US20030165809A1

US20030165809A1 - MARKs as modifiers of the p53 pathway and methods of use

Info

Publication number: US20030165809A1
Application number: US10/161,565
Authority: US
Inventors: Lori Friedman; Gregory Plowman; Marcia Belvin; Helen Francis-Lang; Danxi Li; Roel Funke; Mario Lioubin
Original assignee: Exelixis Inc
Current assignee: Exelixis Inc
Priority date: 2001-06-05
Filing date: 2002-06-03
Publication date: 2003-09-04
Also published as: WO2002099042A2; WO2002099044A2; US20060160764A1; WO2002099041A2; JP2005501528A; EP1401861A2; WO2002099054A2; EP1404374A4; EP1402067A4; AU2002312255A1; WO2002099047A2; CA2449289A1; WO2002099049A3; WO2002099058A2; AU2002345497A1; WO2002099050A2; WO2002099048A3; WO2002099046A3; JP2004536297A; US20030036076A1

Abstract

Human MARK genes are identified as modulators of the p53 pathway, and thus are therapeutic targets for disorders associated with defective p53 function. Methods for identifying modulators of p53, comprising screening for agents that modulate the activity of MARK are provided.

Description

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applications 60/296,076 filed Jun. 5, 2001, 60/328,605 filed Oct. 10, 2001, and 60/357,253 filed Feb. 15, 2002. The contents of the prior applications are hereby incorporated in their entirety.[0001]

BACKGROUND OF THE INVENTION

The p53 gene is mutated in over 50 different types of human cancers, including familial and spontaneous cancers, and is believed to be the most commonly mutated gene in human cancer (Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al., Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of mutations in the p53 gene are missense mutations that alter a single amino acid that inactivates p53 function. Aberrant forms of human p53 are associated with poor prognosis, more aggressive tumors, metastasis, and short survival rates (Mitsudomi et al., Clin Cancer Res 2000 Oct; 6(10):4055-63; Koshland, Science (1993) 262:1953).

The human p53 protein normally functions as a central integrator of signals including DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8). In response to these signals, p53 protein levels are greatly increased with the result that the accumulated p53 activates cell cycle arrest or apoptosis depending on the nature and strength of these signals. Indeed, multiple lines of experimental evidence have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997) 88:323-331). For example, homozygous p53 “knockout” mice are developmentally normal but exhibit nearly 100% incidence of neoplasia in the first year of life (Donehower et al., Nature (1992) 356:215-221).

The biochemical mechanisms and pathways through which p53 functions in normal and cancerous cells are not fully understood, but one clearly important aspect of p53 function is its activity as a gene-specific transcriptional activator. Among the genes with known p53-response elements are several with well-characterized roles in either regulation of the cell cycle or apoptosis, including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2 (Levine, Cell (1997) 88:323-331).

Microtubules have a central role in the regulation of cell shape and polarity during differentiation, chromosome partitioning at mitosis, and intracellular transport. Microtubules undergo rearrangements involving rapid transitions between stable and dynamic states during these processes. Microtubule affinity regulating kinases (MARK) are a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption (Drewes, G., et al. (1997) Cell 89:297-308).

Microtubule affinity regulating kinase 1 (MARK1) is a serine/threonine kinase that phosphorylates microtubule-associated protein tau, leading to disruption of microtubules. It shares 90% amino acid homology with the rat version of MARK1, and demonstrates ubiquitous expression with highest levels in testis and brain (Nagase, T. et al. (2000) DNA Res. 7:143-150).

EMK1 (MARK2) is a serine/threonine protein kinase with two isoforms, which differ by the presence or absence of a 162-bp alternative exon (Espinosa, L. and Navarro, E. (1998) Cytogenet. Cell Genet. 81:278-282). Both human isoforms are coexpressed in a number of cell lines and tissues, with the highest expression found in heart, brain, placenta, skeletal muscle, and pancreas, and at lower levels in lung, liver, and kidney (Inglis, J. et al. (1993) Mammalian Genome 4:401-403). Due to the physical location of this gene, 11q12-q13, EMK1 is a candidate gene for carcinogenic events (Courseaux, A. et al. (1995) Mammalian Genome 6:311-312), and has been associated with colon and prostate cancer (Moore, T. M., et al. (2000) J Biol Chem 275:4311-22; Navarro, E., et al. (1999) Biochim Biophys Acta 1450:254-64).

Microtubule affinity regulating kinase 3 (MARK3) was originally identified as a marker (KP78) induced by treatment with DNA damaging agents. The loss of MARK3 was associated with carcinogenesis in the pancreas (Parsa, I. (1988) Cell Growth Differ. 9:197-208). MARK3 may be involved in cell cycle regulation, and alterations in the MARK3 gene may lead to carcinogenesis. MARK 3 is ubiquitously expressed throughout human tissues, with an additional 3.0 Kb transcript present in the heart (Peng, C. et al. (1998) Cell Growth Differ. 9:197-208).

MAP/microtubule affinity-regulating kinase like 1 (MARKL1) has two isoforms (Nagase, T. et al. (2001) DNA Res. 8:85-95), is activated by the beta-catenin/Tcf complex in hepatic cell lines, and may be involved in hepatic carcinogenesis (Kato, T. et al. (2001). Neoplasia 3:4-9).

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, has 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 GM. 1996 Cell 86:521-529; Wassarman DA, et al., 1995 Curr Opin Gen Dev 5:44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18:261-284). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. 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 the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as p53, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.

All references cited herein, including sequence information in referenced Genbank identifier numbers and website references, are incorporated herein in their entireties.

SUMMARY OF THE INVENTION

We have discovered genes that modify the p53 pathway in Drosophila, and identified their human orthologs, hereinafter referred to as MARK. The invention provides methods for utilizing these p53 modifier genes and polypeptides to identify candidate therapeutic agents that can be used in the treatment of disorders associated with defective p53 function. Preferred MARK-modulating agents specifically bind to MARK polypeptides and restore p53 function. Other preferred MARK-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MARK gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).

MARK-specific modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with a MARK polypeptide or nucleic acid. In one embodiment, candidate p53 modulating agents are tested with an assay system comprising a MARK polypeptide or nucleic acid. Candidate agents that produce a change in the activity of the assay system relative to controls are identified as candidate p53 modulating agents. The assay system may be cell-based or cell-free. MARK-modulating agents include MARK related proteins (e.g. dominant negative mutants, and biotherapeutics); MARK-specific antibodies; MARK-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind MARK or compete with MARK binding target. In one specific embodiment, a small molecule modulator is identified using a kinase 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.

In another embodiment, candidate p53 pathway modulating agents are further tested using a second assay system that detects changes in the p53 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 p53 pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).

The invention further provides methods for modulating the p53 pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MARK 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 the p53 pathway.

DETAILED DESCRIPTION OF THE INVENTION

Genetic screens were designed to identify modifiers of the p53 pathway in Drosophila in which p53 was overexpressed in the wing (Ollmann M, et al., Cell 2000 101:91-101). The KP78a gene was identified as a modifier of the p53 pathway. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, microtubule affinity regulator kinase (MARK) genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective p53 signaling pathway, such as cancer.

In vitro and in vivo methods of assessing MARK function are provided herein. Modulation of the MARK or their respective binding partners is useful for understanding the association of the p53 pathway and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for p53 related pathologies. MARK-modulating agents that act by inhibiting or enhancing MARK expression, directly or indirectly, for example, by affecting a MARK function such as enzymatic (e.g., catalytic) or binding activity, can be identified using methods provided herein. MARK modulating agents are useful in diagnosis, therapy and pharmaceutical development.

Nucleic Acids and Polypeptides of the Invention

Sequences related to MARK nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) number) as GI#s 9845486 (SEQ ID NO:1), 9845488 (SEQ ID NO:2), 18578044 (SEQ ID NO:3), 14250621 (SEQ ID NO:6), 15042610 (SEQ ID NO:7), 8923921 (SEQ ID NO:8), 17445805 (SEQ ID NO:9), 7959214 (SEQ ID NO:11), 14042208 (SEQ ED NO:12), 3089348 (SEQ ID NO:13), 4505102 (SEQ ID NO:14), 5714635 (SEQ ID NO:15), 18448970 (SEQ ID NO:18), 13366083 (SEQ ID NO:19), 14017936 (SEQ ID NO:22), and 16555377 (SEQ ID NO:23) for nucleic acid, and GI#s 9845487 (SEQ ID NO:24), 8923922 (SEQ ID NO:25), 3089349 (SEQ ID NO:26), 4505103 (SEQ ID NO:27), 13366084 (SEQ ID NO:28) and 13899225 (SEQ ID NO:29) for polypeptides. Additionally, nucleic acid sequences of SEQ ID NOs:4, 5, 16, 17, 20, 21, and novel nucleic acid sequence of SEQ ID NO:10 can also be used in the invention.

MARKs are kinase proteins with kinase and UBA/TS-N domains. The term “MARK polypeptide” refers to a full-length MARK protein or a functionally active fragment or derivative thereof. A “functionally active” MARK fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type MARK protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind natural cellular substrates, etc. The functional activity of MARK 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, New Jersey) and as further discussed below. For purposes herein, functionally active fragments also include those fragments that comprise one or more structural domains of a MARK, such as a kinase domain or a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2; http:I/pfam.wustl.edu). For example, the proten kinase domains of MARKs from GI#s 9845487 (SEQ ID NO:24), 8923922 (SEQ ID NO:25), 4505103 (SEQ ID NO:27), and 13899225 (SEQ ID NO:29) is located at approximately amino acid residues 20 to 271, 60 to 311, 56 to 307, and 59 to 310, respectively (PFAM 00069). Further, the ubiquitin associated (UBA/TS-N) domains of MARKs from GI#s 9845487 (SEQ ID NO:24), 8923922 (SEQ ID NO:25), 4505103 (SEQ ID NO:27and 13899225 (SEQ ID NO:29) is located at approximately amino acid residues 291 to 330, 331 to 370, 327 to 366, and 330 to 369, respectively (PFAM 00627). Methods for obtaining MARK 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 any one of SEQ ID NOs:24, 25, 26, 27, 28, or 29 (a MARK). In further preferred embodiments, the fragment comprises the entire kinase (functionally active) domain.

The term “MARK nucleic acid” refers to a DNA or RNA molecule that encodes a MARK polypeptide. Preferably, the MARK 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 MARK. 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 speciation, 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; http://blast.wustl.edufblast/README.html) 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.

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.

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 http:Hlwww.ebi.ac.uk/MPsrch/; 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. 0. 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.”

Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of any of SEQ ID NOs:1 through 23. 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 any one of SEQ ID NOs:1 through 23 under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6X single strength citrate (SSC) (1X SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X 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 6X SSC, 1X 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 0.2X SSC and 0.1% SDS (sodium dodecyl sulfate).

In other embodiments, moderately stringent hybridization conditions are used that comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5X SSC, 50 mM Tris—HCl (pH7.5), 5mM EDTA, 0.1% PVP, 0.1 % Ficoll, 1 % BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20h at 40° C. in a solution containing 35% formamide, 5X SSC, 50 mM Tris—HCl (pH7.5), SmM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 lkg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C. in a solution containing 2X SSC and 0.1% SDS.

Alternatively, low stringency conditions can be used that comprise:incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5X 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.

Isolation, Production. Expression, and Mis-expression of MARK Nucleic Acids and Polypeptides

MARK nucleic acids and polypeptides, useful for identifying and testing agents that modulate MARK function and for other applications related to the involvement of MARK in the p53 pathway. MARK 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 a MARK protein for assays used to assess MARK 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, 2 ^ndedition, 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 MARK is expressed in a cell line known to have defective p53 function (e.g. SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29 and DLD-1 colon cancer cells, among others, available from American Type Culture Collection (ATCC), Manassas, Va.). The recombinant cells are used in cell-based screening assay systems of the invention, as described further below.

The nucleotide sequence encoding a MARK polypeptide can be inserted into any appropriate expression vector. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native MARK 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. A host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.

To detect expression of the MARK gene product, the expression vector can comprise a promoter operably linked to a MARK 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 MARK gene product based on the physical or functional properties of the MARK protein in in vitro assay systems (e.g. immunoassays).

The MARK 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).

Once a recombinant cell that expresses the MARK 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, cite purification reference). Alternatively, native MARK 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.

The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of MARK or other genes associated with the p53 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).

Genetically Modified Animals

Animal models that have been genetically modified to alter MARK expression may be used in in vivo assays to test for activity of a candidate p53 modulating agent, or to further assess the role of MARK in a p53 pathway process such as apoptosis or cell proliferation. Preferably, the altered MARK expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal MARK expression. The genetically modified animal may additionally have altered p53 expression (e.g. p53 knockout). Preferred genetically modified animals are mammals such as primates, rodents (preferably mice), cows, horses, goats, sheep, pigs, dogs and cats. 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.

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).

In one embodiment, the transgenic animal is a “knock-out” animal having a heterozygous or homozygous alteration in the sequence of an endogenous MARK gene that results in a decrease of MARK function, preferably such that MARK 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 MARK gene is used to construct a homologous recombination vector suitable for altering an endogenous MARK 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).

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 MARK gene, e.g., by introduction of additional copies of MARK, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MARK gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. The knock-in can be homozygous or heterozygous.

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).

The genetically modified animals can be used in genetic studies to further elucidate the p53 pathway, as animal models of disease and disorders implicating defective p53 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 MARK function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MARK expression that receive candidate therapeutic agent.

In addition to the above-described genetically modified animals having altered MARK function, animal models having defective p53 function (and otherwise normal MARK function), can be used in the methods of the present invention. For example, a p53 knockout mouse can be used to assess, in vivo, the activity of a candidate p53 modulating agent identified in one of the in vitro assays described below. p53 knockout mice are described in the literature (Jacks et al., Nature 2001;410:1111-1116, 1043-1044; Donehower et al., supra). Preferably, the candidate p53 modulating agent when administered to a model system with cells defective in p53 function, produces a detectable phenotypic change in the model system indicating that the p53 function is restored, i.e., the cells exhibit normal cell cycle progression.

Modulating Agents

The invention provides methods to identify agents that interact with and/or modulate the function of MARK and/or the p53 pathway. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the p53 pathway, as well as in further analysis of the MARK protein and its contribution to the p53 pathway. Accordingly, the invention also provides methods for modulating the p53 pathway comprising the step of specifically modulating MARK activity by administering a MARK-interacting or -modulating agent.

In a preferred embodiment, MARK-modulating agents inhibit or enhance MARK activity or otherwise affect normal MARK function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a further preferred embodiment, the candidate p53 pathway—modulating agent specifically modulates the function of the MARK. The phrases “specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the MARK polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the MARK. The term also encompasses modulating agents that alter the interaction of the MARK with a binding partner or substrate (e.g. by binding to a binding partner of a MARK, or to a protein/binding partner complex, and inhibiting function).

Preferred MARK-modulating agents include small molecule compounds; MARK-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., 19 ^thedition.

Small Molecule Modulators

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 less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500. 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 MARK 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 MARK-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).

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 p53 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.

Protein Modulators

Specific MARK-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the p53 pathway and related disorders, as well as in validation assays for other MARK-modulating agents. In a preferred embodiment, MARK-interacting proteins affect normal MARK function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, MARK-interacting proteins are useful in detecting and providing information about the function of MARK proteins, as is relevant to p53 related disorders, such as cancer (e.g., for diagnostic means).

An MARK-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with a MARK, such as a member of the MARK pathway that modulates MARK expression, localization, and/or activity. MARK-modulators include dominant negative forms of MARK-interacting proteins and of MARK proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous MARK-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 SF et al., Gene (2000) 250:1-14; Drees BL 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 3 ^rd, Trends Genet (2000) 16:5-8).

An MARK-interacting protein may be an exogenous protein, such as a MARK-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). MARK antibodies are further discussed below.

In preferred embodiments, a MARK-interacting protein specifically binds a MARK protein. In alternative preferred embodiments, a MARK-modulating agent binds a MARK substrate, binding partner, or cofactor.

Antibodies

In another embodiment, the protein modulator is a MARK specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MARK modulators. The antibodies can also be used in dissecting the portions of the MARK pathway responsible for various cellular responses and in the general processing and maturation of the MARK.

Antibodies that specifically bind MARK polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of MARK polypeptide, and more preferably, to human MARK. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′).sub.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 MARK which are particularly antigenic can be selected, for example, by routine screening of MARK 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 shown in any of SEQ ID NOs:24, 25, 26, 27, 28, or 29. Monoclonal antibodies with affinities of 10 ⁸M⁻¹preferably 10⁹M⁻¹to 10¹⁰M⁻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 MARK or substantially purified fragments thereof. If MARK fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of a MARK protein. In a particular embodiment, MARK-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.

The presence of MARK-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MARK polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.

Chimeric antibodies specific to MARK 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-detenmining 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 MS, 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).

MARK-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).

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).

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).

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 w 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).

Nucleic Acid Modulators

Other preferred MARK-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit MARK activity. Preferred nucleic acid modulators interfere with the function of the MARK nucleic acid such as DNA replication, transcription, translocation of the MARK RNA to the site of protein translation, translation of protein from the MARK RNA, splicing of the MARK RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the MARK RNA.

In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to a MARK mRNA to bind to and prevent translation, preferably by binding to the 5′ untranslated region. MARK-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.

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. Nos. 5,235,033; and 5,378,841).

Alternative preferred MARK 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).

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 JF, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36:1923-1937; Tonkinson JL et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the invention, a MARK-specific nucleic acid modulator is used in an assay to further elucidate the role of the MARK in the p53 pathway, and/or its relationship to other members of the pathway. In another aspect of the invention, a MARK-specific antisense oligomer is used as a therapeutic agent for treatment of p53-related disease states.

Assay Systems

The invention provides assay systems and screening methods for identifying specific modulators of MARK 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 MARK nucleic acid or protein. In general, secondary assays further assess the activity of a MARK modulating agent identified by a primary assay and may confirm that the modulating agent affects MARK in a manner relevant to the p53 pathway. In some cases, MARK modulators will be directly tested in a secondary assay.

In a preferred embodiment, the screening method comprises contacting a suitable assay system comprising a MARK polypeptide with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. kinase 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 MARK activity, and hence the p53 pathway.

Primary Assays

The type of modulator tested generally determines the type of primary assay.

Primary Assays for Small Molecule Modulators

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 GS 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, immunogenicty and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, colorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.

Cell-based screening assays usually require systems for recombinant expression of MARK 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 MARK-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the MARK protein may be assayed by various known methods such as substrate processing (e.g. ability of the candidate MARK-specific binding agents to function as negative effectors in MARK-expressing cells), binding equilibrium constants (usually at least about 10 ⁷M⁻¹, preferably at least about 10⁸M⁻¹, more preferably at least about 10⁹M⁻¹), and immunogenicity (e.g. ability to elicit MARK 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.

The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a MARK polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The MARK polypeptide can be full length or a fragment thereof that retains functional MARK activity. The MARK polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The MARK polypeptide is preferably human MARK, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of MARK interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MARK-specific binding activity, and can be used to assess normal MARK gene function.

Suitable assay formats that may be adapted to screen for MARK 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).

A variety of suitable assay systems may be used to identify candidate MARK and p53 pathway modulators (e.g. U.S. Pat. No. 6,165,992 (kinase assays); U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosis assays); U.S. Pat. No. 6,020,135 (p53 modulation), among others). Specific preferred assays are described in more detail below.

Kinase assays. In some preferred embodiments the screening assay detects the ability of the test agent to modulate the kinase activity of a MARK polypeptide. In further embodiments, a cell-free kinase assay system is used to identify a candidate p53 modulating agent, and a secondary, cell-based assay, such as an apoptosis or hypoxic induction assay (described below), may be used to further characterize the candidate p53 modulating agent. Many different assays for kinases have been reported in the literature and are well known to those skilled in the art (e.g. U.S. Pat. No. 6,165,992; Zhu et al., Nature Genetics (2000) 26:283-289; and WO0073469). Radioassays, which monitor the transfer of a gamma phosphate are frequently used. For instance, a scintillation assay for p56 (lck) kinase activity monitors the transfer of the gamma phosphate from gamma— ³³P ATP to a biotinylated peptide substrate; the substrate is captured on a streptavidin coated bead that transmits the signal (Beveridge M et al., J Biomol Screen (2000) 5:205-212). This assay uses the scintillation proximity assay (SPA), in which only radio-ligand bound to receptors tethered to the surface of an SPA bead are detected by the scintillant immobilized within it, allowing binding to be measured without separation of bound from free ligand.

Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates. For instance, the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity by ligand stimulating the intact receptor in cultured cells, then capturing solubilized receptor with specific antibodies and quantifying phosphorylation via phosphotyrosine ELISA (Sadick M D, Dev Biol Stand (1999) 97:121-133).

Another example of antibody based assays for protein kinase activity is TRF (time-resolved fluorometry). This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence (Braunwalder AF, et al., Anal Biochem Jul. 1, 1996; 238(2):159-64).

Apoptosis assays. 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). An apoptosis assay system may comprise a cell that expresses a MARK, and that optionally has defective p53 function (e.g. p53 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 p53 modulating agents. In some embodiments of the invention, an apoptosis assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using a cell-free assay system. An apoptosis assay may also be used to test whether MARK function plays a direct role in apoptosis. For example, an apoptosis assay may be performed on cells that over- or under-express MARK relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the MARK plays a direct role in the apoptotic response. Apoptosis assays are described further in U.S. Pat. No. 6,133,437.

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.

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 L S 3800 Liquid Scintillation Counter).

Cell proliferation may also be assayed by colony formation in soft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). For example, cells transformed with MARK are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.

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 a MARK may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson).

Accordingly, a cell proliferation or cell cycle assay system may comprise a cell that expresses a MARK, and that optionally has defective p53 function (e.g. p53 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 p53 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 p53 modulating agents that is initially identified using another assay system such as a cell-free kinase assay system. A cell proliferation assay may also be used to test whether MARK 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 MARK relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the MARK plays a direct role in cell proliferation or cell cycle.

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 a MARK, and that optionally has defective p53 function (e.g. p53 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 p53 modulating agents. In some embodiments of the invention, the angiogenesis assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using another assay system. An angiogenesis assay may also be used to test whether MARK function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over- or under-express MARK relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the MARK plays a direct role in angiogenesis.

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 glyolytic enzymes and VEGF. Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with MARK 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 a MARK, and that optionally has a mutated p53 (e.g. p53 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 p53 modulating agents. In some embodiments of the invention, the hypoxic induction assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using another assay system. A hypoxic induction assay may also be used to test whether MARK 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 MARK relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the MARK plays a direct role in hypoxic induction.

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.

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.

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. May-Jun. 12, 2001 (3):346-53).

Primary Assays for Antibody Modulators

For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the MARK 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 MARK-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.

Primary Assays for Nucleic Acid Modulators

For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance MARK gene expression, preferably mRNA expression. In general, expression analysis comprises comparing MARK expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express MARK) 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 MARK 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 D H 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 MARK 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).

Secondary Assays

Secondary assays may be used to further assess the activity of MARK-modulating agent identified by any of the above methods to confirm that the modulating agent affects MARK in a manner relevant to the p53 pathway. As used herein, MARK-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 MARK.

Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express MARK) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate MARK-modulating agent results in changes in the p53 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 p53 or interacting pathways.

