CA2473499A1 - Minrs as modifiers of insulin receptor signaling and methods of use - Google Patents

Abstract

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

Description

MINRs AS MODIFIERS OF INSULIN RECEPTOR SIGNALING
AND METHODS OF USE
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional patent applications 60/354,824 filed 2/6/2002, 60/358,217 filed 2/20/2002, 60/358,189 filed 2/20/2002, 60/358,126 filed 2/20/2002, 60/358,995 filed 2/21/2002, 60/358,756 filed 2/21/2002, 60/358,765 filed 2121/2002, 60/359,531 filed 2/25/2002, 60/360,222 filed 2/26/2002, 60/360,224 filed 2/26/2002, 60/360,167 filed 2/26/2002, and 60/360,166 filed 2/26/2002. The contents of the prior applications are hereby incorporated in their entirety.
BACKGROUND OF THE INVENTION
Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, In Endocrinology, DeGroot, eds. Philadelphia, Saunders:
1263-1289).
In humans, insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels, which normally occur following a meal. The immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver. A longer-term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue. Insulin can exert other actions beyond these "classic" metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal retention of sodium, and promoting production of androgens by the ovary.
Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance. Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, In Joslin's Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea & Febiger, pp. 201-215; Kahn et al., 1996, Annu. Rev. Med.
47:509-531; Kahn, 1998, Cell 92:593-596). Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension.

