
ctgtggtggt tggtagagcc 6300aggctaggag gaatacctgt gggagttgtt gctgtagaaa
cccgaacagt agaactaagt 6360atcccagctg atccagcaaa cctggattct gaagccaaga
taatccagca ggctggccag 6420gtttggttcc cagattctgc gtttaagacg tatcaggcca
tcaaggactt caaccgggaa 6480gggctgcctc tgatggtctt tgccaactgg agaggcttct
ctggtggaat gaaagatatg 6540tacgaccaag tgctgaagtt tggtgcttac attgtggatg
gcttgaggga gtgctgccag 6600cctgtgctgg tttacattcc tccccaggct gagctgcggg
gtggctcctg ggtggtgatt 6660gactcctcca tcaacccccg gcacatggag atgtatgctg
accgagaaag caggggatct 6720gttctggagc cagaagggac agtagaaatc aaattccgca
gaaaggatct ggtgaaaacc 6780atgcgtcggg tggacccagt ctacatccac ttggctgagc
gattggggac cccagagcta 6840agcacagctg agcggaagga gttggagaac aagttgaagg
agcgggagga attcctaatt 6900cccatttacc atcaggtagc cgtgcagttt gctgacttgc
acgacacacc aggccggatg 6960caggagaagg gtgttattag cgatatcctg gattggaaaa
catcccgtac cttcttctac 7020tggcggctga ggcgtcttct gctggaggac ctggtcaaga
agaaaatcca caatgccaac 7080cctgagctga ctgatggcca gattcaagcc atgttaaggc
gctggtttgt ggaagtggaa 7140ggaacagtga aggcttatgt ttgggacaat aataaggatc
tggcggagtg gctagagaaa 7200cagctgacag aggaggatgg tgttcactcg gtaatagagg
aaaacatcaa atgcatcagc 7260agagactacg tcctcaagca aatccgcagc ttggtccagg
ccaatccaga ggttgccatg 7320gattccatca tccatatgac gcagcacata tcacccactc
agcgagcaga agtcatacgg 7380atcctctcca caatggattc cccttccacg taggaagagc
ttcctgcctg tccctgccct 7440gtctctggag aaaagggcta gagctgcctt ttacaactgt
aaccactgta atgagaaggc 7500acaggagacc cagcactgga gtcaaatggc attttacttc
ctctcgtttc aggttatgca 7560tgacatcctg ggatgtaaga tcacagaatc cccctccagc
ccaccagtca cacctacccc 7620attcagtatt tattaccctg gccaggccta gtcctccact
ccctgcacag gactgagaag 7680gcaatgaaag gtacaaacat gtaccatgag gtcttactaa
ccaaagtagg gctgcccctc 7740ctgtcctgac agccccttgg cctcccagca tggggaagcg
tgaggagttg cccagcagtg 7800agcagccccc ctcactcctg gccccatgag ccgcagccac
aggcagcaga ggagggctaa 7860ggagaggagg aagcctcaag tccattgttt attaccccga
ctcttagccc agcacacagt 7920aggcactgga gaggaatgat tcccagttta accacactac
ggtacctttt atgaagaaaa 7980attagagcat aaaatctact acaagctcca taggaactca
aagatgaggg caaaactgtg 8040agccaagaag cagagaaaga aaatagaacc agttattctt
gatttagggg acctcaacct 8100tgggttcagt ctctgaggac aaagggaaag gtagttgttg
gcctgcctct cgcctgcacg 8160tcactgctgg actagctgtc gcatgtggct gggagctgca
aggccagtgc ttgaggggcc 8220ccagcagttc cacaggtggt gaagcctgag ttggcagagg
aggagccaga agagaactgc 8280cctttctgca ctggtggaaa ctagttattt atgccatgtg
gagagccagt gagatagata 8340gatagtctgt ttgttttgag gacttggaaa gttgttccta
tgaagcctgg agcttggatg 8400gttttgagag gttaatggtg cctccacact cactcttccc
tagttccagg attactgtcc 8460tagcagctaa cggttctact ctcttcccca gagtgtagac
aggcagcagg tctccccaca 8520gctctgaaag gaccctggtg acagctacac cctcagcacc
aggagctggc cttcctgatg 8580agggaggctt ccaggaaaca cagaatccac atgaccttaa
gattatttac aactcagtca 8640tggtgctgct gtcctccagg cttactggcc cctcctgact
ggcatcaggg gcttcctcag 8700gtggtggaga gagtttactt tcaacaacta gtttattcaa
gaaaagaact tactgattcc 8760tctgttccta aagcaagagt ggcaggtgat cagggctggt
gtagcatccg gttcctttag 8820tgcagctaac tgcatttgtc actgatgacc aaggaggaaa
tcactaagac atttgagaag 8880cagtggtatg aacgttcttg gacaagccac agttctgagc
cttaaccctg tagtttgcac 8940acaagaacga gctccacctc cccttcttca ggaggaatct
gtgcggatag attggctgga 9000cttttcaatg gttctgggtt gcaggtgggc actgtatggc
tgggtatgga gcggacagcc 9060cccaggagtc agagcctcag cccggctgcc ctggtggaag
gtacaggtgt tcagcacctt 9120cagaaaaggg cataaagtgg tgggggacaa ttctcagtcc
aggaaaatgc attgaccatt 9180gctggctatt tgcttaccta gtaagaattg gattcatttt
tgaccagatt attcttctat 9240gcttttttgc aataaatcaa atcccacata tctacaagtg
gtatgaagtc ctgcaccccc 9300caggaggcct gtccaggcat gtcttcagag gcagggtggg
ttacactcat ttacctcccc 9360tctccccacc aaattatgac acaaacgagt atgtttcctc
tctagaaccc tgtaatgcct 9420cctcccccat ccccagagct ccttactgta ggtcttaccc
tggacaagga ttttttcaag 9480ttggaggcac agaacatgag caatctgaca ttcccacagc
ccctcaaaca tgcaaggcta 9540ctaaggcagg aggagtataa atgatggata ttgaccaaga
cctgcttgga cggagaccgc 9600catattatct gttctcttcg ttcacaaaac agccttcact
tgtctcagaa tttgatggac 9660acatactgtg atgagcagga gcttcagatg cactctttac
acattttgtt gaaataaacc 9720tctacatttg tagaagaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaa 9766510692DNAHomo sapiens 5ggcccccgcc gggtgctgag
cgcccgccac cgccctcttg gccttttccc ggtccccgcc 60gcgcgcctgc tgctgtcccc
gtgcccgagg actgggagga tggggctgag ccgcttgcct 120gacttttgat ccgaccagta
atcactttgc ccgtgtggcg cgtggcccgt tgcctgaggc 180ttcctggcgc gtggggctgt
ttggtcccct ccggagtgcg gtgaggcggg ccgagccggg 240actgcctggg tttggggata
acgttcccat ctccacccct gttgcagcaa gggaaattga 300ggctgaggga actgggccca
gggacggcga gccgtggctg cctctccagt ccgggccccg 360aagggctgct cgtggatgaa
ccagactgat tttaaggggt gaagagggtg cgtttcaatc 420agatgcttct ggaacgtcga
aattgtcttc tttggaaaga accatcccct ctttgggctt 480cagaggccca aattgaggcg
caatgatgag aggatgtggt ggtctactct gatgtcaatc 540ttgagggcta gggtcttgct
atgtcaccca gcctggagtg gaattgtgtg atcaagactc 600actgaagcct ccaactcctg
ggctcaagca atcctcccac ctcagccacc caagtagctg 660ggactacagg ctggagtgca
atggcatgat cttggctcac tgcaacctcc gcctcccgga 720ttcaacggat tctcctgcct
cagcctcctg agtagctggg attataggtg cctgccacaa 780cacccaggac cagacttatg
aagtggcctt cctgaggaga ttttctgacc ttatggtatg 840aagttccttc aggctcttaa
ttctggataa actggagtga ttaacaagag caaagagaca 900tagtattcag ataaggactt
tgtgtcttat ttttctttta tatcacaact ggagccacag 960ttgtgggcaa ggcactgact
ttcttgatga attctgtcca tctgtaaagc agctatatat 1020cccactggag gctttgcctc
tcagagacaa cgtgaagacg gataagcagg catcaggagc 1080agagtggcct ctcagctggt
cagattctcc gaagaaggat cctctaggct gggcacggtg 1140gctcacacct ataatcccag
cactttggga ggccgaggca ggtggatcac gaggtcagga 1200gttcgaggcc agcctggcca
atatggtctt tctggaagtg gatatctact cagacagtaa 1260gaattataag agctgtaaga
gctcattttg gaggaataat ggatgaacca tctcccttgg 1320cccaacctct ggagctgaac
cagcactctc gattcataat aggttctgtg tctgaagata 1380actcagagga tgagatcagc
aacctggtga agttggacct actggaggag aaggagggct 1440ccttgtcacc tgcttctgtt
ggctcagata cactctctga tttggggatc tctagcctac 1500aggatggctt ggccttgcac
ataaggtcca gcatgtctgg cttgcaccta gtaaagcagg 1560gccgagacag aaagaaaata
gattctcaac gagatttcac tgtggcttct ccagcagaat 1620ttgttactcg ctttggggga
aataaagtga ttgagaaggt tcttattgct aacaatggca 1680ttgcagcagt gaaatgcatg
cggtctatcc gtaggtggtc ttatgaaatg tttcgaaatg 1740aacgtgcaat tagattcgtt
gtcatggtca cacctgaaga ccttaaagcc aatgcagaat 1800acattaagat ggcagatcac
tatgtgccag tgcctggagg accaaacaac aacaactatg 1860caaatgtgga attaattctt
gatattgcta aaaggatccc agtacaagca gtgtgggctg 1920gctggggtca tgcttctgag
aatcccaaac taccggaact tctcttgaaa aatggcattg 1980ccttcatggg tcctccaagc
caggccatgt gggctttagg ggataagatt gcatcttcca 2040tagtggctca aactgcaggt
atcccaactc ttccctggag cggcagtggt cttcgtgtgg 2100actggcagga aaatgatttt
tcaaaacgta tcttaaatgt tccccaggag ctatatgaaa 2160aaggttatgt gaaagatgtg
gatgatgggc tacaggcagc tgaggaagtt ggatatccag 2220taatgatcaa ggcctcagag
ggaggaggag ggaagggaat tagaaaagtc aacaatgcag 2280atgacttccc taatctcttc
agacaggttc aagctgaagt tcctggatct cccatatttg 2340tgatgagact agccaaacaa
tctcgtcatc tggaggtgca gatcttagcg gaccaatatg 2400gcaatgctat ctctttgttt
ggtcgtgatt gctctgtaca acgcaggcat cagaagatta 2460ttgaagaagc acctgctact
attgctactc cagcagtatt tgaacacatg gaacagtgtg 2520cggtgaaact tgccaaaatg
gtgggttatg tgagtgctgg gactgtggaa tacctgtaca 2580gccaggatgg cagcttctac
tttctggaat tgaatcctcg gctgcaggta gagcaccctt 2640gtacagagat ggtggctgat
gtcaatctcc ctgcagcaca gctccagatt gccatgggga 2700ttcctctata tagaatcaag
gatatccgta tgatgtatgg ggtatctccc tggggtgatt 2760ctcccattga ttttgaagat
tctgcacacg ttccttgtcc aaggggccat gttattgctg 2820ctcggatcac tagtgaaaat
ccagatgagg gttttaagcc cagctcagga acagttcagg 2880agctaaattt ccgcagcaat
aagaatgttt ggggatattt cagtgttgct gctgcagggg 2940gacttcatga atttgctgat
tctcagtttg gtcactgctt ttcttgggga gaaaacagag 3000aagaggcaat ttcaaacatg
gtggtggctt tgaaggagct gtctattcgg ggtgactttc 3060gaactacagt tgaatacctg
atcaaattgt tagagactga aagctttcag atgaacagaa 3120ttgatactgg ctggctggac
agactgatag cagaaaaagt acaggctgag cgacctgaca 3180ccatgttggg ggttgtgtgt
ggtgccctcc acgtggcaga tgtgagcctg cggaatagcg 3240tctctaactt ccttcactcc
ttagaaaggg gtcaagtcct tcctgctcat acacttctga 3300atacagtaga tgttgaactt
atctatgagg gagtcaagta tgtacttaag gtgactcgac 3360agtcccccaa ctcctatgtg
gtgatcatga atggctcatg tgtagaagta gatgtacatc 3420ggctgagtga cggtggactg
ctcttgtcct atgatggcag cagttatact acgtatatga 3480aagaggaagt ggatagatat
cgcatcacaa ttggcaataa aacctgtgtg tttgagaagg 3540aaaatgaccc atcggtgatg
cgctcacctt ctgctgggaa gttaatccag tacattgtag 3600aagatggagg tcatgtgttt
gccggccagt gctatgctga gattgaggta atgaagatgg 3660taatgacctt aacagctgtg
gagtctggct gtatccatta cgtcaagcga cctggagcag 3720ctcttgaccc tggctgtgta
ctagccaaaa tgcaactgga caaccccagc aaggttcagc 3780aggctgaact tcacacaggt
agtctgccac ggatccagag cacggcactc agaggcgaga 3840aactccatcg agtgttccat
tatgtcctgg ataatctggt caatgtaatg aatggatact 3900gccttccaga tcctttcttt
agcagcaagg taaaagactg ggtagagcga ttgatgaaaa 3960ccctcagaga tccctccctg
cctctcctag aattgcaaga tattatgacc agtgtgtctg 4020gccgcattcc ccccaatgtg
gagaagtcta tcaagaagga aatggctcag tatgctagca 4080acatcacatc agtcctctgt
cagtttccca gccagcagat tgcaaacatc ctagatagcc 4140atgcagctac attgaaccgg
aaatctgaac gggaagtctt ctttatgaat actcagagca 4200ttgttcagct ggtacagagg
taccgaagtg gcatccgagg ccacatgaag gctgtggtga 4260tggatctgct ccggcagtac
ctgcgagtag agacacaatt ccagaatggt cactatgaca 4320aatgtgtatt cgccctccga
gaagagaata aaagtgacat gaacactgta ctgaactaca 4380tcttctctca cgctcaagtc
accaagaaga atcttctggt cacaatgctt attgatcagt 4440tgtgtggccg ggaccctact
ctcactgatg agctgctgaa tattctcaca gagctaactc 4500aactcagtaa gaccaccaat
gccaaagtag cacttcgagc acgccaggtt cttattgcct 4560cccatttgcc atcatatgag
cttcgccata accaagtaga gtctatcttc ctatcagcta 4620ttgacatgta tggacatcaa
ttttgcattg agaacctgca gaaactcatc ctatcagaaa 4680catctatttt tgatgtccta
ccaaacttct tctatcacag caaccaagta gtgaggatgg 4740cagctctgga ggtgtatgtt
cgaagggctt atattgccta tgaacttaac agcgtacaac 4800accgccagct taaggacaac
acctgtgtgg tggaattcca gttcatgctg cccacatctc 4860atccaaacag agggaacatc
cctacgctaa acagaatgtc cttctcctcc aacctcaacc 4920actatggcat gacccatgta
gctagtgtca gcgatgtact gttggacaac tcattcactc 4980caccttgtca gcggatgggc
ggaatggtct cttttcggac ttttgaagat tttgtcagga 5040tctttgatga agtgatgggc
tgcttctctg actccccacc ccagagtccc acattccctg 5100aggcaggtca cacgtctctt
tatgatgagg ataaggttcc cagggatgaa ccaattcaca 5160ttctcaatgt ggctatcaag
actgactgtg atattgagga tgacaggctg gcagctatgt 5220tcagagaatt tacccagcaa
aataaagcta ccctggttga ccatgggatc cggcgcctta 5280ctttcctggt tgcacaaaag
gatttcagaa agcaggtcaa ctatgaggtg gatcggagat 5340ttcatagaga attccctaaa
ttttttacat tccgagcaag ggataagttt gaggaggatc 5400gtatctatcg tcatctggag
cctgctctgg ctttccagtt agagctgaac cggatgagaa 5460attttgacct cactgccatt
ccatgtgcta atcacaagat gcacctgtat ctcggggcag 5520ccaaggtgga agtgggcaca
gaagtgacag actacaggtt ctttgttcgt gcaatcatca 5580ggcattctga tctggtcacc
aaggaagctt cttttgaata tctgcaaaat gaaggggagc 5640ggctactcct ggaagccatg
gatgagttgg aagttgcttt taacaataca aatgtccgca 5700ctgactgtaa ccacatcttc
ctcaactttg tgcccacggt tatcatggac ccatcaaaga 5760ttgaggaatc cgtgcggagc
atggtaatgc ggtatggaag tcgcctgtgg aaattgcgcg 5820tcctccaggc agaactgaaa
atcaacattc gcctgacgcc aactggaaaa gcaattccca 5880tccgcctctt cctgacaaac
gagtctggct attacttgga tatcagccta tacaaggaag 5940tgactgactc caggacagca
cagatcatgt ttcaggcata tggagacaaa cagggaccac 6000tgcatggaat gttaatcaat
actccatatg tgaccaaaga cctgctgcaa tcaaagaggt 6060tccaggcaca atccttaggg
acaacataca tatatgatat cccagagatg tttcggcagt 6120ccctgatcaa actctgggag
tctatgtcca ctcaagcatt tcttccatct ccccctctgc 6180cttctgacat gctgacttac
actgaactgg tactggatga tcaaggtcag ctggtccaca 6240tgaacaggct tccaggagga
aatgagattg gcatggtagc ttggaaaatg acctttaaaa 6300gtcctgaata tccagaaggc
cgagatatca ttgttattgg caatgacatc acataccgaa 6360ttgggtcctt tgggcctcaa
gaggatttgt tatttctcag agcttccgaa cttgctaggg 6420cagaaggtat tccacgcatc
tatgtatcag ccaacagtgg agcaagaatc ggactggcag 6480aagaaattcg ccatatgttt
catgtggcct gggtagatcc tgaggatcct tacaagggat 6540acaggtattt atatctgact
cctcaagatt ataagagagt cagtgctctc aactctgtcc 6600attgtgaaca cgtggaagat
gaaggagaat ccaggtacaa gataacagat attattggga 6660aagaagaggg aattggaccc
gagaaccttc gaggttctgg aatgattgct ggagaatcct 6720cattggccta taatgagatc
attaccatca gcctggtgac gtgccgggcc attgggattg 6780gggcttacct tgtccggctg
ggacagagaa ccatccaggt tgagaattct cacttaattc 6840taacaggagc tggagccctc
aacaaagtcc tcgggcggga agtgtacacc tccaataacc 6900agctgggggg catccagatt
atgcacaaca atggggtgac ccactgcact gtgtgtgatg 6960actttgaagg ggttttcact
gtcctgcact ggctgtctta catgcccaag agcgtgcaca 7020gttcagttcc tcttctgaac
tcaaaggatc ctatagacag aatcatcgag tttgttccca 7080caaagacccc atacgatcct
cgatggatgc tagcaggccg tcctcaccca acccaaaaag 7140gtcagtggtt gagtggcttt
tttgactatg gatctttctc agagattatg cagccctggg 7200cacagactgt ggtggttggt
agagccaggc taggaggaat acctgtggga gttgttgctg 7260tagaaacccg aacagtagaa
ctaagtatcc cagctgatcc agcaaacctg gattctgaag 7320ccaagataat ccagcaggct
ggccaggttt ggttcccaga ttctgcgttt aagacgtatc 7380aggccatcaa ggacttcaac
cgggaagggc tgcctctgat ggtctttgcc aactggagag 7440gcttctctgg tggaatgaaa
gatatgtacg accaagtgct gaagtttggt gcttacattg 7500tggatggctt gagggagtgc
tgccagcctg tgctggttta cattcctccc caggctgagc 7560tgcggggtgg ctcctgggtg
gtgattgact cctccatcaa cccccggcac atggagatgt 7620atgctgaccg agaaagcagg
ggatctgttc tggagccaga agggacagta gaaatcaaat 7680tccgcagaaa ggatctggtg
aaaaccatgc gtcgggtgga cccagtctac atccacttgg 7740ctgagcgatt ggggacccca
gagctaagca cagctgagcg gaaggagttg gagaacaagt 7800tgaaggagcg ggaggaattc
ctaattccca tttaccatca ggtagccgtg cagtttgctg 7860acttgcacga cacaccaggc
cggatgcagg agaagggtgt tattagcgat atcctggatt 7920ggaaaacatc ccgtaccttc
ttctactggc ggctgaggcg tcttctgctg gaggacctgg 7980tcaagaagaa aatccacaat
gccaaccctg agctgactga tggccagatt caagccatgt 8040taaggcgctg gtttgtggaa
gtggaaggaa cagtgaaggc ttatgtttgg gacaataata 8100aggatctggc ggagtggcta
gagaaacagc tgacagagga ggatggtgtt cactcggtaa 8160tagaggaaaa catcaaatgc