Cell-Based Assays

Cell based assays may use a variety of mammalian cell lines known to have defective p53 function (e.g. SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT -29 and DLD-1 colon cancer cells, among others, available from American Type Culture Collection (ATCC), Manassas, Va.). Cell based assays may detect endogenous p53 pathway activity or may rely on recombinant expression of p53 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

A variety of non-human animal models of normal or defective p53 pathway may be used to test candidate MARK modulators. Models for defective p53 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 p53 pathway. Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.

In a preferred embodiment, p53 pathway activity is assessed by monitoring neovascularization and angiogenesis. Animal models with defective and normal p53 are used to test the candidate modulator's affect on MARK 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 MARK. 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.

In another preferred embodiment, the effect of the candidate modulator on MARK is assessed via tumorigenicity assays. In one example, xenograft human tumors are 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 MARK endogenously are injected in the flank, 1×10 ⁵to 1×10⁷cells 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 maybe 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.

Diagnostic and Therapeutic Uses

Specific MARK-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the p53 pathway, such as angiogenic, apoptotic, or cell proliferation disorders. Accordingly, the invention also provides methods for modulating the p53 pathway in a cell, preferably a cell pre-determnined to have defective p53 function, comprising the step of administering an agent to the cell that specifically modulates MARK activity. Preferably, the modulating agent produces a detectable phenotypic change in the cell indicating that the p53 function is restored, i.e., for example, the cell undergoes normal proliferation or progression through the cell cycle.

The discovery that MARK is implicated in p53 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 p53 pathway and for the identification of subjects having a predisposition to such diseases and disorders.

Various expression analysis methods can be used to diagnose whether MARK 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 p53 signaling that express a MARK, are identified as amenable to treatment with a MARK modulating agent. In a preferred application, the p53 defective tissue overexpresses a MARK 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 MARK cDNA sequences as probes, can determine whether particular tumors express or overexpress MARK. Alternatively, the TaqMane is used for quantitative RT-PCR analysis of MARK expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).

Various other diagnostic methods may be performed, for example, utilizing reagents such as the MARK oligonucleotides, and antibodies directed against a MARK, as described above for:(1) the detection of the presence of MARK gene mutations, or the detection of either over- or under-expression of MARK niRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of MARK gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by MARK.

Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease in a patient, the method comprising:a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MARK expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of disease. 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

The following experimental section and examples are offered by way of illustration and not by way of limitation. [0113]
I. Drosophila p53 Screen [0114]
The Drosophila p53 gene was overexpressed specifically in the wing using the vestigial margin quadrant enhancer. Increasing quantities of Drosophila p53 (titrated using different strength transgenic inserts in 1 or 2 copies) caused deterioration of normal wing morphology from mild to strong, with phenotypes including disruption of pattern and polarity of wing hairs, shortening and thickening of wing veins, progressive crumpling of the wing and appearance of dark “death” inclusions in wing blade. In a screen designed to identify enhancers and suppressors of Drosophila p53, homozygous females carrying two copies of p53 were crossed to 5663 males carrying random insertions of a piggyBac transposon (Fraser M et al., Virology (1985) 145:356-361). Progeny containing insertions were compared to non-insertion-bearing sibling progeny for enhancement or suppression of the p53 phenotypes. Sequence information surrounding the piggyBac insertion site was used to identify the modifier genes. Modifiers of the wing phenotype were identified as members of the p53 pathway. kp78a was a suppressor of the wing phenotype. Human orthologs of the modifiers are referred to herein as MARK. [0115]
BLAST analysis (Altschul et al., supra) was employed to identify Targets from Drosophila modifiers. For example, representative sequences from MARK GI# 9845487 (SEQ ID NO:24), GI# 8923922(SEQ ID NO:25), GI# 4505103 (SEQ ID NO:27), and GI#13899225 (SEQ ID NO:29) share 43%, 65%, 65% and 45% amino acid identity, respectively, with the Drosophila kp78a. [0116]
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; http://pfam.wustl.edu), SMART (Ponting CP, 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. Nov. 10, 2000(11):1679-89) programs. For example, the proten kinase domains of MARKs from GI#s 9845487 (SEQ ID NO:24), 8923922 (SEQ ID NO:25), 4505103 (SEQ ID NO:27), and 13899225 (SEQ ID NO:29) is located at approximately amino acid residues 20 to 271, 60 to 311, 56 to 307, and 59 to 310, respectively (PFAM 00069). Further, the ubiquitin associated (UBA/TS-N) domains of MARKs from GI#s 9845487 (SEQ ID NO:24), 8923922 (SEQ ID NO:25), 4505103 (SEQ ID NO:27), and 13899225 (SEQ ID NO:29) is located at approximately amino acid residues 291 to 330, 331 to 370, 327 to 366, and 330 to 369, respectively (PFAM 00627). Still further, the kinase associated domains from MARKs of GI#s9845487 (SEQ ID NO:24), 8923922 (SEQ ID NO:25), and 4505103 (SEQ ID NO:27) are located at approximately amino acid residues 696 to 745, 746 to 795, and 664 to 713, respectively (PFAM 02149). [0117]
II. High-Throughput In Vitro Fluorescence Polarization Assay [0118]
Fluorescently-labeled MARK 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 MARK activity. [0119]
III. High-Throughput In Vitro Binding Assay. [0120]
[0121] ³³P-labeled MARK peptide is added in an assay buffer (100 mM KC1, 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 p53 modulating agents.
IV. Immunoprecipitations and Immunoblotting [0122]
For coprecipitation of transfected proteins, 3×10[0123] ⁶appropriate recombinant cells containing the MARK 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.
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). [0124]
V. Kinase Assay [0125]
A purified or partially purified MARK is diluted in a suitable reaction buffer, e.g., 50 mM Hepes, pH 7.5, containing magnesium chloride or manganese chloride (1-20 mM) and a peptide or polypeptide substrate, such as myelin basic protein or casein (1-10 μug/ml). The final concentration of the kinase is 1-20 nM. The enzyme reaction is conducted in microtiter plates to facilitate optimization of reaction conditions by increasing assay throughput. A 96-well microtiter plate is employed using a final volume 30-100 μl. The reaction is initiated by the addition of [0126] ³³P-gamma-ATP (0.5 μCi/ml) and incubated for 0.5 to 3 hours at room temperature. Negative controls are provided by the addition of EDTA, which chelates the divalent cation (Mg2⁺ or Mn²⁺) required for enzymatic activity. Following the incubation, the enzyme reaction is quenched using EDTA. Samples of the reaction are transferred to a 96-well glass fiber filter plate (MultiScreen, Millipore). The filters are subsequently washed with phosphate-buffered saline, dilute phosphoric acid (0.5%) or other suitable medium to remove excess radiolabeled ATP. Scintillation cocktail is added to the filter plate and the incorporated radioactivity is quantitated by scintillation counting (Wallac/Perkin Elmer). Activity is defined by the amount of radioactivity detected following subtraction of the negative control reaction value (EDTA quench).
VI. Expression Analysis [0127]
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, and Ambion. [0128]
TaqMan analysis was used to assess expression levels of the disclosed genes in various samples. [0129]
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 4304965 of Applied Biosystems (Foster City, Calif., http://www.appliedbiosvstems.com/). [0130]
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. [0131]
Taqman reactions were carried out following manufacturer's protocols, in 25 pll 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). [0132]
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. [0133]

Results are shown in Table 1. Data presented in bold indicate that greater than 50% of tested tumor samples of the tissue type indicated in row 1 exhibited over expression of the gene listed in column 1, relative to normal samples. Underlined data indicates that between 25% to 49% of tested tumor samples exhibited over expression. 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 1


—	breast	.	.	colon	.	.	lung	.	.	ovary

GI#9845486 (SEQ ID NO: 1)	7	11	.	8	30	.	8	13	.	5	7
GI#9845488 (SEQ ID NO: 2)	1	11	.	4	30	.	0	13	.	1	7
GI#8923921 (SEQ ID NO: 8)	2	11	.	7	30	.	6	13	.	0	7
GI#3089348 (SEQ ID NO: 13)	2	11	.	2	30	.	0	13	.	2	7
GI#13366083 (SEQ ID NO: 19)	2	11	.	2	30	.	5	13	.	1	7