Within the pharmaceutical industry, there is interest in understanding the molecular mechanisms that connect lipid defects and insulin resistance.
Hyperlipidemia and elevation of free fatty acid levels correlate with "Metabolic Syndrome,"
defined as the linkage between several diseases, including obesity and insulin resistance, which often occur in the same patients and which are major risk factors for development of Type 2 diabetes and cardiovascular disease. Current research suggests that the control of lipid levels, in addition to glucose levels, may be required to treat Type 2 Diabetes, heart disease, and other manifestations of Metabolic Syndrome (Santomauro AT et al., Diabetes (1999) 48:1836-1841).
The ability to manipulate and screen the genomes of model organisms such as Drosophila and C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation of genes, pathways, and cellular processes, have direct relevance to more complex vertebrate organisms.
Identification of novel functions of genes involved in particular pathways in such model organisms can directly contribute to the understanding of the correlative pathways in mammals and of methods of modulating them (Dulubova I, et al, J Neurochem 2001 Apr;77(1):229-38;
Cai T, et al., Diabetologia 2001 Jan;44(1):81-8; Pasquinelli AE, et al., Nature. 2000 Nov 2;408(6808):37-8; Ivanov IP, et al., EMBO J 2000 Apr 17;19(8):1907-17; Vajo Z
et al., Mamm Genome 1999 Oct;lO(10):1000-4; Miklos GL and Rubin GM, Cell 1996, 86:521-529; Mechler BM et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res.
37: 33-74; Watson KL., et al., 1994 J Cell Sci. 18: 19-33; Miklos GL, and Rubin GM.
1996 Cell 86:521-529; Wassarman DA, et al., 1995 Curr Opin Gen Dev 5: 44-50;
and Booth DR. 1999 Cancer Metastasis Rev. 18: 261-284). While Drosophila and C.
elegans are not susceptible to human pathologies, various experimental models can mimic the pathological states. A correlation between the pathology model and the modified expression of a Drosophila or C. elegans gene can identify the association of the human ortholog with the human disease.
In one example, a genetic screen is performed in an invertebrate model organism displaying a mutant (generally visible or selectable) phenotype due to mis-expression -generally reduced, enhanced or ectopic expression - of a known gene (the "genetic entry point"). Additional genes are mutated in a random or targeted manner. When an additional gene mutation changes the original mutant phenotype, this gene is identified as a "modifier" that directly or indirectly interacts with the genetic entry point and its associated pathway. If the genetic entry point is an ortholog of a human gene associated with a human pathology, such as lipid metabolic disorders, the screen can identify modifier genes that are candidate targets for novel therapeutics.
Genetic screens may utilize RNA interference (RNAi) techniques, whereby introduction of exogenous double stranded (ds) RNA disrupts the activity of genes containing homologous sequences and induce specific loss-of-function phenotypes (Fire et al., 1998, Nature391:806-811). Suitable methods for introduction of dsRNA
into an animal include injection, feeding, and bathing (Tabara et al, 1998, Science 282:430-431).
RNAi has further been shown to produce specific gene disruptions in cultured Drosophila and mammalian cells (Paddison et al., Proc Natl Acad Sci U S A published Jan 29, 2002 as 10.1073/pnas.032652399; Clemens et al., 2000, Proc Natl Acad Sci U S A
97:6499-503; Wojcik and DeMartino, J Biol Chem, published Dec 5, 2001 as 10.1074/jbc.M109996200; Goto et al., 2001, Biochem J 360:167-72; Elbashir et al., 2001, Nature 411:494-8). ' The insulin receptor (INR) signaling pathway has been extensively studied in C.
elegans. Signaling through daf 2, the C. elegans INR ortholog, mediates various events, including reproductive growth and normal adult life span (see, e.g., US PAT NO
6,225,120; Tissenbaum HA and Ruvkun G, 1998, Genetics 148:703-17; Ogg S and Ruvkun G, 1998, Mol Cell 2:887-93; Lin K et al, 2001, Nat Genet 28:139-45).
NOT2 and S. Cerevisiae ortholog CDC36 are part of a complex of proteins that interact with the Polymerase II holoenzyme to regulate gene expression. The complex contains CCR4, CAF and NOT family proteins, among others. The NOT proteins likely restrict access of TATA box proteins to noncanonical TATAAs. Loss of NOT2 can result in the derepression of genes (Benson et al. 1998, EMBO 17:6714-6722;
Collart et al. 1994, Genes Dev. 8:525-537; Liu, et. al. 2001, J. Biol. Chem. 276: 7541-7548). The Regena (Rga) gene of Drosophila is an ortholog of NOT2, and was originally identified in a Drosophila screen for genes modifying the expression of the white eye color gene.
Regena was shown to affect the expression of four of seven genes tested, which suggested that it is involved in general regulation of gene expression.
Expression of the RP49 ribosomal gene was unaffected by mutations in Rga. Based on sequence similarity and functional similarity, Rga was shown to be the homolog of the yeast gene CDC36/NOT2 (Frolov et al, 1998, Genetics 148: 317-329).
Myotubularins (MYT) belong to a conserved family of proteins from several organisms, including human, Drosophila, and C. elegans (Laporte et al. 1998, Hum.
Molec. Genet. 7:1703-1712; Laporte et al., 2001 Trends in Genetics 17:221-228). The human family consists of at least 10 genes, and Drosophila and C. elegans each have 6 myotubularin related genes. Myotubularins have active site residues that are consistent with both protein and lipid phosphatase activity, and have been shown to have these activities biochemically (Laporte et al. 1998, 2001). In addition, it has been suggested based on experimental evidence in yeast that myotubularin might down regulate kinase activity. In yeast, myotubularin has a strong preference for PtdIns3P
as a substrate (Taylor et al. 2000, Proc. Natl. Acad. Sci. USA 97:8910-8915). Conserved residues in the catalytic domain are consistent with its activity as a monophosphoinositide phosphatase, and mutation of these residues abolishes lipid phosphatase activity in vitro (Taylor et al., 2000; Laporte et al., 2001). In addition, a mutant form of human myotubularin, when introduced into yeast, co-immunoprecipitated with the yeast kinase, suggesting that myotubularin might directly affect PI-3 kinase activity (Blondeau et al. 2000, Hum. Mol. Genet. 9: 2223-2229). The Drosophila myotubularin gene of GI
17737395 falls into the human MTM1/MTMR2 subgroup and it is the only Drosophila gene in this subgroup. MTM1 mutations are associated with the disease X-linked myotubular myopathy (Laporte et al. 1996, Nat. Genet. 13:175-182), which results in the disorganization of muscle fibers. The mutations that have been found in MTM1 in patients are missense mutations that, for the most part, affect residues that are conserved between the human and the Drosophila protein. Mutations in MTMR2 result in Charcot-Marie-Tooth disease, which affects the myelination of motor and sensory neurons (Bolino et al. 2000, Nat. Genet. 25:17-19).
DNMTl is an enzyme that maintains mammalian DNA methylation and is also a component of a repressive transcriptional complex. DNMT associated protein (DMAP1) was identified in a yeast two-hybrid screen for proteins that interact with DNMT1.
DMAP1 has intrinsic transcriptional repressive activity and also binds to the tumor suppressor gene TSG101. TSG101 has been shown to act as a transcriptional co-repressor involved in the silencing of nuclear hormone induced genes, and also may function in late endosomal trafficking (Roundtree et al., 2000, Nature Genetics 25:269-277).
Tuberous sclerosis (TCS) complex in humans is a disease that results in the formation of benign tumors in many tissues (Cheadle et al 2000, Hum. Genet.
107:97-114). These tumors contain differentiated cells, but these cells are much larger than normal. This disorder manifests itself most severely in the central nervous system, which can result in epilepsy, retardation and autism, and is caused by mutations in either the TSC1 or TSC2 genes (Consortium T.E.C.T.S., 1993, Cell 75:1305-1315; van Slegtenhorst et al. 1997, Science 277:805-808). TSC1 encodes hamartin, TSC2 encodes tuberin, and there is evidence that the human proteins interact in vitro (Plank et al 1998, Cancer Res. 58: 4766-4770; van Slegtenhorst et al 1998, Hum. Mol. Genet.
7:1053-1057). Tuberin, the TSC2 protein product contains coiled-coil domains, as well as a predicted GTPase activating protein (GAP) domain, and has GAP activity in vitro (Wienecke et al 1995, J. Biol. Chem. 270:16409-16414). The Rap/ran-GAP domain is also found in the GTPase activating protein (GAP) responsible for the activation of nuclear Ras-related regulatory proteins Rapl, Rsrl and Ran in vitro , which affects cell cycle progression. Gigas (GIG) is the Drosophila ortholog of TCS2. GIG loss-of-function mutants display a range of phenotypes, depending on the strength of the mutant allele, including larval lethality and various neuroanatomical and behavioral defects (Meinertzhagen, 1994, J. Neurogenet 9:157-176; Canal et al. 1998, J. Neurosci 18:999-1008; Acebes and Ferrus 2001, J. Neurosci 21:6264-6273). In addition, cells in a GIG
mutant differentiate normally, but are 2-3 times the normal size.
Overexpression of the Drosophila TSC1 and TSC2 (GIG) genes leads to a reduction in cell size, number and organ size (Potter et al. 2001, Cell 105:357-368; Tapon et al. 2001). Genetic experiments in the fly have demonstrated that the TSC1 and TSC2 GIG genes act together to antagonize insulin receptor signaling (Gao et al. 2001, Genes and Dev. 15:1383-1392;
Potter et al. 2001; Tapon et al. 2001, Cell 105:345-355). One copy of a GIG
loss of function allele is sufficient to rescue the lethality associated with fly insulin receptor mutants. Genetic data indicate that TSC1 and TSC2 (GIG) likely function downstream of Akt, and upstream of S6 kinase in the same pathway as these genes, or in a parallel pathway.
RAB 5 is a member of the Ras superfamily of GTPases, which have been implicated in vesicle trafficking (Somsel Rodman and Wandinger-Ness, 2000, J.
Cell Sci.
113:183-192). The endocytic pathway is important for uptake of nutrients, regulation of cell surface receptors, the recycling of proteins used in the secretory pathway. RABS is associated with the clathrin-coated vesicles and early endosomes and functions to regulate endocytic internalization and early endosome fusion (Woodman, 2000, Traffic 9:695-701 ). The FYVE-domain protein Rabenosyn-5 has been shown to be an effector of RabS and Rab4, physically connecting early endosomes and receptor recycling to the cell surface (De Renzis et al., 2002, Nat. Cell Biol. 4:124-133). Insulin-responsive tissues express several Rab isoforms, including Rab3b, Rab4, RabS, and RabB. Of these isoforms, only Rab4 has been shown to play a role in mediating insulin actions within the cell, including insulin-stimulated GLUT4 translocation to the cell membrane (Knight et al., 2000, Endocrinology 141:208-218). There is some evidence that membrane association of RabS is altered in skeletal muscle isolated from insulin resistant and Type 2 diabetic patients (Bao et al, 1998, Horm. Metab. Res. 30:656-662).
Drosophila SNAP is an ortholog of human alpha-Soluble NSF gene (alpha-SNAP
or "aSNAP). In Drosophila, SNAP is known to be a part of the conserved SNARE
complex necessary for secretory vesicle fusion with the plasma membrane (Ordway et al., 1994, PNAS USA 91:5715-5719). There are no loss-of-function mutations reported in Drosophila, but mutations in NSF, the primary protein SNAP is responsible for recruiting, are defective in motor behavior and display paralysis (Littleton et al. 1998, Neuron 21: 401-413). In vertebrates, it has been demonstrated that SNAPs play a role in the association of the SNARE complex in trans during vesicle docking (Xu et al. 1999, EMBO J. 18: 3293-3304). SNAPs are responsible for recruiting and stimulating NSF, the ATPase responsible for disassembly and recycling of the SNARE complex (Sudlow et al.
1996, FEBS Lett 393: 185-188; Barnard et al 1997, J. Cell Biology 139: 875-883;
Cheatham 2000, Trends in Endocrinol. Metab. 11:356-361). Together, SNAP and NSF
are responsible for increasing the rate of exocytosis dramatically. It has been shown that although beta-SNAP in vertebrates is similar to alpha-SNAP, alpha-SNAP
increases exocytosis more than beta-SNAP (Xu et al. 2002, J. Neurosci 22:53-61).
Mutational analysis of alpha-SNAP shows a requirement for Leucine 294. alpha-SNAP (L294A) acted as a dominant mutant by associating with the SNARE complex and NSF
normally but blocking the ATPase dependent stimulation of exocytosis by exogenous alpha-SNAP
(Barnard et al 1997, supra).
CAF-1 (catabolite repressor protein (CCR4)-associative factor 1), also known as a CCR4-NOT transcription complex subunit 7, is a component of a complex of proteins that interact with the RNA polymerise II holoenzyme to regulate gene expression (Albert \\
et al., 2000, Nucleic Acids Res. 28:809-817). The complex also contains CCR4 and NOT proteins, among others. In addition to the global regulation of RNA
polymerise II
transcription, CAF-1 may also regulate gene expression by regulating early ribosome assembly (Schaper et al., 2001, Curr. Biol. 11:1885-1890). CCR4 and CAF-1 are also components of the major cytoplasmic mRNA deadenylase in S. cerevisiae, and may function in early steps of mRNA turnover by initiating the shortening of the poly(A) tail (Tucker et al., Cell 104:377-386).
VAMPS are members of the SNARE protein family, which are critical proteins in membrane fusion for both regulated and constitutive vesicle trafficking. VAP33 (VAMP-associated proteins of 33 kDa) proteins bind VAMPs and SNARES (Weir et al.
2001, Biochem Biophys Res Commun 286:616-21). Mammalian VAP33 (VAP-A) is widely expressed in multiple tissues and appears to be associated with the ER
and microtubules, as well as trafficking vesicles (Weir et al. 1998, Biochem. J.
333:247-251).
There are three known human isoforms of VAP33. VAP-A and -B are encoded by distinct genes and are approximately 60% identical; VAP-C is a splice variant of VAP-B, which lacks the C-terminal transmembrane domain (Nishimura et al. 1999, Biochem.
Biophys. Res. Commun. 254:21-26). VAP33 has been shown to play a pivotal role in insulin-stimulated GLUT4 translocation to the cell surface in L6 myoblasts and 3T3-Ll adipocytes (Foster et al. 2000, Traffic 6:512-521). There is also evidence that the yeast homolog SCS2 is required for inositol metabolism (Kagiwada et al. 1998, J.
Bacteriol.
180:1700-1708).
PP2 (also called PP2A) is a serine/threonine protein phosphatase that has been implicated in dephosphorylation of the proteins Akt and Gsk3-beta (Ivaska et al. 2002, Mol Cell Biol 22:1352-1359); dephophorylation of Gsk leads to increased glycogen synthase activity. Additional reports show that the insulin resistance mediated by ceramide induce a PP2 activity and can be relieved by treatment with a PP2 inhibitor okadaic acid (Teruel et al. 2001, Diabetes 50:2563-2571). Finally there is evidence that PP2 stimulates Acetyl CoA Carboxylase, an enzyme that catalyzes the production of long chain fatty acids, which may regulate insulin secretion (Kowluru et al. 2001, Diabetes 50:1580-1587). PP2 also appears to inhibit Acyl CoA: cholesterol acyltransferase (ACAT) and cholesterol ester synthesis (Hernandez et al. 1997, Biochim Biophys Acta 1349:233-41). Drosophila MTS (microtubule star) is an ortholog of PP2, and plays an essential role in spindle formation, where it is critical for the attachment of microtubules to the kinetochore during mitosis (Smith et al. 1996, J. Cell Sci. 109:3001-3012), and mouse PP2 is necessary for meiosis (Lu et al 2002, Biol Reprod. 66(1):29-37).
It has been speculated that the MTS/PP2 requirement is due to the hyperphosphorylation and inactivation of the Tau protein, which associates with and promotes stabilization of microtubules (Brandt and Ixe 1993, J Neurochem. 61:997-1005; Planel et al.
2001, J.
Biol. Chem. 276(36):34298-34306).
CSNK1, a serine/threonine protein kinase, belongs to a family of mammalian casein kinase I genes, producing multiple isoforms. Family members contain a highly conserved 290-residue N-terminal catalytic domain coupled to a variable C-terminal region. The C-terminal region serves to promote differential subcellular localization of individual isoforms and to modulate enzyme activity (Mashhoon, et al. 2000, J
Biol Chem 275: 20052-20060). CSNK1 appears to play a role in the regulation of circadian rhythms, intracellular trafficking, DNA repair, cellular morphology, and protein stabilization (Liu et al. 2001, Proc Natl Acad Sci 98:11062-11068). CSNK1 also has been shown to be involved in the regulation of eIF2B in coordination with GSK3 as part of an insulin signaling response (Wang et al. 2001, EMBO 20:4349-4359).
Drosophila GISH (Gilgamesh) is an ortholog of human CSNK1, and has been characterized as being part of a repulsive signaling mechanism that coordinates glial migration and neuronal development in the eye (Hummel, et al. 2002, Neuron 33:193-203).
ERF1 (eucaryotic release factor 1) is responsible for terminating protein biosynthesis. Termination of protein biosynthesis and release of the nascent polypeptide chain are signaled by the presence of an in-frame stop codon at the aminoacyl site of the ribosome. ERF1 recognizes the stop codon and promotes the hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA (Frolova et al.
1994, Nature 372: 701-703). The crystal structure of the release factor has been determined, the overall shape and dimensions of ERF1 resemble a tRNA molecule, with domains designated 1, 2, and 3 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively (Song et al. 2000, Cell 100: 311-321).