atcagcagag actacgtcct caagcaaatc cgcagcttgg 8220tccaggccaa tccagaggtt
gccatggatt ccatcatcca tatgacgcag cacatatcac 8280ccactcagcg agcagaagtc
atacggatcc tctccacaat ggattcccct tccacgtagg 8340aagagcttcc tgcctgtccc
tgccctgtct ctggagaaaa gggctagagc tgccttttac 8400aactgtaacc actgtaatga
gaaggcacag gagacccagc actggagtca aatggcattt 8460tacttcctct cgtttcaggt
tatgcatgac atcctgggat gtaagatcac agaatccccc 8520tccagcccac cagtcacacc
taccccattc agtatttatt accctggcca ggcctagtcc 8580tccactccct gcacaggact
gagaaggcaa tgaaaggtac aaacatgtac catgaggtct 8640tactaaccaa agtagggctg
cccctcctgt cctgacagcc ccttggcctc ccagcatggg 8700gaagcgtgag gagttgccca
gcagtgagca gcccccctca ctcctggccc catgagccgc 8760agccacaggc agcagaggag
ggctaaggag aggaggaagc ctcaagtcca ttgtttatta 8820ccccgactct tagcccagca
cacagtaggc actggagagg aatgattccc agtttaacca 8880cactacggta ccttttatga
agaaaaatta gagcataaaa tctactacaa gctccatagg 8940aactcaaaga tgagggcaaa
actgtgagcc aagaagcaga gaaagaaaat agaaccagtt 9000attcttgatt taggggacct
caaccttggg ttcagtctct gaggacaaag ggaaaggtag 9060ttgttggcct gcctctcgcc
tgcacgtcac tgctggacta gctgtcgcat gtggctggga 9120gctgcaaggc cagtgcttga
ggggccccag cagttccaca ggtggtgaag cctgagttgg 9180cagaggagga gccagaagag
aactgccctt tctgcactgg tggaaactag ttatttatgc 9240catgtggaga gccagtgaga
tagatagata gtctgtttgt tttgaggact tggaaagttg 9300ttcctatgaa gcctggagct
tggatggttt tgagaggtta atggtgcctc cacactcact 9360cttccctagt tccaggatta
ctgtcctagc agctaacggt tctactctct tccccagagt 9420gtagacaggc agcaggtctc
cccacagctc tgaaaggacc ctggtgacag ctacaccctc 9480agcaccagga gctggccttc
ctgatgaggg aggcttccag gaaacacaga atccacatga 9540ccttaagatt atttacaact
cagtcatggt gctgctgtcc tccaggctta ctggcccctc 9600ctgactggca tcaggggctt
cctcaggtgg tggagagagt ttactttcaa caactagttt 9660attcaagaaa agaacttact
gattcctctg ttcctaaagc aagagtggca ggtgatcagg 9720gctggtgtag catccggttc
ctttagtgca gctaactgca tttgtcactg atgaccaagg 9780aggaaatcac taagacattt
gagaagcagt ggtatgaacg ttcttggaca agccacagtt 9840ctgagcctta accctgtagt
ttgcacacaa gaacgagctc cacctcccct tcttcaggag 9900gaatctgtgc ggatagattg
gctggacttt tcaatggttc tgggttgcag gtgggcactg 9960tatggctggg tatggagcgg
acagccccca ggagtcagag cctcagcccg gctgccctgg 10020tggaaggtac aggtgttcag
caccttcaga aaagggcata aagtggtggg ggacaattct 10080cagtccagga aaatgcattg
accattgctg gctatttgct tacctagtaa gaattggatt 10140catttttgac cagattattc
ttctatgctt ttttgcaata aatcaaatcc cacatatcta 10200caagtggtat gaagtcctgc
accccccagg aggcctgtcc aggcatgtct tcagaggcag 10260ggtgggttac actcatttac
ctcccctctc cccaccaaat tatgacacaa acgagtatgt 10320ttcctctcta gaaccctgta
atgcctcctc ccccatcccc agagctcctt actgtaggtc 10380ttaccctgga caaggatttt
ttcaagttgg aggcacagaa catgagcaat ctgacattcc 10440cacagcccct caaacatgca
aggctactaa ggcaggagga gtataaatga tggatattga 10500ccaagacctg cttggacgga
gaccgccata ttatctgttc tcttcgttca caaaacagcc 10560ttcacttgtc tcagaatttg
atggacacat actgtgatga gcaggagctt cagatgcact 10620ctttacacat tttgttgaaa
taaacctcta catttgtaga agaaaaaaaa aaaaaaaaaa 10680aaaaaaaaaa aa
106926850DNAHomo sapiens
6cgcagcccgc gactcctccc ccgccgcacc tcgtggcccc cgcttctccc tagtcccgct
60agaggggctc gcgctccgcc acccgcccgc ccgcccgccc agcgctgcct ggcgcctcac
120ctcgccgaga ccccctaatc cgcgccaaag agccgccggt cgcgcgcgcc tccgggcttt
180gcgtgcgcgc ccccgggcgg cgcagtggcg agcgcgccgc gtgccgcgcg cccgccgcct
240ccgcccctcg gccgtggagg cccccgccgg gtgctgagcg cccgccaccg ccctcttggc
300cttttcccgg tccccgccgc gcgcctgctg ctgtccccgt gcccgaggac tgggaggatg
360gggctgagcc gcttgcctga cttttgatcc gaccagtaat cactttgccc gtgtggcgcg
420tggcccgttg cctgaggctt cctggcgcgt ggggctgttt ggtcccctcc ggagtgcggt
480gaggcgggcc gagccgggac tgcctgggtt tggggataac gttcccatct ccacccctgt
540tgcagcaagg gaaattgagg ctgagggaac tgggcccagg gacggcgagc cgtggctgcc
600tctccagtcc gggccccgaa gggctgctcg tggatgaacc agactgattt taaggggtga
660agagggtgcg tttcaatcag atgcttctgg aacgtcgaaa ttgtcttctt tggaaagaac
720catcccctct ttgggcttca gaggcccaaa ttgaggcgca atgatgagag gatgtggtgg
780tctactctga tgtcaatctt gagggctagg tatgtcccaa gatctcttat tactgctgtt
840aatgatgatt
8507600DNAHomo sapiens 7cgggaccgct tcccccactc ccctctccgc caggaggagg
agccgagggg ttggccccgt 60acccggctgt ggcagaaggg gccgcgaagg ccgggccagg
ggcgccacga ctgcccaggg 120cccgcccgcg gcccgcccgc ggcccgcccg ccctctagcc
gctgggcccc gaatggcaga 180tccgcgctcg gaccatcggc atcgcctcac atcgctccgc
cccgcgccgc cctcccattg 240gctgccgctg agccctcggg cccgcgcctc gccccccggc
ggccctggca gcgccgcagc 300ccggagcggg gtcggaggtg aacggcctgg agtaaccccg
gacgtagtca cctcatggag 360ctcgccggct gaggtgggag acaggggcgg ggcgggggcg
ggtcaggccc ctgaagcccc 420gccccttctc cgtgtgccgg gccggcctgg tgctgcacgc
ctgtcagcca tcgcccgagc 480cgccggcgtc tcctcccgcc cagcagttcc tccacgcagg
ggctccggaa tcgcccgacc 540gcacacgttg ccaccctgag gtgaggtgag ggccggccca
gaacctgtga ccggggacct 60081530DNAHomo sapiens 8agctggttgt catgttgggt
ggcctgattg agttgctttc aaatttgggc aacttgagtc 60tttacaactt aagttgcccg
aatgtccttc agatcaatgt ttgtttgttg taatcattta 120tcttttatct gattatttaa
cttctatcta tgctccaggt catttgtgta cttctgtttt 180tgaaggtttt tctggaagct
taatatattc tcagcgtggt tggcatcaga ttgttaacta 240tttctatcca gcattttgat
gggaataaat tttttatggt tcatgattca ggttagattt 300ggtgtacagg cctgaggact
tgcctcagga agttgtcttt ctatagattc taagccttta 360gggtcttttg ccattgattt
ttctcctaat acacagaata tcatgagcat ctacttaagt 420aaaagctaaa aacaggaaga
tattacaaga taacattcag ctagtgttta tttctttaag 480agatctgtct gtggcaaatt
tgctttcata gtaatatatg tgtctcagta agtagtcttt 540ctaggcagga tttttcttcc
ctgcttaagg gggttagacc tccaaaatat ttgtcagggt 600ctcttttcct tttaagggtt
taacccacaa tcttttctgt atccgttttg taccagctcc 660attcctattc tcaagctttg
gtgcataaaa ccttagttta ggtgtctata tataccacta 720ctttggcctg aaaattagaa
tcgtggctca cacttgattc taagttccct agtagtggcg 780tgtttccttt ctacgtgctt
actttcccac ctgacctgct tttccccacc gtgcagtctg 840ataattggta ttatttgttt
tgattggatc tcagaaatta cctctttaga ccgagctgtg 900ctcctccctc tggatttcat
gagcccatta ttttcttggc ttaattgtcc ttgtaaattt 960ggtctttttg tcctgggctt
tccctgtcat gtgctcctgg cagccaggag aggcagtgga 1020gcttttctcc gttgactgtc
agaagaaagt gactgttgtt gaaaatgttt gctgcaacta 1080aaggagagct gctaagatct
actttctagt taaacaagct tttatatttc tccccggctc 1140ctctcagttc ccttgggaaa
tcaactaatt gtgtgtgtaa gctaagtgtg tggaggagag 1200aagatgcaaa aaggaagaga
aaggtgggga aagaaggaaa ttgggaacaa ttagacaaaa 1260agactatagt ttgaggccat
caggagtatt ctagtctgac tgaagccagg cagaaggcac 1320agtatctctt gtcctcctgg
gttttaagca gctgcaacag gaggaggaac tgcggcttgt 1380gtttacaagg ccgaaatctg
aagaaaaagc ttccattctt ttcccatctt gatatggagg 1440gttctccaga ggaaaataag
gaaatgagat attacatgct tcaaaggtaa gtgttaggct 1500accacatttg ggtaatatat
atatctttaa 153097041DNAHomo sapiens
9atggatgaac catctccctt ggcccaacct ctggagctga accagcactc tcgattcata
60ataggttctg tgtcagaaga taactcagag gatgagatca gcaacctggt gaagctggac
120ctactggagg agaaggaggg ctccttgtca cctgcttctg ttggctcaga tacactctct
180gatttgggga tctctgccct acaggatggc ttggccttgc acataaggtc cagctggtct
240ggcttgcacc tagtaaagca gggcggagac agaaagaaaa tagattctca acgagatttc
300actgtggctt ctccagcaga atttgttact cgctttgggg gaaataaagt gattgagaag
360gttctcatcg ctaacaatgg cattgcagca gtgaaatgca tgcggtctat ccgtaggtgg
420tcttatgaaa tgtttcgaaa tgaacgtgca attagattcg ttgtcatggt cacacctgaa
480gaccttaaag ccaatgcaga atacattaag atggcagatc actatgtgcc ggtgcctgga
540ggagcaaaca acaacaacta tgcaaatgtg gaattaattc ttgatattgc taaaaggatc
600ccagtgcaag cagtgtgggc tggctggggt catgcttctg agaatcccaa actaccggaa
660cttctcttga aaaatggcat tgccttcatg ggtcctccaa accaggccat gtgggcttta
720ggggataaga ttgcatcttc catagtggct caaactgcag gtatcccaac tcttccctgg
780agcggcagtg gtcttcgtgt ggactggcag gaaaatgatt tttcaaaacg tatcttaaat
840gttccccagg agctatatga aaaaggttat gtgaaagatg tggatgatgg gctaaaggca
900gctgagaagg ttggatatcc agtaatgatc aaggcctcag agggaggagg agggaaggga
960attagaaaag ttaacaatgc agatgacttc cctaatctct tcagacaggt tcaagctgaa
1020gttcctggat ctcccatatt tgtgatgaga ctagccaaac aatctcgtca tctggaggtg
1080cagatcttag cggaccaata tggcaatgct atctctttgt ttggtcgtga ttgctctgta
1140caacgcaggc atcagaagat tattgaagaa gcacctgcta ctattgctac tccagcagta
1200tttgaacaca tggaacagtg tgcggtgaaa cttgccaaaa tggtgggtta tgtgagtgct
1260gggactgtgg aatacctgta cagccaggat ggcagcttct actttctaga attgaaccct
1320cggctacagg ttgaacaacc ttgtacagag atggtggctg atgtcaatct ccccgcagca
1380cagctccaga ttgccatggg gattcctcta tatagaatca aggatatccg tatgatgtat
1440ggggtatctc cttggggtga ttctcccatt gattttgaaa attctgctca cgttccttgt
1500ccaaggggcc atgttattgc tgctcggatc actagtgaaa atccagatga gggttttaag
1560cccagctcag gaacagttca ggagctaaat ttccgcagca ataagaatgt ttggggatat
1620ttcagtgttg ctgctgcagg gggacttcat gaatttgctg gttctcagtt tggtcactgc
1680ttttcttggg gagaaaacag agaagaagca atttcaaaca tggtggtggc attgaaggag
1740ctgtctattc ggggtgactt tcgaactaca gttgaatacc tgatcaaatt gttagagact
1800gaaagctttc aaatgaacag aattgatact ggctggctgg acagactgat agcagaaaaa
1860gtacgggctg agcgtcctga caccatgttg ggggttgtgt gtggtgccct ccacgtcgga
1920gatgtgagcc tgcgaaatag cgtctctaac ttccttcact ccttagaaag gggtcaagtc
1980cttccggctc atacacttct gaatacagta gatgttgaac ttatctatga gggagtcaag
2040tatgtactta aggtgactcg acagtccccc aactcctatg tggtgatcat gaatggctca
2100tgtgtagaag tagatgtaca tcggctgagt gacggtggac tgctcttgtc ctatgatggc
2160agcagttaca ccacgtatat gaaggaggaa gtagacagat atcgcatcac aattggcaat
2220aaaacctgtg tgtttgagaa ggaaaatgac ccatcggtga tgcgctcacc ttctgctggg
2280aagttaatcc agtacattgt agaagatgga ggtcatgtgt ttgccggcca gtgctatgca
2340gagattgagg taatgaagat ggtaatgact ttgacagctg tggagtctgg ctgtatccat
2400tacgtcaagc gtcctggagc agctcttgac cctggctgtg tactcgccaa aatgcaactg
2460gacaacccca gcaaggttca gcaggctgaa cttcacacag gtagtctgcc acggatccag
2520agcaccgctc tccgaggcga gaagctccat cgagtgttcc actatgtcct ggataatctg
2580gtcaatgtaa tgaatggata ctgccttcca gatcctttct ttagcagcaa ggtaaaagac
2640tgggtagaac gattgatgaa aaccctcaga gatccctccc tgcctctcct agaattgcaa
2700gatattatga ccagtgtgtc tggccgcatt ccccccaatg tggagaagtc tatcaagaag
2760gaaatggctc agtatgctag caacatcaca tcagtcctct gtcagtttcc cagccagcag
2820attgcaaaca tcctagatag ccatgcagct acattgaacc ggaaatctga acgggaagtc
2880ttctttatga atactcagag cattgtccag ctggtacaga ggtaccgaag tggcatccga
2940ggccacatga aggctgtggt gatggatctg ctccggcagt acctgcgagt agagacacaa
3000ttccagaatg gtcactatga caaatgtgta ttcgcccttc gagaagagaa taagagtgac
3060atgaacactg tactgaacta catcttctct cacgctcaag tcaccaagaa gaatcttctg
3120gtcacaatgc ttattgatca gttgtgtggc cgggacccta ctctaactga tgagctgctg
3180agtattctca cagagctaac tcaactcagt aagaccacca atgccaaagt agcacttcga
3240gcacgccagg ttcttattgc ctcccatttg ccatcatatg acgttcgcca taaccaagta
3300gagtctatct tcctatcagc tattgacatg tatggacatc aattttgcat tgagaacctg
3360cagaaactca tcctatcaga aacatctatt tttgatgtcc taccaaactt cttctatcac
3420agcaaccaag tagtgaggat ggcagctctg gaggtgtacg ttcgaagggc ttatattgcc
3480tatgaactta acagcgtaca acaccgccag cttaaggaca acacctgcgt ggtggaattc
3540cagttcatgc tgcccacatc tcatccaaac agagggaaca tccctacgct aaacagaatg
3600tccttctcct ccaacctcaa ccactatggc atgacccatg tagctagtgt cagcgatgtt
3660ctgttggaca acgccttcac accaccttgt caacgcatgg gcggaatggt ctcttttcgg
3720acttttgaag attttgtcag gatctttgat gaagtaatgg gctgcttctg tgactcccca
3780ccccagagtc ccacattccc tgaggcaggt cacacgtctc tttatgatga ggataaggtt
3840cccagggatg aaccaattca cattctcaat gtggctatca agactgacgg tgatattgag
3900gatgacaggc tggcagctat gttcagagaa ttcacccagc aaaataaagc taccctggct
3960gaccatggga tccggcgcct gactttcctg gttgcacaaa aggatttcag aaagcaggtc
4020aactatgagg tggatcggag atttcataga gaattcccta aattttttac attccgagca
4080agggataagt ttgaggagga tcgtatctat cgtcatctgg agcctgctct ggctttccag
4140ttagagctga accggatgag aaattttgac ctcactgcca ttccatgtgc taatcacaag
4200atgcacctgt atctcggggc agccaaggtg gaagtgggca cagaagtgac agactacagg
4260ttctttgttc gtgcaatcat caggcattct gatctggtca ccaaggaagc ttcttttgaa
4320tatctgcaaa gtgaagggga gcggctactc ctggaagcca tggatgagtt ggaagttgct
4380tttaacaata caaatgtccg cactgactgt aaccacatcc tcctcaactt tgtgcccacg
4440gttatcatgg acccatcaaa gattgaggaa tccgtgcgga gcatggtaat gcggtatgga
4500agtcgcctgt ggaaattgcg cgtcctccag gcagaactga aaatcaacat tcgcctgacg
4560ccaactggaa aagcaattcc catccgcctt ttcctgacaa acgagtctgg ctattacttg
4620gatatcagcc tatacaagga agtgactgac tccaggacag cacagatcat gtttcaggca
4680tatggagaca agcagggacc actgcatgga atgttaatca atactccata tgtgaccaaa
4740gacctgctgc aatcaaagag gttccaggca caatccttag ggacaacgta catatatgat
4800atcccagaga tgtttcggca gtccctgatc aaactctggg agtctatgtc aactcaagca
4860tttcttccat ctccccctct gccttctgac atgctgactt acactgaact ggtactggat
4920gatcaaggtc agctggtcca catgaacagg cttccaggag gaaatgagat tggcatggta
4980gcttggaaaa tgagccttaa aagtcctgaa tatccagaag gccgagatgt tattgttatt
5040ggcaatgaca ttacataccg aattgggtcc tttgggcctc aagaagattt gttatttctc
5100agagcttccg aacttgctag ggccgaaggc attccacgca tctatgtatc agccaacagt
5160ggagcaagaa tcggactggc agaagaaatt cgccatatgt ttcatgtggc ctgggtagat
5220tctgaggatc cttacaaggg atacaggtat ttatatctga ctcctcaaga ttataagaga
5280gtcggtgctc tcaactctgt ccattgtgaa cacgtggaag atgaaggaga atccaggtac
5340aagataacag atattattgg gaaagaagag ggaattggac ccgagaacct tcgaggttct
5400ggaatgatcg ctggagaatc ctcattggcc tataatgaga tcattaccat cagcctggtg
5460acgtcccggg ccattgggat tggggcttac cttgtccggc tgggacagag aaccatccag
5520gttgagaatt ctcacttgat tctaacagga gctggagccc tcaacaaagt cctcgggcgg
5580gaagtgtaca cctccaataa ccagctgggg ggcatccaga ttacgcacaa caatggggtg
5640acccactgca ctgtgtgtga cggctttgaa ggggttttca ctgtcctgca ctggctgtct
5700tacatgccca agagcgtaca cagttcagtt cctcttctga actcaaagga tcctatagac
5760agaatcatcg agtttgttcc cacaaagacc ccatatgatc ctcgatggat gctagcaggc
5820cgccctcacc caacccaaaa aggtcagtgg ttgagtggct tttttgacta tggatctttc
5880tcagagatta tgcagccctg ggcacagacg gtggtggttg gtagagccag gttaggggga
5940atacctgtgg gagttgttgc tgtagaaacc cggacagtag aactaagtgt accagctgat
6000ccagcaaacc tggattctga agccaagata atccagcacg ccggccaagt ttggtttccg
6060gattctgcgt ttaagacgta tcaggccatc aaggacttca accgggaagg gctacctcta
6120atggtctttg ccaactggag aggcttctct ggtggaatga aagatatgta tcaccaagtg