[0135]
1 29 1 2946 DNA Homo sapiens 1 tcctggaatt gcacgcgctt cctgaccacc aggctctggc ccttgagaag ccagcggggc 60 tttgtccctg ttgctctcct tgccaaaccc agtctctctg ctagtggtgg tttcggttgc 120 gacaccgtcc aggttcccag gcaggaaccg ctcggcctgg ctgcttagct acttttcact 180 gaggaggtgg tggaaggtgt cgcctgctct ggctgagtaa gggtggctgg ctgagccggc 240 agcccccgcc ctaggcctgg ctcttcccgg cctctgtact ttgccctcgc tgcctgacag 300 gttctgctgt gggctctgct gaatggaagt cgctggtagt ccttttccct ttctccagtc 360 ggcccacctt gggacacctt gactccaagc ccagcagtaa gtccaacatg attcggggcc 420 gcaactcagc cacctctgct gatgagcagc cccacattgg aaactaccgg ctcctcaaga 480 ccattggcaa gggtaatttt gccaaggtga agttggcccg acacatcctg actgggaaag 540 aggtagctgt gaagatcatt gacaagactc aactgaactc ctccagcctc cagaaactat 600 tccgcgaagt aagaataatg aaggttttga atcatcccaa catagttaaa ttatttgaag 660 tgattgagac tgagaaaacg ctctaccttg tcatggagta cgctagtggc ggagaggtat 720 ttgattacct agtggctcat ggcaggatga aagaaaaaga ggctcgagcc aaattccgcc 780 agatagtgtc tgctgtgcag tactgtcacc agaagtttat tgtccataga gacttaaagg 840 cagaaaacct gctcttggat gctgatatga acatcaagat tgcagacttt ggcttcagca 900 atgaattcac ctttgggaac aagctggaca ccttctgtgg cagtccccct tatgctgccc 960 cagaactctt ccagggcaaa aaatatgatg gacccgaggt ggatgtgtgg agcctaggag 1020 ttatcctcta tacactggtc agcggatccc tgccttttga tggacagaac ctcaaggagc 1080 tgcgggaacg ggtactgagg gggaaatacc gtattccatt ctacatgtcc acggactgtg 1140 aaaacctgct taagaaattt ctcatcctta atcccagcaa gagaggcact ttagagcaaa 1200 tcatgaaaga tcgatggatg aatgtgggtc acgaagatga tgaactaaag ccttacgtgg 1260 agccactccc tgactacaag gacccccggc ggacagagct gatggtgtcc atgggttata 1320 cacgggaaga gatccaggac tcgctggtgg gccagagata caacgaggtg atggccacct 1380 atctgctcct gggctacaag agctccgagc tggaaggcga caccatcacc ctgaaacccc 1440 ggccttcagc tgatctaacc aatagcagcg cccaattccc atcccacaag gtacagcgaa 1500 gcgtgtcggc caatcccaag cagcggcgct tcagcgacca ggctggtcct gccattccca 1560 cctctaattc ttactctaag aagactcaga gtaacaacgc agaaaataag cggcctgagg 1620 aggaccggga gtcagggcgg aaagccagca gcacagccaa ggtgcctgcc agccccctgc 1680 ccggtctgga gaggaagaag accaccccaa ccccctccac gaacagcgtc ctctccacca 1740 gcacaaatcg aagcaggaat tccccacttt tggagcgggc cagcctcggc caggcctcca 1800 tccagaatgg caaagacagc ctaaccatgc cagggtcccg ggcctccacg gcttctgctt 1860 ctgccgcagt ctctgcggcc cggccccgcc agcaccagaa atccatgtcg gcctccgtgc 1920 accccaacaa ggcctctggg ctgcccccca cggagagtaa ctgtgaggtg ccgcggccca 1980 gcacagcccc ccagcgtgtc cctgttgcct ccccatccgc ccacaacatc agcagcagtg 2040 gtggagcccc agaccgaact aacttccccc ggggtgtgtc cagccgaagc accttccatg 2100 ctgggcagct ccgacaggtg cgggaccagc agaatttgcc ctacggtgtg accccagcct 2160 ctccctctgg ccacagccag ggccggcggg gggcctctgg gagcatcttc agcaagttca 2220 cctccaagtt tgtacgcagg aacctgaatg aacctgaaag caaagaccga gtggagacgc 2280 tcagacctca cgtggtgggc agtggcggca acgacaaaga aaaggaagaa tttcgggagg 2340 ccaagccccg ctccctccgc ttcacgtgga gtatgaagac cacgagctcc atggagccca 2400 acgagatgat gcgggagatc cgcaaggtgc tggacgcgaa cagctgccag agcgagctgc 2460 atgagaagta catgctgctg tgcatgcacg gcacgccggg ccacgaggac ttcgtgcagt 2520 gggagatgga ggtgtgcaaa ctgccgcggc tctctctcaa cggggttcga tttaagcgga 2580 tatcgggcac ctccatggcc ttcaaaaaca ttgcctccaa aatagccaac gagctgaagc 2640 tttaacaggc tgccaggagc gggggcggcg ggggcgggcc agctggacgg gctgccggcc 2700 gtgcgccgcc ccacctgggc gagactgcag cgatggattg gtgtgtctcc ctgctggcac 2760 ttctcccctc cctggccctt ctcagttttc tcccacattc acccctgccc agagattccc 2820 ccttctcctc tcccctactg gaggcaaagg aaggggaggg tggatggggg ggcagggctc 2880 cccctcggta ctgcggttgc acagagtatt tcgcctaaac caagaaattt tttattacca 2940 aaaaga 2946 2 2784 DNA Homo sapiens 2 tcctggaatt gcacgcgctt cctgaccacc aggctctggc ccttgagaag ccagcggggc 60 tttgtccctg ttgctctcct tgccaaaccc agtctctctg ctagtggtgg tttcggttgc 120 gacaccgtcc aggttcccag gcaggaaccg ctcggcctgg ctgcttagct acttttcact 180 gaggaggtgg tggaaggtgt cgcctgctct ggctgagtaa gggtggctgg ctgagccggc 240 agcccccgcc ctaggcctgg ctcttcccgg cctctgtact ttgccctcgc tgcctgacag 300 gttctgctgt gggctctgct gaatggaagt cgctggtagt ccttttccct ttctccagtc 360 ggcccacctt gggacacctt gactccaagc ccagcagtaa gtccaacatg attcggggcc 420 gcaactcagc cacctctgct gatgagcagc cccacattgg aaactaccgg ctcctcaaga 480 ccattggcaa gggtaatttt gccaaggtga agttggcccg acacatcctg actgggaaag 540 aggtagctgt gaagatcatt gacaagactc aactgaactc ctccagcctc cagaaactat 600 tccgcgaagt aagaataatg aaggttttga atcatcccaa catagttaaa ttatttgaag 660 tgattgagac tgagaaaacg ctctaccttg tcatggagta cgctagtggc ggagaggtat 720 ttgattacct agtggctcat ggcaggatga aagaaaaaga ggctcgagcc aaattccgcc 780 agatagtgtc tgctgtgcag tactgtcacc agaagtttat tgtccataga gacttaaagg 840 cagaaaacct gctcttggat gctgatatga acatcaagat tgcagacttt ggcttcagca 900 atgaattcac ctttgggaac aagctggaca ccttctgtgg cagtccccct tatgctgccc 960 cagaactctt ccagggcaaa aaatatgatg gacccgaggt ggatgtgtgg agcctaggag 1020 ttatcctcta tacactggtc agcggatccc tgccttttga tggacagaac ctcaaggagc 1080 tgcgggaacg ggtactgagg gggaaatacc gtattccatt ctacatgtcc acggactgtg 1140 aaaacctgct taagaaattt ctcatcctta atcccagcaa gagaggcact ttagagcaaa 1200 tcatgaaaga tcgatggatg aatgtgggtc acgaagatga tgaactaaag ccttacgtgg 1260 agccactccc tgactacaag gacccccggc ggacagagct gatggtgtcc atgggttata 1320 cacgggaaga gatccaggac tcgctggtgg gccagagata caacgaggtg atggccacct 1380 atctgctcct gggctacaag agctccgagc tggaaggcga caccatcacc ctgaaacccc 1440 ggccttcagc tgatctaacc aatagcagcg cccaattccc atcccacaag gtacagcgaa 1500 gcgtgtcggc caatcccaag cagcggcgct tcagcgacca ggctggtcct gccattccca 1560 cctctaattc ttactctaag aagactcaga gtaacaacgc agaaaataag cggcctgagg 1620 aggaccggga gtcagggcgg aaagccagca gcacagccaa ggtgcctgcc agccccctgc 1680 ccggtctgga gaggaagaag accaccccaa ccccctccac gaacagcgtc ctctccacca 1740 gcacaaatcg aagcaggaat tccccacttt tggagcgggc cagcctcggc caggcctcca 1800 tccagaatgg caaagacagc acagcccccc agcgtgtccc tgttgcctcc ccatccgccc 1860 acaacatcag cagcagtggt ggagccccag accgaactaa cttcccccgg ggtgtgtcca 1920 gccgaagcac cttccatgct gggcagctcc gacaggtgcg ggaccagcag aatttgccct 1980 acggtgtgac cccagcctct ccctctggcc acagccaggg ccggcggggg gcctctggga 2040 gcatcttcag caagttcacc tccaagtttg tacgcaggaa cctgaatgaa cctgaaagca 2100 aagaccgagt ggagacgctc agacctcacg tggtgggcag tggcggcaac gacaaagaaa 2160 aggaagaatt tcgggaggcc aagccccgct ccctccgctt cacgtggagt atgaagacca 2220 cgagctccat ggagcccaac gagatgatgc gggagatccg caaggtgctg gacgcgaaca 2280 gctgccagag cgagctgcat gagaagtaca tgctgctgtg catgcacggc acgccgggcc 2340 acgaggactt cgtgcagtgg gagatggagg tgtgcaaact gccgcggctc tctctcaacg 2400 gggttcgatt taagcggata tcgggcacct ccatggcctt caaaaacatt gcctccaaaa 2460 tagccaacga gctgaagctt taacaggctg ccaggagcgg gggcggcggg ggcgggccag 2520 ctggacgggc tgccggccgt gcgccgcccc acctgggcga gactgcagcg atggattggt 2580 gtgtctccct gctggcactt ctcccctccc tggcccttct cagttttctc ccacattcac 2640 ccctgcccag agattccccc ttctcctctc ccctactgga ggcaaaggaa ggggagggtg 2700 gatggggggg cagggctccc cctcggtact gcggttgcac agagtatttc gcctaaacca 2760 agaaattttt tattaccaaa aaga 2784 3 3103 DNA Homo sapiens misc_feature (2941)..(2941) “n” is A, C, G, or T 3 cggtggtggc ggccatgttg ggagcagcag gtccggcggc ggctgcctgt gtgccgggcg 60 cggagcagtg ccgctgaggg caggggagga gcgaggcagg cggccggctg cggcggcaga 120 gagtaggcgg agcggcgcgg cccggccgaa aggcggcaca gcccagccgg gggtcggggg 180 ggtgcggtcc ggagccgctc ggagccggcg cggcctagcc cgagcggcgc atccccgggc 240 tggcgtgagc ggctgcccgg cctccccgca cccccggccg gggcccatgc ggcgggtgct 300 cctgctgtga gaagccccgc ccggccgggc tccgcgcctt cccttccctc ccttcctcca 360 agcttctcgg ttccctcccc cgagataccg gcgccatgtc cagcgctcgg acccccctac 420 ccacgctgaa cgagagggac acggagcagc ccaccttggg acaccttgac tccaagccca 480 gcagtaagtc caacatgatt cggggccgca actcagccac ctctgctgat gagcagcccc 540 acattggaaa ctaccggctc ctcaagacca ttggcaaggg taattttgcc aaggtgaagt 600 tggcccgaca catcctgact gggaaagagg tagctgtgaa gatcattgac aagactcaac 660 tgaactcctc cagcctccag aaactattcc gcgaagtaag aataatgaag gttttgaatc 720 atcccaacat agttaaatta tttgaagtga ttgagactga gaaaacgctc taccttgtca 780 tggagtacgc tagtggcgga gaggtatttg attacctagt ggctcatggc aggatgaaag 840 aaaaagaggc tcgagccaaa ttccgccaga tagtgtctgc tgtgcagtac tgtcaccaga 900 agtttattgt ccatagagac ttaaaggcag aaaacctgct cttggatgct gatatgaaca 960 tcaagattgc agactttggc ttcagcaatg aattcacctt tgggaacaag ctggacacct 1020 tctgtggcag tcccccttat gctgccccag aactcttcca gggcaaaaaa tatgatggac 1080 ccgaggtgga tgtgtggagc ctaggagtta tcctctatac actggtcagc ggatccctgc 1140 cttttgatgg acagaacctc aaggagctgc gggaacgggt actgagggga aaataccgta 1200 ttccattcta catgtccacg gactgtgaaa acctgcttaa gaaatttctc attcttaatc 1260 ccagcaagag aggcacttta gagcaaatca tgaaagatcg atggatgaat gtgggtcacg 1320 aagatgatga actaaagcct tacgtggagc cactccctga ctacaaggac ccccggcgga 1380 cagagctgat ggtgtccatg ggttatacac gggaagagat ccaggactcg ctggtgggcc 1440 agagatacaa cgaggtgatg gccacctatc tgctcctggg ctacaagagc tccgagctgg 1500 aaggcgacac catcaccctg aaaccccggc cttcagctga tctgaccaat agcagcgccc 1560 catccccatc ccacaaggta cagcgcagcg tgtcggccaa tcccaagcag cggcgcttca 1620 gcgaccaggc tggtcctgcc attcccacct ctaattctta ctctaagaag actcagagta 1680 acaacgcaga aaataagcgg cctgaggagg accgggagtc agggcggaaa gccagcagca 1740 cagccaaggt gcctgccagc cccctgcccg gtctggagag gaagaagacc accccaaccc 1800 cctccacgga acagcgtcct ctccaccagc acaaatcgaa gcaggaattc cccacttttg 1860 gagcgggcca gcctcggcca ggcctccatc cagaatggca aagacagcct aaccatgcca 1920 gggtcccggg cctccacggc ttctgcttct gccgcagtct ctgcggcccg gccccgccag 1980 caccagaaat ccatgtcggc ctccgtgcac cccaacaagg cctctgggct gccccccacg 2040 gagagtaact gtgaggtgcc gcggcccagc acagcccccc agcgtgtccc tgttgcctcc 2100 ccatccgccc acaacatcag cagcagtggt ggagccccag accgaactaa cttcccccgg 2160 ggtgtgtcca gccgaagcac cttccatgct gggcagctcc gacaggtgcg ggaccagcag 2220 aatttgccct acggtgtgac cccagcctct ccctctggcc acagccaggg ccggcggggg 2280 gcctctggga gcatcttcag caagttcacc tccaagtttg tacgcagaaa tctgtctttc 2340 aggtttgcca gaaggaacct gaatgaacct gaaagcaaag accgagtgga gacgctcaga 2400 cctcacgtgg tgggcagtgg cggcaacgac aaagaaaagg aagaatttcg ggaggccaag 2460 ccccgctccc tccgcttcac gtggagtatg aagaccacga gctccatgga gcccaacgag 2520 atgatgcggg agatccgcaa ggtgctggac gcgaacagct gccagagcga gctgcatgag 2580 aagtacatgc tgctgtgcat gcacggcacg ccgggccacg aggacttcgt gcagtgggag 2640 atggaggtgt gcaaactgcc gcggctctct ctcaacgggg ttcgatttaa gcggatatcg 2700 ggcacctcca tggccttcaa aaacattgcc tccaaaatag ccaacgagct gaagctttaa 2760 caggctgcca ggagcggggg cggcgggggg cgggccagct ggacgggctg ccggccgctg 2820 cgccgcccca cctgggcgag actgcagcga tggattggtg tgtctcccct gctggcactt 2880 ctcccctccc tggcccttct cagttttctc ttacatgttt gtggggggtg ggagattgtt 2940 ntccagcacc ccacattcac ccctgcccag agattccccc ttctcctctc ccctactgga 3000 ggcaaaggaa ggggagggtg gatggggggg cagggctccc cctcggtact gcggttgcac 3060 agagtatttc gcctaaacca agaaattttt tattaccaaa aag 3103 4 2086 DNA Homo sapiens 4 agtccaacat gattcggggc cgcaactcag ccacctctgc tgatgagcag ccccacattg 60 gaaactaccg gctcctcaag accattggca agggtaattt tgccaaggtg aagttggccc 120 gacacatcct gactgggaaa gaggtagctg tgaagatcat tgacaagact caactgaact 180 cctccagcct ccagaaacta ttccgcgaag taagaataat gaaggttttg aatcatccca 240 acatagttaa attatttgaa gtgattgaga ctgagaaaac gctctacctt gtcatggagt 300 acgctagtgg cggagaggta tttgattacc tagtggctca tggcaggatg aaagaaaaag 360 aggctcgagc caaattccgc cagatagtgt ctgctgtgca gtactgtcac cagaagttta 420 ttgtccatag agacttaaag gcagaaaacc tgctcttgga tgctgatatg aacatcaaga 480 ttgcagactt tggcttcagc aatgaattca cctttgggaa caagctggac accttctgtg 540 gcagtccccc ttatgctgcc ccagaactct tccagggcaa aaaatatgat ggacccgagg 600 tggatgtgtg gagcctagga gttatcctct atacactggt cagcggatcc ctgccttttg 660 atggacagaa cctcaaggag ctgcgggaac gggtactgag gggaaaatac cgtattccat 720 tctacatgtc cacggactgt gaaaacctgc ttaagaaatt tctcattctt aatcccagca 780 agagaggcac tttagagcaa atcatgaaag atcgatggat gaatgtgggt cacgaagatg 840 atgaactaaa gccttacgtg gagccactcc ctgactacaa ggacccccgg cggacagagc 900 tgatggtgtc catgggttat acacgggaag agatccagga ctcgctggtg ggccagagat 960 acaacgaggt gatggccacc tatctgctcc tgggctacaa gagctccgag ctggaaggcg 1020 acaccatcac cctgaaaccc cggccttcag ctgatctgac caatagcagc gccccatccc 1080 catcccacaa ggtacagcgc agcgtgtcgg ccaatcccaa gcagcggcgc ttcagcgacc 1140 aggctggtcc tgccattccc acctctaatt cttactctaa gaagactcag agtaacaacg 1200 cagaaaataa gcggcctgag gaggaccggg agtcagggcg gaaagccagc agcacagcca 1260 aggtgcctgc cagccccctg cccggtctgg agaggaagaa gaccacccca accccctcca 1320 cgaacagcgt cctctccacc agcacaaatc gaagcaggaa ttccccactt ttggagcggg 1380 ccagcctcgg ccaggcctcc atccagaatg gcaaagacag cacagccccc cagcgtgtcc 1440 ctgttgcctc cccatccgcc cacaacatca gcagcagtgg tggagcccca gaccgaacta 1500 acttcccccg gggtgtgtcc agccgaagca ccttccatgc tgggcagctc cgacaggtgc 1560 gggaccagca gaatttgccc tacggtgtga ccccagcctc tccctctggc cacagccagg 1620 gccggcgggg ggcctctggg agcatcttca gcaagttcac ctccaagttt gtacgcagga 1680 acctgaatga acctgaaagc aaagaccgag tggagacgct cagacctcac gtggtgggca 1740 gtggcggcaa cgacaaagaa aaggaagaat ttcgggaggc caagccccgc tccctccgct 1800 tcacgtggag tatgaagacc acgagctcca tggagcccaa cgagatgatg cgggagatcc 1860 gcaaggtgct ggacgcgaac agctgccaga gcgagctgca tgagaagtac atgctgctgt 1920 gcatgcacgg cacgccgggc cacgaggact tcgtgcagtg ggagatggag gtgtgcaaac 1980 tgccgcggct ctctctcaac ggggttcgat ttaagcggat atcgggcacc tccatggcct 2040 tcaaaaacat tgcctccaaa atagccaacg agctgaagct ttaaca 2086 5 2248 DNA Homo sapiens 5 agtccaacat gattcggggc cgcaactcag ccacctctgc tgatgagcag ccccacattg 60 gaaactaccg gctcctcaag accattggca agggtaattt tgccaaggtg aagttggccc 120 gacacatcct gactgggaaa gaggtagctg tgaagatcat tgacaagact caactgaact 180 cctccagcct ccagaaacta ttccgcgaag taagaataat gaaggttttg aatcatccca 240 acatagttaa attatttgaa gtgattgaga ctgagaaaac gctctacctt gtcatggagt 300 acgctagtgg cggagaggta tttgattacc tagtggctca tggcaggatg aaagaaaaag 360 aggctcgagc caaattccgc cagatagtgt ctgctgtgca gtactgtcac cagaagttta 420 ttgtccatag agacttaaag gcagaaaacc tgctcttgga tgctgatatg aacatcaaga 480 ttgcagactt tggcttcagc aatgaattca cctttgggaa caagctggac accttctgtg 540 gcagtccccc ttatgctgcc ccagaactct tccagggcaa aaaatatgat ggacccgagg 600 tggatgtgtg gagcctagga gttatcctct atacactggt cagcggatcc ctgccttttg 660 atggacagaa cctcaaggag ctgcgggaac gggtactgag gggaaaatac cgtattccat 720 tctacatgtc cacggactgt gaaaacctgc ttaagaaatt tctcattctt aatcccagca 780 agagaggcac tttagagcaa atcatgaaag atcgatggat gaatgtgggt cacgaagatg 840 atgaactaaa gccttacgtg gagccactcc ctgactacaa ggacccccgg cggacagagc 900 tgatggtgtc catgggttat acacgggaag agatccagga ctcgctggtg ggccagagat 960 acaacgaggt gatggccacc tatctgctcc tgggctacaa gagctccgag ctggaaggcg 1020 acaccatcac cctgaaaccc cggccttcag ctgatctgac caatagcagc gccccatccc 1080 catcccacaa ggtacagcgc agcgtgtcgg ccaatcccaa gcagcggcgc ttcagcgacc 1140 aggctggtcc tgccattccc acctctaatt cttactctaa gaagactcag agtaacaacg 1200 cagaaaataa gcggcctgag gaggaccggg agtcagggcg gaaagccagc agcacagcca 1260 aggtgcctgc cagccccctg cccggtctgg agaggaagaa gaccacccca accccctcca 1320 cgaacagcgt cctctccacc agcacaaatc gaagcaggaa ttccccactt ttggagcggg 1380 ccagcctcgg ccaggcctcc atccagaatg gcaaagacag cctaaccatg ccagggtccc 1440 gggcctccac ggcttctgct tctgccgcag tctctgcggc ccggccccgc cagcaccaga 1500 aatccatgtc ggcctccgtg caccccaaca aggcctctgg gctgcccccc acggagagta 1560 actgtgaggt gccgcggccc agcacagccc cccagcgtgt ccctgttgcc tccccatccg 1620 cccacaacat cagcagcagt ggtggagccc cagaccgaac taacttcccc cggggtgtgt 1680 ccagccgaag caccttccat gctgggcagc tccgacaggt gcgggaccag cagaatttgc 1740 cctacggtgt gaccccagcc tctccctctg gccacagcca gggccggcgg ggggcctctg 1800 ggagcatctt cagcaagttc acctccaagt ttgtacgcag gaacctgaat gaacctgaaa 1860 gcaaagaccg agtggagacg ctcagacctc acgtggtggg cagtggcggc aacgacaaag 1920 aaaaggaaga atttcgggag gccaagcccc gctccctccg cttcacgtgg agtatgaaga 1980 ccacgagctc catggagccc aacgagatga tgcgggagat ccgcaaggtg ctggacgcga 2040 acagctgcca gagcgagctg catgagaagt acatgctgct gtgcatgcac ggcacgccgg 2100 gccacgagga cttcgtgcag tgggagatgg aggtgtgcaa actgccgcgg ctctctctca 2160 acggggttcg atttaagcgg atatcgggca cctccatggc cttcaaaaac attgcctcca 2220 aaatagccaa cgagctgaag ctttaaca 2248 6 2701 DNA Homo sapiens 6 ggcacgaggg ctgaacgaga gggacacgga gcagcccacc ttgggacacc ttgactccaa 60 gcccagcagt aagtccaaca tgattcgggg ccgcaactca gccacctctg ctgatgagca 120 gccccacatt ggaaactacc ggctcctcaa gaccattggc aagggtaatt ttgccaaggt 180 gaagttggcc cgacacatcc tgactgggaa agaggtagct gtgaagatca ttgacaagac 240 tcaactgaac tcctccagcc tccagaaact attccgcgaa gtaagaataa tgaaggtttt 300 gaatcatccc aacatagtta aattatttga agtgattgag actgagaaaa cgctctacct 360 tgtcatggag tacgctagtg gcggagaggt atttgattac ctagtggctc atggcaggat 420 gaaagaaaaa gaggctcgag ccaaattccg ccagatagtg tctgctgtgc agtactgtca 480 ccagaagttt attgtccata gagacttaaa ggcagaaaac ctgctcttgg atgctgatat 540 gaacatcaag attgcagact ttggcttcag caatgaattc acctttggga acaagctgga 600 caccttctgt ggcagtcccc cttatgctgc cccagaactc ttccagggca aaaaatatga 660 tggacccgag gtggatgtgt ggagcctagg agttatcctc tatacactgg tcagcggatc 720 cctgcctttt gatggacaga acctcaagga gctgcgggaa cgggtactga ggggaaaata 780 ccgtattcca ttctacatgt ccacggactg tgaaaacctg cttaagaaat ttctcattct 840 taatcccagc aagagaggca ctttagagca aatcatgaaa gatcgatgga tgaatgtggg 900 tcacgaagat gatgaactaa agccttacgt ggagccactc cctgactaca aggacccccg 960 gcggacagag ctgatggtgt ccatgggtta tacacgggaa gagatccagg actcgctggt 1020 gggccagaga tacaacgagg tgatggccac ctatctgctc ctgggctaca agagctccga 1080 gctggaaggc gacaccatca ccctgaaacc ccggccttca gctgatctga ccaatagcag 1140 cgccccatcc ccatcccaca aggtacagcg cagcgtgtcg gccaatccca agcagcggcg 1200 cttcagcgac caggcagctg gtcctgccat tcccacctct aattcttact ctaagaagac 1260 tcagagtaac aacgcagaaa ataagcggcc tgaggaggac cgggagtcag ggcggaaagc 1320 cagcagcaca gccaaggtgc ctgccagccc cctgcccggt ctggagagga agaagaccac 1380 cccaaccccc tccacgaaca gcgtcctctc caccagcaca aatcgaagca ggaattcccc 1440 acttttggag cgggccagcc tcggtcaggc ctccatccag aatggcaaag acagcctaac 1500 catgccaggg tcccgggcct ccacggcttc tgcttctgcc gcagtctctg cggcccggcc 1560 ccgccagcac cagaaatcca tgtcggcctc cgtgcacccc aacaaggcct ctgggctgcc 1620 ccccacggag agtaactgtg aggtgccgcg gcccagcaca gccccccagc gtgtccctgt 1680 tgcctcccca tccgcccaca acatcagcag cagtggtgga gccccagacc gaactaactt 1740 cccccggggt gtgtccagcc gaagcacctt ccatgctggg cagctccgac aggtgcggga 1800 ccagcagaat ttgccctacg gtgtgacccc agcctctccc tctggccaca gccagggccg 1860 gcggggggcc tctgggagca tcttcagcaa gttcacctcc aagtttgtac gcagaaatct 1920 gtctttcagg tttgccagaa ggaacctgaa tgaacctgaa agcaaagacc gagtggagac 1980 gctcagacct cacgtggtgg gcagtggcgg caacgacaaa gaaaaggaag aatttcggga 2040 ggccaagccc cgctccctcc gcttcacgtg gagtatgaag accacgagct ccatggagcc 2100 caacgagatg atgcgggaga tccgcaaggt gctggacgcg aacagctgcc agagcgagct 2160 gcatgagaag tacatgctgc tgtgcatgca cggcacgccg ggccacgagg acttcgtgca 2220 gtgggagatg gaggtgtgca aactgccgcg gctctctctc aacggggttc gatttaagcg 2280 gatatcgggc acctccatgg ccttcaaaaa cattgcctcc aaaatagcca acgagctgaa 2340 gctttaacag gctgccagga gcgggggcgg cgggggcggg ccagctggac gggctgccgg 2400 ccgctgcgcc gccccacctg ggcgagactg cagcgatgga ttggtgtgtc tcccctgctg 2460 gcacttctcc cctccctggc ccttctcagt tttctcttac atgtttgtgg ggggtgggag 2520 attgttctcc agccccccac attcacccct gcccagagat tcccccttct cctctcccct 2580 actggaggca aaggaagggg agggtggatg ggggggcagg gctccccctc ggtactgcgg 2640 ttgcacagag tatttcgcct aaaccaagaa attttttatt accaaaaaaa aaaaaaaaaa 2700 a 2701 7 2112 DNA Homo sapiens 7 cccagcagta agtccaacat gattcggggc cgcaactcag ccacctctgc tgatgagcag 60 ccccacattg gaaactaccg gctcctcaag accattggca agggtaattt tgccaaggtg 120 aagttggccc gacacatcct gactgggaaa gaggtagctg tgaagatcat tgacaagact 180 caactgaact cctccagcct ccagaaacta ttccgcgaag taagaataat gaaggttttg 240 aatcatccca acatagttaa attatttgaa gtgattgaga ctgagaaaac gctctacctt 300 gtcatggagt acgctagtgg cggagaggta tttgattacc tagtggctca tggcaggatg 360 aaagaaaaag aggctcgagc caaattccgc cagatagtgt ctgctgtgca gtactgtcac 420 cagaagttta ttgtccatag agacttaaag gcagaaaacc tgctcttgga tgctgatatg 480 aacatcaaga ttgcagactt tggcttcagc aatgaattca cctttgggaa caagctggac 540 accttctgtg gcagtccccc ttatgctgcc ccagaactct tccagggcaa aaaatatgat 600 ggacccgagg tggatgtgtg gagcctagga gttatcctct atacactggt cagcggatcc 660 ctgccttttg atggacagaa cctcaaggag ctgcgggaac gggtactgag gggaaaatac 720 cgtattccat tctacatgtc cacggactgt gaaaacctgc ttaagaaatt tctcattctt 780 aatcccagca agagaggcac tttagagcaa atcatgaaag atcgatggat gaatgtgggt 840 cacgaagatg atgaactaaa gccttacgtg gagccactcc ctgactacaa ggacccccgg 900 cggacagagc tgatggtgtc catgggttat acacgggaag agatccagga ctcgctggtg 960 ggccagagat acaacgaggt gatggccacc tatctgctcc tgggctacaa gagctccgag 1020 ctggaaggcg acaccatcac cctgaaaccc cggccttcag ctgatctgac caatagcagc 1080 gccccatccc catcccacaa ggtacagcgc agcgtgtcgg ccaatcccaa gcagcggcgc 1140 ttcagcgacc