All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.
SUMMARY OF THE INVENTION
We have discovered genes that modify the INR pathway in Drosophila cells, and identified their human orthologs, hereinafter referred to as Modifiers of insulin receptor signaling (MINK). The invention provides methods for utilizing these INR
modifier genes and polypeptides to identify MINK-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired INR function and/or MINK function. Preferred M)IVR-modulating agents specifically bind to NIINR polypeptides and restore INR function. Other preferred MINK-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MINR gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
MINK modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an MINK polypeptide or nucleic acid. In one embodiment, candidate NBNR modulating agents are tested with an assay system comprising a MINK polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate INR
modulating agents. The assay system may be cell-based or cell-free. MINR-modulating agents include MINK related proteins (e.g. dominant negative mutants, and biotherapeutics); MINK -specific antibodies; MINK -specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with MINK or compete with MINK binding partner (e.g. by binding to an MINK
binding partner). In one specific embodiment, a small molecule modulator is identified using a binding assay. In specific embodiments, the screening assay system is selected from a hepatic lipid accumulation assay, a plasma lipid accumulation assay, an adipose lipid accumulation assay, a plasma glucose level assay, a plasma insulin level assay, and insulin sensitivity assay.
In another embodiment, candidate INR pathway modulating agents are further tested using a second assay system that detects changes in activity associated with INR
signaling. 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 INR
pathway.
The invention further provides methods for modulating the MINK function and/or the INR pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a NNIINNR 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 INR
pathway.
DETAILED DESCRIPTION OF THE INVENTION
We used a cellular RNAi screen to identify modifiers of the INR pathway and signaling activity. Modulators of the INR pathway were identified, followed by identification of their orthologs. Accordingly, modifiers of insulin receptor signaling (MINK) genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of disorders related to INR signaling. In one example, therapy involves increasing signaling through INR in order to treat pathologies related to diabetes and/or metabolic syndrome.
The invention provides in vitro and in vivo methods of assessing MINK
function, and methods of modulating (generally inhibiting or agonizing) MINR activity, which are useful for further elucidating INR signaling and for developing diagnostic and therapeutic modalities for pathologies associated with INR signaling. As used herein, pathologies associated with INR signaling encompass pathologies where INR signaling contributes to maintaining the healthy state, as well as pathologies whose course may be altered by modulation of the 1NR signaling.
MINR nucleic acids and nolyueutides Sequences related to n~NR nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) or RefSeq number), and shown in Table 1 (Example 1).
The term "MINK polypeptide" refers to a full-length NBNR protein or a fragment or derivative thereof that is "functionally active," meaning that the NBNR
protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type MINK protein. As one example, a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for generation of inhibitory antibodies, etc, as discussed further below. Preferably, a functionally active NBNR fragment or derivative displays one or more biological activities associated with MINK proteins such as enzymatic activity, signaling activity, ability to bind natural cellular substrates, etc. In one embodiment, a functionally active MINK
polypeptide is a NBNR derivative capable of rescuing defective endogenous MINK activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. If MINK fragments are used in assays to identify modulating agents, the fragments preferably comprise a M1NR domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprise at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of a MINK
protein. A preferred MINR fragment comprises a catalytic domain. Functional domains can be identified using the PFAM program (Bateman A et al., 1999 Nucleic Acids Res 27:260-262; website at pfam.wustl.edu).
The term "MINK nucleic acid" refers to a DNA or RNA molecule that encodes a MINK polypeptide. Preferably, the MINK polypeptide or nucleic acid or fragment thereof is from a human, but it can be an ortholog or derivative thereof with at least 70%, preferably with at least 80%, preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with a human MINK. Methods of identifying the human orthologs of these genes are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences.
Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL
(Thompson JD 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 C. elegans, 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 specified subject sequence, or a specified portion thereof, 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.Oa19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410;
http://blast.wustl.edu/blast/README.html) with 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; 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" (website at www.psc.edu) and references cited therein.; W.R.
Pearson, 1991, Genomics 11:635-650). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). 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 a MINK. 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 a NIINR under stringent hybridization conditions that are: 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
NaCI, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 p.g/ml hernng sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, 1X Denhardt's solution, 100 ~.g/ml yeast tRNA and 0.05%
sodium pyrophosphate; and washing of filters at 65° C for lh in a solution containing O.1X SSC
and 0.1 % SDS (sodium dodecyl sulfate). In other embodiments, moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM
Tris-HCl (pH7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ~,g/ml denatured salmon sperm DNA; hybridization for 18-20h at 40° C in a solution containing 35%
formamide, 5X SSC, 50 mM Tris-HCI (pH7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 p,g/ml salmon sperm DNA, and 10% (wtlvol) dextrin 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 are: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextrin sulfate, and 20 ~,g/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to hours; and washing of filters in 1 x SSC at about 37° ~C for 1 hour.
Isolation, Production, Expression, and Mis-expression of MINR Nucleic Acids and Polypeptides MINR nucleic acids and polypeptides, useful for identifying and testing agents that modulate MINK function and for other applications related to the involvement of MINK in INR signaling. MINK nucleic acids 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 polymerise chain reaction (PCR) are well known in the art.
A wide variety of methods are available for obtaining MINK polypeptides. In general, the intended use for the polypeptide will dictate the particulars of expression, production, and purification methods. For instance, production of polypeptides for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of polypeptides for antibody generation may require structural integrity of particular epitopes. Expression of polypeptides to be purified for screening or antibody production may require the addition of specific tags (i.e., generation of fusion proteins). Overexpression of a MINK polypeptide for cell-based assays used to assess MINK function, such as involvement in tubulogenesis, 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 therefor may be used (e.g., Higgins SJ and Hames BD (eds.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999;
Stanbury PF et al., Principles of Fermentation Technology, 2°d edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE et al, Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York; U.S. Pat. No. 6,165,992).
The nucleotide sequence encoding a NNIINNR polypeptide can be inserted into any appropriate vector for expression of the inserted protein-coding sequence. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native N>ZNR 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.
The N>ZNR polypeptide may be optionally expressed as a fusion or chimeric product, joined via a peptide bond to a heterologous protein sequence. In one application the heterologous sequence encodes a transcriptional reporter gene (e.g., GFP
or other fluorescent proteins, luciferase, beta-galactosidase, etc.). A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame 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).
An NBNR polypeptide can be isolated and purified using standard methods (e.g.
ion exchange, affinity, and gel exclusion chromatography; centrifugation;
differential solubility; electrophoresis). Alternatively, native MINK 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 MINK or other genes associated with INR
signaling. 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 The methods of this invention may use non-human animals that have been genetically modified to alter expression of MINK and/or other genes known to be involved in INR signaling. Preferred genetically modified animals are mammals, particularly mice or rats. Preferred non-mammalian species include Zebrafish, C.
elegans, and Drosophila. Preferably, the altered MINK or other gene expression results in a detectable phenotype, such as modified levels of INR signaling, modified levels of plasma glucose or insulin, or modified lipid profile as compared to control animals having normal expression of the altered gene. The genetically modified animals can be used to further elucidate INR signaling, in animal models of pathologies associated with INR signaling, and for in vivo testing of candidate therapeutic agents, as described below.
Preferred genetically modified animals are transgenic, at least a portion of their cells harboring non-native nucleic acid that is present either as a stable genomic insertion or as an extra-chromosomal element, which is typically mosaic. Preferred transgenic animals have germ-line insertions that are stably transmitted to all cells of progeny animals.
Non-native nucleic acid is introduced into host animals by any expedient method.
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., IRI. 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 MINK
gene that results in a decrease of 1~~NR function, preferably such that MINR
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 n~NR gene is used to construct a homologous recombination vector suitable for altering an endogenous MINK
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 MH et al., (1994) Scan J Immunol 40:257-264;
Declerck PJ 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 MINK gene, e.g., by introduction of additional copies of MINR, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MINK 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 INR pathway, as animal models of disease and disorders implicating defective INR 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 NN1TNNR function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered NNIINNR
expression that receive candidate therapeutic agent.
In addition to the above-described genetically modified animals having altered NBNR function, animal models having defective INR function (and otherwise normal MINK function), can be used in the methods of the present invention. For example, a INR knockout mouse can be used to assess, in vivo, the activity of a candidate INR
modulating agent identified in one of the in vitro assays described below.
Preferably, the candidate INR modulating agent when administered to a model system with cells defective in INR function, produces a detectable phenotypic change in the model system indicating that the INR function is restored.
MINK Modulating agents The invention provides methods to identify agents that interact with and/or modulate the function of MINK and/or INR signaling. Such agents are useful in a variety of diagnostic and therapeutic applications associated with INR signaling, as well as in further analysis of the MINR protein and its contribution to 1NR signaling.
Accordingly, the invention also provides methods for modulating INR signaling comprising the step of specifically modulating MINK activity by administering a MINK-interacting or -modulating agent.
As used herein, a "MINK-modulating agent" is any agent that modulates NBNR
function, for example, an agent that interacts with MINR to inhibit or enhance MINK
activity or otherwise affect normal N>INR function. MINK function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the MINK - modulating agent specifically modulates the function of the MINK. The phrases "specific modulating agent", "specifically modulates", etc., are used herein to refer to modulating agents that directly bind to the NBNR polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the NBNR. These phrases also encompasses modulating agents that alter the interaction of the MINK with a binding partner, substrate, or cofactor (e.g. by binding to a binding partner of an 1~~NR, or to a protein/binding partner complex, and altering MINR function). In a further preferred embodiment, the MINR-modulating agent is a modulator of the 1NR pathway (e.g. it restores and/or upregulates INR function) and thus is also a INR-modulating agent.
Preferred MINK-modulating agents include small molecule chemical agents, NIINR-interacting proteins, including antibodies and other biotherapeutics, and nucleic acid modulators, including antisense oligomers and RNA. 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 Garners or excipients. Techniques for formulation and administration of the compounds may be found in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton, PA, 19'h edition.
Small Molecule Modulators 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 MINK
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 MINK-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, 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 INR signaling. 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 NBNR-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the INR pathway and related disorders, as well as in validation assays for other MINK-modulating agents. In a preferred embodiment, MII~R-interacting proteins affect normal MINR function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, MINR-interacting proteins are useful in detecting and providing information about the function of MINK proteins, as is relevant to 11VR
related disorders, such as diabetes (e.g., for diagnostic means).
A MINK-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an MINK, such as a member of the MINK
pathway that modulates MINR expression, localization, and/or activity. NBNR-modulators include dominant negative forms of MINK-interacting proteins and of MINK proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous N1ZNR-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 JR 3'a, Trends Genet (2000) 16:5-8).
An NBNR-interacting protein may be an exogenous protein, such as an NBNR-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, NY: Cold Spring Harbor Laboratory Press). MINK antibodies are further discussed below.
In preferred embodiments, a MINK-interacting protein specifically binds an MINK protein. In alternative preferred embodiments, a MINK-modulating agent binds an NIINR substrate, binding partner, or cofactor.
Antibodies In another embodiment, the protein modulator is an MINR specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MINK modulators. The antibodies can also be used in dissecting the portions of the MINK pathway responsible for various cellular responses and in the general processing and maturation of the MINK.
Antibodies that specifically bind MINK polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of NIINR
polypeptide, and more preferably, to human T~NR. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
Epitopes of MINR which are particularly antigenic can be selected, for example, by routine screening of N>INR polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hope and Wood (1981), Proc. Nati.
Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;
Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence of a MINK.
Monoclonal antibodies with affinities of 108 M-1 preferably 109 M-~ to 10'° M~1, or stronger can be made by standard procedures as described (Harlow and Lane, supra;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577). Antibodies may be generated against crude cell extracts of MINR or substantially purified fragments thereof.
If MINK fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an MINK protein. In a particular embodiment, N>INR-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 MINK-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MINK polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.
Chimeric antibodies specific to MINK 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 (Mornson et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann LM, 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 SL. 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).