6180ctgaagtttg gtgcttacat tgtggatggc ttgcgggaat gttcccagcc tgtgctggtc
6240tacattcctc cccaggctga gctgcggggt ggctcctggg tggtgatcga cccaaccatc
6300aatcctcggc acatggagat gtatgctgac cgagaaagca ggggatctgt tctggagcca
6360gaagggacgg tagaaatcaa attccgcaga aaggatctgg tgaaaaccat gcgtcgggtg
6420gacccagtct acatccactt ggctgagcga ttggggaccc cagagctgag cccaactgag
6480cggaaggagt tggagagcaa gttgaaggag cgggaggaat tcctaattcc aatttaccat
6540caggtagccg tgcagtttgc tgacttgcac gacactccag gccggatgca ggagaagggt
6600gttattagcg atatcctgga ttggaaaaca tcccgtacct tcttctactg gcgactgagg
6660cgtcttctgc tggaggacct ggtcaagaag aaaatccaca gtgccaaccc tgagctgact
6720gatggccaga ttcaagccat gttaagacgc tggtttgtgg aagtggaagg aacagtgaag
6780gcttatgttt gggacaataa taaggatctg gcggagtggc tagagaaaca gctgacagag
6840gaggatggtg ttcactcggt aatagaggaa aacatcaaat gcatcagcag agactacgtc
6900ctcaagcaaa tccgcagctt ggtccaggcc aatccagagg ttgccatgga ttccatcatc
6960catatgacgc agcacatatc acccactcag cgggcagaag tcataaggat cctttccact
7020atggactccc cttctacgta g
7041107041DNAHomo sapiens 10atggatgaac catctccctt ggcccaacct ctggagctga
accagcactc tcgattcata 60ataggttctg tgtctgaaga taactcagag gatgagatca
gcaacctggt gaagttggac 120ctactggagg agaaggaggg ctccttgtca cctgcttctg
ttggctcaga tacactctct 180gatttgggga tctctagcct acaggatggc ttggccttgc
acataaggtc cagcatgtct 240ggcttgcacc tagtaaagca gggccgagac agaaagaaaa
tagattctca acgagatttc 300actgtggctt ctccagcaga atttgttact cgctttgggg
gaaataaagt gattgagaag 360gttcttattg ctaacaatgg cattgcagca gtgaaatgca
tgcggtctat ccgtaggtgg 420tcttatgaaa tgtttcgaaa tgaacgtgca attagattcg
ttgtcatggt cacacctgaa 480gaccttaaag ccaatgcaga atacattaag atggcagatc
actatgtgcc agtgcctgga 540ggaccaaaca acaacaacta tgcaaatgtg gaattaattc
ttgatattgc taaaaggatc 600ccagtacaag cagtgtgggc tggctggggt catgcttctg
agaatcccaa actaccggaa 660cttctcttga aaaatggcat tgccttcatg ggtcctccaa
gccaggccat gtgggcttta 720ggggataaga ttgcatcttc catagtggct caaactgcag
gtatcccaac tcttccctgg 780agcggcagtg gtcttcgtgt ggactggcag gaaaatgatt
tttcaaaacg tatcttaaat 840gttccccagg agctatatga aaaaggttat gtgaaagatg
tggatgatgg gctacaggca 900gctgaggaag ttggatatcc agtaatgatc aaggcctcag
agggaggagg agggaaggga 960attagaaaag tcaacaatgc agatgacttc cctaatctct
tcagacaggt tcaagctgaa 1020gttcctggat ctcccatatt tgtgatgaga ctagccaaac
aatctcgtca tctggaggtg 1080cagatcttag tggaccaata tggcaatgct atctctttgt
ttggtcgtga ttgctctgta 1140caacgcaggc atcagaagat tattgaagaa gcacctgcta
ctattgctac tccagcagta 1200tttgaacaca tggaacagtg tgcggtgaaa cttgccaaaa
tggtgggtta tgtgagtgct 1260gggactgtgg aatacctgta cagccaggat ggcagcttct
actttctgga attgaatcct 1320cggctgcagg tagagcaccc ttgtacagag atggtggctg
atgtcaatct ccctgcagca 1380cagctccaga ttgccatggg gattcctcta tatagaatca
aggatatccg tatgatgtat 1440ggggtatctc cctggggtga ttctcccatt gattttgaag
attctgcaca cgttccttgt 1500ccaaggggcc atgttattgc tgctcggatc actagtgaaa
atccagatga gggttttaag 1560cccagctcag gaacagttca ggagctaaat ttccgcagca
ataagaatgt ttggggatat 1620ttcagtgttg ctgctgcagg gggacttcat gaatttgctg
attctcagtt tggtcactgc 1680ttttcttggg gagaaaacag agaagaggca atttcaaaca
tggtggtggc tttgaaggag 1740ctgtctattc ggggtgactt tcgaactaca gttgaatacc
tgatcaaatt gttagagact 1800gaaagctttc agatgaacag aattgatact ggctggctgg
acagactgat agcagaaaaa 1860gtacaggctg agcgacctga caccatgttg ggggttgtgt
gtggtgccct ccacgtggca 1920gatgtgagcc tgcggaatag cgtctctaac ttccttcact
ccttagaaag gggtcaagtc 1980cttcctgctc atacacttct gaatacagta gatgttgaac
ttatctatga gggagtcaag 2040tatgtactta aggtgactcg acagtccccc aactcctatg
tggtgatcat gaatggctca 2100tgtgtagaag tagatgtaca tcggctgagt gacggtggac
tgctcttgtc ctatgatggc 2160agcagttata ctacgtatat gaaagaggaa gtggatagat
atcgcatcac aattggcaat 2220aaaacctgtg tgtttgagaa ggaaaatgac ccatcggtga
tgcgctcacc ttctgctggg 2280aagttaatcc agtacattgt agaagatgga ggtcatgtgt
ttgccggcca gtgctatgct 2340gagattgagg taatgaagat ggtaatgacc ttaacagctg
tggagtctgg ctgtatccat 2400tacgtcaagc gacctggagc agctcttgac cctggctgta
tactagccaa aatgcaactg 2460gacaacccca gcaaggttca gcaggctgaa cttcacacag
gtagtctgcc acggatccag 2520agcacggcac tcagaggcga gaaactccat cgagtgttcc
attatgtcct ggataatctg 2580gtcaatgtaa tgaatggata ctgccttcca gatcctttct
ttagcagcaa ggtaaaagac 2640tgggtagagc gattgatgaa aaccctcaga gatccctccc
tgcctctcct agaattgcaa 2700gatattatga ccagtgtgtc tggccgcatt ccccccaatg
tggagaagtc tatcaagaag 2760gaaatggctc agtatgctag caacatcaca tcagtcctct
gtcagtttcc cagccagcag 2820attgcaaaca tcctagatag ccatgcagct acattgaacc
ggaaatctga acgggaagtc 2880ttctttatga atactcagag cattgttcag ctggtacaga
ggtaccgaag tggcatccga 2940ggccacatga aggctgtggt gatggatctg ctccggcagt
acctgcgagt agagacacaa 3000ttccagaatg gtcactatga caaatgtgta ttcgccctcc
gagaagagaa taaaagtgac 3060atgaacactg tactgaacta catcttctct cacgctcaag
tcaccaagaa gaatcttctg 3120gtcacaatgc ttattgatca gttgtgtggc cgggacccta
ctctcactga tgagctgctg 3180aatattctca cagagctaac tcaactcagt aagaccacca
atgccaaagt agcacttcga 3240gcacgccagg ttcttattgc ctcccatttg ccatcatatg
agcttcgcca taaccaagta 3300gagtctatct tcctatcagc tattgacatg tatggacatc
aattttgcat tgagaacctg 3360cagaaactca tcctatcaga aacatctatt tttgatgtcc
taccaaactt cttctatcac 3420agcaaccaag tagtgaggat ggcagctctg gaggtgtatg
ttcgaagggc ttatattgcc 3480tatgaactta acagcgtaca acaccgccag cttaaggaca
acacctgtgt ggtggaattc 3540cagttcatgc tgcccacatc tcatccaaac agagggaaca
tccctacgct aaacagaatg 3600tccttctcct ccaacctcaa ccactatggc atgacccatg
tagctagtgt cagcgatgta 3660ctgttggaca actcattcac tccaccttgt cagcggatgg
gcggaatggt ctcttttcgg 3720acttttgaag attttgtcag gatctttgat gaagtgatgg
gctgcttctc tgactcccca 3780ccccagagtc ccacattccc tgaggcaggt cacacgtctc
tttatgatga ggataaggtt 3840cccagggatg aaccaattca cattctcaat gtggctatca
agactgactg tgatattgag 3900gatgacaggc tggcagctat gttcagagaa tttacccagc
aaaataaagc taccctggtt 3960gaccatggga tccggcgcct tactttcctg gttgcacaaa
aggatttcag aaagcaggtc 4020aactatgagg tggatcggag atttcataga gaattcccta
aattttttac attccgagca 4080agggataagt ttgaggagga tcgtatctat cgtcatctgg
agcctgctct ggctttccag 4140ttagagctga accggatgag aaattttgac ctcactgcca
ttccatgtgc taatcacaag 4200atgcacctgt atctcggggc agccaaggtg gaagtgggca
cagaagtgac agactacagg 4260ttctttgttc gtgcaatcat caggcattct gatctggtca
ccaaggaagc ttcttttgaa 4320tatctgcaaa atgaagggga gcggctactc ctggaagcca
tggatgagtt ggaagttgct 4380tttaacaata caaatgtccg cactgactgt aaccacatct
tcctcaactt tgtgcccacg 4440gttatcatgg acccatcaaa gattgaggaa tccgtgcgga
gcatggtaat gcggtatgga 4500agtcgcctgt ggaaattgcg cgtcctccag gcagaactga
aaatcaacat tcgcctgacg 4560ccaactggaa aagcaattcc catccgcctc ttcctgacaa
acgagtctgg ctattacttg 4620gatatcagcc tatacaagga agtgactgac tccaggacag
cacagatcat gtttcaggca 4680tatggagaca aacagggacc actgcatgga atgttaatca
atactccata tgtgaccaaa 4740gacctgctgc aatcaaagag gttccaggca caatccttag
ggacaacata catatatgat 4800atcccagaga tgtttcggca gtccctgatc aaactctggg
agtctatgtc cactcaagca 4860tttcttccat ctccccctct gccttctgac atgctgactt
acactgaact ggtactggat 4920gatcaaggtc agctggtcca catgaacagg cttccaggag
gaaatgagat tggcatggta 4980gcttggaaaa tgacctttaa aagtcctgaa tatccagaag
gccgagatat cattgttatt 5040ggcaatgaca tcacataccg aattgggtcc tttgggcctc
aagaggattt gttatttctc 5100agagcttccg aacttgctag ggcagaaggt attccacgca
tctatgtatc agccaacagt 5160ggagcaagaa tcggactggc agaagaaatt cgccatatgt
ttcatgtggc ctgggtagat 5220cctgaggatc cttacaaggg atacaggtat ttatatctga
ctcctcaaga ttataagaga 5280gtcagtgctc tcaactctgt ccattgtgaa cacgtggaag
atgaaggaga atccaggtac 5340aagataacag atattattgg gaaagaagag ggaattggac
ccgagaacct tcgaggttct 5400ggaatgattg ctggagaatc ctcattggcc tataatgaga
tcattaccat cagcctggtg 5460acgtgccggg ccattgggat tggggcttac cttgtccggc
tgggacagag aaccatccag 5520gttgagaatt ctcacttaat tctaacagga gctggagccc
tcaacaaagt cctcgggcgg 5580gaagtgtaca cctccaataa ccagctgggg ggcatccaga
ttatgcacaa caatggggtg 5640acccactgca ctgtgtgtga tgactttgaa ggggttttca
ctgtcctgca ctggctgtct 5700tacatgccca agagcgtgca cagttcagtt cctcttctga
actcaaagga tcctatagac 5760agaatcatcg agtttgttcc cacaaagacc ccatacgatc
ctcgatggat gctagcaggc 5820cgtcctcacc caacccaaaa aggtcagtgg ttgagtggct
tttttgacta tggatctttc 5880tcagagatta tgcagccctg ggcacagact gtggtggttg
gtagagccag gctaggagga 5940atacctgtgg gagttgttgc tgtagaaacc cgaacagtag
aactaagtat cccagctgat 6000ccagcaaacc tggattctga agccaagata atccagcagg
ctggccaggt ttggttccca 6060gattctgcgt ttaagacgta tcaggccatc aaggacttca
accgggaagg gctgcctctg 6120atggtctttg ccaactggag aggcttctct ggtggaatga
aagatatgta cgaccaagtg 6180ctgaagtttg gtgcttacat tgtggatggc ttgagggagt
gctgccagcc tgtgctggtt 6240tacattcctc cccaggctga gctgcggggt ggctcctggg
tggtgattga ctcctccatc 6300aacccccggc acatggagat gtatgctgac cgagaaagca
ggggatctgt tctggagcca 6360gaagggacag tagaaatcaa attccgcaga aaggatctgg
tgaaaaccat gcgtcgggtg 6420gacccagtct acatccactt ggctgagcga ttggggaccc
cagagctaag cacagctgag 6480cggaaggagt tggagaacaa gttgaaggag cgggaggaat
tcctaattcc catttaccat 6540caggtagccg tgcagtttgc tgacttgcac gacacaccag
gccggatgca ggagaagggt 6600gttattagcg atatcctgga ttggaaaaca tcccgtacct
tcttctactg gcggctgagg 6660cgtcttctgc tggaggacct ggtcaagaag aaaatccaca
atgccaaccc tgagctgact 6720gatggccaga ttcaagccat gttaaggcgc tggtttgtgg
aagtggaagg aacagtgaag 6780gcttatgttt gggacaataa taaggatctg gcggagtggc
tagagaaaca gctgacagag 6840gaggatggtg ttcactcggt aatagaggaa aacatcaaat
gcatcagcag agactacgtc 6900ctcaagcaaa tccgcagctt ggtccaggcc aatccagagg
ttgccatgga ttccatcatc 6960cgtatgacgc agcacatatc acccactcag cgagcagaag
tcatacggat cctctccaca 7020atggattccc cttccacgtg a
7041119251DNAHomo sapiens 11atggtcttgc ttctttgtct
atcttgtctg attttctcct gtctgacctt ttcctggtta 60aaaatctggg ggaaaatgac
ggactccaag ccgatcacca agagtaaatc agaagcaaac 120ctcatcccga gccaggagcc
ctttccagcc tctgataact caggggagac accgcagaga 180aatggggagg gccacactct
gcccaagaca cccagccagg ccgagccagc ctcccacaaa 240ggccccaaag atgccggtcg
gcggagaaac tccctaccac cctcccacca gaagccccca 300agaaaccccc tttcttccag
tgacgcagca ccctccccag agcttcaagc caacgggact 360gggacacaag gtctggaggc
cacagatacc aatggcctgt cctcctcagc caggccccag 420ggccagcaag ctggctcccc
ctccaaagaa gacaagaagc aggcaaacat caagaggcag 480ctgatgacca acttcatcct
gggctctttt gatgactact cctccgacga ggactctgtt 540gctggctcat ctcgtgagtc
tacccggaag ggcagccggg ccagcttggg ggccctgtcc 600ctggaggctt atctgaccac
aggtgaagct gagacccgcg tccccactat gaggccgagc 660atgtcgggac tccacctggt
gaagagggga cgggaacaca agaagctgga cctgcacaga 720gactttaccg tggcttctcc
cgctgagttt gtcacacgct ttggggggga tcgggtcatc 780gagaaggtgc ttattgccaa
caacgggatt gccgccgtga agtgcatgcg ctccatccgc 840aggtgggcct atgagatgtt
ccgcaacgag cgggccatcc ggtttgttgt gatggtgacc 900cccgaggacc ttaaggccaa
cgcagagtac atcaagatgg cggatcatta cgtccccgtc 960ccaggagggc ccaataacaa
caactatgcc aacgtggagc tgattgtgga cattgccaag 1020agaatccccg tgcaggcggt
gtgggctggc tggggccatg cttcagaaaa ccctaaactt 1080ccggagctgc tgtgcaagaa
tggagttgct ttcttaggcc ctcccagtga ggccatgtgg 1140gccttaggag ataagatcgc
ctccaccgtt gtcgcccaga cgctacaggt cccaaccctg 1200ccctggagtg gaagcggcct
gacagtggag tggacagaag atgatctgca gcagggaaaa 1260agaatcagtg tcccagaaga
tgtttatgac aagggttgcg tgaaagacgt agatgagggc 1320ttggaggcag cagaaagaat
tggttttcca ttgatgatca aagcttctga aggtggcgga 1380gggaagggaa tccggaaggc
tgagagtgcg gaggacttcc cgatcctttt cagacaagta 1440cagagtgaga tcccaggctc
gcccatcttt ctcatgaagc tggcccagca cgcccgtcac 1500ctggaagttc agatcctcgc
tgaccagtat gggaatgctg tgtctctgtt tggtcgcgac 1560tgctccatcc agcggcggca
tcagaagatc gttgaggaag caccggccac catcgccccg 1620ctggccatat tcgagttcat
ggagcagtgt gccatccgcc tggccaagac cgtgggctat 1680gtgagtgcag ggacagtgga
atacctctat agtcaggatg gcagcttcca cttcttggag 1740ctgaatcctc gcttgcaggt
ggaacatccc tgcacagaaa tgattgctga tgttaatctg 1800ccggccgccc agctacagat
cgccatgggc gtgccactgc accggctgaa ggatatccgg 1860cttctgtatg gagagtcacc
atggggagtg actcccattt cttttgaaac cccctcaaac 1920cctcccctcg cccgaggcca
cgtcattgcc gccagaatca ccagcgaaaa cccagacgag 1980ggttttaagc cgagctccgg
gactgtccag gaactgaatt tccggagcag caagaacgtg 2040tggggttact tcagcgtggc
cgctactgga ggcctgcacg agtttgcgga ttcccaattt 2100gggcactgct tctcctgggg
agagaaccgg gaagaggcca tttcgaacat ggtggtggct 2160ttgaaggaac tgtccatccg
aggcgacttt aggactaccg tggaatacct cattaacctc 2220ctggagaccg agagcttcca
gaacaacgac atcgacaccg ggtggttgga ctacctcatt 2280gctgagaaag tgcaggcgga
gaaaccggat atcatgcttg gggtggtatg cggggccttg 2340aacgtggccg atgcgatgtt
cagaacgtgc atgacagatt tcttacactc cctggaaagg 2400ggccaggtcc tcccagcgga
ttcactactg aacctcgtag atgtggaatt aatttacgga 2460ggtgttaagt acattctcaa
ggtggcccgg cagtctctga ccatgttcgt tctcatcatg 2520aatggctgcc acatcgagat
tgatgcccac cggctgaatg atggggggct cctgctctcc 2580tacaatggga acagctacac
cacctacatg aaggaagagg ttgacagtta ccgaattacc 2640atcggcaata agacgtgtgt
gtttgagaag gagaacgatc ctacagtcct gagatccccc 2700tcggctggga agctgacaca
gtacacagtg gaggatgggg gccacgttga ggctgggagc 2760agctacgctg agatggaggt
gatgaagatg atcatgaccc tgaacgttca ggaaagaggc 2820cgggtgaagt acatcaagcg
tccaggtgcc gtgctggaag caggctgcgt ggtggccagg 2880ctggagctcg atgacccttc
taaagtccac ccggctgaac cgttcacagg agaactccct 2940gcccagcaga cactgcccat
cctcggagag aaactgcacc aggtcttcca cagcgtcctg 3000gaaaacctca ccaacgtcat
gagtggcttt tgtctgccag agcccgtttt tagcataaag 3060ctgaaggagt gggtgcagaa
gctcatgatg accctccggc acccgtcact gccgctgctg 3120gagctgcagg agatcatgac
cagcgtggca ggccgcatcc ccgcccctgt ggagaagtct 3180gtccgcaggg tgatggccca
gtatgccagc aacatcacct cggtgctgtg ccagttcccc 3240agccagcaga tagccaccat
cctggactgc catgcagcca ccctgcagcg gaaggctgat 3300cgagaggtct tcttcatcaa
cacccagagc atcgtgcagt tggtccagag ataccgcagc 3360gggatccgcg gctatatgaa
aacagtggtg ttggatctcc tgagaagata cttgcgtgtt 3420gagcaccatt ttcagcaagc
ccactacgac aagtgtgtga taaacctcag ggagcagttc 3480aagccagaca tgtcccaggt
gctggactgc atcttctccc acgcacaggt ggccaagaag 3540aaccagctgg tgatcatgtt
gatcgatgag ctgtgtggcc cagacccttc cctgtcggac 3600gagctgatct ccatcctcaa
cgagctcact cagctgagca aaagcgagca ctgcaaagtg 3660gccctcagag cccggcagat
cctgattgcc tcccacctcc cctcctacga gctgcggcat 3720aaccaggtgg agtccatttt
cctgtctgcc attgacatgt acggccacca gttctgcccc 3780gagaacctca agaaattaat