aggctggtcc tgccattccc acctctaatt cttactctaa gaagactcag 1200 agtaacaacg cagaaaataa gcggcctgag gaggaccggg agtcagggcg gaaagccagc 1260 agcacagcca aggtgcctgc cagccccctg cccggtctgg agaggaagaa gaccacccca 1320 accccctcca cgaacagcgt cctctccacc agcacaaatc gaagcaggaa ttccccactt 1380 ttggagcggg ccagcctcgg ccaggcctcc atccagaatg gcaaagacag cacagccccc 1440 cagcgtgtcc ctgttgcctc cccatccgcc cacaacatca gcagcagtgg tggagcccca 1500 gaccgaacta acttcccccg gggtgtgtcc agccgaagca ccttccatgc tgggcagctc 1560 cgacaggtgc gggaccagca gaatttgccc tacggtgtga ccccagcctc tccctctggc 1620 cacagccagg gccggcgggg ggcctctggg agcatcttca gcaagttcac ctccaagttt 1680 gtacgcagga acctgaatga acctgaaagc aaagaccgag tggagacgct cagacctcac 1740 gtggtgggca gtggcggcaa cgacaaagaa aaggaagaat ttcgggaggc caagccccgc 1800 tccctccgct tcacgtggag tatgaagacc acgagctcca tggagcccaa cgagatgatg 1860 cgggagatcc gcaaggtgct ggacgcgaac agctgccaga gcgagctgca tgagaagtac 1920 atgctgctgt gcatgcacgg cacgccgggc cacgaggact tcgtgcagtg ggagatggag 1980 gtgtgcaaac tgccgcggct ctctctcaac ggggttcgat ttaagcggat atcgggcacc 2040 tccatggcct tcaaaaacat tgcctccaaa atagccaacg agctgaagct ttaacaggct 2100 gccaggagcg gg 2112 8 2965 DNA Homo sapiens 8 cgggcaaccg cctcgcccga agccctccct cgttactgtc cgcatacccc ggcggcgccg 60 ccgcggaaag cggctccccc tcctcttact ccgcgtcctc ttccctcttt cccccgccgg 120 ggcacgcttg ttgcaccgtc ccgcggcctg cgggagccgc tcgccccgga cttgagctcg 180 cgtacgaccc atttcctgtc gccccccgga gcccgcacca cagcccggcc ggtctagacc 240 ccggcagacc ccgctggccg cacaaaatgt cggcccggac gccattgccg acggtgaacg 300 agcgggacac ggtaaatcat acgactgtgg atggatatac tgaaccacac atccagccta 360 ccaagtcgag tagcagacag aacatccccc ggtgtagaaa ctccattacg tcagcaacag 420 atgaacagcc tcacattgga aattaccgtt tacaaaaaac aatagggaag ggaaattttg 480 ccaaagtcaa attggcaaga cacgttctaa ctggtagaga ggttgctgtg aaaataatag 540 acaaaactca gctaaatcct accagtctac aaaagttatt tcgagaagta cgaataatga 600 agatactgaa tcatcctaat atagtaaaat tgtttgaagt tattgaaaca gagaagactc 660 tctatttagt catggaatac gcgagtgggg gtgaagtatt tgattactta gttgcccatg 720 gaagaatgaa agagaaagag gcccgtgcaa aatttaggca gattgtatct gctgtacagt 780 attgtcatca aaagtacatt gttcaccgtg atcttaaggc tgaaaacctt ctccttgatg 840 gtgatatgaa tattaaaatt gctgactttg gttttagtaa tgaatttaca gttgggaaca 900 aattggacac attttgtgga agcccaccct atgctgctcc cgagcttttc caaggaaaga 960 agtatgatgg gcctgaagtg gatgtgtgga gtctgggcgt cattctctat acattagtca 1020 gtggctcctt gcctttcgat ggccagaatt taaaggaact gcgagagcga gttttacgag 1080 ggaagtaccg tattcccttc tatatgtcca cagactgtga aaatcttctg aagaaattat 1140 tagtcctgaa tccaataaag agaggcagct tggaacaaat aatgaaagat cgatggatga 1200 atgttggtca tgaagaggaa gaactaaagc catatactga gcctgatccg gatttcaatg 1260 acacaaaaag aatagacatt atggtcacca tgggctttgc acgagatgaa ataaatgatg 1320 ccttaataaa tcagaagtat gatgaagtta tggctactta tattcttcta ggtagaaaac 1380 cacctgaatt tgaaggtggt gaatcgttat ccagtggaaa cttgtgtcag aggtcccggc 1440 ccagtagtga cttaaacaac agcactcttc agtcccctgc tcacctgaag gtccagagaa 1500 gtatctcagc aaatcagaag cagcggcgtt tcagtgatca tgctggtcca tccattcctc 1560 ctgctgtatc atataccaaa agacctcagg ctaacagtgt ggaaagtgaa cagaaagagg 1620 agtgggacaa agatgtggct cgaaaacttg gcagcacaac agttggatca aaaagcgaga 1680 tgactgcaag ccctcttgta gggccagaga ggaaaaaatc ttcaactatt ccaagtaaca 1740 atgtgtattc tggaggtagc atggcaagaa ggaatacata tgtctgtgaa aggaccacag 1800 atcgatacgt agcattgcag aatggaaaag acagcagcct tacggagatg tctgtgagta 1860 gcatatcttc tgcaggctct tctgtggcct ctgctgtccc ctcagcacga ccccgccacc 1920 agaagtccat gtccacttct ggtcatccta ttaaagtcac actgccaacc attaaagacg 1980 gctctgaagc ttaccggcct ggtacaaccc agagagtgcc tgctgcttcc ccatctgctc 2040 acagtattag tactgcgact ccagaccgga cccgttttcc ccgagggagc tcaagccgaa 2100 gcactttcca tggtgaacag ctccgggagc gacgcagcgt tgcttataat gggccacctg 2160 cttcaccatc ccatgaaacg ggtgcatttg cacatgccag aaggggaacg tcaactggta 2220 taataagcaa aatcacatcc aaatttgttc gcagggatcc aagtgaaggc gaagccagtg 2280 gcagaaccga cacctcaaga agtacatcag gggaaccaaa agaaagagac aaggaagagg 2340 gtaaagattc taagccgcgt tctttgcggt tcacatggag tatgaagacc actagttcaa 2400 tggaccctaa tgacatgatg agagaaatcc gaaaagtgtt agatgcaaat aactgtgatt 2460 atgagcaaaa agagagattt ttgcttttct gtgtccatgg agacgctaga caggatagcc 2520 tcgtgcagtg ggagatggaa gtctgcaagt tgccacgact gtcacttaat ggggttcgct 2580 tcaagcgaat atctgggaca tctattgcct ttaagaacat tgcatcaaaa atagcaaatg 2640 agcttaagct gtaaagaagt ccaaatttac aggttcaggg aagatacata catatatgag 2700 gtacagtttt tgaatgtact ggtaatgcct aatgtggtct gcctgtgaat ctccccatgt 2760 agaatttgcc cttaatgcaa taaggttata catagttatg aactgtaaaa ttaaagtcag 2820 tatgaactat aataaatatc tgtagcttaa aaagtaggtt cacatgtaca ggtaagtata 2880 ttgtgtattt ctgttcattt tctgttcata gagttgtata ataaaacatg attgcttaaa 2940 aacttgaaaa aaaaaaaaaa aaaaa 2965 9 3210 DNA Homo sapiens 9 ggcgcggcgg cggcggtggc tgtgaccgcg cggaccgagc cgagacattc gcgccggggg 60 atcgggcgcc gccgccgctg ggccccgggc gcgtggatgc ggctgggtcg ggcggcgccg 120 tacacctgag gcggagaacg gggcgcggcg cgggtgacgc tgtcagggcc gcggttcctg 180 acgcccaggc gctcgccagg acgagccagg cagtgatttg aggcaccggc ttcaccttca 240 cccatggtcc ggagagccta gcggggctcg ccaccgcctc ccggctcccc ttccacgcct 300 catcctgcca gcctcgccgc cccgccagcg ccgggcaacc gcctcgcccg aagccctccc 360 tcgttactgt ccgcataccc cggcggcgcc gccgcgggaa gcggctcccc ctcctcttcc 420 tccgcgtcct cttccctctt tcccccgccg gggccgcttg ttgcaccgcc ccgcggcctg 480 cgggagccgc tcgccccggc cttgtgctcg cgtccgcacc cctttcctgt cgccccccgg 540 ggcccgcacc acagcccggc cggcgagacc ccggccagac cccgctgccc gcacaaaatg 600 tcggcccgga cgccattgcc gacggtgaac gagcgggaca cggaaaatca tacatctgtg 660 gatggatata ctgaaccaca catccagcct accaagtcga gtagcagaca gaacatcccc 720 cggtgtagaa actccattac gtcagcaaca gatgaacagc ctcacattgg aaattaccgt 780 ttacaaaaaa caatagggaa gggaaatttt gccaaagtca aattggcaag acacgttcta 840 actggtagag aggttgctgt gaaaataata gacaaaactc agctaaatcc taccagtcta 900 caaaagttat ttcgagaagt acgaataatg aagatactga atcatcctaa tataggtgaa 960 gtatttgatt acttagttgc ccatggaaga atgaaagaga aagaggcccg tgcaaaattt 1020 aggcagattg tatctgctgt acagtattgt catcaaaagt acattgttca ccgtgatctt 1080 aagctgaaaa ccttctcctt gatggtgata tgaatattaa aattgctgac tttggtttta 1140 gtaatgaatt tacagttggg aacaaattgg acacattttg tggaagccca ccctatgctg 1200 ctcccgagct tttccaagga aagaagtatg atgggcctga agtggatgtg tggagtctgg 1260 gcgtcattct ctatacatta gtcagtggct ccttgccttt cgatggccag aatttaaagg 1320 aactgcgaga gcgagtttta cgagggaagt accgtattcc cttctatatg tccacagact 1380 gtgaaaatct tctgaagaaa ttattagtcc tgaatccaat aaagagaggc agcttggaac 1440 aaataatgaa agatcgatgg atgaatgttg gtcatgaaga ggaagaacta aagccatata 1500 ctgagcctga tccggatttc aatgacacaa aaagaataga cattatggtc accatgggct 1560 ttgcacgaga tgaaataaat gatgccttaa taaatcagaa gtatgatgaa gttatggcta 1620 cttatattct tctaggtaga aaaccacctg aatttgaagg tggtgaatcg ttatccagtg 1680 gaaacttgtg tcagaggtcc cggcccagta gtgacttaaa caacagcact cttcagtccc 1740 ctgctcacct gaaggtccag agaagtatct cagcaaatca gaagcagcgg cgtttcagtg 1800 atcatgctgg tccatccatt cctcctgctg tatcatatac caaaagacct caggctaaca 1860 gtgtggaaag tgaacagaaa gaggagtggg acaaagatgt ggctcgaaaa cttggcagca 1920 caacagttgg atcaaaaagc gagatgactg caagccctct tgtagggcca gagaggaaaa 1980 aatcttcaac tattccaagt aacaatgtgt attctggagg tagcatggca agaaggaata 2040 catatgtctg tgaaaggacc acagatcgat acgtagcatt gcagaatgga aaagacagca 2100 gccttacgga gatgtctgtg agtagcatat cttctgcagg ctcttctgtg gcctctgctg 2160 tcccctcagc acgaccccgc caccagaagt ccatgtccac ttctggtcat cctattaaag 2220 tcacactgcc aaccattaaa gacggctctg aagcttaccg gcctggtaca acccagagag 2280 tgcctgctgc ttccccatct gctcacagta ttagtactgc gactccagac cggacccgtt 2340 ttccccgagg gagctcaagc cgaagcactt tccatggtga acagctccgg gagcgacgca 2400 gcgttgctta taatgggcca cctgcttcac catcccatga aacgggtgca tttgcacatg 2460 ccagaagggg aacgtcaact ggtataataa gcaaaatcac atccaaattt gttcgcaggg 2520 atccaagtga aggcgaagcc agtggcagaa ccgacacctc aagaagtaca tcaggggaac 2580 caaaagaaag agacaaggaa gagggtaaag attctaagcc gcgttctttg cggttcacat 2640 ggagtatgaa gaccactagt tcaatggacc ctaatgacat gatgagagaa atccgaaaag 2700 tgttagatgc aaataactgt gattatgagc aaaaagagag atttttgctt ttctgtgtcc 2760 atggagacgc tagacaggat agcctcgtgc agtgggagat ggaagtctgc aagttgccac 2820 gactgtcact taatggggtt cgcttcaagc gaatatctgg gacatctatt gcctttaaga 2880 acattgcatc aaaaatagca aatgagctta agctgtaaag aagtccaaat ttacaggttc 2940 agggaagata catacatata tgaggtacag tttttgaatg tactggtaat gcctaatgtg 3000 gtctgcctgt gaatctcccc atgtagaatt tgcccttaat gcaataaggt tatacatagt 3060 tatgaactgt aaaattaaag tcagtatgaa ctataataaa tatctgtagc ttaaaaagta 3120 ggttcacatg tacaggtaag tatattgtgt atttctgttc attttctgtt catagagttg 3180 tataataaaa catgattgct taaaaacttg 3210 10 2505 DNA Homo sapiens 10 gctggccgca caaaatgtcg gcccggacgc cattgccgac ggtgaacgag cgggacacgg 60 aaaatcatac atctgtggat ggatatactg aaccacacat ccagcctacc aagtcgagta 120 gcagacagaa catcccccgg tgtagaaact ccattacgtc agcaacagat gaacagcctc 180 acattggaaa ttaccgttta caaaaaacaa tagggaaggg aaattttgcc aaagtcaaat 240 tggcaagaca cgttctaact ggtagagagg ttgctgtgaa aataatagac aaaactcagc 300 taaatcctac cagtctacaa aagttatttc gagaagtacg aataatgaag atactgaatc 360 atcctaatat agtaaaattg tttgaagtta ttgaaacaga gaagactctc tatttagtca 420 tggaatacgc gagtgggggt gaagtatttg attacttagt tgcccatgga agaatgaaag 480 agaaagaggc ccgtgcaaaa tttaggcaga ttgtatctgc tgtacagtat tgtcatcaaa 540 agtacattgt tcaccgtgat cttaaggctg aaaaccttct ccttgatggt gatatgaata 600 ttaaaattgc tgactttggt tttagtaatg aatttacagt tgggaacaaa ttggacacat 660 tttgtggaag cccaccctat gctgctcccg agcttttcca aggaaagaag tatgatgggc 720 ctgaagtgga tgtgtggagt ctgggcgtca ttctctatac attagtcagt ggctccttgc 780 ctttcgatgg ccagaattta aaggaactgc gagagcgagt tttacgaggg aagtaccgta 840 ttcccttcta tatgtccaca gactgtgaaa atcttctgaa gaaattatta gtcctgaatc 900 caataaagag aggcagcttg gaacaaataa tgaaagatcg atggatgaat gttggtcatg 960 aagaggaaga actaaagcca tatactgagc ctgatccgga tttcaatgac acaaaaagaa 1020 tagacattat ggtcaccatg ggctttgcac gagatgaaat aaatgatgcc ttaataaatc 1080 agaagtatga tgaagttatg gctacttata ttcttctagg tagaaaacca cctgaatttg 1140 aaggtggtga atcgttatcc agtggaaact tgtgtcagag gtcccggccc agtagtgact 1200 taaacaacag cactcttcag tcccctgctc acctgaaggt ccagagaagt atctcagcaa 1260 atcagaagca gcggcgtttc agtgatcatg ctggtccatc cattcctcct gctgtatcat 1320 ataccaaaag acctcaggct aacagtgtgg aaagtgaaca gaaagaggag tgggacaaag 1380 atgtggctcg aaaacttggc agcacaacag ttggatcaaa aagcgagatg actgcaagcc 1440 ctcttgtagg gccagagagg aaaaaatctt caactattcc aagtaacaat gtgtattctg 1500 gaggtagcat ggcaagaagg aatacatatg tctgtgaaag gaccacagat cgatacgtag 1560 cattgcagaa tggaaaagac agcagcctta cggagatgtc tgtgagtagc atatcttctg 1620 caggctcttc tgtggcctct gctgtcccct cagcacgacc ccgccaccag aagtccatgt 1680 ccacttctgg tcatcctatt aaagtcacac tgccaaccat taaagacggc tctgaagctt 1740 accggcctgg tacaacccag agagtgcctg ctgcttcccc atctgctcac agtattagta 1800 ctgcgactcc agaccggacc cgttttcccc gagggagctc aagccgaagc actttccatg 1860 gtgaacagct ccgggagcga cgcagcgttg cttataatgg gccacctgct tcaccatccc 1920 atgaaacggg tgcatttgca catgccagaa ggggaacgtc aactggtata ataagcaaaa 1980 tcacatccaa atttgttcgc agggatccaa gtgaaggcga agccagtggc agaaccgaca 2040 cctcaagaag tacatcaggg gaaccaaaag aaagagacaa ggaagagggt aaagattcta 2100 agccgcgttc tttgcggttc acatggagta tgaagaccac tagttcaatg gaccctaatg 2160 acatgatgag agaaatccga aaagtgttag atgcaaataa ctgtgattat gagcaaaaag 2220 agagattttt gcttttctgt gtccatggag acgctagaca ggatagcctc gtgcagtggg 2280 agatggaagt ctgcaagttg ccacgactgt cacttaatgg ggttcgcttc aagcgaatat 2340 ctgggacatc tattgccttt aagaacattg catcaaaaat agcaaatgag cttaagctgt 2400 aaagaagtcc aaatttacag gttcagggaa gatacataca tatatgaggt acagtttttg 2460 aatgtactgg taatgcctaa tgtggtctgc ctgtgaatct cccca 2505 11 4638 DNA Homo sapiens 11 ggcgcggcgg cggcggtggc tgtgaccgcg cggaccgagc cgagacattc gcgccggggg 60 atcgggcgcc gccgccgctg ggccccgggc gcgtggatgc ggctgggtcg ggcggcgccg 120 tacacctgag gcggagaacg gggcgcggcg cgggtgacgc tgtcagggcc gcggttcctg 180 acgcccaggc gctcgccagg acgagccagg cagtgatttg aggcaccggc ttcaccttca 240 cccatggtcc ggagagccta gcggggctcg ccaccgcctc ccggctcccc ttccacgcct 300 catcctgcca gcctcgccgc cccgccagcg ccgggcaacc gcctcgcccg aagccctccc 360 tcgttactgt ccgcataccc cggcggcgcc gccgcgggaa gcggctcccc ctcctcttcc 420 tccgcgtcct cttccctctt tcccccgccg gggccgcttg ttgcaccgcc ccgcggcctg 480 cgggagccgc tcgccccggc cttgtgctcg cgtccgcacc cctttcctgt cgccccccgg 540 ggcccgcacc acagcccggc cggcgagacc ccggccagac cccgctgccc gcacaaaatg 600 tcggcccgga cgccattgcc gacggtgaac gagcgggaca cggaaaatca tacatctgtg 660 gatggatata ctgaaccaca catccagcct accaagtcga gtagcagaca gaacatcccc 720 cggtgtagaa actccattac gtcagcaaca gatgaacagc ctcacattgg aaattaccgt 780 ttacaaaaaa caatagggaa gggaaatttt gccaaagtca aattggcaag acacgttcta 840 actggtagag aggttgctgt gaaaataata gacaaaactc agctaaatcc taccagtcta 900 caaaagttat ttcgagaagt acgaataatg aagatactga atcatcctaa tataggtgaa 960 gtatttgatt acttagttgc ccatggaaga atgaaagaga aagaggcccg tgcaaaattt 1020 aggcagattg tatctgctgt acagtattgt catcaaaagt acattgttca ccgtgatctt 1080 aaggctgaaa accttctcct tgatggtgat atgaatatta aaattgctga ctttggtttt 1140 agtaatgaat ttacagttgg gaacaaattg gacacatttt gtggaagccc accctatgct 1200 gctcccgagc ttttccaagg aaagaagtat gatgggcctg aagtggatgt gtggagtctg 1260 ggcgtcattc tctatacatt agtcagtggc tccttgcctt tcgatggcca gaatttaaag 1320 gaactgcgag agcgagtttt acgagggaag taccgtattc ccttctatat gtccacagac 1380 tgtgaaaatc ttctgaagaa attattagtc ctgaatccaa taaagagagg cagcttggaa 1440 caaataatga aagatcgatg gatgaatgtt ggtcatgaag aggaagaact aaagccatat 1500 actgagcctg atccggattt caatgacaca aaaagaatag acattatggt caccatgggc 1560 tttgcacgag atgaaataaa tgatgcctta ataaatcaga agtatgatga agttatggct 1620 acttatattc ttctaggtag aaaaccacct gaatttgaag gtggtgaatc gttatccagt 1680 ggaaacttgt gtcagaggtc ccggcccagt agtgacttaa acaacagcac tcttcagtcc 1740 cctgctcacc tgaaggtcca gagaagtatc tcagcaaatc agaagcagcg gcgtttcagt 1800 gatcatgctg gtccatccat tcctcctgct gtatcatata ccaaaagacc tcaggctaac 1860 agtgtggaaa gtgaacagaa agaggagtgg gacaaagatg tggctcgaaa acttggcagc 1920 acaacagttg gatcaaaaag cgagatgact gcaagccctc ttgtagggcc agagaggaaa 1980 aaatcttcaa ctattccaag taacaatgtg tattctggag gtagcatggc aagaaggaat 2040 acatatgtct gtgaaaggac cacagatcga tacgtagcat tgcagaatgg aaaagacagc 2100 agccttacgg agatgtctgt gagtagcata tcttctgcag gctcttctgt ggcctctgct 2160 gtcccctcag cacgaccccg ccaccagaag tccatgtcca cttctggtca tcctattaaa 2220 gtcacactgc caaccattaa agacggctct gaagcttacc ggcctggtac aacccagaga 2280 gtgcctgctg cttccccatc tgctcacagt attagtactg cgactccaga ccggacccgt 2340 tttccccgag ggagctcaag ccgaagcact ttccatggtg aacagctccg ggagcgacgc 2400 agcgttgctt ataatgggcc acctgcttca ccatcccatg aaacgggtgc atttgcacat 2460 gccagaaggg gaacgtcaac tggtataata agcaaaatca catccaaatt tgttcgcaga 2520 agtacatcag gggaaccaaa agaaagagac aaggaagagg gtaaagattc taagccgcgt 2580 tctttgcggt tcacatggag tatgaagacc actagttcaa tggaccctaa tgacatgatg 2640 agagaaatcc gaaaagtgtt agatgcaaat aactgtgatt atgagcaaaa agagagattt 2700 ttgcttttct gtgtccatgg agacgctaga caggatagcc tcgtgcagtg ggagatggaa 2760 gtctgcaagt tgccacgact gtcacttaat ggggttcgct tcaagcgaat atctgggaca 2820 tctattgcct ttaagaacat tgcatcaaaa atagcaaatg agcttatgct gtaaagaagt 2880 ccaaatttac aggttcaggg aagatacata catatatgag gtacagtttt tgaatgtact 2940 ggtaatgcct aatgtggtct gcctgtgaat ctccccatgt agaatttgcc cttaatgcaa 3000 taaggttata catagttatg aactgtaaaa ttaaagtcag tatgaactat aataaatatc 3060 tgtagcttaa aaagtaggtt cacatgtaca ggtaagtata ttgtgtattt ctgttcattt 3120 tctgttcata gagttgtata ataaaacatg attgcttaaa aacttgtata gttgtctaga 3180 tttctgcacc tgaatgtatg tttgatgctt tgatttgaaa atgttcttcc ctgttattta 3240 cattctggtg ggtttttaaa attcttacct ccatcatgca attttgaaaa ttgtgtccag 3300 aattaaaagt gcatagaaat agcctttaca attgtagcat ggacctttaa aaattgtttt 3360 aaaatcttat ttaaatttaa accagaagct gaaaaataga tcagctttat tatacacaaa 3420 attattactg cttatctttg ctcttttcct tgttatcccg caaggtttag ttgagaagat 3480 acaaaatgtt tacagtgttg gcacttagag tttttaaatt caagtacatg aaattcagta 3540 atagcattgc cttgagctaa ctaggaagta ccgggaaaaa agttaaatct acatcaagtt 3600 tcttttgaac tttgaagtgt tttctgaccc actgctaact gtagcaacaa aatttaaaag 3660 aaaaaaaaca tactttatct ggctattata acataaactg tcacgtaggt ttgctgcctt 3720 cagaataccg caatttaatt gcgggaatat aataatattg ggactgtttc acagcacaaa 3780 ctcatcttta cagtgttgat caatgcatca gttaagaaat aatgccacct caggaattaa 3840 ctggcattgg gaacatttgc ctcattctcc tgctatcctc ttcattcacc cctgccactg 3900 taatatctat aagtacttaa gagacttgtg agcaaaacat actatttata acagtatatg 3960 attgatttat gcttatgtgg ttgttcagtt tgttcccatg taactcgttt gttttaaata 4020 ttttgccaga tttcttgtat ttattccaca tcattatgcc tataatgtgc cgctttgtga 4080 ttgggcattt gcctactttt ctttcataat tagtgatata tgcgatgtaa aaccactagt 4140 aaaggtacat tttaatactt gttattttat actgaattag ccttggaggt tgactgtgca 4200 atgttattta ctgttgtaat tactgtaata ccaacatatg ggccccatct gcacactcct 4260 gaaaaacaga aagtgtattc aaattttatc agtttaaaga aaataaagct gtgataaata 4320 ctgtaattcc aacctacatt agaaggtcta agtgtaggtg atgtgccatt ccataatggc 4380 ttccagacta gggtgaattt tatgttctgt actgtactgt gatgtagctt tcttctgtaa 4440 cagttatgtt ttaaaattaa gtgagttttt tttttgcctt agcaaagggt ggtgtttgaa 4500 aaaaaaaatg tgtagcccct ttttaaccta gtgttcattc aaaaaaaaat tgatgcaaat 4560 ctttattcac tttcactggt gcacactgaa attttacttg aacagttctc ataataaagc 4620 acttgtcttt tgctcttt 4638 12 2720 DNA Homo sapiens 12 tcatggaata cgcgagtggg ggtgaagtat ttgattactt agttgcccat ggaagaatga 60 aagagaaaga ggcccgtgca aaatttaggc agattgtatc tgctgtacag tattgtcatc 120 aaaagtacat tgttcaccgt gatcttaagg ctgaaaacct tctccttgat ggtgatatga 180 atattaaaat tgctgacttt ggttttagta atgaatttac agttgggaac aaattggaca 240 cattttgtgg aagcccaccc tatgctgctc ccgagctttt ccaaggaaag aagtatgatg 300 gtcctgaagt ggatgtgtgg agtctgggcg tcattctcta tacattagtc agtggctcct 360 tgcctttcga tggccagaat ttaaaggaac tgcgagagcg agttttacga gggaagtacc 420 gtattccctt ctatatgtcc acagactgtg aaaatcttct gaagaaatta ttagtcctga 480 atccaataaa gagaggcagc ttggaacaaa taatgaaaga tcgatggatg aatgttggtc 540 atgaagagga agaactaaag ccatatactg agcctgatcc ggatttcaat gacacaaaaa 600 gaatagacat tatggtcacc atgggctttg cacgagatga aataaatgat gccttaataa 660 atcagaagta tgatgaagtt atggctactt atattcttct aggtagaaaa ccacctgaat 720 ttgaaggtgg tgaatcgtta tccagtggaa acttgtgtca gaggtcccgg cccagtagtg 780 acttaaacaa cagcactctt cagtcccctg ctcacctgaa ggtccagaga agtatctcag 840 caaatcagaa gcagcggcgt ttcagtgatc atgctggtcc atccattcct cctgctgtat 900 catataccaa aagacctcag gctaacagtg tggaaagtga acagaaagag gagtgggaca 960 aagatgtggc tcgaaaactt ggcagcacaa cagttggatc aaaaagcgag atgactgcaa 1020 gccctcttgt agggccagag aggaaaaaat cttcaactat tccaagtaac aatgtgtatt 1080 ctggaggtag catggcaaga aggaatacat atgtctgtga aaggaccaca gatcgatacg 1140 tagcattgca gaatggaaaa aacagcagcc ttacggagat gtctgtgagt agcatatctt 1200 ctgcaggctc ttctgtggcc tctgctgccc cctcagcacg accccgccac cagaagtcca 1260 tgtccacttc tggtcatcct attaaagtca cactgccaac cattaaagac ggctctgaag 1320 cttaccggcc tggtacaacc cagagagtgc ctgctgcttc cccatctgct cacagtatta 1380 gtactgcgac tccagaccgg acccgttttc cccgagggag ctcaagccga agcactttcc 1440 atggtgaaca gctccgggag cgacgcagcg ttgcttataa tgggccacct gcttcaccat 1500 cccatgaaac gggtgcattt gcacatgcca gaaggggaac gtcaactggt ataataagca 1560 aaatcacatc caaatttgtt cgcagaagta catcagggga accaaaagaa agagacaagg 1620 aagagggtaa agattctaag ccgcgttctt tgcggttcac atggagtatg aagaccacta 1680 gttcaatgga ccctaatgac atgatgagag aaatccgaaa agtgttagat gcaaataact 1740 gtgattatga gcaaaaagag agatttttgc ttttctgtgt ccatggagac gctagacagg 1800 atagcctcgt gcagtgggag atggaagtct gcaagttgca cgactgtcac ttaatggggt 1860 tcgcttcaag cgaatatctg ggacatctat tgcctttaag aacattgcat caaaaatagc 1920 aaatgagctt aagctgtaaa gaagtccaaa tttacaggtt cagggaagat acatacatat 1980 atgaggtaca gtttttgaat gtactggtaa tgcctaatgt ggtctgcctg tgaatctccc 2040 catgtagaat ttgcccttaa tgcaataagg ttatacatag ttatgaactg taaaattaaa 2100 gtcagtatga actataataa atatctgtag cttaaaaagt aggttcacat gtacaggtaa 2160 gtatattgtg tatttctgtt cattttctgt tcatagagtt gtataataaa acatgattgc 2220 ttaaaaactt gtatagttgt ctagatttct gcacctgaat gtatgtttga tgctttgatt 2280 tgaaaatgtt cttccctgtt atttacattc cggtgggttt ttaaaattct tacctccatc 2340 atgcaatttt gaaaattgtg tccagaatta aaagtgcata gaaatagcct ttacaattgt 2400 agcatggacc tttaaaaatt gttttaaaat cttatttaaa tttaaaccag aagctgaaaa 2460 atagatcagc tttattatac acaaaattat tactgcttat ctttgctctt ttccttgtta 2520 tcccgcaagg tttagttgag aagatacaaa atgtttacag tgttggcact tagagttttt 2580 aaattcaagt acatgaaatt cagtaatagc attgccttga gctaactagg aagtaccggg 2640 aaaaaagtta aatctacatc aagtttcttt tgaactttga agtgttttct gacccactgc 2700 taactgtagc aacaaaattt 2720 13 2698 DNA Homo sapiens 13 gagctgaaat tcgcggtgcg acgggaggga gtggagaagg aggtgagggg gcccaggatc 60 