MINK-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 (IJ.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 im the range of about 0.1 mg/kg -to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to aboutl0 mg/ml.
Immunotherapeutic methods are further described in the literature (US Pat. No.

5,859,206; W00073469).
Nucleic Acid Modulators Other preferred MINK-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA)~ which generally inhibit NIINR
activity. Preferred antisense oligomers interfere with the function of NIINR
nucleic acids, such as DNA replication, transcription, MINK RNA translocation, translation of protein from the MINK RNA, RNA splicing, and any catalytic activity in which the MINK
RNA
participates.
In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to a MINK mRNA to bind to and prevent translation from the MINK mRNA, preferably by binding to the 5' untranslated region. MINR-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, a chimeric mixture of DNA and RNA, 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 phosphorothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which containing 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 inter-subunit linkages. Methods of producing and using PMOs and other antisense oligonucleotides are well known in the art (e.g. see W099/18193; Summerton J, and Welter D, Antisense Nucleic Acid Drug Dev 1997, 7:187-95; Probst JC, Methods 2000, 22:271-281; US PAT NO: 5,325,033; US PAT NO: 5,378,841).
Alternative preferred MINK 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); W00129058; W09932619; Elbashir SM, 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 specifically inhibit gene expression, are often used to elucidate the function of particular genes (see, e.g., US 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 humans and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF et al, 1993, J Med Chem 36:1923-1937;
Tonkinson JL et al., 1996, Cancer Invest 14:54-65). Accordingly, in one aspect of the invention, a MINR-specific antisense oligomer is used in an assay to further elucidate the function of MINK in INR signaling. Zebrafish is a particularly useful model for the study of INR signaling using antisense oligomers. For example, PMOs are used to selectively inactive one or more genes in vivo in the Zebrafish embryo. By injecting PMOs into Zebrafish'at the 1-16 cell stage candidate targets emerging from the Drosophila screens are validated in this vertebrate model system. In another aspect of the invention, PMOs are used to screen the Zebrafish genome for identification of other therapeutic modulators of 1NR signaling. In a further aspect of the invention, a N>ZNR-specific antisense oligomer is used as a therapeutic agent for treatment of metabolic pathologies.
Assay Systems The invention provides assay systems and screening methods for identifying specific modulators of MINK 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 or events. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the NBNR nucleic acid or protein. In general, secondary assays further assess the activity of a N>INR-modulating agent identified by a primary assay and may confirm that the modulating agent affects MINK in a manner relevant to INR signaling. In some cases, MINK-modulators will be directly tested in a "secondary assay," without having been identified or confirmed in a "primary assay."
In a preferred embodiment, the assay system comprises contacting a suitable assay system comprising a MINK polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity, which is based on the particular molecular event the assay system detects. The method further comprises detecting the same type of activity in the presence of a candidate agent (" the agent-biased activity of the system"). A
difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates MINR activity, and hence INR signaling. A statistically significant difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates MINK activity, and hence the INR signaling. The MINK
polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above 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 cellular extracts, 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.
In a 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 PR, Nat Struct Biol (2000) 7:730-4; Fernandes PB, Curr Opin Chem Biol (1998) 2:597-603;
Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4:445-451).
Suitable assay formats that may be adapted to screen for MINK modulators are known in the art.
Binding Assays. A variety of assays are available to detect the activity of proteins that have specific binding activity. Exemplary assays use fluorescence polarization, fluorescence polarization, and laser scanning techniques to measure binding of fluorescently labeled proteins, peptides, or other molecules (Lynch BA et al., 1999, Anal Biochem 275:62-73; Li HY, 2001, J Cell Biochem 80:293-303; Zuck P et al., Proc Natl Acad Sci USA 1999, 96: 11122-11127). In another example, binding activity is detected using the scintillation proximity assay (SPA), which uses a biotinylated peptide probe captured on a streptavidin coated SPA bead and a radio-labeled partner molecule.
The assay specifically detects the radio-labeled protein bound to the peptide probe via scintillant immobilized within the SPA bead (Sonatore LM et al., 1996, Anal Biochem 240:289-297).
Transcriptional activity assays. In one example, transcriptional activity is detected using quantitative RT-PCR (e.g., using the TaqMan~, PE Applied Biosystems).
In another example, a transcriptional reporter (e.g., luciferase, GFP, beta-galactosidase, etc.) operably linked to a responsive gene regulatory sequence is used (e.g., Berg M et al, 2000, J Biomol Screen, 5:71-76). Proteins that are part of a transcriptional complex may also be assayed for binding activity (i.e., to other members of the complex).
Phosphatase assays. Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate.
Since phosphatases act in opposition to kinases, appropriate assays monitor the removal of a phosphate from a protein substrate. In one example, the dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which in turn renders the cleaved substrate significantly more fluorescent (Nishikata M
et al., Biochem J (1999) 343:35-391). In another example, fluorescence polarization monitors direct binding of the phosphatase with the target; increasing concentrations of phosphatase increases the rate of dephosphorylation, leading to a change in polarization (Parker GJ et al., (2000) J Biomol Screen 5:77-88). Other appropriate assays for may monitor lipid phosphatase activity and may use labeled, such as fluorescently labeled or radio-labeled substrates to detect removal of a phosphate from a phosphatidylinositol substrate. In one example, an assay uses "FlashPlate" technology (U.S. Patent No.
5,972,595), in which a radio-labeled hydrophobic substrate is immobilized on a solid support in each well of a multi-well plate. Dephosphorylation of the substrate is measured as a decrease in bound radioactivity, which is detected by the close proximity of the scintillant. Other assays for detecting phosphoinositide phosphatase activity are known in the art (see, e.g., US PAT NOs: Patent 6,001,354 and 6,238,903).