actttcggaa acaaccatct tcgacgtcct gcctactttc 3840ttctatcacg caaacaaagt
cgtgtgcatg gcgtccttgg aggtttacgt gcggaggggc 3900tacatcgcct atgagttaaa
cagcctgcag caccggcagc tcccggacgg cacctgcgtg 3960gtagaattcc agttcatgct
gccgtcctcc cacccaaacc ggatgaccgt gcccatcagc 4020atcaccaacc ctgacctgct
gaggcacagc acagagctct tcatggacag cggcttctcc 4080ccactgtgcc agcgcatggg
agccatggta gccttcagga gattcgagga cttcaccaga 4140aattttgatg aagtcatctc
ttgcttcgcc aacgtgccca aagacacccc cctcttcagc 4200gaggcccgca cctccctata
ctccgaggat gactgcaaga gcctcagaga agagcccatc 4260cacattctga atgtgtccat
ccagtgtgca gaccacctgg aggatgaggc actggtgccg 4320attttacgga cattcgtaca
gtccaagaaa aatatccttg tggattatgg actccgacga 4380atcacattct tgattgccca
agagaaagaa tttcccaagt ttttcacatt cagagcaaga 4440gatgagtttg cagaagatcg
catttaccgt cacttggaac ctgccctggc cttccagctg 4500gaacttaacc ggatgcgtaa
cttcgatctg accgccgtgc cctgtgccaa ccacaagatg 4560cacctttacc tgggtgctgc
caaggtgaag gaaggtgtgg aagtgacgga ccataggttc 4620ttcatccgcg ccatcatcag
gcactctgac ctgatcacaa aggaagcctc cttcgaatac 4680ctgcagaacg agggtgagcg
gctgctcctg gaggccatgg acgagctgga ggtggcgttc 4740aataacacca gcgtgcgcac
cgactgcaac cacatcttcc tcaacttcgt gcccactgtc 4800atcatggacc ccttcaagat
cgaggagtcc gtgcgctaca tggttatgcg ctacggcagc 4860cggctgtgga aactccgtgt
gctacaggct gaggtcaaga tcaacatccg ccagaccacc 4920accggcagtg ccgttcccat
ccgcctgttc atcaccaatg agtcgggcta ctacctggac 4980atcagcctct acaaagaagt
gactgactcc agatctggaa atatcatgtt tcactccttc 5040ggcaacaagc aagggcccca
gcacgggatg ctgatcaata ctccctacgt caccaaggat 5100ctgctccagg ccaagcgatt
ccaggcccag accctgggaa ccacctacat ctatgacttc 5160ccggaaatgt tcaggcaggc
tctctttaaa ctgtggggct ccccagacaa gtatcccaaa 5220gacatcctga catacactga
attagtgttg gactctcagg gccagctggt ggagatgaac 5280cgacttcctg gtggaaatga
ggtgggcatg gtggccttca aaatgaggtt taagacccag 5340gagtacccgg aaggacggga
tgtgatcgtc atcggcaatg acatcacctt tcgcattgga 5400tcctttggcc ctggagagga
ccttctgtac ctgcgggcat ccgagatggc ccgggcagag 5460ggcattccca aaatttacgt
ggcagccaac agtggcgccc gtattggcat ggcagaggag 5520atcaaacaca tgttccacgt
ggcttgggtg gacccagaag acccccacaa aggatttaaa 5580tacctgtacc tgactcccca
agactacacc agaatcagct ccctgaactc cgtccactgt 5640aaacacatcg aggaaggagg
agagtccaga tacatgatca cggatatcat cgggaaggat 5700gatggcttgg gcgtggagaa
tctgaggggc tcaggcatga ttgctgggga gtcctctctg 5760gcttacgaag agatcgtcac
cattagcttg gtgacctgcc gagccattgg gattggggcc 5820tacttggtga ggctgggcca
gcgagtgatc caggtggaga attcccacat catcctcaca 5880ggagcaagtg ctctcaacaa
ggtcctggga agagaggtct acacatccaa caaccagctg 5940ggtggcgttc agatcatgca
ttacaatggt gtctcccaca tcaccgtgcc agatgacttt 6000gagggggttt ataccatcct
ggagtggctg tcctatatgc caaaggataa tcacagccct 6060gtccctatca tcacacccac
tgaccccatt gacagagaaa ttgaattcct cccatccaga 6120gctccctacg acccccggtg
gatgcttgca ggaaggcctc acccaactct gaagggaacg 6180tggcagagcg gattctttga
ccacggcagt ttcaaggaaa tcatggcacc ctgggcgcag 6240accgtggtga caggacgagc
aaggcttggg gggattcccg tgggagtgat tgctgtggag 6300acacggactg tggaggtggc
agtccctgca gaccctgcca acctggattc tgaggccaag 6360ataattcagc aggcaggaca
ggtgtggttc ccagactcag cctacaaaac cgcccaggcc 6420atcaaggact tcaaccggga
gaagttgccc ctgatgatct ttgccaactg gagggggttc 6480tccggtggca tgaaagacat
gtatgaccag gtgctgaagt ttggagccta catcgtggac 6540ggccttagac aatacaaaca
gcccatcctg atctatatcc cgccctatgc ggagctccgg 6600ggaggctcct gggtggtcat
agatgccacc atcaacccgc tgtgcataga aatgtatgca 6660gacaaagaga gcaggggtgg
tgttctggaa ccagagggga cagtggagat taagttccga 6720aagaaagatc tgataaagtc
catgagaagg atcgatccag cttacaagaa gctcatggaa 6780cagctagggg aacctgatct
ctccgacaag gaccgaaagg acctggaggg ccggctaaag 6840gctcgcgagg acctgctgct
ccccatctac caccaggtgg cggtgcagtt cgccgacttc 6900catgacacac ccggccggat
gctggagaag ggcgtcatat ctgacatcct ggagtggaag 6960accgcacgca ccttcctgta
ttggcgtctg cgccgcctcc tcctggagga ccaggtcaag 7020caggagatcc tgcaggccag
cggggagctg agtcacgtgc atatccagtc catgctgcgt 7080cgctggttcg tggagacgga
gggggctgtc aaggcctact tgtgggacaa caaccaggtg 7140gttgtgcagt ggctggaaca
gcactggcag gcaggggatg gcccgcgctc caccatccgt 7200gagaacatca cgtacctgaa
gcacgactct gtcctcaaga ccatccgagg cctggttgaa 7260gaaaaccccg aggtggccgt
ggactgtgtg atatacctga gccagcacat cagcccagct 7320gagcgggcgc aggtcgttca
cctgctgtct accatggaca gcccggcctc cacctgaccg 7380tggcccgccc agccactccc
gggaccacgg caaaaggaac cacccagacc caccacccgt 7440acaccctcag cagaccctga
agacttgctt ttaaacaaag aaaatcctgg gcacttctgc 7500agggctgctg gttccgagct
gacacccgtc ttaacaaaag gcccaggagt gcctcttcca 7560aacaaaaaca gcctcctctc
catagctggg aagtttattt tgttttgtct ctgaagacag 7620cagttttatt gcatcactaa
atctaatcaa gctaaaacat ccctgtttcc ttttgcaaaa 7680cagtgcctgg catgtgggat
ccaggcgttc tttaggatcc ttggatacca catcgtgaaa 7740tcttttattt ttttactctg
agaccagcac cagatgtaag taagcatctc atatatttca 7800gccaaataaa tgggccaagg
gaaaaaaata tatatatata gacaggacta gagaaaacct 7860atttttgtaa tgatgtttct
ttggatactg tctagtcacc cagaaaaatg tatggatgaa 7920tttttttttt tttttttgag
acaaagtctc actgtgtcat gcaggctgga gtgcagtggc 7980atgatctcac tgcaacctcc
atctcctgtc tcagcctcct agataactgg gattacaggt 8040gcccaccacc atgcccggct
aatttttgta tttttggtag agacagagtt tcaccaggtt 8100ggtcaggctg gtctcaaact
cttgacttca ggtaatccac ccaccttggc ctcccaaagt 8160gctgggatta caggcatgag
ccaccatctt cagccagatg atttttttat tgagagagtg 8220aaatgctatt ttgttcccca
aatggcgcta gtgaatcact aggagggtcc cactgatagg 8280ccatgtttag cactggttgc
cagggattct ctttttgaga gagggaaagc aaaatgaatg 8340gaagtaccca gctggaggtt
tcagggcttc tggaggatgc tctcgcatag ctcgaggtcc 8400tctgcccacc tcttctctcc
aaggaaaatg aggactgccc cttccccctg caggattggc 8460ccccagcctg cgcatgcacc
ctcctcttgc ccaagtgggg agcacagagg cggagaggaa 8520tcccttacca cacccacggc
ccagcttgct cacgagtgtc acctctgtga cggtcaccac 8580tgctcccttg gagggccact
tgagttactg ttgcttcctc gcctgctggc ttgatgagca 8640ccgatggtgg gatctgaccc
cgaggggcag agctgtcggt gactgaggac tggactgtgg 8700tgaccatgcc gatttgctca
gggagaacgt tgcaatgcac ccagcagctc ctggctctgc 8760aggcggcaca gcctggggcc
ctgtgatcct ctggtttctt ccattggggc ggagtcgggg 8820gtggagggag ctggccacaa
cccactgctc tgatgggtgg tttgtccaag gatgctgaat 8880gtaatgcctg gtcaatgtgg
aagcccatga ggttgcccag ggaagcctcc aaaagctggg 8940atgcttgagg gtatccaagt
tgaaaaagac aaaatctgac catcagccag tgacagtcct 9000ggcaaatgaa ggtggggcgg
ggcagtgagg ggtgggagaa ggtgaatgat tcattattcc 9060accccgaggt ttgctggggt
gaggggaaga atcgatgctg ctttgggaac tgaaggtttt 9120tctgttggga aggccctctt
ggttttggag agaaagacaa gttatgagta gctgctaccc 9180tggaacggtg ggcagagagc
ctactaggaa atgtgcagaa taaactattt tttgaaggaa 9240aaaaaaaaaa a
9251127452DNAHomo sapiens
12atggtcttgc ttctttgtct atcttgtctg attttctcct gtctgacctt ttcctggtta
60aaaatctggg agaaaatgac ggactccaag ccgatcacca agagtaaatc agaagcaaac
120ctcatcccga gccaggagcc ctttccagcc tctgataact caggggagac accgcagaga
180aatggggagg gccacactct gcacaaagac acccagccag gccgagccca gcctcccaca
240aaggcccaaa gatccggtcg gcggagaaac tccctaccac cctcccgcca gaagccccca
300agaaaccccc tttcttccag tgacgcagca ccctccccag agcttcaagc caacgggact
360gggacacaag gtctggaggc cacagatacc aatggcctgt cctcctcagc caggccccag
420ggcagcaagc tggtcccctc caaagaagac aagaagcagg caaacatcaa gaggcagctg
480atgaccaact tcatcctggg ctcttttgat gactactcct ccgacgagga ctctgttgct
540ggctcatctc gtgagtctac ccggaagggc agccgggcca gcttgggggc cctgtccctg
600gaggcttatc tgaccacagg tgaagctgag acccgcgtcc ccactatgag gccgagcatg
660tcgggactcc acctggtgaa gaggggacgg gaacacaaga agctggacct gcacagagac
720tttaccgtgg cttctcccgc tgagtttgtc acacgctttg ggggggatcg ggtcatcgag
780aaggtgctta ttgccaacaa cgggattgcc gctgtgaagt gcatgcgctc catccgcagg
840tgggcctatg agatgttccg caacgagcgg gccatccggt ttgttcgcat ggtgaccccc
900gaggacctta aggccaacgc agagtacatc aagatggcgg atcattacgg gcccgcccca
960ggagggccca ataacaacaa ctatgccaac gtggagctga ttgtggacat tgccaagaga
1020atcccgttgc aggcggtgtg ggctggctgg ggccatgctt tagaaaaccc taaacttccg
1080gagctgctgt gcaagaatgg agttgctttc ttaggccctc ccaggttgag gccaatggtg
1140ggtctaggag ataagatcgc ctccaccgtt gtcgcccaga cgctacaggt cccaaccctg
1200cccaggagtg gaagcgccct gacagtggag tggacagaag atgatctgca gcagggaaaa
1260agaatcagtg tcccagaaga tgtttatgac aagggttgcg tgaaagacgt agatgagggc
1320ttggaggcag cagaaagaat tggttttcca ttgatgatca aagcttctga aggtggcgga
1380gggaagggaa tccgggaaac tgagagtgcg gaggacttcc cgatcctttt cagacaagta
1440cagagtgaga tcccaggctc gcccatcttt ctcatgaagc tggcccagca cgcccgtcac
1500ctggaagttc agatcctcgc tgaccagtat gggaatgctg tgtctctgtt tggtcgcgac
1560tgctccatcc agcggcggca tcagaagatc gttgaggaag caccggccac catcgcgccg
1620ctggccatat tcgagttcat ggagcagtgt gccattcgcc tggccaagac cgtgggctat
1680gtgagtgcag ggacagtgga atacctctat agtcaggatg gtagcttcca cttcttggag
1740ctgaatcctc gcttgcaggt ggaacatccc tgcacagaaa tgattgctga cgttaatctg
1800ccggccgccc agctacagat cgccatgggt gccccactgc accggctgaa agatatccgg
1860cttctgtatg gagagtcacc ctggggagac tccccaattt cttttgaaaa ctcagctcat
1920ctcccctgcc cccgaggcca cgtcattgcc accagaatca ccagcgaaaa cccagacgag
1980ggttttaagc cgagctccgg gactgtccag gaactgaatt tccggagcag caagaacgtc
2040tggggttact tcacggtggc cgctactgga ggcctgcacg agtttgcgat ttcccagttt
2100gggcactgct tctcctgggg agagaaccgg aaagaggcca tttcgaacat ggtggtggct
2160ttgaaggaac tgtccctccg aggcgacttt aggactaccg tggaatacct cattaacctc
2220ctggagaccg agagcttcca gaacaactac atcgacaccg ggtggttgga ctacctcatt
2280gctgagaaag tgcaaaagaa accgaatatc atgcttgggg tggtatgcgg ggcccttgaa
2340cgtggagatg cgatgttcag aacgtgcatg acagatttct tacactccct ggaaaggggc
2400caggtcctcc cagcggattc actactgaac ctcgtagatg tggaattaat ttacgagggt
2460gtaaagtaca ttctaaaggt gacccggcag tctctgacca tgttcgttct catcatgaat
2520ggctgccaca tcgagattga tgcccaccgg ctgaatgatg gggggctcct gctctcctac
2580aatgggaaca gctacaccac ctacatgaag gaagaggttg acagttaccg taccatcggc
2640aataagacgt gtgtttttga gaaggagaac gatcctacag tcctgagatc cccctcggct
2700gggaagctga cacagatcac agtggaggat gggggccacg ttgaggctgg gagacgctac
2760gctgagatgg aggtgatgaa gatgatcatg accctgaacg ttcaggaaag aggccgggtg
2820aagtacatca agcgtccagg tgcggtgctg gaagcaggct gcgtggtggc caggctggag
2880ctcgatgacc cttctaaagt ccacccggct gaaccgttca caggagaact ccctgcccag
2940cagaacactg ccgacctcgg aaagaaactg cacagggtct tccacagcgt cctgggaagc
3000ctcaccaacg tcatgagtgg cttttgtctg ccagagccgt tttttagcat aaagctgaag
3060gagtgggtgc agaagctcat gatgaccctc cggcacccgt cactgctgct ggacgtgcag
3120gagatcatga ccagtcgtgc aggccgcatc cccccccctg ttgagaagtc tgtccgcaag
3180gtgatggccc agtatgccag caacatcacc tcggtgctgt gccagttccc cagccagcag
3240atagccacca tcctggactg ccatgcagcc accctgcagc ggaaggctga tcgagaggtc
3300ttcttcatca acacccagag catggtgcag ttggtccaga ggtaccgaag tggaatccgc
3360ggtcatatga aaacagtggt gatcgatctc ttgagaagat acttgcgtgt tgagaccatt
3420ttcggcaagg caagagatgc tgatgccaac tccagtggga tggtgggggg cgtgaggagc
3480ctgagcttta cctctgtgtg ggtggttttg tctcccccag cccactacga caagtgtgtg
3540ataaacctca gggaacagtt caagccagac atgtcccagg tgctggactg catcttctcc
3600cacgcacagg tgaccaagaa gaaccagctg gtgatcatgt tgatcgatga gctgtgtggc
3660ccagaccctt ccctgtcgga cgagctgatc tccatcctca acgagctcac tcagctgagc
3720aaaagcgagc actgcaaagt ggccctcaga gcccggcaga tcctgatcgc ctccccctcc
3780tacgagctgc ggcataacca ggtggagtcc attttcctgt ctgccattga catgtacggc
3840caccagttct gccccgagaa cctccagaaa ttaatacttt cggaaacaac catcttcgac
3900gtcctgaata ctttcttcta tcacgcaaac aaagtcgtgt gcatggcgtc cttggaggtt
3960tacgtggggg gggcttacat cgcctatgtg ttaaacagcc tgcagcaccg gcagctcccg
4020gacggcacct gcgtggtaga attccagttc atgctgccgt cctcccaccc aaaccggatg
4080accgtgccca tcagcatcac caaccctgac ctgctgaggc acacgacaga gctcttcatg
4140gacagcggct tctccccact gtgccagcgc atgggagcca tggtagcctt caggagattc
4200gaggacttca ccagaaattt tgatgaagtc atctcttgct tcgccaacgt gccgaaagac
4260ccccccctct tcagcgaggc ccgcacctcc ctatactccg aggatgactg caagagcctc
4320agagaagagc ccatccacat tctgaatgtg tccatccagt gtgcggacca cctggaggat
4380gaggcactgg tgccgatttt acgtacattc gtacagtcca agaaaaatat ccttgtggat
4440tatggactcc gacgaatccc attcttgatt gcccaagaga aagaatttcc caagtttttc
4500acattcagag caagagatga gtttgcagaa gatcgcattt accgtcactt ggaacctgcc
4560ctggctttcc agctggaact caaccggatg cgtaacttcg atctgaccgc cgtgccctgt
4620gccaaccaca agatgcacct ttacctgggt gctgccaagg tggaaggaag gtatgaagtg
4680acggaccata ggttcttcat ccgtgccatc atcaggcact ctgacctgat cacaaaggaa
4740gcctccttcg aatacctgca gaacgagggt gagcggctgc tcctggaggc catggacgag
4800ctggaggtgg cgttcaataa caccaacgtg cgcaccgact gcaaccacat cttcctcaac
4860ttcgtgccca ctgtcatcat ggaccccaac aagatcgagg agtccgtgcg ctacatggtt
4920atgcgctacg gcagccggct gtggaaactc cgtgtgctac aggctgaggt caagatcaac
4980atccgccaga ccaccaccgg cagtgccgtt cccatccgcc tgttcatcac caatgagtcg
5040ggctactacc tggacatcag cctctacaaa gaagtgactg actccagatc tggaaatatc
5100atgtttcact ccttcggcaa caagcaaggg ccccagcacg ggatgctgat caatactccc
5160tacgtcacca aggatctgct ccaggccaag cgattccagg cccagaccct gggaaccacc
5220tacatctatg acttcccgga aatgttcagg caggctctct ttaaactgtg gggctcccca
5280gacaagtatc ccaaagacat cctgacatac actgaattag tgttggactc tcagggccag
5340ctggtggaga tgaaccgact tcctggtgga aatgaggtgg gcatggtggc cttcaaaatg
5400aggtttaaga cccaggagta cccggaagga cgggatgtga tcgtcatcgg caatgacatc
5460acctttcgca ttggatcctt tggccctgga gaggaccttc tgtacctgcg ggcatccgag
5520atggcccggg cagaggcgat tcccaaaatt tacgtggcag ccaacagtgg cgcccgtatt
5580ggcatggcag aggagatcaa acacatgttc cacgtggctt gggtggaccc agaagacccc
5640cacaaaggat ttaaatacct gtacctgact ccccaagact acaccagaat cagctccctg
5700aactccgtcc actgtaaaca catcgaggaa ggaggagagt ccagatacat gatcacggat
5760atcatcggga aggatgatgg cttgggcgtg gagaatctga ggggctcagg catgattgct
5820ggggagtcct ctctggctta cgaagagatc gtcaccatta gcttggtgac ctgccgagcc
5880attgggattg gggcctactt ggtgaggctg ggccagcgag tgatccaggt ggagaattcc
5940cacatcatcc tcacaggagc aagtgctctc aacaaggtcc tgggaagaga ggtctacaca
6000tccaacaacc agctgggtgg cgttcagatc atgcattaca atggtgtctc ccacatcacc
6060gtgccagatg actttgaggg ggtttatacc atcctggagt ggctgtccta tatgccaaag
6120gataatcaca gccctgtccc tatcatcaca cccactgacc ccattgacag agaaattgaa
6180ttcctcccat ccagagctcc ctacgacccc cggtggatgc ttgcaggaag gcctcaccca