gcggggcgcc ctgaggcaag gggacgccgg tgggtcgaag cgcagcccgc cgcccgcagg 120 ctcggctccg ccactgccgc cctcccggtc tcctcgcctc gggcgccgag gcagggagag 180 aatgagcccc gggacccgcc gggggacggc ccgggccagg cccgggatct agaacggccg 240 tagggggaag ggagccgccc tccccacggc gccttttcgg aactgccgtg gactcgagga 300 cgctggtcgc cggcctccta gggctgtgct gttttgtttt gaccctcgca ttgtgcagaa 360 ttaaagtgca gtaaaatgtc cactaggacc ccattgccaa cggtgaatga acgagacact 420 gaaaaccaca cgtcacatgg agatgggcgt caagaagtta cctctcgtac cagccgctca 480 ggagctcggt gtagaaactc tatagcctcc tgtgcagatg aacaacctca catcggaaac 540 tacagactgt tgaaaacaat cggcaagggg aattttgcaa aagtaaaatt ggcaagacat 600 atccttacag gcagagaggt tgcaataaaa ataattgaca aaactcagtt gaatccaaca 660 agtctacaaa agctcttcag agaagtaaga ataatgaaga ttttaaatca tcccaatata 720 gtgaagttat tcgaagtcat tgaaactgaa aaaacactct acctaatcat ggaatatgca 780 agtggaggtg aagtatttga ctatttggtt gcacatggca ggatgaagga aaaagaagca 840 agatctaaat ttagacagat tgtgtctgca gttcaatact gccatcagaa acggatcgta 900 catcgagacc tcaaggctga aaatctattg ttagatgccg atatgaacat taaaatagca 960 gatttcggtt ttagcaatga atttactgtt ggcggtaaac tcgacacgtt ttgtggcagt 1020 cctccatacg cagcacctga gctcttccag ggcaagaaat atgacgggcc agaagtggat 1080 gtgtggagtc tgggggtcat tttatacaca ctagtcagtg gctcacttcc ctttgatggg 1140 caaaacctaa aggaactgag agagagagta ttaagaggga aatacagaat tcccttctac 1200 atgtctacag actgtgaaaa ccttctcaaa cgtttcctgg tgctaaatcc aattaaacgc 1260 ggcactctag agcaaatcat gaaggacagg tggatcaatg cagggcatga agaagatgaa 1320 ctcaaaccat ttgttgaacc agagctagac atctcagacc aaaaaagaat agatattatg 1380 gtgggaatgg gatattcaca agaagaaatt caagaatctc ttagtaagat gaaatacgat 1440 gaaatcacag ctacatattt gttattgggg agaaaatctt cagagctgga tgctagtgat 1500 tccagttcta gcagcaatct ttcacttgct aaggttaggc cgagcagtga tctcaacaac 1560 agtactggcc agtctcctca ccacaaagtg cagagaagtg tttcttcaag ccaaaagcaa 1620 agacgctaca gtgaccatgc tggaccagct attccttctg ttgtggcgta tccgaaaagg 1680 agtcagacaa gcactgcaga tggtgacctc aaagaagatg gaatttcctc ccggaaatca 1740 agtggcagtg ctgttggagg aaagggaatt gctccagcca gtcccatgct tgggaatgca 1800 agtaatccta ataaggcgga tattcctgaa cgcaagaaaa gctccactgt ccctagtagt 1860 aacacagcat ctggtggaat gacacgacga aatacttatg tttgcagtga gagaactaca 1920 gctgatagac actcagtgat tcagaatggc aaagaaaaca gcactattcc tgatcagaga 1980 actccagttg cttcaacaca cagtatcagt agtgcagcca ccccagatcg aatccgcttc 2040 ccaagaggca ctgccagtcg tagcactttc cacggccagc cccgggaacg gcgaaccgca 2100 acatataatg gccctcctgc ctctcccagc ctgtcccatg aagccacacc attgtcccag 2160 actcgaagcc gaggctccac taatctcttt agtaaattaa cttcaaaact cacaaggagt 2220 cgcaatgtat ctgctgagca aaaagatgaa aacaaagaag caaagcctcg atccctacgc 2280 ttcacctgga gcatgaaaac cactagttca atggatcccg gggacatgat gcgggaaatc 2340 cgcaaagtgt tggacgccaa taactgcgac tatgagcaga gggagcgctt cttgctcttc 2400 tgcgtccacg gagatgggca cgcggagaac ctcgtgcagt gggaaatgga agtgtgcaag 2460 ctgccaagac tgtctctgaa cggggtccgg tttaagcgga tatcggggac atccatagcc 2520 ttcaaaaata ttgcttccaa aattgccaat gagctaaagc tgtaacccag tgattatgat 2580 gtaaattaag tagcaagtaa agtgttttcc tgaacactga tggaaatgta tagaataata 2640 tttaggcaat aacgtctgca tcttctaaat catgaaatta aagtctgagg acgagagc 2698 14 2914 DNA Homo sapiens 14 gacggcccgg gccaggcccg ggatctagaa cggccgtagg gggaagggag ccgccctccc 60 cacggcgcct tttcggaact gccgtggact cgaggacgct ggtcgccggc ctcctagggc 120 tgtgctgttt tgttttgacc ctcgcattgt gcagaattaa agtgcagtaa aatgtccact 180 aggaccccat tgccaacggt gaatgaacga gacactgaaa accacacgtc acatggagat 240 gggcgtcaag aagttacctc tcgtaccagc cgctcaggag ctcggtgtag aaactctata 300 gcctcctgtg cagatgaaca acctcacatc ggaaactaca gactgttgaa aacaatcggc 360 aaggggaatt ttgcaaaagt aaaattggca agacatatcc ttacaggcag agaggttgca 420 ataaaaataa ttgacaaaac tcagttgaat ccaacaagtc tacaaaagct cttcagagaa 480 gtaagaataa tgaagatttt aaatcatccc aatatagtga agttattcga agtcattgaa 540 actcaaaaaa cactctacct aatcatggaa tatgcaagtg gaggtaaagt atttgactat 600 ttggttgcac atggcaggat gaaggaaaaa gaagcaagat ctaaatttag acagattgtg 660 tctgcagttc aatactgcca tcagaaacgg atcgtacatc gagacctcaa ggctgaaaat 720 ctattgttag atgccgatat gaacattaaa atagcagatt tcggttttag caatgaattt 780 actgttggcg gtaaactcga cacgttttgt ggcagtcctc catacgcagc acctgagctc 840 ttccagggca agaaatatga cgggccagaa gtggatgtgt ggagtctggg ggtcatttta 900 tacacactag tcagtggctc acttcccttt gatgggcaaa acctaaagga actgagagag 960 agagtattaa gagggaaata cagaattccc ttctacatgt ctacagactg tgaaaacctt 1020 ctcaaacgtt tcctggtgct aaatccaatt aaacgcggca ctctagagca aatcatgaag 1080 gacaggtgga tcaatgcagg gcatgaagaa gatgaactca aaccatttgt tgaaccagag 1140 ctagacatct cagaccaaaa aagaatagat attatggtgg gaatgggata ttcacaagaa 1200 gaaattcaag aatctcttag taagatgaaa tacgatgaaa tcacagctac atatttgtta 1260 ttggggagaa aatcttcaga ggttaggccg agcagtgatc tcaacaacag tactggccag 1320 tctcctcacc acaaagtgca gagaagtgtt tcttcaagcc aaaagcaaag acgctacagt 1380 gaccatgctg gaccaggtat tccttctgtt gtggcgtatc cgaaaaggag tcagaccagc 1440 actgcagata gtgacctcaa agaagatgga atttcctccc ggaaatcaac tggcagtgct 1500 gttggaggaa agggaattgc tccagccagt cccatgcttg ggaatgcaag taatcctaat 1560 aaggcggata ttcctgaacg caagaaaagc tccactgtcc ctagtagtaa cacagcatct 1620 ggtggaatga cacgacgaaa tacttatgtt tgcagtgaga gaactacaga tgatagacac 1680 tcagtgattc agaatggcaa agaaaacagc actattcctg atcagagaac tccagttgct 1740 tcaacacaca gtatcagtag tgcagccacc ccagatcgaa tccgcttccc aagaggcact 1800 gccagtcgta gcactttcca cggccagccc cgggaacggc gaaccgcaac atataatggc 1860 cctcctgcct ctcccagcct gtcccatgaa gccacaccat tgtcccagac tcgaagccga 1920 ggctccacta ctctctttag taaattaact tcaaaactca caaggagtcg caatgtatct 1980 gctaagcaaa aagatgaaaa caaagaagca aagcctcgat ccctacgctt cacctggagc 2040 atgaaaacca ctagttcaat ggatcccggg gacatgatgc gggaaatccg caaagtgttg 2100 gacgccaata actgcgacta tgagcagagg gagcgcttct tgctcttctg cgtccacgga 2160 gatgggcacg cggagaacct cgtgcagtgg gaaatggaag tgtgcaagct gccaagactg 2220 tctctgaacg gggtccggtt taagcggata tcggggacat ccatagcctt caaaaatatt 2280 gcttccaaaa ttgccaatga gctaaagctg taacccagtg attatgatgt aaattaagta 2340 gcaagtaaag tgttttcctg aacactgatg gaaatgtata gaataatatt taggcaataa 2400 cgtctgcatc ttctaaatca tgaaattaaa gtctgaggac gagagcacgc ctgggagcga 2460 aagctggcct tttttctacg aatgcactac attaaagatg tgcaacctat gcgccccctg 2520 ccctacttcc gttaccctga gagtcggcgt gtggccccat ctccatgtgc ctcccgtctg 2580 ggtgggtgtg agagtggacg gtatgtgtgt gaagtggtgt atatggaagc atctccctac 2640 actggcagcc agtcattact agtacctctg cgggagatca tccggtgcta aaacattaca 2700 gttgccaagg aggaaaatac tgaatgactg ctaagaatta accttaagac cagttcatag 2760 ttaatacagg tttacagttc atgcctgtgg ttttgtgttt gttgttttgt gtttttttag 2820 tgcaaaaggt ttaaatttat agttgtgaac attgcttgtg tgtgtttttc taagtagatt 2880 cacaagataa ttaaaaattc actttttctc aggt 2914 15 3895 DNA Homo sapiens 15 ctgcaggaat tccgatcctt ccgcaggttc acctacggaa accttgttac gacttttact 60 tcctctagat agtcaagttc gaccgtcttc tcagcgctcc gccagggccg tgggccgacc 120 ccggcggggc cgatccgagg gcctcactaa accatccaat cggtagtagc gacgggcggt 180 gtgtacaaag ggcagggact taatcaacgc aagcttatga cccgcactta ctgggaattc 240 ctcgttcatg gggaataatt gcaatccccg atccccatca cgaatggggt tcaacgggtt 300 acccgcgcct gccggcgtag ggtaggcaca cgctgagcca gtcagtgtag cgcgcgtgca 360 gccccggaca tctaagggca tcacagacct gttattgctc aatctcgggt ggctgaacgc 420 cacttgtccc tctaagaagt tgggggacgc cgaccgctcg ggggtcgcgt aactagttag 480 catgccagag tctcgttcgt tatcggaatt aaccagacaa atcgctccac caactaagaa 540 cggccatgca ccaccaccca cggaatcgag aaagagctat caatctgtca atcctgtccg 600 tgtccgggcc gggtgaggtt tcccgtgttg agtcaaatta agccgcaggc tccactcctg 660 gtggtgccct tccgtcaatt cctttaagtt tcagctttgc aaccatactc cccccggaac 720 ccaaagactt tggtttcccg gaagctgccc ggcgggtcat gggaataacg ccgccgcatc 780 gccggtcggc atcgtttatg gtcggaacta cgacggtatc tgatcgtctt cgaacctccg 840 actttcgttc ttgattaatg aaaacattct tggcaaatgc tttcgctctg gtccgtcttg 900 cgccggtcca agaatttcgg aattccgcag cggcggccag cagggcggag gctgaggcag 960 caagctcgct agagagggag aagcagtcgg gcgcaggcgc ctcctccgca gcccgctcca 1020 tggtcggcgc ccacagcccg cggcggcctg tcttgcgctc cacttccttc acatcctcct 1080 ccgcctcctc gttttcaggc gccgccggcg gcgctgtgtg gaggcccgcg agctgaaatt 1140 cgcggtgcga cgggagggag tggagaagga ggtgaggggg cccaggatcg cggggcgccc 1200 tgaggcaagg ggacgccggc gggccgaagc gcagcccgcc gcccgcaggc tcggctccgc 1260 cactgccgcc ctcccggtct cctcgcctcg gccgccgagg cagggagaga atgagccccg 1320 ggacccgccg ggggacggcc cgggccaggc ccgggatcta gacggccgta gggggaaggg 1380 agccgccctc cccacggcgc cttttcggaa ctgccgtgga ctcgaggacg ctggtcgccg 1440 gcctcctagg gctgtgctgt tttgttttga ccctcgcatt gtgcagaatt aaagtgcagt 1500 aaaatgtcca ctaggacccc attgccaacg gtgaatgaac gagacactga aaaccacacg 1560 tcacatggag atgggcgtca agaagttacc tctcgtacca gccgctcagg agctcggtgt 1620 agaaactcta tagcctcctg tgcagatgaa caacctcaca tcggaaacta cagactgttg 1680 aaaacaatcg gcaaggggaa ttttgcaaaa gtaaaattgg caagacatat ccttacaggc 1740 agagaggttg caataaaaat aattgacaaa actcagttga atccaacaag tctacaaaag 1800 ctcttcagag aagtaagaat aatgaagatt ttaaatcatc ccaatatagt gaagttattc 1860 gaagtcattg aaactgaaaa aacactctac ctaatcatgg aatatgcaag tggaggtgaa 1920 gtatttgact atttggttgc acatggcaag atgaaggaaa aagaagcaag atctaaattt 1980 agacagggtt gtcaagctgg acagactatt aaagttcaag tctcctttga tttgcttagt 2040 ctgatgttta catttattgt gtctgcagtt caatactgcc atcagaaacg gatcgtacat 2100 cgagacctca aggctgaaaa tctattgtta gatgccgata tgaacattaa aatagcagat 2160 ttcggtttta gcaatgaatt tactgttggc ggtaaactcg acacgttttg tggcagtcct 2220 ccatacgcag cacctgagct cttccagggc aagaaatatg acgggccaga agtggatgtg 2280 tggagtctgg gggtcatttt atacacacta gtcagtggct cacttccctt tgatgggcaa 2340 aacctaaagg aactgagaga gagagtatta agagggaaat acagaattcc cttctacatg 2400 tctacagact gtgaaaacct tctcaaacgt ttcctggtgc taaatccaat taaacgcggc 2460 actctagagc aaatcatgaa ggacaggtgg atcaatgcag ggcatgaaga agatgaactc 2520 aaaccatttg ttgaaccaga gctagacatc tcagaccaaa aaagaataga tattatggtg 2580 ggaatgggat attcacaaga agaaattcaa gaatctctta gtaagatgaa atacgatgaa 2640 atcacagcta catatttgtt attggggaga aaatcttcag agctggatgc tagtgattcc 2700 agttctagca gcaatctttc acttgctaag gttaggccga gcagtgatct caacaacagt 2760 actggccagt ctcctcacca caaagtgcag agaagtgttt cttcaagcca aaagcaaaga 2820 cgctacagtg accatgctgg accagctatt ccttctgttg tggcgtatcc gaaaaggagt 2880 cagacaagca ctgcagatgg tgacctcaaa gaagatggaa tttcctcccg gaaatcaagt 2940 ggcagtgctg ttggaggaaa gggaattgct ccagccagtc ccatgcttgg gaatgcaagt 3000 aatcctaata aggcggatat tcctgaacgc aagaaaagct ccactgtccc tagtagtaac 3060 acagcatctg gtggaatgac acgacgaaat acttatgttt gcagtgagag aactacagct 3120 gatagacact cagtgattca gaatggcaaa gaaaacagca ctattcctga tcagagaact 3180 ccagttgctt caacacacag tatcagtagt gcagccaccc cagatcgaat ccgcttccca 3240 agaggcactg ccagtcgtag cactttccac ggccagcccc gggaacggcg aaccgcaaca 3300 tataatggcc ctcctgcctc tcccagcctg tcccatgaag ccacaccatt gtcccagact 3360 cgaagccgag gctccactaa tctctttagt aaattaactt caaaactcac aaggagtcgc 3420 aatgtatctg ctgagcaaaa agatgaaaac aaagaagcaa agcctcgatc cctacgcttc 3480 acctggagca tgaaaaccac tagttcaatg gatcccgggg acatgatgcg ggaaatccgc 3540 aaagtgttgg acgccaataa ctgcgactat gagcagaggg agcgcttctt gctcttctgc 3600 gtccacggag atgggcacgc ggagaacctc gtgcagtggg aaatggaagt gtgcaagctg 3660 ccaagactgt ctctgaacgg ggtccggttt aagcggatat cggggacatc catagccttc 3720 aaaaatattg cttccaaaat tgccaatgag ctaaagctgt aacccagtga ttatgatgta 3780 aattaagtag caagtaaagt gttttcctga acactgatgg aaatgtatag aataatattt 3840 aggcaataac gtctgcatct tctaaatcat gaaattaaag tctgaggacg agagc 3895 16 2145 DNA Homo sapiens 16 atgtccacta ggaccccatt gccaacggtg aatgaacgag acactgaaaa ccacacgtca 60 catggagatg ggcgtcaaga agttacctct cgtaccagcc gctcaggagc tcggtgtaga 120 aactctatag cctcctgtgc agatgaacaa cctcacatcg gaaactacag actgttgaaa 180 acaatcggca aggggaattt tgcaaaagta aaattggcaa gacatatcct tacaggcaga 240 gaggttgcaa taaaaataat tgacaaaact cagttgaatc caacaagtct acaaaagctc 300 ttcagagaag taagaataat gaagatttta aatcatccca atatagtgaa gttattcgaa 360 gtcattgaaa ctgaaaaaac actctaccta atcatggaat atgcaagtgg aggtgaagta 420 tttgactatt tggttgcaca tggcaggatg aaggaaaaag aagcaagatc taaatttaga 480 cagattgtgt ctgcagttca atactgccat cagaaacgga tcgtacatcg agacctcaag 540 gctgaaaatc tattgttaga tgccgatatg aacattaaaa tagcagattt cggttttagc 600 aatgaattta ctgttggcgg taaactcgac acgttttgtg gcagtcctcc atacgcagca 660 cctgagctct tccagggcaa gaaatatgac gggccagaag tggatgtgtg gagtctgggg 720 gtcattttat acacactagt cagtggctca cttccctttg atgggcaaaa cctaaaggaa 780 ctgagagaga gagtattaag agggaaatac agaattccct tctacatgtc tacagactgt 840 gaaaaccttc tcaaacgttt cctggtgcta aatccaatta aacgcggcac tctagagcaa 900 atcatgaagg acaggtggat caatgcaggg catgaagaag atgaactcaa accatttgtt 960 gaaccagagc tagacatctc agaccaaaaa agaatagata ttatggtggg aatgggatat 1020 tcacaagaag aaattcaaga atctcttagt aagatgaaat acgatgaaat cacagctaca 1080 tatttgttat tggggagaaa atcttcagag gttaggccga gcagtgatct caacaacagt 1140 actggccagt ctcctcacca caaagtgcag agaagtgttt cttcaagcca aaagcaaaga 1200 cgctacagtg accatgctgg accagctatt ccttctgttg tggcgtatcc gaaaaggagt 1260 cagaccagca ctgcagatag tgacctcaaa gaagatggaa tttcctcccg gaaatcaagt 1320 ggcagtgctg ttggaggaaa gggaattgct ccagccagtc ccatgcttgg gaatgcaagt 1380 aatcctaata aggcggatat tcctgaacgc aagaaaagct ccactgtccc tagtagtaac 1440 acagcatctg gtggaatgac acgacgaaat acttatgttt gcagtgagag aactacagct 1500 gatagacact cagtgattca gaatggcaaa gaaaacagca ctattcctga tcagagaact 1560 ccagttgctt caacacacag tatcagtagt gcagccaccc cagatcgaat ccgcttccca 1620 agaggcactg ccagtcgtag cactttccac ggccagcccc gggaacggcg aaccgcaaca 1680 tataatggcc ctcctgcctc tcccagcctg tcccatgaag ccacaccatt gtcccagact 1740 cgaagccgag gctccactaa tctctttagt aaattaactt caaaactcac aaggagtcgc 1800 aatgtatctg ctgagcaaaa agatgaaaac aaagaagcaa agcctcgatc cctacgcttc 1860 acctggagca tgaaaaccac tagttcaatg gatcccgggg acatgatgcg ggaaatccgc 1920 aaagtgttgg acgccaataa ctgcgactat gagcagaggg agcgcttctt gctcttctgc 1980 gtccacggag atgggcacgc ggagaacctc gtgcagtggg aaatggaagt gtgcaagctg 2040 ccaagactgt ctctgaacgg ggtccggttt aagcggatat cggggacatc catagccttc 2100 aaaaatattg cttccaaaat tgccaatgag ctaaagctgt aaccc 2145 17 2193 DNA Homo sapiens 17 atgtccacta ggaccccatt gccaacggtg aatgaacgag acactgaaaa ccacacgtca 60 catggagatg ggcgtcaaga agttacctct cgtaccagcc gctcaggagc tcggtgtaga 120 aactctatag cctcctgtgc agatgaacaa cctcacatcg gaaactacag actgttgaaa 180 acaatcggca aggggaattt tgcaaaagta aaattggcaa gacatatcct tacaggcaga 240 gaggttgcaa taaaaataat tgacaaaact cagttgaatc caacaagtct acaaaagctc 300 ttcagagaag taagaataat gaagatttta aatcatccca atatagtgaa gttattcgaa 360 gtcattgaaa ctgaaaaaac actctaccta atcatggaat atgcaagtgg aggtgaagta 420 tttgactatt tggttgcaca tggcaggatg aaggaaaaag aagcaagatc taaatttaga 480 cagattgtgt ctgcagttca atactgccat cagaaacgga tcgtacatcg agacctcaag 540 gctgaaaatc tattgttaga tgccgatatg aacattaaaa tagcagattt cggttttagc 600 aatgaattta ctgttggcgg taaactcgac acgttttgtg gcagtcctcc atacgcagca 660 cctgagctct tccagggcaa gaaatatgac gggccagaag tggatgtgtg gagtctgggg 720 gtcattttat acacactagt cagtggctca cttccctttg atgggcaaaa cctaaaggaa 780 ctgagagaga gagtattaag agggaaatac agaattccct tctacatgtc tacagactgt 840 gaaaaccttc tcaaacgttt cctggtgcta aatccaatta aacgcggcac tctagagcaa 900 atcatgaagg acaggtggat caatgcaggg catgaagaag atgaactcaa accatttgtt 960 gaaccagagc tagacatctc agaccaaaaa agaatagata ttatggtggg aatgggatat 1020 tcacaagaag aaattcaaga atctcttagt aagatgaaat acgatgaaat cacagctaca 1080 tatttgttat tggggagaaa atcttcagag ctggatgcta gtgattccag ttctagcagc 1140 aatctttcac ttgctaaggt taggccgagc agtgatctca acaacagtac tggccagtct 1200 cctcaccaca aagtgcagag aagtgtttct tcaagccaaa agcaaagacg ctacagtgac 1260 catgctggac cagctattcc ttctgttgtg gcgtatccga aaaggagtca gaccagcact 1320 gcagatagtg acctcaaaga agatggaatt tcctcccgga aatcaagtgg cagtgctgtt 1380 ggaggaaagg gaattgctcc agccagtccc atgcttggga atgcaagtaa tcctaataag 1440 gcggatattc ctgaacgcaa gaaaagctcc actgtcccta gtagtaacac agcatctggt 1500 ggaatgacac gacgaaatac ttatgtttgc agtgagagaa ctacagctga tagacactca 1560 gtgattcaga atggcaaaga aaacagcact attcctgatc agagaactcc agttgcttca 1620 acacacagta tcagtagtgc agccacccca gatcgaatcc gcttcccaag aggcactgcc 1680 agtcgtagca ctttccacgg ccagccccgg gaacggcgaa ccgcaacata taatggccct 1740 cctgcctctc ccagcctgtc ccatgaagcc acaccattgt cccagactcg aagccgaggc 1800 tccactaatc tctttagtaa attaacttca aaactcacaa ggagtcgcaa tgtatctgct 1860 gagcaaaaag atgaaaacaa agaagcaaag cctcgatccc tacgcttcac ctggagcatg 1920 aaaaccacta gttcaatgga tcccggggac atgatgcggg aaatccgcaa agtgttggac 1980 gccaataact gcgactatga gcagagggag cgcttcttgc tcttctgcgt ccacggagat 2040 gggcacgcgg agaacctcgt gcagtgggaa atggaagtgt gcaagctgcc aagactgtct 2100 ctgaacgggg tccggtttaa gcggatatcg gggacatcca tagccttcaa aaatattgct 2160 tccaaaattg ccaatgagct aaagctgtaa ccc 2193 18 3373 DNA Homo sapiens 18 caggcgcctc ctccgcagcc cgctccatgg tcggcgccca cagcccgcgg cggcctgtct 60 tgcgctccac ttccttcaca tcctcctccg cctcctcgtt ttcaggcgcc gccggcggcg 120 ctgtgtggag gcccgcgagc tgaaattcgc ggtgcgacgg gagggagtgg agaaggaggt 180 gagggggccc aggatcgcgg ggcgccctga ggcaagggga cgccggcggg ccgaagcgca 240 gcccgccgcc cgcaggctcg gctccgccac tgccgccctc ccggtctcct cgcctcggcc 300 gccgaggcag ggagagaatg agccccggga cccgccgggg acggcccggg ccaggcccgg 360 gatctagaac ggccgtaggg ggaagggagc cgccctcccc acggcgcctt ttcggaactg 420 ccgtggactc gaggacgctg gtcgccggcc tcctagggct gtgctgtttt gttttgaccc 480 tcgcattgtg cagaattaaa gtgcagtaaa atgtccacta ggaccccatt gccaacggtg 540 aatgaacgag acactgaaaa ccacacgtca catggagatg ggcgtcaaga agttacctct 600 cgtaccagcc gctcaggagc tcggtgtaga aactctatag cctcctgtgc agatgaacaa 660 cctcacatcg gaaactacag actgttgaaa acaatcggca aggggaattt tgcaaaagta 720 aaattggcaa gacatatcct tacaggcaga gaggttgcaa taaaaataat tgacaaaact 780 cagttgaatc caacaagtct acaaaagctc ttcagagaag taagaataat gaagatttta 840 aatcatccca atatagtgaa gttattcgaa gtcattgaaa ctgaaaaaac actctaccta 900 atcatggaat atgcaagtgg aggtaaagta tttgactatt tggttgcaca tggcaggatg 960 aaggaaaaag aagcaagatc taaatttaga cagattgtgt ctgcagttca atactgccat 1020 cagaaacgga tcgtacatcg agacctcaag gctgaaaatc tattgttaga tgccgatatg 1080 aacattaaaa tagcagattt cggttttagc aatgaattta ctgttggcgg taaactcgac 1140 acgttttgtg gcagtcctcc atacgcagca cctgagctct tccagggcaa gaaatatgac 1200 gggccagaag tggatgtgtg gagtctgggg gtcattttat acacactagt cagtggctca 1260 cttccctttg atgggcaaaa cctaaaggaa ctgagagaga gagtattaag agggaaatac 1320 agaattccct tctacatgtc tacagactgt gaaaaccttc tcaaacgttt cctggtgcta 1380 aatccaatta aacgcggcac tctagagcaa atcatgaagg acaggtggat caatgcaggg 1440 catgaagaag atgaactcaa accatttgtt gaaccagagc tagacatctc agaccaaaaa 1500 agaatagata ttatggtggg aatgggatat tcacaagaag aaattcaaga atctcttagt 1560 aagatgaaat acgatgaaat cacagctaca tatttgttat tggggagaaa atcttcagag 1620 ctggatgcta gtgattccag ttctagcagc aatctttcac ttgctaaggt taggccgagc 1680 agtgatctca acaacagtac tggccagtct cctcaccaca aagtgcagag aagtgtttct 1740 tcaagccaaa agcaaagacg ctacagtgac catgctggac cagctattcc ttctgttgtg 1800 gcgtatccga aaaggagtca gaccagcact gcagatagtg acctcaaaga agatggaatt 1860 tcctcccgga aatcaagtgg cagtgctgtt ggaggaaagg gaattgctcc agccagtccc 1920 atgcttggga atgcaagtaa tcctaataag gcggatattc ctgaacgcaa gaaaagctcc 1980 actgtcccta gtagtaacac agcatctggt ggaatgacac gacgaaatac ttatgtttgc 2040 agtgagagaa ctacagctga tagacactca gtgattcaga atggcaaaga aaacagcact 2100 attcctgatc agagaactcc agttgcttca acacacagta tcagtagtgc agccacccca 2160 gatcgaatcc gcttcccaag aggcactgcc agtcgtagca ctttccacgg ccagccccgg 2220 gaacggcgaa ccgcaacata taatggccct cctgcctctc ccagcctgtc ccatgaagcc 2280 acaccattgt cccagactcg aagccgaggc tccactaatc tctttagtaa attaacttca 2340 aaactcacaa ggagaaacat gtcattcagg tttatcaaaa ggcttccaac tgaatatgag 2400 aggaacggga gatatgaggg ctcaagtcgc aatgtatctg ctgagcaaaa agatgaaaac 2460 aaagaagcaa agcctcgatc cctacgcttc acctggagca tgaaaaccac tagttcaatg 2520 gatcccgggg acatgatgcg ggaaatccgc aaagtgttgg acgccaataa ctgcgactat 2580 gagcagaggg agcgcttctt gctcttctgc gtccacggag atgggcacgc ggagaacctc 2640 gtgcagtggg aaatggaagt gtgcaagctg ccaagactgt ctctgaacgg ggtccggttt 2700 aagcggatat cggggacatc catagccttc aaaaatattg cttccaaaat tgccaatgag 2760 ctaaagctgt aacccagtga ttatgatgta aattaagtag caagtaaagt gttttcctga 2820 acactgatgg aaatgtatag aataatattt aggcaataac gtctgcatct tctaaatcat 2880 gaaattaaag tctgaggacg agagcacgcc tgggagcgaa agctggcctt ttttctacga 2940 atgcactaca ttaaagatgt gcaacctatg cgccccctgc cctacttccg ttaccctgag 3000 agtcggcgtg tggccccatc tccatgtgcc tcccgtctgg gtgggtgtga gagtggacgg 3060 tatgtgtgtg aagtggtgta tatggaagca tctccctaca ctggcagcca gtcattacta 3120 gtacctctgc gggagatcat ccggtgctaa aacattacag ttgccaagga ggaaaatact 3180 gaatgactgc taagaattaa ccttaagacc agttcatagt taatacaggt ttacagttca 3240 tgcctgtggt tttgtgtttg ttgttttgtg tttttttagt gcaaaaggtt taaatttata 3300 gttgtgaaca ttgcttgtgt gtgtttttct aagtagattc acaagataat taaaaattca 3360 ctttttctca ggt 3373 19 3609 DNA Homo sapiens misc_feature (3606)..