GAP assays. GAP proteins stimulate GTP hydrolysis to GDP. Exemplary assays may monitor GAP activity, for instance, via a GTP hydrolysis assay using labeled GTP
(e.g., Jones S et al., Molec Biol Cell (1998) 9:2819-2837). Alternative assays may detect GAP function in endosome trafficking by monitoring movement of a cargo molecule, which may be labeled (Sonnichsen et al., 2000, J Cell Biol 149:901-14).
Kinase assays. Preferred assays detect kinase activity, the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine or threonine residue in a protein substrate. Radioassays, which monitor the transfer from [gamma-32P or 33P]ATP, may be used to assay kinase activity. Separation of the phospho-labeled product from the remaining radio-labeled ATP can be accomplished by various methods including SDS-polyacrylamide gel electrophoresis, filtration using glass fiber filters or other matrices which bind peptides or proteins, and adsorption/binding of peptide or protein substrates to solid-phase matrices allowing removal of remaining radiolabeled ATP by washing. In one example, a scintillation assay 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 MD, 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 1996 Jul 1;238(2):159-64). Generic assays may be established for protein kinases that rely upon the phosphorylation of substrates such as myelein basic protein, casein, histone, or synthetic peptides such as polyGlutamate/Tyrosine and radiolabeled ATP.
Release factor activity assays. Appropriate assays may detect in vitro release factor activity (see, e.g., Seit-Nebi et al. 2001, Nucleic Acids Res 29:3982-7; Frolova et al. 1994, Nature 372:701-3; Caskey et al. 1974, Methods Enzymol 30:293-303).
Cell-based screening assays usually require systems for recombinant expression of N11NR and any auxiliary proteins demanded by the particular assay. Cell-free assays often use recombinantly produced purified or substantially purified proteins.
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 NIINR-interacting proteins are used in screening assays, the binding specificity of the interacting protein to the MINK protein may be assayed by various known methods, including binding equilibrium constants (usually at least about 107 M-1, preferably at least about 10g M-I, more preferably at least about 109 M-1), and immunogenic properties. 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 NBNR polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The MINK polypeptide can be full length or a fragment thereof that retains functional MINK activity. The MINK
polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The MINR polypeptide is preferably human MIIVR, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of MINR interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MINK -specific binding activity, and can be used to assess normal NBNR gene function.
Certain screening assays may also be used to test antibody and nucleic acid modulators; for nucleic acid modulators, appropriate assay systems involve MINK
mRNA expression.

Primary assays for antibody modulators For antibody modulators, appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the MINK 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 methods for detecting MINK-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 MINK gene expression, preferably mRNA expression. In general, expression analysis comprises comparing MINR expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express MINK) 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 MINK
mRNA
expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley &
Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, ACurr 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 NBNR
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 a MINR-modulating agent identified by any of the above methods to confirm that the modulating agent affects MINK in a manner relevant to INR signaling. As used herein, MINR-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 modulator on a particular genetic or biochemical pathway or to test the specificity of the modulator's interaction with MINK.
Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express MINK) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate MINK-modulating agent results in changes in INR signaling, in comparison to untreated (or mock- or placebo-treated) cells or animals. Changes in INR signaling may be detected as modifications to INR
pathway components, or changes in their expression or activity. Assays may also detect an output of normal or defective INR signaling, used herein to encompass immediate outputs, such as glucose uptake, or longer-term effects, such as changes in glycogen and triglycerides metabolism, adipocyte differentiation, or development of diabetes or other INR-related pathologies. Certain assays use sensitized genetic backgrounds, used herein to describe cells or animals engineered for altered expression of genes in the INR or interacting pathways, or pathways associated with INR signaling or an output of INR
signaling.
Cell-based assays Cell-based assays may use a variety of insulin-sensitive mammalian cells and may detect endogenous INR signaling or may rely on recombinant expression of INR
and/or other INR pathway components. Exemplary insulin-sensitive cells include adipocytes, hepatocytes, and pancreatic beta cells. Suitable adipocytes include 3T3 L1 cells, which are most commonly used for insulin sensitivity assays, as well as primary cells from mice or human biopsy. Suitable hepatocytes include the rat hepatoma H4-II-E cell line.
Suitable beta cells include rat INS-1 cells with optimized glucose-sensitive insulin secretion (such as clone 823-13, Hohmeier et al., 2000, Diabetes 49:424).
Other suitable cells include muscle cells, such as L6 myotubes, and CHO cells engineered to over-express INR. For certain assay systems it may be useful to treat cells with factors such as glucosamine, free fatty acids or TNF alpha, which induce an insulin resistant state.
Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.

Cell based assays generally test whether treatment of insulin responsive cells with the MINK - modulating agent alters INR signaling in response to insulin stimulation ("insulin sensitivity"); such assays are well-known in the art (see, e.g., Sweeney et al., 1999, J Biol Chem 274:10071). In a preferred embodiment, assays are performed to determine whether inhibition of MINK function increases insulin sensitivity.
In one example, INR signaling is assessed by measuring expression of insulin-responsive genes. Hepatocytes are preferred for these assays. Many insulin responsive genes are known (e.g., p85 PI3 kinase, hexokinase II, glycogen synthetase, lipoprotein lipase, etc; PEPCK is specifically down-regulated in response to INR
signaling). Any available means for expression analysis, as previously described, may be used.
Typically, mRNA expression is detected. In a preferred application, Taqman analysis is used to directly measure mRNA expression. Alternatively, expression is indirectly monitored from a transgenic reporter construct comprising sequences encoding a reporter gene (such as luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.) under control of regulatory sequences (e.g., enhancer/promoter regions) of an insulin responsive gene. Methods for making and using reporter constructs are well known.
INR signaling may also be detected by measuring the activity of components of the INR-signaling pathway, which are well-known in the art (see, e.g., Kahn and Weir, Eds., Joslin's Diabetes Mellitus, Williams & Wilkins, Baltimore, MD, 1994).
Suitable assays may detect phosphorylation of pathway members, including IRS, PI3K, Akt, GSK3 etc., for instance, using an antibody that specifically recognizes a phosphorylated protein. Assays may also detect a change in the specific signaling activity of pathway components (e.g., kinase activity of PI3K, GSK3, Akt, etc.). Kinase assays, as well as methods for detecting phosphorylated protein substrates, are well known in the art (see, e.g., Ueki K et al, 2000, Mol Cell Bio1;20:8035-46).
In another example, assays measure glycogen synthesis in response to insulin stimulation, preferably using hepatocytes. Glycogen synthesis may be assayed by various means, including measurement of glycogen content, and determination of glycogen synthase activity using labeled, such as radio-labeled, glucose (see, e.g., Aiston S and Agius L, 1999, Diabetes 48:15-20; Rother KI et al., 1998, J Biol Chem 273:17491-7).