6240actctgaagg gaacgtggca gagcggattc tttgaccacg gcagtttcaa ggaaatcatg
6300gcaccctggg cgcagaccgt ggtgacagga cgagcaaggc ttggggggat tcccgtggga
6360gtgattgctg tggagacacg gactgtggag gtggcagtcc ctgcagaccc tgccaacctg
6420gattctgagg ccaagataat tcagcaggca ggacaggtgt ggttcccaga ctcagcctac
6480aaaaccgccc aggccatcaa ggacttcaac cgggagaagt tgcccctgat gatctttgcc
6540aactggaggg ggttctccgg tggcatgaaa gacatgtatg accaggtgct gaagtttgga
6600gcctacatcg tggacggcct tagacaatac aaacagccca tcctgatcta tatccgccct
6660atgcgggagc tccggggagg ctcctgggtg gtcatagatg ccaccatcaa cccgctgtgc
6720atagaaatgt atgcagacaa agagagcagg ggtggtgttc tggaaccaga ggggacagtg
6780gagattaagt tccgaaagga agatctgata aagtccatga gaaggatcga tccagcttac
6840aagaagctca tggaacagct aggggaacct gatctctccg acaaggaccg aaaggacctg
6900gagggccggc taaaggctcg cgaggacctg ctgctcccca tctaccacca ggtggcggtg
6960cagttcgccg acttccatga cacacccggc cggatgctgg agaagggcgt catatctgac
7020atcctggagt ggaagaccgc acgcaccttc ctgtattggc gtctgcgccg cctcctcctg
7080gaggaccagg tcaagcagga gatcctgcag gccagcgggg agctgagtca cgtgcatatc
7140cagtccatgc tgcgtcgctg gttcgtggag acggaggggg ctgtcaaggc ctacttgtgg
7200gacaacaacc aggtggttgt gcagtggctg gaacagcact ggcaggcagg ggatggcccg
7260cgctccacca tccgtgagaa catcacgtac ctgaagcacg actctgtcct caagaccatc
7320cgaggcctgg ttgaagaaaa ccccgaggtg gccgtggact gtgtgatata cctgagccag
7380cacatcagcc cagctgagcg ggcgcaggtc gttcacctgc tgtctaccat ggacagcccg
7440gcctccacct ga
7452132359DNAHomo sapiens 13cagttcatgc tgccgtcctc ccacccaaac cggatgaccg
tgcccatcaa catcaccaac 60cctgacctgc tgaggcacag cacagagctc ttcatggaca
gcggcttctc cccactgtgc 120cagcgcatgg gagccatggt agccttcagg agattcgagg
acttcaccag aaattttggt 180gaagtcatct cttgcttcgc caacgtgccc aaagacaccc
ccctcttcag cgaggcccgc 240acctccctat actccgagga tgactgcaag agcctcagag
aagagcccat ccacattctg 300aatatgtcca tccagtgtgc agaccacctg gaggatgagg
cactggtgcc gattttacgg 360acattcgtac agtccaagaa aaatatcctt gtggattatg
gactccgacg aatcacattc 420ttgattgccc aagagaaaga atttcccaag tttttcacat
tcagagcaag agatgagttt 480gcagaagatc gcatttaccg tcacttggaa cctgccctgg
ccttccagct ggaactcaac 540cggatgcgta acttcgatct gaccgccgtg ccctgtgcca
accacaagat gcacctttac 600ctgggtgctg ccaaggtgaa ggaaggtgtg gaagtgacgg
accataggtt cttcatccgc 660gccatcatca ggcactctga cctgatcaca aaggaagcct
ccttcgaata cctgcagaac 720gagggtgagc ggctgctcct ggaggccatg gacgagctgg
aggtggcgtt caataacacc 780agcgtgcgca ccgactgcaa ccacatcttc ctcaacttcg
tgcccactgt catcatggac 840cccttcaaga tcgaggagtc cgtgcgctac atggttatgc
gctacggcag ccggctgtgg 900aaactccgtg tgctacaggc tgaggtcaag atcaacatcc
gccagaccac caccggcagt 960gccgttccca tccgcctgtt catcaccaat gagtcgggct
actacctgga catcagcctc 1020tacaaagaag tgactgactc cagatctgga aatatcatgt
ttcactcctt cggcaacaag 1080caagggcccc agcacgggat gctgatcaat actccctacg
tcaccaagga tctgctccag 1140gccaagcgat tccaggccca gaccctggga accacctaca
tctatgactt cccggaaatg 1200ttcaggcagg ctctctttaa actgtggggc tccccagaca
agtatcccaa agacatcctg 1260acatacactg aattagtgtt ggactctcag ggccagctgg
tggagatgaa ccgacttcct 1320ggtggaaatg aggtgggcat ggtggccttc aaaatgaggt
ttaagaccca ggagtacccg 1380gaaggacggg atgtgatcgt catcggcaat gacatcacct
ttcgcattgg atcctttggg 1440cctggagagg accttctcta cctccgggca tccgagatgg
cgcggccaga ggcgattccc 1500aaaatttacg tggcagccaa cagtggcgcc cgtattggca
tggcagagga gatcaaacac 1560atgttccacg tggcttgggt ggacccagaa gacccccaca
aaggatttaa atacctgtac 1620ctgactcccc aagactacac cagaatcagc tccctgaact
ccgtccactg taaacacatc 1680gaggaaggag gagagtccag atacatcatg accgatatca
tcgggaagga tgatggcttg 1740ggcgtggaga atctgagggg ctcaggcatg attgctgggg
agtcctctct cgcttacgaa 1800gagatcgtca ccattagctt ggtgacctgc cgagccattg
ggattggggc ctacttggtg 1860aggctgggcc agcgagtgat ccaggtggag aattcccaca
tcatcctcac aggagcaagt 1920gctctcaaca aggtcctggg aagagaggtc tacacatcca
acaaccagct cggtggcgtt 1980cagatcatgc attacaatgg tgtctcccac atcaccgtgc
cagatgactt tgagggggtt 2040tataccatcc tggagtggct gtcctatatg ccaaaggata
atcacagccc tgtccctatc 2100atcacaccca ctgaccccat tgacagagaa attgaattcc
tcccatccag agctccctac 2160gacccccggt ggatgcttgc aggaaggcct cacccaactc
tgaagggaac ctggcagagc 2220ggattctttg accacggcag tttcaaggaa atcatggcac
cctgggcgca gaccgtggtg 2280acaggacgag caaggcttgg ggggattccc gtgggagtga
ttgctgtgga gacacggact 2340gtggaggtgg cagtccctg
2359147496DNAHomo sapiens 14cagctagcag gcttagattc
aggccctcag caaacaagga acctggaaaa tgtaaccctg 60aatgcacggt ggggaggaca
tggcaagaga aaagcggcag gaataaagtg attttctgaa 120tggtcttgct tctttgtcta
tcttgtctga ttttctcctg tctgaccttt tcctggttaa 180aaatctgggg gaaaatgacg
gactccaagc cgatcaccaa gagtaaatca gaagcaaacc 240tcatcccgag ccaggagccc
tttccagcct ctgataactc aggggagaca ccgcagagaa 300atggggaggg ccacactctg
cccaagacac ccagccaggc cgagccagcc tcccacaaag 360gccccaaaga tgccggtcgg
cggagaaact ccctaccacc ctcccaccag aagcccccaa 420gaaaccccct ttcttccagt
gacgcagcac cctccccaga gcttcaagcc aacgggattg 480ggacacaagg tctggaggcc
acagatacca atggcctgtc ctcctcagcc aggccccagg 540gccagcaagc tggctccccc
tccaaagaag acaagaagca ggcaaacatc aagaggcagc 600tgatgaccaa cttcatcctg
ggctcttttg atgactactc ctctgacgag gactctgttg 660ctggctcatc tcgtgagtct
acccggaagg gcagccgggc cagcttgggg gccctgtccc 720tggaggctta tctgaccaca
ggtgaagctg agacccgcgt ccccactatg aggccgagca 780tgtcgggact ccacctggtg
aagaggggac gggaacacaa gaagctggac ctgcacagag 840actttaccgt ggcttctccc
gctgagtttg tcacacgctt tgggggggat cgggtcatcg 900agaaggtgct tattgccaac
aacgggattg ccgccgtgaa gtgcatgcgc tccatccgca 960ggtgggccta tgagatgttc
cgcaacgagc gggccatccg gtttgttgtg atggtgaccc 1020ccgaggacct taaggccaac
gcagagtaca tcaagatggc ggatcattac gtccccgtcc 1080caggagggcc caataacaac
aactatgcca acgtggagct gattgtggac attgccaaga 1140gaatccccgt gcaggcggtg
tgggctggct ggggccatgc ttcagaaaac cctaaacttc 1200cggagctgct gtgcaagaat
ggagttgctt tcttaggccc tcccagtgag gccatgtggg 1260ccttaggaga taagatcgcc
tccaccgttg tcgcccagac gctacaggtc ccaaccctgc 1320cctggagtgg aagcggcctg
acagtggagt ggacagaaga tgatctgcag cagggaaaaa 1380gaaccagtgt cccagaagat
gtttatgaca agggttgcgt gaaagacgta gatgagggct 1440tggaggcagc agaaagaatt
ggttttccat tgatgatcaa agcttctgaa ggtggcggag 1500ggaagggaat ccggaaggct
gagagtgcgg aggacttccc gatccttttc agacaagtac 1560agagtgagat cccaggctcg
cccatctttc tcatgaagct ggcccagcac gcccgtcacc 1620tggaagttca gatcctcgct
gaccagtatg ggaatgctgt gtctctgttt ggtcgcgact 1680gctccatcca gcggcggcat
cagaagatcg ttgaggaagc accggccacc atcgccccgc 1740tggccatatt cgagttcatg
gagcagtgtg ccatccgcct ggccaagacc gtgggctatg 1800tgagtgcagg gacagtggaa
tacctctata gtcaggatgg cagcttccac ttcttggagc 1860tgaatcctcg cttgcaggtg
gaacatccct gcacagaaat gattgctgac gttaatctgc 1920cggccgccca gctacagatc
gccatgggcg tgccactgca ccggctgaag gatatccggc 1980ttctgtatgg agagtcacca
tggggagtga ctcccatttc ttttgaaacc ccctcaaacc 2040ctcccctcgc ccgaggccac
gtcattgccg ccagaatcac cagcgaaaac ccagacgagg 2100gttttaagcc gagctccggg
actgtccagg aactgaattt ccggagcagc aagaacgtgt 2160ggggttactt cagcgtggcc
gctactggag gcctgcacga gtttgcggat tcccaatttg 2220ggcactgctt ctcctgggga
gagaaccggg aagaggccat ttcgaacatg gtggtggctt 2280tgaaggaact gtccatccga
ggtgacttta ggactaccgt ggaatacctc attaacctcc 2340tggagaccga gagcttccag
aacaacgaca tcgacaccgg gtggttggac tacctcattg 2400ctgagaaagt gcaggcggag
aaaccggata tcatgcttgg ggtggtatgc ggggccttga 2460acgtggccga tgcgatgttc
agaacgtgca tgacagattt cttacactcc ctggaaaggg 2520gccaggtcct cccagcggat
tcactactga acctcgtaga tgtggaatta atttacggag 2580gtgttaagta cattctcaag
gtggcccggc agtctctgac catgttcgtt ctcatcatga 2640atggctgcca catcgagatt
gatgcccacc ggctgaatga tggggggctc ctgctctcct 2700acaatgggaa cagctacacc
acctacatga aggaagaggt tgacagttac cgaattacca 2760tcggcaataa gacgtgtgtg
tttgagaagg agaacgatcc tacagtcctg agatccccct 2820cggctgggaa gctgacacag
tacacagtgg aggatggggg ccacgttgag gctgggagca 2880gctacgctga gatggaggtg
atgaagatga tcatgaccct gaacgttcag gaaagaggcc 2940gggtgaagta catcaagcgt
ccaggtgccg tgctggaagc aggctgcgtg gtggccaggc 3000tggagctcga tgacccttct
aaagtccacc cggctgaacc gttcacagga gaactccctg 3060cccagcagac actgcccatc
ctcggagaga aactgcacca ggtcttccac agcgtcctgg 3120aaaacctcac caacgtcatg
agtggctttt gtctgccaga gcccgttttt agcataaagc 3180tgaaggagtg ggtgcagaag
ctcatgatga ccctccggca cccgtcactg ccgctgctgg 3240agctgcagga gatcatgacc
agcgtggcag gccgcatccc cgcccctgtg gagaagtctg 3300tccgcagggt gatggcccag
tatgccagca acatcacctc ggtgctgtgc cagttcccca 3360gccagcagat agccaccatc
ctggactgcc atgcagccac cctgcagcgg aaggctgatc 3420gagaggtctt cttcatcaac
acccagagca tcgtgcagtt ggtccagaga taccgcagcg 3480ggatccgcgg ctatatgaaa
acagtggtgt tggatctcct gagaagatac ttgcgtgttg 3540agcaccattt tcagcaagcc
cactacgaca agtgtgtgat aaacctcagg gagcagttca 3600agccagacat gtcccaggtg
ctggactgca tcttctccca cgcacaggtg gccaagaaga 3660accagctggt gatcatgttg
atcgatgagc tgtgtggccc agacccttcc ctgtcggacg 3720agctgatctc catcctcaac
gagctcactc agctgagcaa aagcgagcac tgcaaagtgg 3780ccctcagagc ccggcagatc
ctgattgcct cccacctccc ctcctacgag ctgcggcata 3840accaggtgga gtccattttc
ctgtctgcca ttgacatgta cggccaccag ttctgccccg 3900agaacctcaa gaaattaata
ctttcggaaa caaccatctt cgacgtcctg cctactttct 3960tctatcacgc aaacaaagtc
gtgtgcatgg cgtccttgga ggtttacgtg cggaggggct 4020acatcgccta tgagttaaac
agcctgcagc accggcagct cccggacggc acctgcgtgg 4080tagaattcca gttcatgctg
ccgtcctccc acccaaaccg gatgaccgtg cccatcagca 4140tcaccaaccc tgacctgctg
aggcacagca cagagctctt catggacagc ggcttctccc 4200cactgtgcca gcgcatggga
gccatggtag ccttcaggag attcgaggac ttcaccagaa 4260attttgatga agtcatctct
tgcttcgcca acgtgcccaa agacaccccc ctcttcagcg 4320aggcccgcac ctccctatac
tccgaggatg actgcaagag cctcagagaa gagcccatcc 4380acattctgaa tgtgtccatc
cagtgtgcag accacctgga ggatgaggca ctggtgccga 4440ttttacggac attcgtacag
tccaagaaaa atatccttgt ggattatgga ctccgacgaa 4500tcacattctt gattgcccaa
gagaaagaat ttcccaagtt tttcacattc agagcaagag 4560atgagtttgc agaagatcgc
atttaccgtc acttggaacc tgccctggcc ttccagctgg 4620aactcaaccg gatgcgtaac
ttcgatctga ccgccgtgcc ctgtgccaac cacaagatgc 4680acctttacct gggtgctgcc
aaggtgaagg aaggtgtgga agtgacggac cataggttct 4740tcatccgcgc catcatcagg
cactctgacc tgatcacaaa ggaagcctcc ttcgaatacc 4800tgcagaacga gggtgagcgg
ctgctcctgg aggccatgga cgagctggag gtggcgttca 4860ataacaccag cgtgcgcacc
gactgcaacc acatcttcct caacttcgtg cccactgtca 4920tcatggaccc cttcaagatc
gaggagtccg tgcgctacat ggttatgcgc tacggcagcc 4980ggctgtggaa actccgtgtg
ctacaggctg aggtcaagat caacatccgc cagaccacca 5040ccggcagtgc cgttcccatc
cgcctgttca tcaccaatga gtcgggctac tacctggaca 5100tcagcctcta caaagaagtg
actgactcca gatctggaaa tatcatgttt cactccttcg 5160gcaacaagca agggccccag
cacgggatgc tgatcaatac tccctacgtc accaaggatc 5220tgctccaggc caagcgattc
caggcccaga ccctgggaac cacctacatc tatgacttcc 5280cggaaatgtt caggcaggct
ctctttaaac tgtggggctc cccagacaag tatcccaaag 5340acatcctgac atacactgaa
ttagtgttgg actctcaggg ccagctggtg gagatgaacc 5400gacttcctgg tggaaatgag
gtgggcatgg tggccttcaa aatgaggttt aagacccagg 5460agtacccgga aggacgggat
gtgatcgtca tcggcaatga catcaccttt cgcattggat 5520cctttggccc tggagaggac
cttctgtacc tgcgggcatc cgagatggcc cgggcagagg 5580gcattcccaa aatttacgtg
gcagccaaca gtggcgcccg tattggcatg gcagaggaga 5640tcaaacacat gttccacgtg
gcttgggtgg acccagaaga cccccacaaa ggatttaaat 5700acctgtacct gactccccaa
gactacacca gaatcagctc cctgaactcc gtccactgta 5760aacacatcga ggaaggagga
gagtccagat acatgatcac ggatatcatc gggaaggatg 5820atggcttggg cgtggagaat
ctgaggggct caggcatgat tgctggggag tcctctctgg 5880cttacgaaga gatcgtcacc
attagcttgg tgacctgtcg agccattggg attggggcct 5940acttggtgag gctgggccag
cgagtgatcc aggtggagaa ttcccacatc atcctcacag 6000gagcaagtgc tctcaacaag
gtcctgggaa gagaggtcta cacatccaac aaccagctgg 6060gtggcgttca gatcatgcat
tacaatggtg tctcccacat caccgtgcca gatgactttg 6120agggggttta taccatcctg
gagtggctgt cctatatgcc aaaggataat cacagccctg 6180tccctatcat cacacccact
gaccccattg acagagaaat tgaattcctc ccatccagag 6240ctccctacga cccccggtgg
atgcttgcag gaaggcctca cccaactctg aagggaacgt 6300ggcagagcgg attctttgac
cacggcagtt tcaaggaaat catggcaccc tgggcgcaga 6360ccgtggtgac aggacgagca
aggcttgggg ggattcccgt gggagtgatt gctgtggaga 6420cacggactgt ggaggtggca
gtccctgcag accctgccaa cctggattct gaggccaaga 6480taattcagca ggcaggacag
gtgtggttcc cagactcagc ctacaaaacc gcccaggccg 6540tcaaggactt caaccgggag
aagttgcccc tgatgatctt tgccaactgg agggggttct 6600ccggtggcat gaaagacatg
tatgaccagg tgctgaagtt tggagcctac atcgtggacg 6660gccttagaca atacaaacag
cccatcctga tctatatccc gccctatgcg gagctccggg 6720gaggctcctg ggtggtcata
gatgccacca tcaacccgct gtgcatagaa atgtatgcag 6780acaaagagag caggggtggt
gttctggaac cagaggggac agtggagatt aagttccgaa 6840agaaagatct gataaagtcc
atgagaagga tcgatccagc ttacaagaag ctcatggaac 6900agctagggga acctgatctc
tccgacaagg accgaaagga cctggagggc cggctaaagg 6960ctcgcgagga cctgctgctc
cccatctacc accaggtggc ggtgcagttc gccgacttcc 7020atgacacacc cggccggatg
ctggagaagg gcgtcatatc tgacatcctg gagtggaaga 7080ccgcacgcac cttcctgtat
tggcgtctgc gccgcctcct cctggaggac caggtcaagc 7140aggagatcct gcaggccagc
ggggagctga gtcacgtgca tatccagtcc atgctgcgtc 7200gctggttcgt ggagacggag
ggggctgtca aggcctactt gtgggacaac aaccaggtgg 7260ttgtgcagtg gctggaacag
cactggcagg caggggatgg cccgcgctcc accatccgtg 7320agaacatcac gtacctgaag
cacgactctg tcctcaagac catccgaggc ctggttgaag 7380aaaaccccga ggtggccgtg
gactgtgtga tatacctgag ccagcacatc agcccagctg 7440agcgggcgca ggtcgttcac
ctgctgtcta ccatggacag cccggcctcc acctga 7496155626DNAHomo sapiens
15gactagtatg ggaatgctgt gtctctgttt ggtcgcgact gctccatcca gcggcggcat
60cagaagatcg ttgaggaagc accggccacc atcgccccgc tggccatatt cgagttcatg
120gagcagtgtg ccatccgcct ggccaagacc gtgggctatg tgagtgcagg gacagtggaa
180tacctctata gtcaggatgg cagcttccac ttcttggagc tgaatcctcg cttgcaggtg
240gaacatccct gcacagaaat gattgctgac gttaatctgc cggccgccca gctacagatc
300gccatgggcg tgccactgca ccggctgaag gatatccggc ttctgtatgg agagtcacca
360tggggagtga ctcccatttc ttttgaaacc ccctcaaacc ctcccctcgc ccgatgcgat
420gttcagaacg tgcatgacag atttcttaca ctccctggaa aggggccagg tcctcccagc
480ggattcacta ctgaacctcg tagatgtgga attaatttac ggaggtgtta agtacattct
540caaggtggcc cggcagtctc tgaccatgtt cgttctcatc atgaatggct gccacatcga