(3606) “n” is A, C, G, or T 19 cgcctccctc cgccgccgct tgggccggct ccgcgccccc tccgcggccc ccgcccgccc 60 gcctgcccgc cgcccccatg gcgcccgggg tccccgctgc acggggccac taggaccctc 120 ggcgtccctt cccctccccc gccctgcccc ctctcccgcc gcgcggaccc gggcgttctc 180 ggcgcccagc ttttgagctc gcgtccccag gccggcgggg ggggagggga agagagggga 240 ccctgggacc cccgcccccc ccacccggcc gcccctgccc cccgggaccc ggagaagatg 300 tcttcgcgga cggtgctggc cccgggcaac gatcggaact cggacacgca tggcaccttg 360 ggcagtggcc gctcctcgga caaaggcccg tcctggtcca gccgctcact gggtgcccgt 420 tgccggaact ccatcgcctc ctgtcccgag gagcagcccc acgtgggcaa ctaccgcctg 480 ctgaggacca ttgggaaggg caactctgcc aaagtcaagc tggctcggca catcctcact 540 ggtcgggagg ttgccatcaa gattatcgac aaaacccagc tgaatcccag cagcctgcag 600 aagctgttcc gagaagtccg catcatgaag ggcctaaacc accccaacat cgtgaagctc 660 tttgaggtga ttgagactga gaagacgctg tacctggtga tggagtacgc aagtgctgga 720 gaagtgtttg actacctcgt gtcgcatggc cgcatgaagg agaaggaagc tcgagccaag 780 ttccgacaga ttgtttcggc tgtgcactat tgtcaccaga aaaatattgt acacagggac 840 ctgaaggctg agaacctctt gctggatgcc gaggccaaca tcaagattgc tgactttggc 900 ttcagcaacg agttcacgct gggatcgaag ctggacacgt tctgcgggag ccccccatat 960 gccgccccgg agctgtttca gggcaagaag tacgacgggc cggaggtgga catctggagc 1020 ctgggagtca tcctgtacac cctcgtcagc ggctccctgc ccttcgacgg gcacaacctc 1080 aaggagctgc gggagcgagt actcagaggg aagtaccggg tccctttcta catgtcaaca 1140 gactgtgaga gcatcctgcg gagatttttg gtgctgaacc cagctaaacg ctgtactctc 1200 gagcaaatca tgaaagacaa atggatcaac atcggctatg agggtgagga gttgaagcca 1260 tacacagagc ccgaggagga cttcggggac accaagagaa ttgaggtgat ggtgggtatg 1320 ggctacacac gggaagaaat caaagagtcc ttgaccagcc agaagtacaa cgaagtgacc 1380 gccacctacc tcctgctggg caggaagact gaggagggtg gggaccgggg cgccccaggg 1440 ctggccctgg cacgggtgcg ggcgcccagc gacaccacca acggaacaag ttccagcaaa 1500 ggcaccagcc acagcaaagg gcagcggagt tcctcttcca cctaccaccg ccagcgcagg 1560 catagcgatt tctgtggccc atcccctgca cccctgcacc ccaaacgcag cccgacgagc 1620 acgggggagg cggagctgaa ggaggagcgg ctgccaggcc ggaaggcgag ctgcagcacc 1680 gcggggagtg ggagtcgagg gctgcccccc tccagcccca tggtcagcag cgcccacaac 1740 cccaacaagg cagagatccc agagcggcgg aaggacagca cgagcacccc caacaacctc 1800 cctcctagca tgatgacccg cagaaacacc tacgtttgca cagaacgccc gggggctgag 1860 cgcccgtcac tgttgccaaa tgggaaagaa aacagctcag gcaccccacg ggtgccccct 1920 gcctccccct ccagtcacag cctggcaccc ccatcagggg agcggagccg cctggcacgc 1980 ggttccacca tccgcagcac cttccatggt ggccaggtcc gggaccggcg ggcagggggt 2040 gggggtggtg ggggtgtgca gaatgggccc cctgcctctc ccacactggc ccatgaggct 2100 gcacccctgc ccgccgggcg gccccgcccc accaccaacc tcttcaccaa gctgacctcc 2160 aaactgaccc gaagggttac cctcgatccc tctaaacggc agaactctaa ccgctgtgtt 2220 tcgggcgcct ctctgcccca gggatccaag atcaggtcgc agacgaacct gagagaatcg 2280 ggggacctga ggtcacaagt tgccatctac cttgggatca aacggaaacc gccccccggc 2340 tgctccgatt cccctggagt gtgaagctga ccagctcgcg ccctcctgag gccctgatgg 2400 cagctctgcg ccaggccaca gcagccgccc gctgccgctg ccgccagcca cagccgttcc 2460 tgctggcctg cctgcacggg ggtgcgggcg ggcccgagcc cctgtcccac ttcgaagtgg 2520 aggtctgcca gctgccccgg ccaggcttgc ggggagttct cttccgccgt gtggcgggca 2580 ccgccctggc cttccgcacc ctcgtcaccc gcatctccaa cgacctcgag ctctgagcca 2640 ccacggtccc agggccctta ctcttcctct cccttgtcgc cttcacttct acaggagggg 2700 aaggggccag ggaggggatt ctccctttat catcacctca gtttccctga attatatttg 2760 ggggcaaaga ttgtcccctc tgctgttctc tggggccgct cagcacagaa gaaggatgag 2820 ggggctcagc ggggggagct ggcaccttcc tggagcctcc agccagtcct gtcctccctc 2880 gccctaccaa gagggcacct gaggagactt tggggacagg gcaggggcag ggagggaaac 2940 tgaggaaatc ttccattcct cccaacagct caaaattagg ccttgggcag gggcagggag 3000 agctgctgag cctaaagact ggagaatctg ggggactggg agtgggggtc agagaggcag 3060 attccttccc ctcccgtccc ctcacgctca aacccccact tcctgcccca ggctggcgcg 3120 gggcactttg tacaaatcct tgtaaatacc ccacaccctc ccctctgcaa aggtctcttg 3180 aggagctgcc gctgtcacct acggttttta agttattaca ccccgaccct cctcctgtca 3240 gccccctcac ctgcagcctg ttgcccaata aatttaagag agtccccccc tccccaatgc 3300 tgaccctagg attttccttc cctgccctca cctgcaaatg agttaaagaa gaggcgtggg 3360 aatccaggca gtggtttttc ctttcggagc ctcggttttc tcatctgcag aatgggagcg 3420 gtgggggtgg gaaggtaagg atggtcgtgg aagaaggcag gatggaactc ggcctcatcc 3480 ccgaggcccc agttcctata tcgggccccc cattcatcca ctcacactcc cagccaccat 3540 gttacactgg actctaagcc acttcttact ccagtagtaa atttattgca ataaacaatc 3600 attganccc 3609 20 2085 DNA Homo sapiens 20 agatgtcttc gcggacggtg ctggccccgg gcaacgatcg gaactcggac acgcatggca 60 ccttgggcag tggccgctcc tcggacaaag gcccgtcctg gtccagccgc tcactgggtg 120 cccgttgccg gaactccatc gcctcctgtc ccgaggagca gccccacgtg ggcaactacc 180 gcctgctgag gaccattggg aagggcaact ttgccaaagt caagctggct cggcacatcc 240 tcactggtcg ggaggttgcc atcaagatta tcgacaaaac ccagctgaat cccagcagcc 300 tgcagaagct gttccgagaa gtccgcatca tgaagggcct aaaccacccc aacatcgtga 360 agctctttga ggtgattgag actgagaaga cgctgtacct ggtgatggag tacgcaagtg 420 ctggagaagt gtttgactac ctcgtgtcgc atggccgcat gaaggagaag gaagctcgag 480 ccaagttccg acagattgtt tcggctgtgc actattgtca ccagaaaaat attgtacaca 540 gggacctgaa ggctgagaac ctcttgctgg atgccgaggc caacatcaag attgctgact 600 ttggcttcag caacgagttc acgctgggat cgaagctgga cacgttctgc gggagccccc 660 catatgccgc cccggagctg tttcagggca agaagtacga cgggccggag gtggacatct 720 ggagcctggg agtcatcctg tacaccctcg tcagcggctc cctgcccttc gacgggcaca 780 acctcaagga gctgcgggag cgagtactca gagggaagta ccgggtccct ttctacatgt 840 caacagactg tgagagcatc ctgcggagat ttttggtgct gaacccagct aaacgctgta 900 ctctcgagca aatcatgaaa gacaaatgga tcaacatcgg ctatgagggt gaggagttga 960 agccatacac agagcccgag gaggacttcg gggacaccaa gagaattgag gtgatggtgg 1020 gtatgggcta cacacgggaa gaaatcaaag agtccttgac cagccagaag tacaacgaag 1080 tgaccgccac ctacctcctg ctgggcagga agactgagga gggtggggac cggggcgccc 1140 cagggctggc cctggcacgg gtgcgggcgc ccagcgacac caccaacgga acaagttcca 1200 gcaaaggcac cagccacagc aaagggcagc ggagttcctc ttccacctac caccgccagc 1260 gcaggcatag cgatttctgt ggcccatccc ctgcacccct gcaccccaaa cgcagcccga 1320 cgagcacggg ggaggcggag ctgaaggagg agcggctgcc aggccggaag gcgagctgca 1380 gcaccgcggg gagtgggagt cgagggctgc ccccctccag ccccatggtc agcagcgccc 1440 acaaccccaa caaggcagag atcccagagc ggcggaagga cagcacgagc acccccaaca 1500 acctccctcc tagcatgatg acccgcagaa acacctacgt ttgcacagaa cgcccggggg 1560 ctgagcgccc gtcactgttg ccaaatggga aagaaaacag ctcaggcacc ccacgggtgc 1620 cccctgcctc cccctccagt cacagcctgg cacccccatc aggggagcgg agccgcctgg 1680 cacgcggttc caccatccgc agcaccttcc atggtggcca ggtccgggac cggcgggcag 1740 ggggtggggg tggtgggggt gtgcagaatg ggccccctgc ctctcccaca ctggcccatg 1800 aggctgcacc cctgcccgcc gggcggcccc gccccaccac caacctcttc accaagctga 1860 cctccaaact gacccgaagg gttaccctcg atccctctaa acggcagaac tctaaccgct 1920 gtgtttcggg cgcctctctg ccccagggat ccaagatcag gtcgcagacg aacctgagag 1980 aatcggggga cctgaggtca caagttgcca tctaccttgg gatcaaacgg aaaccgcccc 2040 ccggctgctc cgattcccct ggagtgtgaa gctgaccagc tcgcg 2085 21 2278 DNA Homo sapiens 21 agatgtcttc gcggacggtg ctggccccgg gcaacgatcg gaactcggac acgcatggca 60 ccttgggcag tggccgctcc tcggacaaag gcccgtcctg gtccagccgc tcactgggtg 120 cccgttgccg gaactccatc gcctcctgtc ccgaggagca gccccacgtg ggcaactacc 180 gcctgctgag gaccattggg aagggcaact ttgccaaagt caagctggct cggcacatcc 240 tcactggtcg ggaggttgcc atcaagatta tcgacaaaac ccagctgaat cccagcagcc 300 tgcagaagct gttccgagaa gtccgcatca tgaagggcct aaaccacccc aacatcgtga 360 agctctttga ggtgattgag actgagaaga cgctgtacct ggtgatggag tacgcaagtg 420 ctggagaagt gtttgactac ctcgtgtcgc atggccgcat gaaggagaag gaagctcgag 480 ccaagttccg acagattgtt tcggctgtgc actattgtca ccagaaaaat attgtacaca 540 gggacctgaa ggctgagaac ctcttgctgg atgccgaggc caacatcaag attgctgact 600 ttggcttcag caacgagttc acgctgggat cgaagctgga cacgttctgc gggagccccc 660 catatgccgc cccggagctg tttcagggca agaagtacga cgggccggag gtggacatct 720 ggagcctggg agtcatcctg tacaccctcg tcagcggctc cctgcccttc gacgggcaca 780 acctcaagga gctgcgggag cgagtactca gagggaagta ccgggtccct ttctacatgt 840 caacagactg tgagagcatc ctgcggagat ttttggtgct gaacccagct aaacgctgta 900 ctctcgagca aatcatgaaa gacaaatgga tcaacatcgg ctatgagggt gaggagttga 960 agccatacac agagcccgag gaggacttcg gggacaccaa gagaattgag gtgatggtgg 1020 gtatgggcta cacacgggaa gaaatcaaag agtccttgac cagccagaag tacaacgaag 1080 tgaccgccac ctacctcctg ctgggcagga agactgagga gggtggggac cggggcgccc 1140 cagggctggc cctggcacgg gtgcgggcgc ccagcgacac caccaacgga acaagttcca 1200 gcaaaggcac cagccacagc aaagggcagc ggagttcctc ttccacctac caccgccagc 1260 gcaggcatag cgatttctgt ggcccatccc ctgcacccct gcaccccaaa cgcagcccga 1320 cgagcacggg ggaggcggag ctgaaggagg agcggctgcc aggccggaag gcgagctgca 1380 gcaccgcggg gagtgggagt cgagggctgc ccccctccag ccccatggtc agcagcgccc 1440 acaaccccaa caaggcagag atcccagagc ggcggaagga cagcacgagc acccccaaca 1500 acctccctcc tagcatgatg acccgcagaa acacctacgt ttgcacagaa cgcccggggg 1560 ctgagcgccc gtcactgttg ccaaatggga aagaaaacag ctcaggcacc ccacgggtgc 1620 cccctgcctc cccctccagt cacagcctgg cacccccatc aggggagcgg agccgcctgg 1680 cacgcggttc caccatccgc agcaccttcc atggtggcca ggtccgggac cggcgggcag 1740 ggggtggggg tggtgggggt gtgcagaatg ggccccctgc ctctcccaca ctggcccatg 1800 aggctgcacc cctgcccgcc gggcggcccc gccccaccac caacctcttc accaagctga 1860 cctccaaact gacccgaagg gtcgcagacg aacctgagag aatcggggga cctgaggtca 1920 caagttgcca tctaccttgg gatcaaacgg aaaccgcccc ccggctgctc cgattcccct 1980 ggagtgtgaa gctgaccagc tcgcgccctc ctgaggccct gatggcagct ctgcgccagg 2040 ccacagcagc cgcccgctgc cgctgccgcc agccacagcc gttcctgctg gcctgcctgc 2100 acgggggtgc gggcgggccc gagcccctgt cccacttcga agtggaggtc tgccagctgc 2160 cccggccagg cttgcgggga gttctcttcc gccgtgtggc gggcaccgcc ctggccttcc 2220 gcaccctcgt cacccgcatc tccaacgacc tcgagctctg agccaccacg gtcccagg 2278 22 4917 DNA Homo sapiens 22 agaagatgtc ttcgcggacg gtgctggccc cgggcaacga tcggaactcg gacacgcatg 60 gcaccttggg cagtggccgc tcctcggaca aaggcccgtc ctggtccagc cgctcactgg 120 gtgcccgttg ccggaactcc atcgcctcct gtcccgagga gcagccccac gtgggcaact 180 accgcctgct gaggaccatt gggaagggca actttgccaa agtcaagctg gctcggcaca 240 tcctcactgg tcgggaggtt gccatcaaga ttatcgacaa aacccagctg aatcccagca 300 gcctgcagaa gctgttccga gaagtccgca tcatgaaggg cctaaaccac cccaacatcg 360 tgaagctctt tgaggtgatt gagactgaga agacgctgta cctggtgatg gagtacgcaa 420 gtgctggaga agtgtttgac tacctcgtgt cgcatggccg catgaaggag aaggaagctc 480 gagccaagtt ccgacagatt gtttcggctg tgcactattg tcaccagaaa aatattgtac 540 acagggacct gaaggctgag aacctcttgc tggatgccga ggccaacatc aagattgctg 600 actttggctt cagcaacgag ttcacgctgg gatcgaagct ggacacgttc tgcgggagcc 660 ccccatatgc cgccccggag ctgtttcagg gcaagaagta cgacgggccg gaggtggaca 720 tctggagcct gggagtcatc ctgtacaccc tcgtcagcgg ctccctgccc ttcgacgggc 780 acaacctcaa ggagctgcgg gagcgagtac tcagagggaa gtaccgggtc cctttctaca 840 tgtcaacaga ctgtgagagc atcctgcgga gatttttggt gctgaaccca gctaaacgct 900 gtactctcga gcaaatcatg aaagacaaat ggatcaacat cggctatgag ggtgaggagt 960 tgaagccata cacagagccc gaggaggact tcggggacac caagagaatt gaggtgatgg 1020 tgggtatggg ctacacacgg gaagaaatca aagagtcctt gaccagccag aagtacaacg 1080 aagtgaccgc cacctacctc ctgctgggca ggaagactga ggagggtggg gaccggggcg 1140 ccccagggct ggccctggca cgggtgcggg cgcccagcga caccaccaac ggaacaagtt 1200 ccagcaaagg caccagccac agcaaagggc agcggagttc ctcttccacc taccaccgcc 1260 agcgcaggca tagcgatttc tgtggcccat cccctgcacc cctgcacccc aaacgcagcc 1320 cgacgagcac gggggaggcg gagctgaagg aggagcggct gccaggccgg aaggcgagct 1380 gcagcaccgc ggggagtggg agtcgagggc tgcccccctc cagccccatg gtcagcagcg 1440 cccacaaccc caacaaggca gagatcccag agcggcggaa ggacagcacg agcaccccca 1500 acaacctccc tcctagcatg atgacccgca gaaacaccta cgtttgcaca gaacgcccgg 1560 gggctgagcg cccgtcactg ttgccaaatg ggaaagaaaa cagctcaggc accccacggg 1620 tgccccctgc ctccccctcc agtcacagcc tggcaccccc atcaggggag cggagccgcc 1680 tggcacgcgg ttccaccatc cgcagcacct tccatggtgg ccaggtccgg gaccggcggg 1740 cagggggtgg gggtggtggg ggtgtgcaga atgggccccc tgcctctccc acactggccc 1800 atgaggctgc acccctgccc gccgggcggc cccgccccac caccaacctc ttcaccaagc 1860 tgacctccaa actgacccga agggttaccc tcgatccctc taaacggcag aactctaacc 1920 gctgtgtttc gggcgcctct ctgccccagg gatccaagat caggtcgcag acgaacctga 1980 gagaatcggg ggacctgagg tcacaagttg ccatctacct tgggatcaaa cggaaaccgc 2040 cccccggctg ctccgattcc cctggagtgt gaagctgacc agctcgcgcc ctcctgaggc 2100 cctgatggca gctctgcgcc aggccacagc agccgcccgc tgccgctgcc gccagccaca 2160 gccgttcctg ctggcctgcc tgcacggggg tgcgggcggg cccgagcccc tgtcccactt 2220 cgaagtggag gtctgccagc tgccccggcc aggcttgcgg ggagttctct tccgccgtgt 2280 ggcgggcacc gccctggcct tccgcaccct cgtcacccgc atctccaacg acctcgagct 2340 ctgagccacc acggtcccag ggcccttact cttcctctcc cttgtcgcct tcacttctac 2400 aggaggggaa ggggccaggg aggggattct ccctttatca tcacctcagt ttccctgaat 2460 tatatttggg ggcaaagatt gtcccctctg ctgttctctg gggccgctca gcacagaaga 2520 aggatgaggg ggctcagcgg ggggagctgg caccttcctg gagcctccag ccagtcctgt 2580 cctccctcgc cctaccaaga gggcacctga ggagactttg gggacagggc aggggcaggg 2640 agggaaactg aggaaatctt ccattcctcc caacagctca aaattaggcc ttgggcaggg 2700 gcagggagag ctgctgagcc taaagactgg agaatctggg ggactgggag tgggggtcag 2760 agaggcagat tccttcccct cccgtcccct cacgctcaaa cccccacttc ctgccccagg 2820 ctggcgcggg gcactttgta caaatccttg taaatacccc acaccctccc ctctgcaaag 2880 gtctcttgag gagctgccgc tgtcacctac ggtttttaag ttattacacc ccgaccctcc 2940 tcctgtcagc cccctcacct gcagcctgtt gcccaataaa tttaagagag tccccccctc 3000 cccaatgctg accctaggat tttccttccc tgccctcacc tgcaaatgag ttaaagaaga 3060 ggcgtgggaa tccaggcagt ggtttttcct ttcggagcct cggttttctc atctgcagaa 3120 tgggagcggt gggggtggga aggtaaggat ggtcgtggaa gaaggcagga tggaactcgg 3180 cctcatcccc gaggccccag ttcctatatc gggcccccca ttcatccact cacactccca 3240 gccaccatgt tacactggac tctaagccac ttcttactcc agtagtaaat ttattcaata 3300 aacaatcatt gacccatgcc tactccatgc caggcccagt gctggacaca gagacatgaa 3360 gctctgtctg tgggagacag ggattctgac acagacaccg gacaaaccat tgtcttgggg 3420 agcccagaag agaaagtggg cagggtgggg tcattgggga agatgctcta gaggaattaa 3480 tgctggaatg gggtgttgaa ggatgagtag gagttagtta ggcattgagt ttgccctggg 3540 caaaagccca gaagtgggag tatgtggtat atcttcagag aactgggtaa tttcagtgtg 3600 gctgctgtgt tgggcatgga tggagaatca gcaagagaaa tgctgtatta ggactaataa 3660 tccatctacg ctgcttaagc aaaaaggtat ttgttggttt atgttactta atagtccagg 3720 ggcacctggc ttcaggtagg tttgatccag gcatcaggcc attgcatcta ttttttcagt 3780 gtaagttgaa ttctagtaat ttttatcaag taagggctcc tttcctggtg gcacagatga 3840 cttcagcagt tagaagtttc tatccctcca gctttctgca gcagaaagac cctcattgtc 3900 agtttcccag caaaagtccc agggcagact ctcattggcc caaatgggcc atgtgatttt 3960 ctctaaacca atcactgtga ctctagagtg gccagactca gagctgcact tagtaggggt 4020 tcctcaaagg aaggtcaagt gtcatgagca ggagaaaagg catgggagct ggacagatta 4080 tagtggttga agtctgtgca gtacagaagg gcggagctta ttcacacagc acctttgggg 4140 ccaaaatgaa taagctggac tttctcccca tggcactggg gaaccatgga agttcaggga 4200 acttcaggga agaggcttgg tcaattcctg agagcatcct ctgtgctggg gacacagtgg 4260 taatcaagac agccccaaca ctgccctcat agagctcaca gtccaatgga ggaggcagat 4320 gtgtcctcag gcagcgactg ggcagggctg gtatagggga gtccagaggt gatgcctgcc 4380 tcagccaggg agggcttcct ggaggagaag gagccagcta gacatggata ggagtgcgtt 4440 ttaggcacag caaatggcac atacaagggc cagggagcaa gagagaggac aggtcctcaa 4500 caaatggcat gtgactttgt aagtgtagaa ttgctgtgag gtatggggct aggggcgtca 4560 gtagggcctt gaaggttatg gacaggggcc tgggctttct tccaagggca ctgggggagc 4620 catggcaagg ttgtaggtag ggtagagatg ggcgggtttg tgctatgtgc agggtggaag 4680 ggagggaagt tgacaggtca gaagatcagg aaagaggtcg gggctggaca gatggggaga 4740 gcgcagatag atttaagaga gtcctgtgag gcaaagtggg caggacctgg taacaggtgt 4800 ctggactgtg gctttggctg gctcagaagg tccccactgg cgtgtgtggt ctatgtagcc 4860 tctgggtgtg gagctgggat cttcaactgg ggacagtaca gtaaagaaca tcacagc 4917 23 3226 DNA Homo sapiens 23 gacccggaga agatgtcttc gcggacggtg ctggccccgg gcaacgatcg gaactcggac 60 acgcatggca ccttgggcag tggccgctcc tcggacaaag gcccgtcctg gtccagccgc 120 tcactgggtg cccgttgccg gaactccatc gcctcctgtc ccgaggagca gccccacgtg 180 ggcaactacc gcctgctgag gaccattggg aagggcaact ttgccaaagt caagctggct 240 cggcacatcc tcactggtcg ggaggttgcc atcaagatta tcgacaaaac ccagctgaat 300 cccagcagcc tgcagaagct gttccgagaa gtccgcatca tgaagggcct aaaccacccc 360 aacatcgtga agctctttga ggtgattgag actgagaaga cgctgtacct ggtgatggag 420 tacgcaagtg ctggagaagt gtttgactac ctcgtgtcgc atggccgcat gaaggagaag 480 gaagctcgag ccaagttccg acagattgtt tcggctgtgc actattgtca ccagaaaaat 540 attgtacaca gggacctgaa ggctgagaac ctcttgctgg atgccgaggc caacatcaag 600 attgctgact ttggcttcag caacgagttc acgctgggat cgaagctgga cacgttctgc 660 gggagccccc catatgccgc cccggagctg tttcagggca agaagtacga cgggccggag 720 gtggacatct ggagcctggg agtcatcctg tacaccctcg tcagcggctc cctgcccttc 780 gacgggcaca acctcaagga gctgcgggag cgagtactca gagggaagta ccgggtccct 840 ttctacatgt caacagactg tgagagcatc ctgcggagat ttttggtgct gaacccagct 900 aaacgctgta ctctcgagca aatcatgaaa gacaaatgga tcaacatcgg ctatgagggt 960 gaggagttga agccatacac agagcccgag gaggacttcg gggacaccaa gagaattgag 1020 gtgatggtgg gtatgggcta cacacgggaa gaaatcaaag agtccttgac cagccagaag 1080 tacaacgaag tgaccgccac ctacctcctg ctgggcagga agactgagga gggtggggac 1140 cggggcgccc cagggctggc cctggcacgg gtgcgggcgc ccagcgacac caccaacgga 1200 acaagttcca gcaaaggcac cagccacagc aaagggcagc ggagttcctc ttccacctac 1260 caccgccagc gcaggcatag cgatttctgt ggcccatccc ctgcacccct gcaccccaaa 1320 cgcagcccga cgagcacggg ggaggcggag ctgaaggagg agcggctgcc aggccggaag 1380 gcgagctgca gcaccgcggg gagtgggagt cgagggctgc ccccctccag ccccatggtc 1440 agcagcgccc acaaccccaa caaggcagag atcccagagc ggcggaagga cagcacgagc 1500 acccccaaca acctccctcc tagcatgatg acccgcagaa acacctacgt ttgcacagaa 1560 cgcccggggg ctgagcgccc gtcactgttg ccaaatggga aagaaaacag ctcaggcacc 1620 ccacgggtgc cccctgcctc cccctccagt cacagcctgg cacccccatc aggggagcgg 1680 agccgcctgg cacgcggttc caccatccgc agcaccttcc atggtggcca ggtccgggac 1740 cggcgggcag ggggtggggg tggtgggggt gtgcagaatg ggccccctgc ctctcccaca 1800 ctggcccatg aggctgcacc cctgcccgcc gggcggcccc gccccaccac caacctcttc 1860 accaagctga cctccaaact gacccgaagg gtcgcagacg aacctgagag aatcggggga 1920 cctgaggtca caagttgcca tctaccttgg gatcaaacgg aaaccgcccc ccggctgctc 1980 cgattcccct ggagtgtgaa gctgaccagc tcgcgccctc ctgaggccct gatggcagct 2040 ctgcgccagg ccacagcagc cgcccgctgc cgctgccgcc agccacagcc gttcctgctg 2100 gcctgcctgc acgggggtgc gggcgggccc gagcccctgt cccacttcga agtggaggtc 2160 tgccagctgc cccggccagg cttgcgggga gttctcttcc gccgtgtggc gggcaccgcc 2220 ctggccttcc gcaccctcgt cacccgcatc tccaacgacc tcgagctctg agccaccacg 2280 gtcccaggcc cttatcttct ctcccttgtc gcttcacttc tacaggaggg gaaggggcca 2340 gggaggggat tctcccttta tcatcacctc agtttccctg aattatattt gggggcaaag 2400 attgtcccct ctgctgttct ctggggccgc tcagcacaga agaaggatga gggggctcag 2460 cggggggagc tggcaccttc ctggagcctc cagccagtcc tgtcctccct cgccctacca 2520 agagggcacc tgaggagact ttggggacag ggcaggggca gggagggaaa ctgaggaaat 2580 cttccattcc tcccaacagc tcaaaattag gccttgggca ggggcaggga gagctgctga 2640 gcctaaagac tggagaatct gggggactgg gagtgggggt cagagaggca gattccttcc 2700 cctcccgtcc cctcacgctc aaacccccac ttcctgcccc aggctggcgc ggggcacttt 2760 gtacaaatcc ttgtaaatac cccacacctt cccttctgca aaggtctctt gaggagctgc 2820 cgctgtcacc tacggttttt aagttattac accccgaccc tcctcctgtc agccccctca 2880 cgtgcagcct gttgcccaat aaatttagga gagtcccccc ctccccaatg ctgaccctag 2940 gattttcctt ccctgccctc acctgcaaat gagttaaaga agaggcgtgg gaatccaggc 3000 agtggttttt cctttcggag cctcggtttt ctcatctgca gaatgggagc ggtgggggtg 3060 ggaaggtaag gatggtcgtc caagaaggca ggatggaact cggcctcatc cccgaggccc 3120 cagttcctat atcgggcccc ccattcatcc actcacactc ccagccacca tgttacactg 3180 gactttaagc catttcttac tccagtagta aatttattca ataaac 3226 24 745 PRT Homo sapiens 24 Met Ile Arg Gly Arg Asn Ser Ala Thr Ser Ala Asp Glu Gln Pro His 1 5 10 15 Ile Gly Asn Tyr Arg Leu Leu Lys Thr Ile Gly Lys Gly Asn Phe Ala 20 25 30 Lys Val Lys Leu Ala Arg His Ile Leu Thr Gly Lys Glu Val Ala Val 35 40 45 Lys Ile Ile Asp Lys Thr Gln Leu Asn Ser Ser Ser Leu Gln Lys Leu 50 55 60 Phe Arg Glu Val Arg Ile Met Lys Val Leu Asn His Pro Asn Ile Val 65 70 75 80 Lys Leu Phe Glu Val Ile Glu Thr Glu Lys Thr Leu Tyr Leu Val Met 85 90 95 Glu Tyr Ala Ser Gly Gly Glu Val Phe Asp Tyr Leu Val Ala His Gly 100 105 110 Arg Met Lys Glu Lys Glu Ala Arg Ala Lys Phe Arg Gln Ile Val Ser 115 120 125 Ala Val Gln Tyr Cys His Gln Lys Phe Ile Val His Arg Asp Leu Lys 130 135 140 Ala Glu Asn Leu Leu Leu Asp Ala Asp Met Asn Ile Lys Ile Ala Asp 145 150 155 160 Phe Gly Phe Ser Asn Glu Phe Thr Phe Gly Asn Lys Leu Asp Thr Phe 165 170 175 Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu Leu Phe Gln Gly Lys Lys 180 185 190 Tyr Asp Gly Pro Glu Val Asp Val Trp Ser Leu Gly Val Ile Leu Tyr 195 200 205 Thr Leu Val