Other suitable assays measure cellular uptake of glucose (typically labeled glucose) in response to insulin stimulation. Adipocytes are preferred for these assays.
Assays also measure translocation of glucose transporter (GLUT) 4, which is a primary mediator of insulin-induced glucose uptake, primarily in muscle and adipocytes, and which specifically translocates to the cell surface following insulin stimulation. Such assays may detect endogenous GLUT4 translocation using GLUT4-specific antibodies or may detect exogenously introduced, epitope-tagged GLUT4 using an antibody specific to the particular epitope (see, e.g., Sweeney, 1999, supra; Quon MJ et al., 1994, Proc Natl Acad Sci U S A 91:5587-91).
Other preferred assays detect insulin secretion from beta cells in response to glucose. Such assays typically use ELISA (see, e.g., Bergsten and Hellman, 1993, Diabetes 42:670-4) or radioimmunoassay (RIA; see, e.g., Hohmeier et al., 2000, supra).
Animal Assays A variety of non-human animal models of metabolic disorders may be used to test candidate NBNR modulators. Such models typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in lipid metabolism, adipogenesis, and/or the INR signaling pathway.
Additionally, particular feeding conditions, and/or administration or certain biologically active compounds, may contribute to or create animal models of lipid and/or metabolic disorders. Assays generally required systemic delivery of the candidate modulators, such as by oral administration, injection (intravenous, subcutaneous, intraperitoneous), bolus administration, etc.
In one embodiment, assays use mouse models of diabetes andlor insulin resistance. Mice carrying knockouts of genes in the leptin pathway, such as ob (leptin) or db (leptin receptor), or the INR signaling pathway, such as INR or the insulin receptor substrate (IRS), develop symptoms of diabetes, and show hepatic lipid accumulation (fatty liver) and, frequently, increased plasma lipid levels (Nishina et al., 1994, Metabolism 43:549-553; Michael et al., 2000, Mol Cell 6:87-97; Bruning JC et al., 1998, Mol Cell 2:559-569). Certain susceptible wild type mice, such as C57BIJ6, exhibit similar symptoms when fed a high fat diet (Linton and Fazio, 2001, Current Opinion in Lipidology 12:489-495). Accordingly, appropriate assays using these models test whether administration of a candidate modulator alters, preferably decreases lipid accumulation in the liver. Lipid levels in plasma and adipose tissue may also be tested.
Methods for assaying lipid content, typically by FPLC or colorimetric assays (Shimano H
et al., 1996, J Clin Invest 98:1575-1584; Hasty et al., 2001, J Biol Chem 276:37402-37408), and lipid synthesis, such as by scintillation measurement of incorporation of radio-labeled substrates (Horton JD et al., 1999, J Clin Invest 103:1067-1076), are well known in the art. Other useful assays test blood glucose levels, insulin levels, and insulin sensitivity (e.g., Michael MD, 2000, Molecular Cell 6: 87). Insulin sensitivity is routinely tested by a glucose tolerance test or an insulin tolerance test.
In another embodiment, assays use mouse models of lipoprotein biology and cardiovascular disease. For instance, mouse knockouts of apolipoprotein E
(apoE) display elevated plasma cholesterol and spontaneous arterial lesions (Zhang SH, 1992, Science 258:468-471). Transgenic mice over-expressing cholesterol ester transfer protein (CETP) also display increased plasma lipid levels (specifically, very-low-density lipoprotein [VLDL] and low-density lipoprotein [LDL] cholesterol levels) and plaque formation in arteries (Marotti KR et al., 1993, Nature 364:73-75). Assays using these models may test whether administration of candidate modulators alters plasma lipid levels, such as by decreasing levels of the pro-atherogenic LDL and VLDL, increasing HDL, or by decreasing overall lipid (including trigyceride) levels.
Additionally histological analysis of arterial morphology and lesion formation (i.e., lesion number and size) may indicate whether a candidate modulator can reduce progression and/or severity of atherosclerosis. Numerous other mouse models for atherosclerosis are available, including knockouts of Apo-A1, PPARgamma, and scavenger receptor (SR)-B1 in LDLR- or ApoE-null background (reviewed in, e.g., Glass CK and Witztum JL, 2001, Cell 104:503-516).
In another embodiment, the ability of candidate modulators to alter plasma lipid levels and artherosclerotic progression are tested in mouse models for multiple lipid disorders. For instance, mice with knockouts in both leptin and LDL receptor genes display hypercholesterolemia, hypertriglyceridemia and arterial lesions and provide a model for the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis (Hasty AH et al, 2001, supra.).
Diya nostic Methods The discovery that MINR is implicated in INR signaling provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders associated with INR signaling and for the identification of subjects having a predisposition to such diseases and disorders. Any method for assessing MINK
expression in a sample, as previously described, may be used. Such methods may, for example, utilize reagents such as the MINK oligonucleotides and antibodies directed against NIINR, as described above for: (1) the detection of the presence of MINK gene mutations, or the detection of either over- or under-expression of MINK mRNA
relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of MINK gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in a biological pathway mediated by MINK.
Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in NI)NR
expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MINK expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder. 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.
I. Drosophila cell RNAi screen We used a cellular RNAi screen to identify modifiers of the INR pathway.
Briefly, the screen involved treating cells from the Dmel line, a derivative of the Drosophila S2 cell line that thrives in serum-free media, with dsRNA
corresponding to predicted Drosophila genes, in order to effect disruption of these genes (Adams et al., 2000, Science 287:2185-95). Duplicate wells of cells in a multi-well plate were treated with dsRNA corresponding to individual Drosophila genes (methods were essentially as described in Clemens et al., 2000, supra). Quantitative RT-PCR using TaqMan~
(PE
Applied Biosystems) was used to measure expression of the lactate dehydrogenase ("LDH," GI 1519714; Abu-Shumays and Fristrom, 1997, Dev Genet 20:11-22) gene, which we had previously show to correlate with INR pathway activity.
Specifically, LDH expression was increased when INR pathway activity was increased by RNAi-based knock-down of negative effectors of INR signaling (e.g., PTEN, GSK3beta, and AFX), in the presence or absence of insulin. LDH expression was decreased when INR
pathway activity was decreased by RNAi-based knock-down of positive effectors of INR
signaling (e.g., INR, IRS, AKT). Accordingly, lactate dehydrogenase expression was used as a surrogate for lNR pathway activity. The screen identified "modifier"
genes, whose knock-down by RNAi produced a changes in LDH expression. Genes whose disruption by RNAi produced an increase in LDH expression were identified as candidate negative effectors of INR pathway activity, while those whose disruption decreased LDH
expression were candidate positive effectors. Potential modifiers were retested in triplicate in a confirmation experiment using RT-PCR analysis of LDH, as well as a sodium/phosphate cotransporter ("CG 4726," GI 10727399; amino acid sequence in GI
7296119), whose expression was also found to decrease following RNAi-based disruption of INR. The dsRNA used for the confirmation experiment was produced from a PCR product generated using different primers to the candidate modifier gene than were used to produce the original result. Table 1 lists the modifiers and their orthologs.
Table 1 MINR MINR NA MINR MINR AA MINR Modifier Modifier symbol GI# NA SEQ GI# AA SEQ name GI#
ID NO: ID NO:

~

ERF1 4759033 16 4759034 32 CG5605 7296284;

II. Huh-Throug-put In Vitro Fluorescence Polarization Assay Fluorescently-labeled nRNR peptide/substrate are added to each well of a 96-well microtiter plate, along with a test compound of choice in a test buffer (10 mM
HEPES, 10 mM NaCI, 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 MINK activity.
III. High-Throughput In Vitro Binding Assay.
s3P-labeled MINK peptide is added in an assay buffer (100 mM KCI, 20 mM
HEPES pH 7.6, 1 mM MgCl2, 1% glycerol, 0.5070 NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a compound of interest 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.
IV. Immunoprecipitations and Immunoblottine For coprecipitation of transfected proteins, 3 x 106 appropriate cells 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 NaCI, 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 x g for 15 min. The cell lysate are incubated with 25 ~,1 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 directly 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).
VI. Kinase assay A purified or partially purified MINR 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 p,g/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 ~1. The reaction is initiated by the addition of 33P-gamma-ATP (0.5 p.Ci/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).