600gattgatgcc caccggctga atgatggggg gctcctgctc tcctacaatg ggaacagcta
660caccacctac atgaaggaag aggttgacag ttaccgaatt accatcggca ataagacgtg
720tgtgtttgag aaggagaacg atcctacagt cctgagatcc ccctcggctg ggaagctgac
780acagtacaca gtggaggatg ggggccacgt tgaggctggg agcagctacg ctgagatgga
840ggtgatgaag atgatcatga ccctgaacgt tcaggaaaga ggccgggtga agtacatcaa
900gcgtccaggt gccgtgctgg aagcaggctg cgtggtggcc aggctggagc tcgatgaccc
960ttctaaagtc cacccggctg aaccgttcac aggagaactc cctgcccagc agacactgcc
1020catcctcgga gagaaactgc accaggtctt ccacagcgtc ctggaaaacc tcaccaacgt
1080catgagtggc ttttgtctgc cagagcccgt ttttagcata aagctgaagg agtgggtgca
1140gaagctcatg atgaccctcc ggcacccgtc actgccgctg ctggagctgc aggagatcat
1200gaccagcgtg gcaggccgca tccccgcccc tgtggagaag tctgtccgca gggtgatggc
1260ccagtatgcc agcaacatca cctcggtgct gtgccagttc cccagccagc agatagccac
1320catcctggac tgccatgcag ccaccctgca gcggaaggct gatcgagagg tcttcttcat
1380caacacccag agcatcgtgc agttggtcca gagataccgc agcgggatcc gcggctatat
1440gaaaacagtg gtgttggatc tcctgagaag atacttgcgt gttgagcacc attttcagca
1500agcccactac gacaagtgtg tgataaacct cagggagcag ttcaagccag acatgtccca
1560ggtgctggac tgcatcttct cccacgcaca ggtggccaag aagaaccagc tggtgatcat
1620gttgatcgat gagctgtgtg gcccagaccc ttccctgtcg gacgagctga tctccatcct
1680caacgagctc actcagctga gcaaaagcga gcactgcaaa gtggccctca gagcccggca
1740gatcctgatt gcctcccacc tcccctccta cgagctgcgg cataaccagg tggagtccat
1800tttcctgtct gccattgaca tgtacggcca ccagttctgc cccgagaacc tcaagaaatt
1860aatactttcg gaaacaacca tcttcgacgt cctgcctact ttcttctatc acgcaaacaa
1920agtcgtgtgc atggcgtcct tggaggttta cgtgcggagg ggctacatcg cctatgagtt
1980aaacagcctg cagcaccggc agctcccgga cagcacctgc gtggtagaat tccagttcat
2040gctgccgtcc tcccacccaa accggatgac cgtgcccatc agcatcacca accctgacct
2100gctgaggcac agcacagagc tcttcatgga cagcggcttc tccccactgt gccagcgcat
2160gggagccatg gtagccttca ggagattcga ggacttcacc agaaattttg atgaagtcat
2220ctcttgcttc gccaacgtgc ccaaagacac ccccctcttc agcgaggccc gcacctccct
2280atactccgag gatgactgca agagcctcag agaagagccc atccacattc tgaatgtgtc
2340catccagtgt gcagaccacc tggaggatga ggcactggtg ccgattttac ggacattcgt
2400acagtccaag aaaaatatcc ttgtggatta tggactccga cgaatcacat tcttgattgc
2460ccaagagaaa gaatttccca agtttttcac attcagagca agagatgagt ttgcagaaga
2520tcgcatttac cgtcacttgg aacctgccct ggccttccag ctggaactca accggatgcg
2580taacttcgat ctgaccgccg tgccctgtgc caaccacaag atgcaccttt acctgggtgc
2640tgccaaggtg aaggaaggtg tggaagtgac ggaccatagg ttcttcatcc gcgccatcat
2700caggcactct gacctgatca caaaggaagc ctccttcgaa tacctgcaga acgagggtga
2760gcggctgctc ctggaggcca tggacgagct ggaggtggcg ttcaataaca ccagcgtgcg
2820caccgactgc aaccacatct tcctcaactt cgtgcccact gtcatcatgg accccttcaa
2880gatcgaggag tccgtgcgct acatggttat gcgctacggc agccggctgt ggaaactccg
2940tgtgctacag gctgaggtca agatcaacat ccgccagacc accaccggca gtgccgttcc
3000catccgcctg ttcatcacca atgagtcggg ctactacctg gacatcagcc tctacaaaga
3060agtgactgac tccagatctg gaaatatcat gtttcactcc ttcggcaaca agcaagggcc
3120ccagcacggg atgctgatca atactcccta cgtcaccaag gatctgctcc aggccaagcg
3180attccaggcc cagaccctgg gaaccaccta catctatgac ttcccggaaa tgttcaggca
3240ggctctcttt aaactgtggg gctccccaga caagtatccc aaagacatcc tgacatacac
3300tgaattagtg ttggactctc agggccagct ggtggagatg aaccgacttc ctggtggaaa
3360tgaggtgggc atggtggcct tcaaaatgag gtttaagacc caggagtacc cggaaggacg
3420ggatgtgatc gtcatcggca atgacatcac ctttcgcatt ggatcctttg gccctggaga
3480ggaccttctg tacctgcggg catccgagat ggcccgggca gagggcattc ccaaaattta
3540cgtggcagcc aacagtggcg cccgtattgg catggcagag gagatcaaac acatgttcca
3600cgtggcttgg gtggacccag aagaccccca caaaggattt aaatacctgt acctgactcc
3660ccaagactac accagaatca gctccctgaa ctccgtccac tgtaaacaca tcgaggaagg
3720aggagagtcc agatacatga tcacggatat catcgggaag gatgatggct tgggcgtgga
3780gaatctgagg ggctcaggca tgattgctgg ggagtcctct ctggcttacg aagagatcgt
3840caccattagc ttggtgacct gccgagccat tgggattggg gcctacttgg tgaggctggg
3900ccagcgagtg atccaggtgg agaattccca catcatcctc acaggagcaa gtgctctcaa
3960caaggtcctg ggaagagagg tctacacatc caacaaccag ctgggtggcg ttcagatcat
4020gcattacaat ggtgtctccc acatcaccgt gccagatgac tttgaggggg tttataccat
4080cctggagtgg ctgtcctata tgccaaagga taatcacagc cctgtcccta tcatcacacc
4140cactgacccc attgacagag aaattgaatt cctcccatcc agagctccct acgacccccg
4200gtggatgctt gcaggaaggc ctcacccaac tctgaaggga acgtggcaga gcggattctt
4260tgaccacggc agtttcaagg aaatcatggc accctgggcg cagaccgtgg tgacaggacg
4320agcaaggctt ggggggattc ccgtgggagt gattgctgtg gagacacgga ctgtggaggt
4380ggcagtccct gcagaccctg ccaacctgga ttctgaggcc aagataattc agcaggcagg
4440acaggtgtgg ttcccagact cagcctacaa aaccgcccag gccatcaagg acttcaaccg
4500ggagaagttg cccctgatga tctttgccaa ctggaggggg ttctccggtg gcatgaaaga
4560catgtatgac caggtgctga agtttggagc ctacatcgtg gacggcctta gacaatacaa
4620acagcccatc ctgatctata tcccgcccta tgcggagctc cggggaggct cctgggtggt
4680catagatgcc accatcaacc cgctgtgcat agaaatgtat gcagacaaag agagcagggg
4740tggtgttctg gaaccagagg ggacagtgga gattaagttc cgaaagaaag atctgataaa
4800gtccatgaga aggatcgatc cagcttacaa gaagctcatg gaacagctag gggaacctga
4860tctctccgac aaggaccgaa aggacctgga gggccggcta aaggctcgcg aggacctgct
4920gctccccatc taccaccagg tggcggtgca gttcgccgac ttccatgaca cacccggccg
4980gatgctggag aagggcgtca tatctgacat cctggagtgg aagaccgcac gcaccttcct
5040gtattggcgt ctgcgccgcc tcctcctgga ggaccaggtc aagcaggaga tcctgcaggc
5100cagcggggag ctgagtcacg tgcatatcca gtccatgctg cgtcgctggt tcgtggagac
5160ggagggggct gtcaaggcct acttgtggga caacaaccag gtggttgtgc agtggctgga
5220acagcactgg caggcagggg atggcccgcg ctccaccatc cgtgagaaca tcacgtacct
5280gaagcacgac tctgtcctca agaccatccg aggcctggtt gaagaaaacc ccgaggtggc
5340cgtggactgt gtgatatacc tgagccagca catcagccca gctgagcggg cgcaggtcgt
5400tcacctgctg tctaccatgg acagcccggc ctccacctga ccgtggccca cccagccact
5460cccgggacca cggcaaaagg aaccacccag acccaccacc cgtacaccct cagcagaccc
5520tgaagacttg cttttaaaca aagaaaatcc tgggcacttc tgcagggctg ctggttccga
5580gctgacaccc gtcttaataa aaggcccagg agtgcctctt ccaaac
5626162268PRTHomo sapiens 16Met Ser Gly Leu His Leu Val Lys Gln Gly Arg
Asp Arg Lys Lys Ile1 5 10
15Asp Ser Gln Arg Asp Phe Thr Val Ala Ser Pro Ala Glu Phe Val Thr
20 25 30Arg Phe Gly Gly Asn Lys Val
Ile Glu Lys Val Leu Ile Ala Asn Asn 35 40
45Gly Ile Ala Ala Val Lys Cys Met Arg Ser Ile Arg Arg Trp Ser
Tyr 50 55 60Glu Met Phe Arg Asn Glu
Arg Ala Ile Arg Phe Val Val Met Val Thr65 70
75 80Pro Glu Asp Leu Lys Ala Asn Ala Glu Tyr Ile
Lys Met Ala Asp His 85 90
95Tyr Val Pro Val Pro Gly Gly Pro Asn Asn Asn Asn Tyr Ala Asn Val
100 105 110Glu Leu Ile Leu Asp Ile
Ala Lys Arg Ile Pro Val Gln Ala Val Trp 115 120
125Ala Gly Trp Gly His Ala Ser Glu Asn Pro Lys Leu Pro Glu
Leu Leu 130 135 140Leu Lys Asn Gly Ile
Ala Phe Met Gly Pro Pro Ser Gln Ala Met Trp145 150
155 160Ala Leu Gly Asp Lys Ile Ala Ser Ser Ile
Val Ala Gln Thr Ala Gly 165 170
175Ile Pro Thr Leu Pro Trp Ser Gly Ser Gly Leu Arg Val Asp Trp Gln
180 185 190Glu Asn Asp Phe Ser
Lys Arg Ile Leu Asn Val Pro Gln Glu Leu Tyr 195
200 205Glu Lys Gly Tyr Val Lys Asp Val Asp Asp Gly Leu
Gln Ala Ala Glu 210 215 220Glu Val Gly
Tyr Pro Val Met Ile Lys Ala Ser Glu Gly Gly Gly Gly225
230 235 240Lys Gly Ile Arg Lys Val Asn
Asn Ala Asp Asp Phe Pro Asn Leu Phe 245
250 255Arg Gln Val Gln Ala Glu Val Pro Gly Ser Pro Ile
Phe Val Met Arg 260 265 270Leu
Ala Lys Gln Ser Arg His Leu Glu Val Gln Ile Leu Ala Asp Gln 275
280 285Tyr Gly Asn Ala Ile Ser Leu Phe Gly
Arg Asp Cys Ser Val Gln Arg 290 295
300Arg His Gln Lys Ile Ile Glu Glu Ala Pro Ala Thr Ile Ala Thr Pro305
310 315 320Ala Val Phe Glu
His Met Glu Gln Cys Ala Val Lys Leu Ala Lys Met 325
330 335Val Gly Tyr Val Ser Ala Gly Thr Val Glu
Tyr Leu Tyr Ser Gln Asp 340 345
350Gly Ser Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu Gln Val Glu His
355 360 365Pro Cys Thr Glu Met Val Ala
Asp Val Asn Leu Pro Ala Ala Gln Leu 370 375
380Gln Ile Ala Met Gly Ile Pro Leu Tyr Arg Ile Lys Asp Ile Arg
Met385 390 395 400Met Tyr
Gly Val Ser Pro Trp Gly Asp Ser Pro Ile Asp Phe Glu Asp
405 410 415Ser Ala His Val Pro Cys Pro
Arg Gly His Val Ile Ala Ala Arg Ile 420 425
430Thr Ser Glu Asn Pro Asp Glu Gly Phe Lys Pro Ser Ser Gly
Thr Val 435 440 445Gln Glu Leu Asn
Phe Arg Ser Asn Lys Asn Val Trp Gly Tyr Phe Ser 450
455 460Val Ala Ala Ala Gly Gly Leu His Glu Phe Ala Asp
Ser Gln Phe Gly465 470 475
480His Cys Phe Ser Trp Gly Glu Asn Arg Glu Glu Ala Ile Ser Asn Met
485 490 495Val Val Ala Leu Lys
Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr 500
505 510Val Glu Tyr Leu Ile Lys Leu Leu Glu Thr Glu Ser
Phe Gln Met Asn 515 520 525Arg Ile
Asp Thr Gly Trp Leu Asp Arg Leu Ile Ala Glu Lys Val Gln 530
535 540Ala Glu Arg Pro Asp Thr Met Leu Gly Val Val
Cys Gly Ala Leu His545 550 555
560Val Ala Asp Val Ser Leu Arg Asn Ser Val Ser Asn Phe Leu His Ser
565 570 575Leu Glu Arg Gly
Gln Val Leu Pro Ala His Thr Leu Leu Asn Thr Val 580
585 590Asp Val Glu Leu Ile Tyr Glu Gly Val Lys Tyr
Val Leu Lys Val Thr 595 600 605Arg
Gln Ser Pro Asn Ser Tyr Val Val Ile Met Asn Gly Ser Cys Val 610
615 620Glu Val Asp Val His Arg Leu Ser Asp Gly
Gly Leu Leu Leu Ser Tyr625 630 635
640Asp Gly Ser Ser Tyr Thr Thr Tyr Met Lys Glu Glu Val Asp Arg
Tyr 645 650 655Arg Ile Thr
Ile Gly Asn Lys Thr Cys Val Phe Glu Lys Glu Asn Asp 660
665 670Pro Ser Val Met Arg Ser Pro Ser Ala Gly
Lys Leu Ile Gln Tyr Ile 675 680
685Val Glu Asp Gly Gly His Val Phe Ala Gly Gln Cys Tyr Ala Glu Ile 690
695 700Glu Val Met Lys Met Val Met Thr
Leu Thr Ala Val Glu Ser Gly Cys705 710
715 720Ile His Tyr Val Lys Arg Pro Gly Ala Ala Leu Asp
Pro Gly Cys Val 725 730
735Leu Ala Lys Met Gln Leu Asp Asn Pro Ser Lys Val Gln Gln Ala Glu
740 745 750Leu His Thr Gly Ser Leu
Pro Arg Ile Gln Ser Thr Ala Leu Arg Gly 755 760
765Glu Lys Leu His Arg Val Phe His Tyr Val Leu Asp Asn Leu
Val Asn 770 775 780Val Met Asn Gly Tyr
Cys Leu Pro Asp Pro Phe Phe Ser Ser Lys Val785 790
795 800Lys Asp Trp Val Glu Arg Leu Met Lys Thr
Leu Arg Asp Pro Ser Leu 805 810
815Pro Leu Leu Glu Leu Gln Asp Ile Met Thr Ser Val Ser Gly Arg Ile
820 825 830Pro Pro Asn Val Glu
Lys Ser Ile Lys Lys Glu Met Ala Gln Tyr Ala 835
840 845Ser Asn Ile Thr Ser Val Leu Cys Gln Phe Pro Ser
Gln Gln Ile Ala 850 855 860Asn Ile Leu
Asp Ser His Ala Ala Thr Leu Asn Arg Lys Ser Glu Arg865
870 875 880Glu Val Phe Phe Met Asn Thr
Gln Ser Ile Val Gln Leu Val Gln Arg 885
890 895Tyr Arg Ser Gly Ile Arg Gly His Met Lys Ala Val
Val Met Asp Leu 900 905 910Leu
Arg Gln Tyr Leu Arg Val Glu Thr Gln Phe Gln Asn Gly His Tyr 915
920 925Asp Lys Cys Val Phe Ala Leu Arg Glu
Glu Asn Lys Ser Asp Met Asn 930 935
940Thr Val Leu Asn Tyr Ile Phe Ser His Ala Gln Val Thr Lys Lys Asn945
950 955 960Leu Leu Val Thr
Met Leu Ile Asp Gln Leu Cys Gly Arg Asp Pro Thr 965
970 975Leu Thr Asp Glu Leu Leu Asn Ile Leu Thr
Glu Leu Thr Gln Leu Ser 980 985
990Lys Thr Thr Asn Ala Lys Val Ala Leu Arg Ala Arg Gln Val Leu Ile
995 1000 1005Ala Ser His Leu Pro Ser
Tyr Glu Leu Arg His Asn Gln Val Glu 1010 1015
1020Ser Ile Phe Leu Ser Ala Ile Asp Met Tyr Gly His Gln Phe
Cys 1025 1030 1035Ile Glu Asn Leu Gln
Lys Leu Ile Leu Ser Glu Thr Ser Ile Phe 1040 1045
1050Asp Val Leu Pro Asn Phe Phe Tyr His Ser Asn Gln Val
Val Arg 1055 1060 1065Met Ala Ala Leu
Glu Val Tyr Val Arg Arg Ala Tyr Ile Ala Tyr 1070
1075 1080Glu Leu Asn Ser Val Gln His Arg Gln Leu Lys
Asp Asn Thr Cys 1085 1090 1095Val Val
Glu Phe Gln Phe Met Leu Pro Thr Ser His Pro Asn Arg 1100
1105 1110Gly Asn Ile Pro Thr Leu Asn Arg Met Ser
Phe Ser Ser Asn Leu 1115 1120 1125Asn
His Tyr Gly Met Thr His Val Ala Ser Val Ser Asp Val Leu 1130
1135 1140Leu Asp Asn Ser Phe Thr Pro Pro Cys
Gln Arg Met Gly Gly Met 1145 1150
1155Val Ser Phe Arg Thr Phe Glu Asp Phe Val Arg Ile Phe Asp Glu
1160 1165 1170Val Met Gly Cys Phe Ser
Asp Ser Pro Pro Gln Ser Pro Thr Phe 1175 1180
1185Pro Glu Ala Gly His Thr Ser Leu Tyr Asp Glu Asp Lys Val
Pro 1190 1195 1200Arg Asp Glu Pro Ile
His Ile Leu Asn Val Ala Ile Lys Thr Asp 1205 1210
1215Cys Asp Ile Glu Asp Asp Arg Leu Ala Ala Met Phe Arg
Glu Phe 1220 1225 1230Thr Gln Gln Asn
Lys Ala Thr Leu Val Asp His Gly Ile Arg Arg 1235
1240 1245Leu Thr Phe Leu Val Ala Gln Lys Asp Phe Arg
Lys Gln Val Asn 1250 1255 1260Tyr Glu
Val Asp Arg Arg Phe His Arg Glu Phe Pro Lys Phe Phe 1265
1270 1275Thr Phe Arg Ala Arg Asp Lys Phe Glu Glu
Asp Arg Ile Tyr Arg 1280 1285 1290His
Leu Glu Pro Ala Leu Ala Phe Gln Leu Glu Leu Asn Arg Met 1295
1300 1305Arg Asn Phe Asp Leu Thr Ala Ile Pro
Cys Ala Asn His Lys Met 1310 1315
1320His Leu Tyr Leu Gly Ala Ala Lys Val Glu Val Gly Thr Glu Val
1325 1330 1335Thr Asp Tyr Arg Phe Phe
Val Arg Ala Ile Ile Arg His Ser Asp 1340 1345
1350Leu Val Thr Lys Glu Ala Ser Phe Glu Tyr Leu Gln Asn Glu
Gly 1355 1360 1365Glu Arg Leu Leu Leu
Glu Ala Met Asp Glu Leu Glu Val Ala Phe 1370 1375
1380Asn Asn Thr Asn Val Arg Thr Asp Cys Asn His Ile Phe
Leu Asn 1385 1390 1395Phe Val Pro Thr
Val Ile Met Asp Pro Ser Lys Ile Glu Glu Ser 1400
1405 1410Val Arg Ser Met Val Met Arg Tyr Gly Ser Arg
Leu Trp Lys Leu 1415 1420 1425Arg Val
Leu Gln Ala Glu Leu Lys Ile Asn Ile Arg Leu Thr Pro 1430
1435 1440Thr Gly Lys Ala Ile Pro Ile Arg Leu Phe
Leu Thr Asn Glu Ser 1445 1450 1455Gly
Tyr Tyr Leu Asp Ile Ser Leu Tyr Lys Glu Val Thr Asp Ser 1460
1465 1470Arg Thr Ala Gln Ile Met Phe Gln Ala
Tyr Gly Asp Lys Gln Gly 1475 1480
1485Pro Leu His Gly Met Leu Ile Asn Thr Pro Tyr Val Thr Lys Asp
1490 1495 1500Leu Leu Gln Ser Lys Arg
Phe Gln Ala Gln Ser Leu Gly Thr Thr 1505 1510
1515Tyr Ile Tyr Asp Ile Pro Glu Met Phe Arg Gln Ser Leu Ile
Lys 1520 1525 1530Leu Trp Glu Ser Met
Ser Thr Gln Ala Phe Leu Pro Ser Pro Pro 1535 1540
1545Leu Pro Ser Asp Met Leu Thr Tyr Thr Glu Leu Val Leu
Asp Asp 1550 1555 1560Gln Gly Gln Leu
Val His Met Asn Arg Leu Pro Gly Gly Asn Glu 1565
1570 1575Ile Gly Met Val Ala Trp Lys Met Thr Phe Lys
Ser Pro Glu Tyr 1580 1585 1590Pro Glu
Gly Arg Asp Ile Ile Val Ile Gly Asn Asp Ile Thr Tyr 1595
1600 1605Arg Ile Gly Ser Phe Gly Pro Gln Glu Asp