Ser Gly Ser Leu Pro Phe Asp Gly Gln Asn Leu Lys Glu 210 215 220 Leu Arg Glu Arg Val Leu Arg Gly Lys Tyr Arg Ile Pro Phe Tyr Met 225 230 235 240 Ser Thr Asp Cys Glu Asn Leu Leu Lys Lys Phe Leu Ile Leu Asn Pro 245 250 255 Ser Lys Arg Gly Thr Leu Glu Gln Ile Met Lys Asp Arg Trp Met Asn 260 265 270 Val Gly His Glu Asp Asp Glu Leu Lys Pro Tyr Val Glu Pro Leu Pro 275 280 285 Asp Tyr Lys Asp Pro Arg Arg Thr Glu Leu Met Val Ser Met Gly Tyr 290 295 300 Thr Arg Glu Glu Ile Gln Asp Ser Leu Val Gly Gln Arg Tyr Asn Glu 305 310 315 320 Val Met Ala Thr Tyr Leu Leu Leu Gly Tyr Lys Ser Ser Glu Leu Glu 325 330 335 Gly Asp Thr Ile Thr Leu Lys Pro Arg Pro Ser Ala Asp Leu Thr Asn 340 345 350 Ser Ser Ala Gln Phe Pro Ser His Lys Val Gln Arg Ser Val Ser Ala 355 360 365 Asn Pro Lys Gln Arg Arg Phe Ser Asp Gln Ala Gly Pro Ala Ile Pro 370 375 380 Thr Ser Asn Ser Tyr Ser Lys Lys Thr Gln Ser Asn Asn Ala Glu Asn 385 390 395 400 Lys Arg Pro Glu Glu Asp Arg Glu Ser Gly Arg Lys Ala Ser Ser Thr 405 410 415 Ala Lys Val Pro Ala Ser Pro Leu Pro Gly Leu Glu Arg Lys Lys Thr 420 425 430 Thr Pro Thr Pro Ser Thr Asn Ser Val Leu Ser Thr Ser Thr Asn Arg 435 440 445 Ser Arg Asn Ser Pro Leu Leu Glu Arg Ala Ser Leu Gly Gln Ala Ser 450 455 460 Ile Gln Asn Gly Lys Asp Ser Leu Thr Met Pro Gly Ser Arg Ala Ser 465 470 475 480 Thr Ala Ser Ala Ser Ala Ala Val Ser Ala Ala Arg Pro Arg Gln His 485 490 495 Gln Lys Ser Met Ser Ala Ser Val His Pro Asn Lys Ala Ser Gly Leu 500 505 510 Pro Pro Thr Glu Ser Asn Cys Glu Val Pro Arg Pro Ser Thr Ala Pro 515 520 525 Gln Arg Val Pro Val Ala Ser Pro Ser Ala His Asn Ile Ser Ser Ser 530 535 540 Gly Gly Ala Pro Asp Arg Thr Asn Phe Pro Arg Gly Val Ser Ser Arg 545 550 555 560 Ser Thr Phe His Ala Gly Gln Leu Arg Gln Val Arg Asp Gln Gln Asn 565 570 575 Leu Pro Tyr Gly Val Thr Pro Ala Ser Pro Ser Gly His Ser Gln Gly 580 585 590 Arg Arg Gly Ala Ser Gly Ser Ile Phe Ser Lys Phe Thr Ser Lys Phe 595 600 605 Val Arg Arg Asn Leu Asn Glu Pro Glu Ser Lys Asp Arg Val Glu Thr 610 615 620 Leu Arg Pro His Val Val Gly Ser Gly Gly Asn Asp Lys Glu Lys Glu 625 630 635 640 Glu Phe Arg Glu Ala Lys Pro Arg Ser Leu Arg Phe Thr Trp Ser Met 645 650 655 Lys Thr Thr Ser Ser Met Glu Pro Asn Glu Met Met Arg Glu Ile Arg 660 665 670 Lys Val Leu Asp Ala Asn Ser Cys Gln Ser Glu Leu His Glu Lys Tyr 675 680 685 Met Leu Leu Cys Met His Gly Thr Pro Gly His Glu Asp Phe Val Gln 690 695 700 Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly Val 705 710 715 720 Arg Phe Lys Arg Ile Ser Gly Thr Ser Met Ala Phe Lys Asn Ile Ala 725 730 735 Ser Lys Ile Ala Asn Glu Leu Lys Leu 740 745 25 795 PRT Homo sapiens 25 Met Ser Ala Arg Thr Pro Leu Pro Thr Val Asn Glu Arg Asp Thr Val 1 5 10 15 Asn His Thr Thr Val Asp Gly Tyr Thr Glu Pro His Ile Gln Pro Thr 20 25 30 Lys Ser Ser Ser Arg Gln Asn Ile Pro Arg Cys Arg Asn Ser Ile Thr 35 40 45 Ser Ala Thr Asp Glu Gln Pro His Ile Gly Asn Tyr Arg Leu Gln Lys 50 55 60 Thr Ile Gly Lys Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His Val 65 70 75 80 Leu Thr Gly Arg Glu Val Ala Val Lys Ile Ile Asp Lys Thr Gln Leu 85 90 95 Asn Pro Thr Ser Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys 100 105 110 Ile Leu Asn His Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr 115 120 125 Glu Lys Thr Leu Tyr Leu Val Met Glu Tyr Ala Ser Gly Gly Glu Val 130 135 140 Phe Asp Tyr Leu Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg 145 150 155 160 Ala Lys Phe Arg Gln Ile Val Ser Ala Val Gln Tyr Cys His Gln Lys 165 170 175 Tyr Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Gly 180 185 190 Asp Met Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr 195 200 205 Val Gly Asn Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala 210 215 220 Pro Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Val 225 230 235 240 Trp Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro 245 250 255 Phe Asp Gly Gln Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly 260 265 270 Lys Tyr Arg Ile Pro Phe Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu 275 280 285 Lys Lys Leu Leu Val Leu Asn Pro Ile Lys Arg Gly Ser Leu Glu Gln 290 295 300 Ile Met Lys Asp Arg Trp Met Asn Val Gly His Glu Glu Glu Glu Leu 305 310 315 320 Lys Pro Tyr Thr Glu Pro Asp Pro Asp Phe Asn Asp Thr Lys Arg Ile 325 330 335 Asp Ile Met Val Thr Met Gly Phe Ala Arg Asp Glu Ile Asn Asp Ala 340 345 350 Leu Ile Asn Gln Lys Tyr Asp Glu Val Met Ala Thr Tyr Ile Leu Leu 355 360 365 Gly Arg Lys Pro Pro Glu Phe Glu Gly Gly Glu Ser Leu Ser Ser Gly 370 375 380 Asn Leu Cys Gln Arg Ser Arg Pro Ser Ser Asp Leu Asn Asn Ser Thr 385 390 395 400 Leu Gln Ser Pro Ala His Leu Lys Val Gln Arg Ser Ile Ser Ala Asn 405 410 415 Gln Lys Gln Arg Arg Phe Ser Asp His Ala Gly Pro Ser Ile Pro Pro 420 425 430 Ala Val Ser Tyr Thr Lys Arg Pro Gln Ala Asn Ser Val Glu Ser Glu 435 440 445 Gln Lys Glu Glu Trp Asp Lys Asp Val Ala Arg Lys Leu Gly Ser Thr 450 455 460 Thr Val Gly Ser Lys Ser Glu Met Thr Ala Ser Pro Leu Val Gly Pro 465 470 475 480 Glu Arg Lys Lys Ser Ser Thr Ile Pro Ser Asn Asn Val Tyr Ser Gly 485 490 495 Gly Ser Met Ala Arg Arg Asn Thr Tyr Val Cys Glu Arg Thr Thr Asp 500 505 510 Arg Tyr Val Ala Leu Gln Asn Gly Lys Asp Ser Ser Leu Thr Glu Met 515 520 525 Ser Val Ser Ser Ile Ser Ser Ala Gly Ser Ser Val Ala Ser Ala Val 530 535 540 Pro Ser Ala Arg Pro Arg His Gln Lys Ser Met Ser Thr Ser Gly His 545 550 555 560 Pro Ile Lys Val Thr Leu Pro Thr Ile Lys Asp Gly Ser Glu Ala Tyr 565 570 575 Arg Pro Gly Thr Thr Gln Arg Val Pro Ala Ala Ser Pro Ser Ala His 580 585 590 Ser Ile Ser Thr Ala Thr Pro Asp Arg Thr Arg Phe Pro Arg Gly Ser 595 600 605 Ser Ser Arg Ser Thr Phe His Gly Glu Gln Leu Arg Glu Arg Arg Ser 610 615 620 Val Ala Tyr Asn Gly Pro Pro Ala Ser Pro Ser His Glu Thr Gly Ala 625 630 635 640 Phe Ala His Ala Arg Arg Gly Thr Ser Thr Gly Ile Ile Ser Lys Ile 645 650 655 Thr Ser Lys Phe Val Arg Arg Asp Pro Ser Glu Gly Glu Ala Ser Gly 660 665 670 Arg Thr Asp Thr Ser Arg Ser Thr Ser Gly Glu Pro Lys Glu Arg Asp 675 680 685 Lys Glu Glu Gly Lys Asp Ser Lys Pro Arg Ser Leu Arg Phe Thr Trp 690 695 700 Ser Met Lys Thr Thr Ser Ser Met Asp Pro Asn Asp Met Met Arg Glu 705 710 715 720 Ile Arg Lys Val Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gln Lys Glu 725 730 735 Arg Phe Leu Leu Phe Cys Val His Gly Asp Ala Arg Gln Asp Ser Leu 740 745 750 Val Gln Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn 755 760 765 Gly Val Arg Phe Lys Arg Ile Ser Gly Thr Ser Ile Ala Phe Lys Asn 770 775 780 Ile Ala Ser Lys Ile Ala Asn Glu Leu Lys Leu 785 790 795 26 729 PRT Homo sapiens 26 Met Ser Thr Arg Thr Pro Leu Pro Thr Val Asn Glu Arg Asp Thr Glu 1 5 10 15 Asn His Thr Ser His Gly Asp Gly Arg Gln Glu Val Thr Ser Arg Thr 20 25 30 Ser Arg Ser Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser Cys Ala Asp 35 40 45 Glu Gln Pro His Ile Gly Asn Tyr Arg Leu Leu Lys Thr Ile Gly Lys 50 55 60 Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His Ile Leu Thr Gly Arg 65 70 75 80 Glu Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn Pro Thr Ser 85 90 95 Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Ile Leu Asn His 100 105 110 Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Glu Lys Thr Leu 115 120 125 Tyr Leu Ile Met Glu Tyr Ala Ser Gly Gly Glu Val Phe Asp Tyr Leu 130 135 140 Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg Ser Lys Phe Arg 145 150 155 160 Gln Ile Val Ser Ala Val Gln Tyr Cys His Gln Lys Arg Ile Val His 165 170 175 Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Asp Met Asn Ile 180 185 190 Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Val Gly Gly Lys 195 200 205 Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu Leu Phe 210 215 220 Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Val Trp Ser Leu Gly 225 230 235 240 Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe Asp Gly Gln 245 250 255 Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys Tyr Arg Ile 260 265 270 Pro Phe Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu Lys Arg Phe Leu 275 280 285 Val Leu Asn Pro Ile Lys Arg Gly Thr Leu Glu Gln Ile Met Lys Asp 290 295 300 Arg Trp Ile Asn Ala Gly His Glu Glu Asp Glu Leu Lys Pro Phe Val 305 310 315 320 Glu Pro Glu Leu Asp Ile Ser Asp Gln Lys Arg Ile Asp Ile Met Val 325 330 335 Gly Met Gly Tyr Ser Gln Glu Glu Ile Gln Glu Ser Leu Ser Lys Met 340 345 350 Lys Tyr Asp Glu Ile Thr Ala Thr Tyr Leu Leu Leu Gly Arg Lys Ser 355 360 365 Ser Glu Leu Asp Ala Ser Asp Ser Ser Ser Ser Ser Asn Leu Ser Leu 370 375 380 Ala Lys Val Arg Pro Ser Ser Asp Leu Asn Asn Ser Thr Gly Gln Ser 385 390 395 400 Pro His His Lys Val Gln Arg Ser Val Ser Ser Ser Gln Lys Gln Arg 405 410 415 Arg Tyr Ser Asp His Ala Gly Pro Ala Ile Pro Ser Val Val Ala Tyr 420 425 430 Pro Lys Arg Ser Gln Thr Ser Thr Ala Asp Gly Asp Leu Lys Glu Asp 435 440 445 Gly Ile Ser Ser Arg Lys Ser Ser Gly Ser Ala Val Gly Gly Lys Gly 450 455 460 Ile Ala Pro Ala Ser Pro Met Leu Gly Asn Ala Ser Asn Pro Asn Lys 465 470 475 480 Ala Asp Ile Pro Glu Arg Lys Lys Ser Ser Thr Val Pro Ser Ser Asn 485 490 495 Thr Ala Ser Gly Gly Met Thr Arg Arg Asn Thr Tyr Val Cys Ser Glu 500 505 510 Arg Thr Thr Ala Asp Arg His Ser Val Ile Gln Asn Gly Lys Glu Asn 515 520 525 Ser Thr Ile Pro Asp Gln Arg Thr Pro Val Ala Ser Thr His Ser Ile 530 535 540 Ser Ser Ala Ala Thr Pro Asp Arg Ile Arg Phe Pro Arg Gly Thr Ala 545 550 555 560 Ser Arg Ser Thr Phe His Gly Gln Pro Arg Glu Arg Arg Thr Ala Thr 565 570 575 Tyr Asn Gly Pro Pro Ala Ser Pro Ser Leu Ser His Glu Ala Thr Pro 580 585 590 Leu Ser Gln Thr Arg Ser Arg Gly Ser Thr Asn Leu Phe Ser Lys Leu 595 600 605 Thr Ser Lys Leu Thr Arg Ser Arg Asn Val Ser Ala Glu Gln Lys Asp 610 615 620 Glu Asn Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe Thr Trp Ser Met 625 630 635 640 Lys Thr Thr Ser Ser Met Asp Pro Gly Asp Met Met Arg Glu Ile Arg 645 650 655 Lys Val Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gln Arg Glu Arg Phe 660 665 670 Leu Leu Phe Cys Val His Gly Asp Gly His Ala Glu Asn Leu Val Gln 675 680 685 Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly Val 690 695 700 Arg Phe Lys Arg Ile Ser Gly Thr Ser Ile Ala Phe Lys Asn Ile Ala 705 710 715 720 Ser Lys Ile Ala Asn Glu Leu Lys Leu 725 27 713 PRT Homo sapiens 27 Met Ser Thr Arg Thr Pro Leu Pro Thr Val Asn Glu Arg Asp Thr Glu 1 5 10 15 Asn His Thr Ser His Gly Asp Gly Arg Gln Glu Val Thr Ser Arg Thr 20 25 30 Ser Arg Ser Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser Cys Ala Asp 35 40 45 Glu Gln Pro His Ile Gly Asn Tyr Arg Leu Leu Lys Thr Ile Gly Lys 50 55 60 Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His Ile Leu Thr Gly Arg 65 70 75 80 Glu Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn Pro Thr Ser 85 90 95 Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Ile Leu Asn His 100 105 110 Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Gln Lys Thr Leu 115 120 125 Tyr Leu Ile Met Glu Tyr Ala Ser Gly Gly Lys Val Phe Asp Tyr Leu 130 135 140 Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg Ser Lys Phe Arg 145 150 155 160 Gln Ile Val Ser Ala Val Gln Tyr Cys His Gln Lys Arg Ile Val His 165 170 175 Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Asp Met Asn Ile 180 185 190 Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Val Gly Gly Lys 195 200 205 Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu Leu Phe 210 215 220 Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Val Trp Ser Leu Gly 225 230 235 240 Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe Asp Gly Gln 245 250 255 Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys Tyr Arg Ile 260 265 270 Pro Phe Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu Lys Arg Phe Leu 275 280 285 Val Leu Asn Pro Ile Lys Arg Gly Thr Leu Glu Gln Ile Met Lys Asp 290 295 300 Arg Trp Ile Asn Ala Gly His Glu Glu Asp Glu Leu Lys Pro Phe Val 305 310 315 320 Glu Pro Glu Leu Asp Ile Ser Asp Gln Lys Arg Ile Asp Ile Met Val 325 330 335 Gly Met Gly Tyr Ser Gln Glu Glu Ile Gln Glu Ser Leu Ser Lys Met 340 345 350 Lys Tyr Asp Glu Ile Thr Ala Thr Tyr Leu Leu Leu Gly Arg Lys Ser 355 360 365 Ser Glu Val Arg Pro Ser Ser Asp Leu Asn Asn Ser Thr Gly Gln Ser 370 375 380 Pro His His Lys Val Gln Arg Ser Val Ser Ser Ser Gln Lys Gln Arg 385 390 395 400 Arg Tyr Ser Asp His Ala Gly Pro Gly Ile Pro Ser Val Val Ala Tyr 405 410 415 Pro Lys Arg Ser Gln Thr Ser Thr Ala Asp Ser Asp Leu Lys Glu Asp 420 425 430 Gly Ile Ser Ser Arg Lys Ser Thr Gly Ser Ala Val Gly Gly Lys Gly 435 440 445 Ile Ala Pro Ala Ser Pro Met Leu Gly Asn Ala Ser Asn Pro Asn Lys 450 455 460 Ala Asp Ile Pro Glu Arg Lys Lys Ser Ser Thr Val Pro Ser Ser Asn 465 470 475 480 Thr Ala Ser Gly Gly Met Thr Arg Arg Asn Thr Tyr Val Cys Ser Glu 485 490 495 Arg Thr Thr Asp Asp Arg His Ser Val Ile Gln Asn Gly Lys Glu Asn 500 505 510 Ser Thr Ile Pro Asp Gln Arg Thr Pro Val Ala Ser Thr His Ser Ile 515 520 525 Ser Ser Ala Ala Thr Pro Asp Arg Ile Arg Phe Pro Arg Gly Thr Ala 530 535 540 Ser Arg Ser Thr Phe His Gly Gln Pro Arg Glu Arg Arg Thr Ala Thr 545 550 555 560 Tyr Asn Gly Pro Pro Ala Ser Pro Ser Leu Ser His Glu Ala Thr Pro 565 570 575 Leu Ser Gln Thr Arg Ser Arg Gly Ser Thr Thr Leu Phe Ser Lys Leu 580 585 590 Thr Ser Lys Leu Thr Arg Ser Arg Asn Val Ser Ala Lys Gln Lys Asp 595 600 605 Glu Asn Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe Thr Trp Ser Met 610 615 620 Lys Thr Thr Ser Ser Met Asp Pro Gly Asp Met Met Arg Glu Ile Arg 625 630 635 640 Lys Val Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gln Arg Glu Arg Phe 645 650 655 Leu Leu Phe Cys Val His Gly Asp Gly His Ala Glu Asn Leu Val Gln 660 665 670 Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly Val 675 680 685 Arg Phe Lys Arg Ile Ser Gly Thr Ser Ile Ala Phe Lys Asn Ile Ala 690 695 700 Ser Lys Ile Ala Asn Glu Leu Lys Leu 705 710 28 688 PRT Homo sapiens 28 Met Ser Ser Arg Thr Val Leu Ala Pro Gly Asn Asp Arg Asn Ser Asp 1 5 10 15 Thr His Gly Thr Leu Gly Ser Gly Arg Ser Ser Asp Lys Gly Pro Ser 20 25 30 Trp Ser Ser Arg Ser Leu Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser 35 40 45 Cys Pro Glu Glu Gln Pro His Val Gly Asn Tyr Arg Leu Leu Arg Thr 50 55 60 Ile Gly Lys Gly Asn Ser Ala Lys Val Lys Leu Ala Arg His Ile Leu 65 70 75 80 Thr Gly Arg Glu Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn 85 90 95 Pro Ser Ser Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Gly 100 105 110 Leu Asn His Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Glu 115 120 125 Lys Thr Leu Tyr Leu Val Met Glu Tyr Ala Ser Ala Gly Glu Val Phe 130 135 140 Asp Tyr Leu Val Ser His Gly Arg Met Lys Glu Lys Glu Ala Arg Ala 145 150 155 160 Lys Phe Arg Gln Ile Val Ser Ala Val His Tyr Cys His Gln Lys Asn 165 170 175 Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Glu 180 185 190 Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Leu 195 200 205 Gly Ser Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro 210 215 220 Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Ile Trp 225 230 235 240 Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe 245 250 255 Asp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys 260 265 270 Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys Glu Ser Ile Leu Arg 275 280 285 Arg Phe Leu Val Leu Asn Pro Ala Lys Arg Cys Thr Leu Glu Gln Ile 290 295 300 Met Lys Asp Lys Trp Ile Asn Ile Gly Tyr Glu Gly Glu Glu Leu Lys 305 310 315 320 Pro Tyr Thr Glu Pro Glu Glu Asp Phe Gly Asp Thr Lys Arg Ile Glu 325 330 335 Val Met Val Gly Met Gly Tyr Thr Arg Glu Glu Ile Lys Glu Ser Leu 340 345 350 Thr Ser Gln Lys Tyr Asn Glu Val Thr Ala Thr Tyr Leu Leu Leu Gly 355 360 365 Arg Lys Thr Glu Glu Gly Gly Asp Arg Gly Ala Pro Gly Leu Ala Leu 370 375 380 Ala Arg Val Arg Ala Pro Ser Asp Thr Thr Asn Gly Thr Ser Ser Ser 385 390 395 400 Lys Gly Thr Ser His Ser Lys Gly Gln Arg Ser Ser Ser Ser Thr Tyr 405 410 415 His Arg Gln Arg Arg His Ser Asp Phe Cys Gly Pro Ser Pro Ala Pro 420 425 430 Leu His Pro Lys Arg Ser Pro Thr Ser Thr Gly Glu Ala Glu Leu Lys 435 440 445 Glu Glu Arg Leu Pro Gly Arg Lys Ala Ser Cys Ser Thr Ala Gly Ser 450 455 460 Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val Ser Ser Ala His 465 470 475 480 Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys Asp Ser Thr Ser 485 490 495 Thr Pro Asn Asn Leu Pro Pro Ser Met Met Thr Arg Arg Asn Thr Tyr 500 505 510 Val Cys Thr Glu Arg Pro Gly Ala Glu Arg Pro Ser Leu Leu Pro Asn 515 520 525 Gly Lys Glu Asn Ser Ser Gly Thr Pro Arg Val Pro Pro Ala Ser Pro 530 535 540 Ser Ser His Ser Leu Ala Pro Pro Ser Gly Glu Arg Ser Arg Leu Ala 545 550 555 560 Arg Gly Ser Thr Ile Arg Ser Thr Phe His Gly Gly Gln Val Arg Asp 565 570 575 Arg Arg Ala Gly Gly Gly Gly Gly Gly Gly Val Gln Asn Gly Pro Pro 580 585 590 Ala Ser Pro Thr Leu Ala His Glu Ala Ala Pro Leu Pro Ala Gly Arg 595 600 605 Pro Arg Pro Thr Thr Asn Leu Phe Thr Lys Leu Thr Ser Lys Leu Thr 610 615 620 Arg Arg Val Thr Leu Asp Pro Ser Lys Arg Gln Asn Ser Asn Arg Cys 625 630 635 640 Val Ser Gly Ala Ser Leu Pro Gln Gly Ser Lys Ile Arg Ser Gln Thr 645 650 655 Asn Leu Arg Glu Ser Gly Asp Leu Arg Ser Gln Val Ala Ile Tyr Leu 660 665 670 Gly Ile Lys Arg Lys Pro Pro Pro Gly Cys Ser Asp Ser Pro Gly Val 675 680 685 29 688 PRT Homo sapiens 29 Met Ser Ser Arg Thr Val Leu Ala Pro Gly Asn Asp Arg Asn Ser Asp 1 5 10 15 Thr His Gly Thr Leu Gly Ser Gly Arg Ser Ser Asp Lys Gly Pro Ser 20 25 30 Trp Ser Ser Arg Ser Leu Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser 35 40 45 Cys Pro Glu Glu Gln Pro His Val Gly Asn Tyr Arg Leu Leu Arg Thr 50 55 60 Ile Gly Lys Gly Asn Ser Ala Lys Val Lys Leu Ala Arg His Ile Leu 65 70 75 80 Thr Gly Arg Glu Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn 85 90 95 Pro Ser Ser Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Gly 100 105 110 Leu Asn His Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Glu 115 120 125 Lys Thr Leu Tyr Leu Val Met Glu Tyr Ala Ser Ala Gly Glu Val Phe 130 135 140 Asp Tyr Leu Val Ser His Gly Arg Met Lys Glu Lys Glu Ala Arg Ala 145 150 155 160 Lys Phe Arg Gln Ile Val Ser Ala Val His Tyr Cys His Gln Lys Asn 165 170 175 Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Glu 180 185 190 Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Leu 195 200 205 Gly Ser Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro 210 215 220 Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Ile Trp 225 230 235 240 Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe 245 250 255 Asp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys 260 265 270 Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys Glu Ser Ile Leu Arg 275 280 285 Arg Phe Leu Val Leu Asn Pro Ala Lys Arg Cys Thr Leu Glu Gln Ile 290 295 300 Met Lys Asp Lys Trp Ile Asn Ile Gly Tyr Glu Gly Glu Glu Leu Lys 305 310 315 320 Pro Tyr Thr Glu Pro Glu Glu Asp Phe Gly Asp Thr Lys Arg Ile Glu 325 330 335 Val Met Val Gly Met Gly Tyr Thr Arg Glu Glu Ile Lys Glu Ser Leu 340 345 350 Thr Ser Gln Lys Tyr Asn Glu Val Thr Ala Thr Tyr Leu Leu Leu Gly 355 360 365 Arg Lys Thr Glu Glu Gly Gly Asp Arg Gly Ala Pro Gly Leu Ala Leu 370 375 380 Ala Arg Val Arg Ala Pro Ser Asp Thr Thr Asn Gly Thr Ser Ser Ser 385 390 395 400 Lys Gly Thr Ser His Ser Lys Gly Gln Arg Ser Ser Ser Ser Thr Tyr 405 410 415 His Arg Gln Arg Arg His Ser Asp Phe Cys Gly Pro Ser Pro Ala Pro 420 425 430 Leu His Pro Lys Arg Ser Pro Thr Ser Thr Gly Glu Ala Glu Leu Lys 435 440 445 Glu Glu Arg Leu Pro Gly Arg Lys Ala Ser Cys Ser Thr Ala Gly Ser 450 455 460 Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val Ser Ser Ala His 465 470 475 480 Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys Asp Ser Thr Ser 485 490 495 Thr Pro Asn Asn Leu Pro Pro Ser Met Met Thr Arg Arg Asn Thr Tyr 500 505 510 Val Cys Thr Glu Arg Pro Gly Ala Glu Arg Pro Ser Leu Leu Pro Asn 515 520 525 Gly Lys Glu Asn Ser Ser Gly Thr Pro Arg Val Pro Pro Ala Ser Pro 530 535 540 Ser Ser His Ser Leu Ala Pro Pro Ser Gly Glu Arg Ser Arg Leu Ala 545 550 555 560 Arg Gly Ser Thr Ile Arg Ser Thr Phe His Gly Gly Gln Val Arg Asp 565 570 575 Arg Arg Ala Gly Gly Gly Gly Gly Gly Gly Val Gln Asn Gly Pro Pro 580 585 590 Ala Ser Pro Thr Leu Ala His Glu Ala Ala Pro Leu Pro Ala Gly Arg 595 600 605 Pro Arg Pro Thr Thr Asn Leu Phe Thr Lys Leu Thr Ser Lys Leu Thr 610 615 620 Arg Arg Val Thr Leu Asp Pro Ser Lys Arg Gln Asn Ser Asn Arg Cys 625 630 635 640 Val Ser Gly Ala Ser Leu Pro Gln Gly Ser Lys Ile Arg Ser Gln Thr 645 650 655 Asn Leu Arg Glu Ser Gly Asp Leu Arg Ser Gln Val Ala Ile Tyr Leu 660 665 670 Gly Ile Lys Arg Lys Pro Pro Pro Gly Cys Ser Asp Ser Pro Gly Val 675 680 685