SEQUENCE LISTING
<110> Exelixis, Inc.
<120> MINRs AS MODIFIERS OF INSULIN RECEPTOR SIGNALING AND METHODS OF
USE
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<150> 60/354,824 <151> 2002-02-06 <150> 60/358,217 <151> 2002-02-20 <150> 60/358,189 <151> 2002-02-20 <150> 60/358,126 <151> 2002-02-20 <150> 60/358,995 <151> 2002-02-21 <150> 60/358,756 <151> 2002-02-21 <150> 60/358,765 <151> 2002-02-21 <150> 60/359,531 <151> 2002-02-25 <150> 60/360,222 <151> 2002-02-26 <150> 60/360,224 <151> 2002-02-26 <150> 60/360,167 <151> 2002-02-26 <150> 60/360,166 <151> 2002-02-26 <160> 32 <170> PatentIn version 3.2 <210> 1 <211> 1759 <212> DNA
<213> Homo Sapiens <400> 1 ggcacgagaa aattcatgcg agggagacgt ggtgggcggt ccttcctgtg acacgaccct 60 tgagtgacag ttctatttga ttgcctccgg tactgtgagg aaaggacacg actctatggt 120 gaggactgat ggacatacat tatctgagaa aagaaactac caggtgacaa acagcatgtt 180 tggtgcttca agaaagaagt ttgtagaggg ggtcgacagt gactaccatg acgaaaacat 240 gtactacagccagtcttctatgtttccacatcggtcagaaaaagatatgctggcatcacc300 atctacatcaggtcagctgtctcagtttggggcaagtttatacgggcaacaaagtgcact360 aggccttccaatgagggggatgagcaacaatacccctcagttaaatcgcagcttatcaca420 aggcactcagttaccgagccacgtcacgccaacaacaggggtaccaacaatgtcacttca480 cacgcctccatctccaagcaggggtattttgcctatgaatcctaggaatatgatgaacca540 ctcccaggttggtcagggcattggaattcctagcaggacaaatagcatgagcagttcagg600 gttaggtagccccaacagaagctcgccaagcataatatgtatgccaaagcagcagccttc660 tcgacagccttttactgtgaacagtatgtctggatttggaatgaacaggaatcaggcatt720 tggaatgaataactccttatcaagtaacatttttaatggaacagacggaagtgaaaatgt780 gacaggattggacctttcagatttcccagcattagcagaccgaaacaggagggaaggaag840 tggtaacccaactccattaataaaccccttggctggaagagctccttatgttggaatggt900 aacaaaaccagcaaatgaacaatcccaggacttctcaatacacaatgaagattttccagc960 attaccaggctccagctataaagatccaacatcaagtaatgatgacagtaaatctaattt1020 gaatacatctggcaagacaacttcaagtacagatggacccaaattccctggagataaaag1080 ttcaacaacacaaaataataaccagcagaaaaaagggatccaggtgttacctgatggtcg1140 ggttactaacattcctcaagggatggtgacggaccaatttggaatgattggcctgttaac1200 atttatcagggcagcagagacagacccaggaatggtacatcttgcattaggaagtgactt1260 aacaacattaggcctcaatctgaactctcctgaaaatctctaccccaaatttgcgtcacc1320 ctgggcatcttcaccttgtcgacctcaagacatagacttccatgttccatctgagtactt1380 aacgaacattcacattagggataagctggctgcaataaaacttggccgatatggtgaaga1440 ccttctcttctatctctattacatgaatggaggagacgtattacaacttttagctgcagt1500 ggagctttttaaccgtgattggagataccacaaagaagaacgagtatggattaccagggc1560 accaggcatggagccaacaatgaaaaccaatacctatgagaggggaacatattacttctt1620 tgactgtcttaactggaggaaagtagctaaggagttccatctggaatatgacaaattaga1680 agaacggcctcacctgccatccaccttcaactacaaccctgctcagcaagccttctaaaa1740 aaaaaaaaaaaaaaaaaaa 1759 <210> 2 <211> 3411 <212> DNA
<213> Homo sapiens <400> 2 gcagccgagc agcctggcaa cggcggtggc gcccggagcc cgagagtttc caggatggct 60 tctgcatcaacttctaaatataattcacactccttggagaatgagtctattaagaggacg120 tctcgagatggagtcaatcgagatctcactgaggctgttcctcgacttccaggagaaaca180 ctaatcactgacaaagaagttatttacatatgtcctttcaatggccccattaagggaaga240 gtttacatcacaaattatcgtctttatttaagaagtttggaaacggattcttctctaata300 cttgatgttcctctgggtgtgatctcgagaattgaaaaaatgggaggcgcgacaagtaga360 ggagaaaattcctatggtctagatattacttgtaaagacatgagaaacctgaggttcgct420 ttgaaacaggaaggccacagcagaagagatatgtttgagatcctcacgagatacgcgttt480 cccctggctcacagtctgccattatttgcatttttaaatgaagaaaagtttaacgtggat540 ggatggacagtttacaatccagtggaagaatacaggaggcagggcttgcccaatcaccat600 tggagaataacttttattaataagtgctatgagctctgtgacacttaccctgctcttttg660 gtggttccgtatcgtgcctcagatgatgacctccggagagttgcaacttttaggtcccga720 aatcgaattccagtgctgtcatggattcatccagaaaataagacggtcattgtgcgttgc780 agtcagcctcttgtcggtatgagtgggaaacgaaataaagatgatgagaaatatctcgat840 gttatcagggagactaataaacaaatttctaaactcaccatttatgatgcaagacccagc900 gtaaatgcagtggccaacaaggcaacaggaggaggatatgaaagtgatgatgcatatcat960 aacgccgaacttttcttcttagacattcataatattcatgttatgcgggaatctttaaaa1020 aaagtgaaggacattgtttatcctaatgtagaagaatctcattggttgtccagtttggag1080 tctactcattggttagaacatatcaagctcgttttgacaggagccattcaagtagcagac1140 aaagtttcttcagggaagagttcagtgcttgtgcattgcagtgacggatgggacaggact1200 gctcagctgacatccttggccatgctgatgttggatagcttctataggagcattgaaggg1260 ttcgaaatactggtacaaaaagaatggataagttttggacataaatttgcatctcgaata1320 ggtcatggtgataaaaaccacaccgatgctgaccgttctcctatttttctccagtttatt1380 gattgtgtgtggcaaatgtcaaaacagttccctacagcttttgaattcaatgaacaattt1440 ttgattataattttggatcatctgtatagttgccgatttggtactttcttattcaactgt1500 gaatctgctcgagaaagacagaaggttacagaaaggactgtttctttatggtcactgata1560 aacagtaataaagaaaaattcaaaaaccccttctatactaaagaaatcaatcgagtttta1620 tatccagttgccagtatgcgtcacttggaactctgggtgaattactacattagatggaac1680 cccaggatcaagcaacaacagccgaatccagtggagcagcgttacatggagctcttagcc1740 ttacgcgacgaatacataaagcggcttgaggaactgcagctcgccaactctgccaagctt1800 tctgatcccccaacttcaccttccagtccttcgcaaatgatgccccatgtgcaaactcac1860 ttctgaggggggaccctggcaccgcattagagctcgaaataaaggcgatagctgactttc1920 atttggggcatttgtaaaaagtagattaaaatatttgcctccatgtagaacttgaactaa1980 cataatcttaaactcttgaatatgtgccttctagaatacatattacaagaaaactacagg2040 gtccacacggcaatcagaagaaaggagctgagatgaggttttggaaaaccctgacacctt2100 taaaaagcagtttttgaaagacaaaatttagatttaatttacgtcttgagaaatactata2160 tatacaatatatatggggggggcttaattgaaacaacattattttaaaatcaaaggggat2220 atatgtttgtggaatggattttcctgaagctgcttaacagttgctttggattctctaaga2280 tgaatccaaatgtgaaagatgcatgttactgccaaaaccaaattgagctcagcttcctag2340 gcattacccaaaagcaaggtgtttaagtaattgccagcttttataccatcatgagtggtg2400 acttaaggagaaatagctgtatagatgagtttttcattatttggaaatttaggggtagaa2460 aatgttttcccctaattttccagagaagcctatttttatatttttaaaaaactgacaggg2520 cccagttaaatatgatttgcattttttaaatttgccagttttattttctaaattctttca2580 tgagcttgcctaaaattcggaatggttttcgggttgtggcaaaccccaaagagagcactg2640 tccaaggatgtcgggagcatcctgctgcttaggggaatgttttcgcaaatgttgctctag2700 tcagtccagctcatctgccaaaatgtagggctaccgtcttggatgcatgagctattgcta2760 gagcatcatccttagaaatcagtgccccagatgtacatgtgttgagcgtattcttgaagt2820 attgtgtttatgcatttcaatttcaatggtgttggcttcccctccccaccccacgcgtgc2880 ataaaaactggttctacaaatttttacttgaagtaccaggccgtttgctttttcaggttg2940 ttttgttttatagtattaagtgaaattttaaatgcacagttctatttgctatctgaacta3000 attcatttattaagtatatttgtaaaagctaaggctcgagttaaaacaatgaagtgtttt3060 acaatgatttgtaaaggactatttataactaatatggttttgttttcaatgaattaagaa3120 agattaaatatatctttgtaaattattttatgtcatagtttaattggtctcccaagtaag3180 acatctcaaatacagtagtataatgtatgaattttgtaagtataagaaattttattagac3240 attctcttactttttgtaaatgctgtaaatatttcataaattaacaaagtgtcactccat3300 aaaaagaaagctaatactaatagcctaaaagattttgtgaaatttcatgaaaacttttta3360 atggcaataatgactaaagacctgctgtaataaatgtattaactgaaacct 3411 <210> 3 <211> 1932 <212> DNA
<213> Homo sapiens <400> 3 atggagacga gctcgagctg cgagagtctt ggctcccagc cggcggcggc tcggccgccc 60 agcgtggact ccttgtccag tgcctccact tctcattcag agaattcagt gcatacaaaa 120 tcagcttctg ttgtatcatc agattccatt tcaacttctg ccgacaactt ttctcctgat 180 ttgagggtcctgagggagtctaacaagttagcagaaatggaagaaccacccttgcttcca240 ggagaaaatattaaagacatggccaaagatgtaacttatatatgtccattcactggcgct300 gtacgaggaactctgactgtcacgaattataggttatatttcaaaagcatggaacgggat360 cccccatttgttttagatgcttcccttggtgtgataaatagagtagaaaaaattggtggt420 gcttctagtcgaggtgaaaattcttatggactagaaactgtgtgtaaggatattaggaat480 ttacgatttgctcataaacctgaggggcggacaagaagatccatatttgagaatctaatg540 aaatatgcatttcctgtctctaataacctgcctctttttgcttttgaatacaaagaagta600 ttccctgaaaatgggtggaagctatatgaccctcttttagagtatagaaggcagggaatt660 ccaaatgaaagctggagaataacaaagataaatgaacgatatgaactttgtgatacatac720 cctgccctcctggttgtgccagcaaatattcctgatgaagaattaaagagagtggcatcc780 ttcagatcaagaggccgtatcccagttttatcatggattcatcctgaaagtcaagccaca840 atcactcggtgtagccagcccatggttggagtgagtggaaagcgaagcaaagaagatgaa900 aaataccttcaagctatcatggattccaatgcccagtctcacaaaatctttatatttgat960 gcccggccaagtgttaatgctgttgccaacaaggcaaagggtggaggttatgaaagtgaa1020 gatgcctatcaaaatgctgaactagttttcctggatatccacaatattcatgttatgaga1080 gaatcattac.gaaaacttaaggagattgtgtaccccaacattgaggaaactcactggttg1140 tctaacttggaatctactcattggctagaacatattaagcttattcttgcaggggctctt1200 aggattgctgacaaggtagagtcagggaagacgtctgtggtagtgcattgcagtgatggt1260 tgggatcgcacagctcagctcacttcccttgccatgctcatgttggatggatactatcga1320 accatccgaggatttgaagtccttgtggagaaagaatggctaagttttggacatcgattt1380 caactaagagttggccatggagataagaaccatgcagatgcagacagatcgcctgttttt1440 cttcaatttattgactgtgtctggcagatgacaagacagtttcctaccgcatttgaattc1500 aatgagtattttctcattaccattttggaccacctatacagctgcttattcggaacattc1560 ctctgtaatagtgaacaacagagaggaaaagagaatcttcctaaaaggactgtgtcactg1620 tggtcttacataaacagccagctggaagacttcactaatcctctctatgggagctattcc1680 aatcatgtcctttatccagtagccagcatgcgccacctagagctctgggtgggatattac1740 ataaggtggaatccacggatgaaaccacaggaacctattcacaacagatacaaagaactt1800 cttgctaaacgagcagagcttcagaaaaaagtagaggaactacagagagagatttctaac1860 cgatcaacctcatcctcagagagagccagctctcctgcacagtgtgtcactcctgtccaa1920 actgttgtatas 1932 <210>