Leu Leu Phe Leu Arg 1610 1615 1620Ala
Ser Glu Leu Ala Arg Ala Glu Gly Ile Pro Arg Ile Tyr Val 1625
1630 1635Ser Ala Asn Ser Gly Ala Arg Ile Gly
Leu Ala Glu Glu Ile Arg 1640 1645
1650His Met Phe His Val Ala Trp Val Asp Pro Glu Asp Pro Tyr Lys
1655 1660 1665Gly Tyr Arg Tyr Leu Tyr
Leu Thr Pro Gln Asp Tyr Lys Arg Val 1670 1675
1680Ser Ala Leu Asn Ser Val His Cys Glu His Val Glu Asp Glu
Gly 1685 1690 1695Glu Ser Arg Tyr Lys
Ile Thr Asp Ile Ile Gly Lys Glu Glu Gly 1700 1705
1710Ile Gly Pro Glu Asn Leu Arg Gly Ser Gly Met Ile Ala
Gly Glu 1715 1720 1725Ser Ser Leu Ala
Tyr Asn Glu Ile Ile Thr Ile Ser Leu Val Thr 1730
1735 1740Cys Arg Ala Ile Gly Ile Gly Ala Tyr Leu Val
Arg Leu Gly Gln 1745 1750 1755Arg Thr
Ile Gln Val Glu Asn Ser His Leu Ile Leu Thr Gly Ala 1760
1765 1770Gly Ala Leu Asn Lys Val Leu Gly Arg Glu
Val Tyr Thr Ser Asn 1775 1780 1785Asn
Gln Leu Gly Gly Ile Gln Ile Met His Asn Asn Gly Val Thr 1790
1795 1800His Cys Thr Val Cys Asp Asp Phe Glu
Gly Val Phe Thr Val Leu 1805 1810
1815His Trp Leu Ser Tyr Met Pro Lys Ser Val His Ser Ser Val Pro
1820 1825 1830Leu Leu Asn Ser Lys Asp
Pro Ile Asp Arg Ile Ile Glu Phe Val 1835 1840
1845Pro Thr Lys Thr Pro Tyr Asp Pro Arg Trp Met Leu Ala Gly
Arg 1850 1855 1860Pro His Pro Thr Gln
Lys Gly Gln Trp Leu Ser Gly Phe Phe Asp 1865 1870
1875Tyr Gly Ser Phe Ser Glu Ile Met Gln Pro Trp Ala Gln
Thr Val 1880 1885 1890Val Val Gly Arg
Ala Arg Leu Gly Gly Ile Pro Val Gly Val Val 1895
1900 1905Ala Val Glu Thr Arg Thr Val Glu Leu Ser Ile
Pro Ala Asp Pro 1910 1915 1920Ala Asn
Leu Asp Ser Glu Ala Lys Ile Ile Gln Gln Ala Gly Gln 1925
1930 1935Val Trp Phe Pro Asp Ser Ala Phe Lys Thr
Tyr Gln Ala Ile Lys 1940 1945 1950Asp
Phe Asn Arg Glu Gly Leu Pro Leu Met Val Phe Ala Asn Trp 1955
1960 1965Arg Gly Phe Ser Gly Gly Met Lys Asp
Met Tyr Asp Gln Val Leu 1970 1975
1980Lys Phe Gly Ala Tyr Ile Val Asp Gly Leu Arg Glu Cys Cys Gln
1985 1990 1995Pro Val Leu Val Tyr Ile
Pro Pro Gln Ala Glu Leu Arg Gly Gly 2000 2005
2010Ser Trp Val Val Ile Asp Ser Ser Ile Asn Pro Arg His Met
Glu 2015 2020 2025Met Tyr Ala Asp Arg
Glu Ser Arg Gly Ser Val Leu Glu Pro Glu 2030 2035
2040Gly Thr Val Glu Ile Lys Phe Arg Arg Lys Asp Leu Val
Lys Thr 2045 2050 2055Met Arg Arg Val
Asp Pro Val Tyr Ile His Leu Ala Glu Arg Leu 2060
2065 2070Gly Thr Pro Glu Leu Ser Thr Ala Glu Arg Lys
Glu Leu Glu Asn 2075 2080 2085Lys Leu
Lys Glu Arg Glu Glu Phe Leu Ile Pro Ile Tyr His Gln 2090
2095 2100Val Ala Val Gln Phe Ala Asp Leu His Asp
Thr Pro Gly Arg Met 2105 2110 2115Gln
Glu Lys Gly Val Ile Ser Asp Ile Leu Asp Trp Lys Thr Ser 2120
2125 2130Arg Thr Phe Phe Tyr Trp Arg Leu Arg
Arg Leu Leu Leu Glu Asp 2135 2140
2145Leu Val Lys Lys Lys Ile His Asn Ala Asn Pro Glu Leu Thr Asp
2150 2155 2160Gly Gln Ile Gln Ala Met
Leu Arg Arg Trp Phe Val Glu Val Glu 2165 2170
2175Gly Thr Val Lys Ala Tyr Val Trp Asp Asn Asn Lys Asp Leu
Ala 2180 2185 2190Glu Trp Leu Glu Lys
Gln Leu Thr Glu Glu Asp Gly Val His Ser 2195 2200
2205Val Ile Glu Glu Asn Ile Lys Cys Ile Ser Arg Asp Tyr
Val Leu 2210 2215 2220Lys Gln Ile Arg
Ser Leu Val Gln Ala Asn Pro Glu Val Ala Met 2225
2230 2235Asp Ser Ile Ile His Met Thr Gln His Ile Ser
Pro Thr Gln Arg 2240 2245 2250Ala Glu
Val Ile Arg Ile Leu Ser Thr Met Asp Ser Pro Ser Thr 2255
2260 2265172483PRTHomo sapiens 17Met Val Leu Leu Leu
Cys Leu Ser Cys Leu Ile Phe Ser Cys Leu Thr1 5
10 15Phe Ser Trp Leu Lys Ile Trp Glu Lys Met Thr
Asp Ser Lys Pro Ile 20 25
30Thr Lys Ser Lys Ser Glu Ala Asn Leu Ile Pro Ser Gln Glu Pro Phe
35 40 45Pro Ala Ser Asp Asn Ser Gly Glu
Thr Pro Gln Arg Asn Gly Glu Gly 50 55
60His Thr Leu His Lys Asp Thr Gln Pro Gly Arg Ala Gln Pro Pro Thr65
70 75 80Lys Ala Gln Arg Ser
Gly Arg Arg Arg Asn Ser Leu Pro Pro Ser Arg 85
90 95Gln Lys Pro Pro Arg Asn Pro Leu Ser Ser Ser
Asp Ala Ala Pro Ser 100 105
110Pro Glu Leu Gln Ala Asn Gly Thr Gly Thr Gln Gly Leu Glu Ala Thr
115 120 125Asp Thr Asn Gly Leu Ser Ser
Ser Ala Arg Pro Gln Gly Ser Lys Leu 130 135
140Val Pro Ser Lys Glu Asp Lys Lys Gln Ala Asn Ile Lys Arg Gln
Leu145 150 155 160Met Thr
Asn Phe Ile Leu Gly Ser Phe Asp Asp Tyr Ser Ser Asp Glu
165 170 175Asp Ser Val Ala Gly Ser Ser
Arg Glu Ser Thr Arg Lys Gly Ser Arg 180 185
190Ala Ser Leu Gly Ala Leu Ser Leu Glu Ala Tyr Leu Thr Thr
Gly Glu 195 200 205Ala Glu Thr Arg
Val Pro Thr Met Arg Pro Ser Met Ser Gly Leu His 210
215 220Leu Val Lys Arg Gly Arg Glu His Lys Lys Leu Asp
Leu His Arg Asp225 230 235
240Phe Thr Val Ala Ser Pro Ala Glu Phe Val Thr Arg Phe Gly Gly Asp
245 250 255Arg Val Ile Glu Lys
Val Leu Ile Ala Asn Asn Gly Ile Ala Ala Val 260
265 270Lys Cys Met Arg Ser Ile Arg Arg Trp Ala Tyr Glu
Met Phe Arg Asn 275 280 285Glu Arg
Ala Ile Arg Phe Val Arg Met Val Thr Pro Glu Asp Leu Lys 290
295 300Ala Asn Ala Glu Tyr Ile Lys Met Ala Asp His
Tyr Gly Pro Ala Pro305 310 315
320Gly Gly Pro Asn Asn Asn Asn Tyr Ala Asn Val Glu Leu Ile Val Asp
325 330 335Ile Ala Lys Arg
Ile Pro Leu Gln Ala Val Trp Ala Gly Trp Gly His 340
345 350Ala Leu Glu Asn Pro Lys Leu Pro Glu Leu Leu
Cys Lys Asn Gly Val 355 360 365Ala
Phe Leu Gly Pro Pro Arg Leu Arg Pro Met Val Gly Leu Gly Asp 370
375 380Lys Ile Ala Ser Thr Val Val Ala Gln Thr
Leu Gln Val Pro Thr Leu385 390 395
400Pro Arg Ser Gly Ser Ala Leu Thr Val Glu Trp Thr Glu Asp Asp
Leu 405 410 415Gln Gln Gly
Lys Arg Ile Ser Val Pro Glu Asp Val Tyr Asp Lys Gly 420
425 430Cys Val Lys Asp Val Asp Glu Gly Leu Glu
Ala Ala Glu Arg Ile Gly 435 440
445Phe Pro Leu Met Ile Lys Ala Ser Glu Gly Gly Gly Gly Lys Gly Ile 450
455 460Arg Glu Thr Glu Ser Ala Glu Asp
Phe Pro Ile Leu Phe Arg Gln Val465 470
475 480Gln Ser Glu Ile Pro Gly Ser Pro Ile Phe Leu Met
Lys Leu Ala Gln 485 490
495His Ala Arg His Leu Glu Val Gln Ile Leu Ala Asp Gln Tyr Gly Asn
500 505 510Ala Val Ser Leu Phe Gly
Arg Asp Cys Ser Ile Gln Arg Arg His Gln 515 520
525Lys Ile Val Glu Glu Ala Pro Ala Thr Ile Ala Pro Leu Ala
Ile Phe 530 535 540Glu Phe Met Glu Gln
Cys Ala Ile Arg Leu Ala Lys Thr Val Gly Tyr545 550
555 560Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr
Ser Gln Asp Gly Ser Phe 565 570
575His Phe Leu Glu Leu Asn Pro Arg Leu Gln Val Glu His Pro Cys Thr
580 585 590Glu Met Ile Ala Asp
Val Asn Leu Pro Ala Ala Gln Leu Gln Ile Ala 595
600 605Met Gly Ala Pro Leu His Arg Leu Lys Asp Ile Arg
Leu Leu Tyr Gly 610 615 620Glu Ser Pro
Trp Gly Asp Ser Pro Ile Ser Phe Glu Asn Ser Ala His625
630 635 640Leu Pro Cys Pro Arg Gly His
Val Ile Ala Thr Arg Ile Thr Ser Glu 645
650 655Asn Pro Asp Glu Gly Phe Lys Pro Ser Ser Gly Thr
Val Gln Glu Leu 660 665 670Asn
Phe Arg Ser Ser Lys Asn Val Trp Gly Tyr Phe Thr Val Ala Ala 675
680 685Thr Gly Gly Leu His Glu Phe Ala Ile
Ser Gln Phe Gly His Cys Phe 690 695
700Ser Trp Gly Glu Asn Arg Lys Glu Ala Ile Ser Asn Met Val Val Ala705
710 715 720Leu Lys Glu Leu
Ser Leu Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr 725
730 735Leu Ile Asn Leu Leu Glu Thr Glu Ser Phe
Gln Asn Asn Tyr Ile Asp 740 745
750Thr Gly Trp Leu Asp Tyr Leu Ile Ala Glu Lys Val Gln Lys Lys Pro
755 760 765Asn Ile Met Leu Gly Val Val
Cys Gly Ala Leu Glu Arg Gly Asp Ala 770 775
780Met Phe Arg Thr Cys Met Thr Asp Phe Leu His Ser Leu Glu Arg
Gly785 790 795 800Gln Val
Leu Pro Ala Asp Ser Leu Leu Asn Leu Val Asp Val Glu Leu
805 810 815Ile Tyr Glu Gly Val Lys Tyr
Ile Leu Lys Val Thr Arg Gln Ser Leu 820 825
830Thr Met Phe Val Leu Ile Met Asn Gly Cys His Ile Glu Ile
Asp Ala 835 840 845His Arg Leu Asn
Asp Gly Gly Leu Leu Leu Ser Tyr Asn Gly Asn Ser 850
855 860Tyr Thr Thr Tyr Met Lys Glu Glu Val Asp Ser Tyr
Arg Thr Ile Gly865 870 875
880Asn Lys Thr Cys Val Phe Glu Lys Glu Asn Asp Pro Thr Val Leu Arg
885 890 895Ser Pro Ser Ala Gly
Lys Leu Thr Gln Ile Thr Val Glu Asp Gly Gly 900
905 910His Val Glu Ala Gly Arg Arg Tyr Ala Glu Met Glu
Val Met Lys Met 915 920 925Ile Met
Thr Leu Asn Val Gln Glu Arg Gly Arg Val Lys Tyr Ile Lys 930
935 940Arg Pro Gly Ala Val Leu Glu Ala Gly Cys Val
Val Ala Arg Leu Glu945 950 955
960Leu Asp Asp Pro Ser Lys Val His Pro Ala Glu Pro Phe Thr Gly Glu
965 970 975Leu Pro Ala Gln
Gln Asn Thr Ala Asp Leu Gly Lys Lys Leu His Arg 980
985 990Val Phe His Ser Val Leu Gly Ser Leu Thr Asn
Val Met Ser Gly Phe 995 1000
1005Cys Leu Pro Glu Pro Phe Phe Ser Ile Lys Leu Lys Glu Trp Val
1010 1015 1020Gln Lys Leu Met Met Thr
Leu Arg His Pro Ser Leu Leu Leu Asp 1025 1030
1035Val Gln Glu Ile Met Thr Ser Arg Ala Gly Arg Ile Pro Pro
Pro 1040 1045 1050Val Glu Lys Ser Val
Arg Lys Val Met Ala Gln Tyr Ala Ser Asn 1055 1060
1065Ile Thr Ser Val Leu Cys Gln Phe Pro Ser Gln Gln Ile
Ala Thr 1070 1075 1080Ile Leu Asp Cys
His Ala Ala Thr Leu Gln Arg Lys Ala Asp Arg 1085
1090 1095Glu Val Phe Phe Ile Asn Thr Gln Ser Met Val
Gln Leu Val Gln 1100 1105 1110Arg Tyr
Arg Ser Gly Ile Arg Gly His Met Lys Thr Val Val Ile 1115
1120 1125Asp Leu Leu Arg Arg Tyr Leu Arg Val Glu
Thr Ile Phe Gly Lys 1130 1135 1140Ala
Arg Asp Ala Asp Ala Asn Ser Ser Gly Met Val Gly Gly Val 1145
1150 1155Arg Ser Leu Ser Phe Thr Ser Val Trp
Val Val Leu Ser Pro Pro 1160 1165
1170Ala His Tyr Asp Lys Cys Val Ile Asn Leu Arg Glu Gln Phe Lys
1175 1180 1185Pro Asp Met Ser Gln Val
Leu Asp Cys Ile Phe Ser His Ala Gln 1190 1195
1200Val Thr Lys Lys Asn Gln Leu Val Ile Met Leu Ile Asp Glu
Leu 1205 1210 1215Cys Gly Pro Asp Pro
Ser Leu Ser Asp Glu Leu Ile Ser Ile Leu 1220 1225
1230Asn Glu Leu Thr Gln Leu Ser Lys Ser Glu His Cys Lys
Val Ala 1235 1240 1245Leu Arg Ala Arg
Gln Ile Leu Ile Ala Ser Pro Ser Tyr Glu Leu 1250
1255 1260Arg His Asn Gln Val Glu Ser Ile Phe Leu Ser
Ala Ile Asp Met 1265 1270 1275Tyr Gly
His Gln Phe Cys Pro Glu Asn Leu Gln Lys Leu Ile Leu 1280
1285 1290Ser Glu Thr Thr Ile Phe Asp Val Leu Asn
Thr Phe Phe Tyr His 1295 1300 1305Ala
Asn Lys Val Val Cys Met Ala Ser Leu Glu Val Tyr Val Gly 1310
1315 1320Gly Ala Tyr Ile Ala Tyr Val Leu Asn
Ser Leu Gln His Arg Gln 1325 1330
1335Leu Pro Asp Gly Thr Cys Val Val Glu Phe Gln Phe Met Leu Pro
1340 1345 1350Ser Ser His Pro Asn Arg
Met Thr Val Pro Ile Ser Ile Thr Asn 1355 1360
1365Pro Asp Leu Leu Arg His Thr Thr Glu Leu Phe Met Asp Ser
Gly 1370 1375 1380Phe Ser Pro Leu Cys
Gln Arg Met Gly Ala Met Val Ala Phe Arg 1385 1390
1395Arg Phe Glu Asp Phe Thr Arg Asn Phe Asp Glu Val Ile
Ser Cys 1400 1405 1410Phe Ala Asn Val
Pro Lys Asp Pro Pro Leu Phe Ser Glu Ala Arg 1415
1420 1425Thr Ser Leu Tyr Ser Glu Asp Asp Cys Lys Ser
Leu Arg Glu Glu 1430 1435 1440Pro Ile
His Ile Leu Asn Val Ser Ile Gln Cys Ala Asp His Leu 1445
1450 1455Glu Asp Glu Ala Leu Val Pro Ile Leu Arg
Thr Phe Val Gln Ser 1460 1465 1470Lys
Lys Asn Ile Leu Val Asp Tyr Gly Leu Arg Arg Ile Pro Phe 1475
1480 1485Leu Ile Ala Gln Glu Lys Glu Phe Pro
Lys Phe Phe Thr Phe Arg 1490 1495
1500Ala Arg Asp Glu Phe Ala Glu Asp Arg Ile Tyr Arg His Leu Glu
1505 1510 1515Pro Ala Leu Ala Phe Gln
Leu Glu Leu Asn Arg Met Arg Asn Phe 1520 1525
1530Asp Leu Thr Ala Val Pro Cys Ala Asn His Lys Met His Leu
Tyr 1535 1540 1545Leu Gly Ala Ala Lys
Val Glu Gly Arg Tyr Glu Val Thr Asp His 1550 1555
1560Arg Phe Phe Ile Arg Ala Ile Ile Arg His Ser Asp Leu
Ile Thr 1565 1570 1575Lys Glu Ala Ser
Phe Glu Tyr Leu Gln Asn Glu Gly Glu Arg Leu 1580
1585 1590Leu Leu Glu Ala Met Asp Glu Leu Glu Val Ala
Phe Asn Asn Thr 1595 1600 1605Asn Val
Arg Thr Asp Cys Asn His Ile Phe Leu Asn Phe Val Pro 1610
1615 1620Thr Val Ile Met Asp Pro Asn Lys Ile Glu
Glu Ser Val Arg Tyr 1625 1630 1635Met
Val Met Arg Tyr Gly Ser Arg Leu Trp Lys Leu Arg Val Leu 1640
1645 1650Gln Ala Glu Val Lys Ile Asn Ile Arg
Gln Thr Thr Thr Gly Ser 1655 1660
1665Ala Val Pro Ile Arg Leu Phe Ile Thr Asn Glu Ser Gly Tyr Tyr
1670 1675 1680Leu Asp Ile Ser Leu Tyr
Lys Glu Val Thr Asp Ser Arg Ser Gly 1685 1690
1695Asn Ile Met Phe His Ser Phe Gly Asn Lys Gln Gly Pro Gln
His 1700 1705 1710Gly Met Leu Ile Asn
Thr Pro Tyr Val Thr Lys Asp Leu Leu Gln 1715 1720
1725Ala Lys Arg Phe Gln Ala Gln Thr Leu Gly Thr Thr Tyr
Ile Tyr 1730 1735 1740Asp Phe Pro Glu
Met Phe Arg Gln Ala Leu Phe Lys Leu Trp Gly 1745
1750 1755Ser Pro Asp Lys Tyr Pro Lys Asp Ile Leu Thr
Tyr Thr Glu Leu 1760 1765 1770Val Leu
Asp Ser Gln Gly Gln Leu Val Glu Met Asn Arg Leu Pro 1775
1780 1785Gly Gly Asn Glu Val Gly Met Val Ala Phe
Lys Met Arg Phe Lys 1790 1795 1800Thr
Gln Glu Tyr Pro Glu Gly Arg Asp Val Ile Val Ile Gly Asn 1805
1810 1815Asp Ile Thr Phe Arg Ile Gly Ser Phe
Gly Pro Gly Glu Asp Leu 1820 1825
1830Leu Tyr Leu Arg Ala Ser Glu Met Ala Arg Ala Glu Ala Ile Pro
1835 1840 1845Lys Ile Tyr Val Ala Ala
Asn Ser Gly Ala Arg Ile Gly Met Ala 1850 1855
1860Glu Glu Ile Lys His Met Phe His Val Ala Trp Val Asp Pro
Glu 1865 1870 1875Asp Pro His Lys Gly
Phe Lys Tyr Leu Tyr Leu Thr Pro Gln Asp 1880 1885
1890Tyr Thr Arg Ile Ser Ser Leu Asn Ser Val His Cys Lys
His Ile 1895 1900 1905Glu Glu Gly Gly
Glu Ser Arg Tyr Met Ile Thr Asp Ile Ile Gly 1910
1915 1920Lys Asp Asp Gly Leu Gly Val Glu Asn Leu Arg
Gly Ser Gly Met 1925 1930 1935Ile Ala
Gly Glu Ser Ser Leu Ala Tyr Glu Glu Ile Val Thr Ile 1940
1945 1950Ser Leu Val Thr Cys Arg Ala Ile Gly Ile
Gly Ala Tyr Leu Val 1955 1960 1965Arg
Leu Gly Gln Arg Val Ile Gln Val Glu Asn Ser His Ile Ile 1970
1975 1980Leu Thr Gly Ala Ser Ala Leu Asn Lys
Val Leu Gly Arg Glu Val 1985 1990
1995Tyr Thr Ser Asn Asn Gln Leu Gly Gly Val Gln Ile Met His Tyr
2000 2005 2010Asn Gly Val Ser His Ile
Thr Val Pro Asp Asp Phe Glu Gly Val 2015 2020
2025Tyr Thr Ile Leu Glu Trp Leu Ser Tyr Met Pro Lys Asp Asn
His 2030 2035 2040Ser Pro Val Pro Ile
Ile Thr Pro Thr Asp Pro Ile Asp Arg Glu 2045 2050
2055Ile Glu Phe Leu Pro Ser Arg Ala Pro Tyr Asp Pro Arg
Trp Met 2060 2065 2070Leu Ala Gly Arg
Pro His Pro Thr Leu Lys Gly Thr Trp Gln Ser 2075
2080 2085Gly Phe Phe Asp His Gly Ser Phe Lys Glu Ile
Met Ala Pro Trp 2090 2095 2100Ala Gln
Thr Val Val Thr Gly Arg Ala Arg Leu Gly Gly Ile Pro 2105
2110 2115Val Gly Val Ile Ala Val Glu Thr Arg Thr
Val Glu Val Ala Val 2120 2125 2130Pro
Ala Asp Pro Ala Asn Leu Asp Ser Glu Ala Lys Ile Ile Gln 2135