Claims

What is claimed is:

1. A method of identifying a candidate p53 pathway modulating agent, said method comprising the steps of:

(a) providing an assay system comprising a purified MARK polypeptide or nucleic acid or a functionally active fragment or derivative thereof;

(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 p53 pathway modulating agent.

2. The method of claim 1 wherein the assay system comprises cultured cells that express the MARK polypeptide.

3. The method of claim 2 wherein the cultured cells additionally have defective p53 function.

4. The method of claim 1 wherein the assay system includes a screening assay comprising a MARK polypeptide, and the candidate test agent is a small molecule modulator.

5. The method of claim 4 wherein the assay is a kinase 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 a MARK polypeptide and the candidate test agent is an antibody.

8. The method of claim 1 wherein the assay system includes an expression assay comprising a MARK 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 p53 pathway modulating agent identified in (c) to a model system comprising cells defective in p53 function and, detecting a phenotypic change in the model system that indicates that the p53 function is restored.

12. The method of claim 11 wherein the model system is a mouse model with defective p53 function.

13. A method for modulating a p53 pathway of a cell comprising contacting a cell defective in p53 function with a candidate modulator that specifically binds to a MARK polypeptide comprising an amino acid sequence selected from group consisting of SEQ ID NOs:24, 25, 26, 27, 28, and 29, whereby p53 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 p53 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 MARK,

(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 p53 pathway modulating agent, and wherein the second assay detects an agent-biased change in the p53 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 p53 pathway gene.

20. A method of modulating p53 pathway in a mammalian cell comprising contacting the cell with an agent that specifically binds a MARK 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 p53 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:

(a) obtaining a biological sample from the patient;

(b) contacting the sample with a probe for MARK expression;

(c) comparing results from step (b) with a control;

(d) 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.