2140 2145Gln Ala Gly Gln Val Trp Phe Pro Asp
Ser Ala Tyr Lys Thr Ala 2150 2155
2160Gln Ala Ile Lys Asp Phe Asn Arg Glu Lys Leu Pro Leu Met Ile
2165 2170 2175Phe Ala Asn Trp Arg Gly
Phe Ser Gly Gly Met Lys Asp Met Tyr 2180 2185
2190Asp Gln Val Leu Lys Phe Gly Ala Tyr Ile Val Asp Gly Leu
Arg 2195 2200 2205Gln Tyr Lys Gln Pro
Ile Leu Ile Tyr Ile Arg Pro Met Arg Glu 2210 2215
2220Leu Arg Gly Gly Ser Trp Val Val Ile Asp Ala Thr Ile
Asn Pro 2225 2230 2235Leu Cys Ile Glu
Met Tyr Ala Asp Lys Glu Ser Arg Gly Gly Val 2240
2245 2250Leu Glu Pro Glu Gly Thr Val Glu Ile Lys Phe
Arg Lys Glu Asp 2255 2260 2265Leu Ile
Lys Ser Met Arg Arg Ile Asp Pro Ala Tyr Lys Lys Leu 2270
2275 2280Met Glu Gln Leu Gly Glu Pro Asp Leu Ser
Asp Lys Asp Arg Lys 2285 2290 2295Asp
Leu Glu Gly Arg Leu Lys Ala Arg Glu Asp Leu Leu Leu Pro 2300
2305 2310Ile Tyr His Gln Val Ala Val Gln Phe
Ala Asp Phe His Asp Thr 2315 2320
2325Pro Gly Arg Met Leu Glu Lys Gly Val Ile Ser Asp Ile Leu Glu
2330 2335 2340Trp Lys Thr Ala Arg Thr
Phe Leu Tyr Trp Arg Leu Arg Arg Leu 2345 2350
2355Leu Leu Glu Asp Gln Val Lys Gln Glu Ile Leu Gln Ala Ser
Gly 2360 2365 2370Glu Leu Ser His Val
His Ile Gln Ser Met Leu Arg Arg Trp Phe 2375 2380
2385Val Glu Thr Glu Gly Ala Val Lys Ala Tyr Leu Trp Asp
Asn Asn 2390 2395 2400Gln Val Val Val
Gln Trp Leu Glu Gln His Trp Gln Ala Gly Asp 2405
2410 2415Gly Pro Arg Ser Thr Ile Arg Glu Asn Ile Thr
Tyr Leu Lys His 2420 2425 2430Asp Ser
Val Leu Lys Thr Ile Arg Gly Leu Val Glu Glu Asn Pro 2435
2440 2445Glu Val Ala Val Asp Cys Val Ile Tyr Leu
Ser Gln His Ile Ser 2450 2455 2460Pro
Ala Glu Arg Ala Gln Val Val His Leu Leu Ser Thr Met Asp 2465
2470 2475Ser Pro Ala Ser Thr
2480182458PRTHomo sapiens 18Met Val Leu Leu Leu Cys Leu Ser Cys Leu Ile
Phe Ser Cys Leu Thr1 5 10
15Phe Ser Trp Leu Lys Ile Trp Gly Lys Met Thr Asp Ser Lys Pro Ile
20 25 30Thr Lys Ser Lys Ser Glu Ala
Asn Leu Ile Pro Ser Gln Glu Pro Phe 35 40
45Pro Ala Ser Asp Asn Ser Gly Glu Thr Pro Gln Arg Asn Gly Glu
Gly 50 55 60His Thr Leu Pro Lys Thr
Pro Ser Gln Ala Glu Pro Ala Ser His Lys65 70
75 80Gly Pro Lys Asp Ala Gly Arg Arg Arg Asn Ser
Leu Pro Pro Ser His 85 90
95Gln Lys Pro Pro Arg Asn Pro Leu Ser Ser Ser Asp Ala Ala Pro Ser
100 105 110Pro Glu Leu Gln Ala Asn
Gly Thr Gly Thr Gln Gly Leu Glu Ala Thr 115 120
125Asp Thr Asn Gly Leu Ser Ser Ser Ala Arg Pro Gln Gly Gln
Gln Ala 130 135 140Gly Ser Pro Ser Lys
Glu Asp Lys Lys Gln Ala Asn Ile Lys Arg Gln145 150
155 160Leu Met Thr Asn Phe Ile Leu Gly Ser Phe
Asp Asp Tyr Ser Ser Asp 165 170
175Glu Asp Ser Val Ala Gly Ser Ser Arg Glu Ser Thr Arg Lys Gly Ser
180 185 190Arg Ala Ser Leu Gly
Ala Leu Ser Leu Glu Ala Tyr Leu Thr Thr Gly 195
200 205Glu Ala Glu Thr Arg Val Pro Thr Met Arg Pro Ser
Met Ser Gly Leu 210 215 220His Leu Val
Lys Arg Gly Arg Glu His Lys Lys Leu Asp Leu His Arg225
230 235 240Asp Phe Thr Val Ala Ser Pro
Ala Glu Phe Val Thr Arg Phe Gly Gly 245
250 255Asp Arg Val Ile Glu Lys Val Leu Ile Ala Asn Asn
Gly Ile Ala Ala 260 265 270Val
Lys Cys Met Arg Ser Ile Arg Arg Trp Ala Tyr Glu Met Phe Arg 275
280 285Asn Glu Arg Ala Ile Arg Phe Val Val
Met Val Thr Pro Glu Asp Leu 290 295
300Lys Ala Asn Ala Glu Tyr Ile Lys Met Ala Asp His Tyr Val Pro Val305
310 315 320Pro Gly Gly Pro
Asn Asn Asn Asn Tyr Ala Asn Val Glu Leu Ile Val 325
330 335Asp Ile Ala Lys Arg Ile Pro Val Gln Ala
Val Trp Ala Gly Trp Gly 340 345
350His Ala Ser Glu Asn Pro Lys Leu Pro Glu Leu Leu Cys Lys Asn Gly
355 360 365Val Ala Phe Leu Gly Pro Pro
Ser Glu Ala Met Trp Ala Leu Gly Asp 370 375
380Lys Ile Ala Ser Thr Val Val Ala Gln Thr Leu Gln Val Pro Thr
Leu385 390 395 400Pro Trp
Ser Gly Ser Gly Leu Thr Val Glu Trp Thr Glu Asp Asp Leu
405 410 415Gln Gln Gly Lys Arg Ile Ser
Val Pro Glu Asp Val Tyr Asp Lys Gly 420 425
430Cys Val Lys Asp Val Asp Glu Gly Leu Glu Ala Ala Glu Arg
Ile Gly 435 440 445Phe Pro Leu Met
Ile Lys Ala Ser Glu Gly Gly Gly Gly Lys Gly Ile 450
455 460Arg Lys Ala Glu Ser Ala Glu Asp Phe Pro Ile Leu
Phe Arg Gln Val465 470 475
480Gln Ser Glu Ile Pro Gly Ser Pro Ile Phe Leu Met Lys Leu Ala Gln
485 490 495His Ala Arg His Leu
Glu Val Gln Ile Leu Ala Asp Gln Tyr Gly Asn 500
505 510Ala Val Ser Leu Phe Gly Arg Asp Cys Ser Ile Gln
Arg Arg His Gln 515 520 525Lys Ile
Val Glu Glu Ala Pro Ala Thr Ile Ala Pro Leu Ala Ile Phe 530
535 540Glu Phe Met Glu Gln Cys Ala Ile Arg Leu Ala
Lys Thr Val Gly Tyr545 550 555
560Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr Ser Gln Asp Gly Ser Phe
565 570 575His Phe Leu Glu
Leu Asn Pro Arg Leu Gln Val Glu His Pro Cys Thr 580
585 590Glu Met Ile Ala Asp Val Asn Leu Pro Ala Ala
Gln Leu Gln Ile Ala 595 600 605Met
Gly Val Pro Leu His Arg Leu Lys Asp Ile Arg Leu Leu Tyr Gly 610
615 620Glu Ser Pro Trp Gly Val Thr Pro Ile Ser
Phe Glu Thr Pro Ser Asn625 630 635
640Pro Pro Leu Ala Arg Gly His Val Ile Ala Ala Arg Ile Thr Ser
Glu 645 650 655Asn Pro Asp
Glu Gly Phe Lys Pro Ser Ser Gly Thr Val Gln Glu Leu 660
665 670Asn Phe Arg Ser Ser Lys Asn Val Trp Gly
Tyr Phe Ser Val Ala Ala 675 680
685Thr Gly Gly Leu His Glu Phe Ala Asp Ser Gln Phe Gly His Cys Phe 690
695 700Ser Trp Gly Glu Asn Arg Glu Glu
Ala Ile Ser Asn Met Val Val Ala705 710
715 720Leu Lys Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr
Thr Val Glu Tyr 725 730
735Leu Ile Asn Leu Leu Glu Thr Glu Ser Phe Gln Asn Asn Asp Ile Asp
740 745 750Thr Gly Trp Leu Asp Tyr
Leu Ile Ala Glu Lys Val Gln Ala Glu Lys 755 760
765Pro Asp Ile Met Leu Gly Val Val Cys Gly Ala Leu Asn Val
Ala Asp 770 775 780Ala Met Phe Arg Thr
Cys Met Thr Asp Phe Leu His Ser Leu Glu Arg785 790
795 800Gly Gln Val Leu Pro Ala Asp Ser Leu Leu
Asn Leu Val Asp Val Glu 805 810
815Leu Ile Tyr Gly Gly Val Lys Tyr Ile Leu Lys Val Ala Arg Gln Ser
820 825 830Leu Thr Met Phe Val
Leu Ile Met Asn Gly Cys His Ile Glu Ile Asp 835
840 845Ala His Arg Leu Asn Asp Gly Gly Leu Leu Leu Ser
Tyr Asn Gly Asn 850 855 860Ser Tyr Thr
Thr Tyr Met Lys Glu Glu Val Asp Ser Tyr Arg Ile Thr865
870 875 880Ile Gly Asn Lys Thr Cys Val
Phe Glu Lys Glu Asn Asp Pro Thr Val 885
890 895Leu Arg Ser Pro Ser Ala Gly Lys Leu Thr Gln Tyr
Thr Val Glu Asp 900 905 910Gly
Gly His Val Glu Ala Gly Ser Ser Tyr Ala Glu Met Glu Val Met 915
920 925Lys Met Ile Met Thr Leu Asn Val Gln
Glu Arg Gly Arg Val Lys Tyr 930 935
940Ile Lys Arg Pro Gly Ala Val Leu Glu Ala Gly Cys Val Val Ala Arg945
950 955 960Leu Glu Leu Asp
Asp Pro Ser Lys Val His Pro Ala Glu Pro Phe Thr 965
970 975Gly Glu Leu Pro Ala Gln Gln Thr Leu Pro
Ile Leu Gly Glu Lys Leu 980 985
990His Gln Val Phe His Ser Val Leu Glu Asn Leu Thr Asn Val Met Ser
995 1000 1005Gly Phe Cys Leu Pro Glu
Pro Val Phe Ser Ile Lys Leu Lys Glu 1010 1015
1020Trp Val Gln Lys Leu Met Met Thr Leu Arg His Pro Ser Leu
Pro 1025 1030 1035Leu Leu Glu Leu Gln
Glu Ile Met Thr Ser Val Ala Gly Arg Ile 1040 1045
1050Pro Ala Pro Val Glu Lys Ser Val Arg Arg Val Met Ala
Gln Tyr 1055 1060 1065Ala Ser Asn Ile
Thr Ser Val Leu Cys Gln Phe Pro Ser Gln Gln 1070
1075 1080Ile Ala Thr Ile Leu Asp Cys His Ala Ala Thr
Leu Gln Arg Lys 1085 1090 1095Ala Asp
Arg Glu Val Phe Phe Ile Asn Thr Gln Ser Ile Val Gln 1100
1105 1110Leu Val Gln Arg Tyr Arg Ser Gly Ile Arg
Gly Tyr Met Lys Thr 1115 1120 1125Val
Val Leu Asp Leu Leu Arg Arg Tyr Leu Arg Val Glu His His 1130
1135 1140Phe Gln Gln Ala His Tyr Asp Lys Cys
Val Ile Asn Leu Arg Glu 1145 1150
1155Gln Phe Lys Pro Asp Met Ser Gln Val Leu Asp Cys Ile Phe Ser
1160 1165 1170His Ala Gln Val Ala Lys
Lys Asn Gln Leu Val Ile Met Leu Ile 1175 1180
1185Asp Glu Leu Cys Gly Pro Asp Pro Ser Leu Ser Asp Glu Leu
Ile 1190 1195 1200Ser Ile Leu Asn Glu
Leu Thr Gln Leu Ser Lys Ser Glu His Cys 1205 1210
1215Lys Val Ala Leu Arg Ala Arg Gln Ile Leu Ile Ala Ser
His Leu 1220 1225 1230Pro Ser Tyr Glu
Leu Arg His Asn Gln Val Glu Ser Ile Phe Leu 1235
1240 1245Ser Ala Ile Asp Met Tyr Gly His Gln Phe Cys
Pro Glu Asn Leu 1250 1255 1260Lys Lys
Leu Ile Leu Ser Glu Thr Thr Ile Phe Asp Val Leu Pro 1265
1270 1275Thr Phe Phe Tyr His Ala Asn Lys Val Val
Cys Met Ala Ser Leu 1280 1285 1290Glu
Val Tyr Val Arg Arg Gly Tyr Ile Ala Tyr Glu Leu Asn Ser 1295
1300 1305Leu Gln His Arg Gln Leu Pro Asp Gly
Thr Cys Val Val Glu Phe 1310 1315
1320Gln Phe Met Leu Pro Ser Ser His Pro Asn Arg Met Thr Val Pro
1325 1330 1335Ile Ser Ile Thr Asn Pro
Asp Leu Leu Arg His Ser Thr Glu Leu 1340 1345
1350Phe Met Asp Ser Gly Phe Ser Pro Leu Cys Gln Arg Met Gly
Ala 1355 1360 1365Met Val Ala Phe Arg
Arg Phe Glu Asp Phe Thr Arg Asn Phe Asp 1370 1375
1380Glu Val Ile Ser Cys Phe Ala Asn Val Pro Lys Asp Thr
Pro Leu 1385 1390 1395Phe Ser Glu Ala
Arg Thr Ser Leu Tyr Ser Glu Asp Asp Cys Lys 1400
1405 1410Ser Leu Arg Glu Glu Pro Ile His Ile Leu Asn
Val Ser Ile Gln 1415 1420 1425Cys Ala
Asp His Leu Glu Asp Glu Ala Leu Val Pro Ile Leu Arg 1430
1435 1440Thr Phe Val Gln Ser Lys Lys Asn Ile Leu
Val Asp Tyr Gly Leu 1445 1450 1455Arg
Arg Ile Thr Phe Leu Ile Ala Gln Glu Lys Glu Phe Pro Lys 1460
1465 1470Phe Phe Thr Phe Arg Ala Arg Asp Glu
Phe Ala Glu Asp Arg Ile 1475 1480
1485Tyr Arg His Leu Glu Pro Ala Leu Ala Phe Gln Leu Glu Leu Asn
1490 1495 1500Arg Met Arg Asn Phe Asp
Leu Thr Ala Val Pro Cys Ala Asn His 1505 1510
1515Lys Met His Leu Tyr Leu Gly Ala Ala Lys Val Lys Glu Gly
Val 1520 1525 1530Glu Val Thr Asp His
Arg Phe Phe Ile Arg Ala Ile Ile Arg His 1535 1540
1545Ser Asp Leu Ile Thr Lys Glu Ala Ser Phe Glu Tyr Leu
Gln Asn 1550 1555 1560Glu Gly Glu Arg
Leu Leu Leu Glu Ala Met Asp Glu Leu Glu Val 1565
1570 1575Ala Phe Asn Asn Thr Ser Val Arg Thr Asp Cys
Asn His Ile Phe 1580 1585 1590Leu Asn
Phe Val Pro Thr Val Ile Met Asp Pro Phe Lys Ile Glu 1595
1600 1605Glu Ser Val Arg Tyr Met Val Met Arg Tyr
Gly Ser Arg Leu Trp 1610 1615 1620Lys
Leu Arg Val Leu Gln Ala Glu Val Lys Ile Asn Ile Arg Gln 1625
1630 1635Thr Thr Thr Gly Ser Ala Val Pro Ile
Arg Leu Phe Ile Thr Asn 1640 1645
1650Glu Ser Gly Tyr Tyr Leu Asp Ile Ser Leu Tyr Lys Glu Val Thr
1655 1660 1665Asp Ser Arg Ser Gly Asn
Ile Met Phe His Ser Phe Gly Asn Lys 1670 1675
1680Gln Gly Pro Gln His Gly Met Leu Ile Asn Thr Pro Tyr Val
Thr 1685 1690 1695Lys Asp Leu Leu Gln
Ala Lys Arg Phe Gln Ala Gln Thr Leu Gly 1700 1705
1710Thr Thr Tyr Ile Tyr Asp Phe Pro Glu Met Phe Arg Gln
Ala Leu 1715 1720 1725Phe Lys Leu Trp
Gly Ser Pro Asp Lys Tyr Pro Lys Asp Ile Leu 1730
1735 1740Thr Tyr Thr Glu Leu Val Leu Asp Ser Gln Gly
Gln Leu Val Glu 1745 1750 1755Met Asn
Arg Leu Pro Gly Gly Asn Glu Val Gly Met Val Ala Phe 1760
1765 1770Lys Met Arg Phe Lys Thr Gln Glu Tyr Pro
Glu Gly Arg Asp Val 1775 1780 1785Ile
Val Ile Gly Asn Asp Ile Thr Phe Arg Ile Gly Ser Phe Gly 1790
1795 1800Pro Gly Glu Asp Leu Leu Tyr Leu Arg
Ala Ser Glu Met Ala Arg 1805 1810
1815Ala Glu Gly Ile Pro Lys Ile Tyr Val Ala Ala Asn Ser Gly Ala
1820 1825 1830Arg Ile Gly Met Ala Glu
Glu Ile Lys His Met Phe His Val Ala 1835 1840
1845Trp Val Asp Pro Glu Asp Pro His Lys Gly Phe Lys Tyr Leu
Tyr 1850 1855 1860Leu Thr Pro Gln Asp
Tyr Thr Arg Ile Ser Ser Leu Asn Ser Val 1865 1870
1875His Cys Lys His Ile Glu Glu Gly Gly Glu Ser Arg Tyr
Met Ile 1880 1885 1890Thr Asp Ile Ile
Gly Lys Asp Asp Gly Leu Gly Val Glu Asn Leu 1895
1900 1905Arg Gly Ser Gly Met Ile Ala Gly Glu Ser Ser
Leu Ala Tyr Glu 1910 1915 1920Glu Ile
Val Thr Ile Ser Leu Val Thr Cys Arg Ala Ile Gly Ile 1925
1930 1935Gly Ala Tyr Leu Val Arg Leu Gly Gln Arg
Val Ile Gln Val Glu 1940 1945 1950Asn
Ser His Ile Ile Leu Thr Gly Ala Ser Ala Leu Asn Lys Val 1955
1960 1965Leu Gly Arg Glu Val Tyr Thr Ser Asn
Asn Gln Leu Gly Gly Val 1970 1975
1980Gln Ile Met His Tyr Asn Gly Val Ser His Ile Thr Val Pro Asp
1985 1990 1995Asp Phe Glu Gly Val Tyr
Thr Ile Leu Glu Trp Leu Ser Tyr Met 2000 2005
2010Pro Lys Asp Asn His Ser Pro Val Pro Ile Ile Thr Pro Thr
Asp 2015 2020 2025Pro Ile Asp Arg Glu
Ile Glu Phe Leu Pro Ser Arg Ala Pro Tyr 2030 2035
2040Asp Pro Arg Trp Met Leu Ala Gly Arg Pro His Pro Thr
Leu Lys 2045 2050 2055Gly Thr Trp Gln
Ser Gly Phe Phe Asp His Gly Ser Phe Lys Glu 2060
2065 2070Ile Met Ala Pro Trp Ala Gln Thr Val Val Thr
Gly Arg Ala Arg 2075 2080 2085Leu Gly
Gly Ile Pro Val Gly Val Ile Ala Val Glu Thr Arg Thr 2090
2095 2100Val Glu Val Ala Val Pro Ala Asp Pro Ala
Asn Leu Asp Ser Glu 2105 2110 2115Ala
Lys Ile Ile Gln Gln Ala Gly Gln Val Trp Phe Pro Asp Ser 2120
2125 2130Ala Tyr Lys Thr Ala Gln Ala Ile Lys
Asp Phe Asn Arg Glu Lys 2135 2140
2145Leu Pro Leu Met Ile Phe Ala Asn Trp Arg Gly Phe Ser Gly Gly
2150 2155 2160Met Lys Asp Met Tyr Asp
Gln Val Leu Lys Phe Gly Ala Tyr Ile 2165 2170
2175Val Asp Gly Leu Arg Gln Tyr Lys Gln Pro Ile Leu Ile Tyr
Ile 2180 2185 2190Pro Pro Tyr Ala Glu
Leu Arg Gly Gly Ser Trp Val Val Ile Asp 2195 2200
2205Ala Thr Ile Asn Pro Leu Cys Ile Glu Met Tyr Ala Asp
Lys Glu 2210 2215 2220Ser Arg Gly Gly
Val Leu Glu Pro Glu Gly Thr Val Glu Ile Lys 2225
2230 2235Phe Arg Lys Lys Asp Leu Ile Lys Ser Met Arg
Arg Ile Asp Pro 2240 2245 2250Ala Tyr
Lys Lys Leu Met Glu Gln Leu Gly Glu Pro Asp Leu Ser 2255
2260 2265Asp Lys Asp Arg Lys Asp Leu Glu Gly Arg
Leu Lys Ala Arg Glu 2270 2275 2280Asp
Leu Leu Leu Pro Ile Tyr His Gln Val Ala Val Gln Phe Ala 2285
2290 2295Asp Phe His Asp Thr Pro Gly Arg Met
Leu Glu Lys Gly Val Ile 2300 2305
2310Ser Asp Ile Leu Glu Trp Lys Thr Ala Arg Thr Phe Leu Tyr Trp
2315 2320 2325Arg Leu Arg Arg Leu Leu
Leu Glu Asp Gln Val Lys Gln Glu Ile 2330 2335
2340Leu Gln Ala Ser Gly Glu Leu Ser His Val His Ile Gln Ser
Met 2345 2350 2355Leu Arg Arg Trp Phe
Val Glu Thr Glu Gly Ala Val Lys Ala Tyr 2360 2365
2370Leu Trp Asp Asn Asn Gln Val Val Val Gln Trp Leu Glu
Gln His 2375 2380 2385Trp Gln Ala Gly
Asp Gly Pro Arg Ser Thr Ile Arg Glu Asn Ile 2390
2395 2400Thr Tyr Leu Lys His Asp Ser Val Leu Lys Thr
Ile Arg Gly Leu 2405 2410 2415Val Glu
Glu Asn Pro Glu Val Ala Val Asp Cys Val Ile Tyr Leu 2420
2425 2430Ser Gln His Ile Ser Pro Ala Glu Arg Ala
Gln Val Val His Leu 2435 2440 2445Leu
Ser Thr Met Asp Ser Pro Ala Ser Thr 2450 2455
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