<211>

<212>
DNA

<213> Sapiens Homo <400>

atggctacgggcgcggatgtacgggacattctagaactcgggggtccagaaggggatgca60 gcctctgggaccatcagcaagaaggacattatcaacccggacaagaaaaaatccaagaag120 tcctctgagacactgactttcaagaggcccgagggcatgcaccgggaagtctatgccttg180 ctctactctgacaagaaggatgcacccccactgctacccagtgacactggccagggatac240 cgtacagtgaaggccaagttgggctccaagaaggtgcggccttggaagtggatgccattc300 accaacccggcccgcaaggacggagcaatgttcttccactggcgacgtgcagcggaggag360 ggcaaggactacccctttgccaggttcaataagactgtgcaggtgcctgtgtactcggag420 caggagtaccagctttatctccacgatgatgcttggactaaggcagaaactgaccacctc480 tttgacctcagccgccgctttgacctgcgttttgttgttatccatgaccggtatgaccac540 cagcagttcaagaagcgttctgtggaagacctgaaggagcggtactaccacatctgtgct600 aagcttgccaacgtgcgggctgtgccaggcacagaccttaagataccagtatttgatgct660 gggcacgaacgacggcggaaggaacagcttgagcgtctctacaaccggaccccagagcag720 gtggcagaggaggagtacctgctacaggagctgcgcaagattgaggcccggaagaaggag780 cgggagaaacgcagccaggacctgcagaagctgatcacagcggcagacaccactgcagag840 cagcggcgcacggaacgcaaggcccccaaaaagaagctaccccagaaaaaggaggctgag900 aagccggctgttcctgagactgcaggcatcaagtttccagacttcaagtctgcaggtgtc960 acgctgcggagccaacggatgaagctgccaagctctgtgggacagaagaagatcaaggcc1020 ctggaacagatgctgctggagcttggtgtggagctgagcccgacacctacggaggagctg1080 gtgcacatgttcaatgagctgcgaagcgacctggtgctgctctacgagctcaagcaggcc1140 tgtgccaactgcgagtatgagctgcagatgctgcggcaccgtcatgaggcactggcccgg1200 gctggtgtgctagggggccctgccacaccagcatcaggcccaggcccggcctctgctgag1260 ccggcagtgactgaacccggacttggtcctgaccccaaggacaccatcattgatgtggtg1320 ggcgcacccctcacgcccaattcgagaaagcgacgggagtcggcctccagctcatcttcc1380 gtgaagaaagccaagaagccgtga 1404 <210> 5 <211> 5411 <212> DNA
<213> Homo Sapiens <400> 5 ggtgcgtcct ggtccaccat ggccaaacca acaagcaaag attcaggctt gaaggagaag 60 tttaagattctgttgggactgggaacaccgaggccaaatcccaggtctgcagagggtaaa120 cagacggagtttatcatcaccgcggaaatactgagagaactgagcatggaatgtggcctc180 aacaatcgcatccggatgatagggcagatttgtgaagtcgcaaaaaccaagaaatttgaa240 gagcacgcagtggaagcactctggaaggcggtcgcggatctgttgcagccggagcggacg300 ctggaggcccggcacgcggtgctggctctgctgaaggccatcgtgcaggggcagggcgag360 cgtttgggggtcctcagagccctcttctttaaggtcatcaaggattacccttccaacgaa420 gaccttcacgaaaggctggaggttttcaaggccctcacagacaatgggagacacatcacc480 tacttggaggaagagctggctgactttgtcctgcagtggatggatgttggcttgtcctcg540 gaattccttctggtgctggtgaacttggtcaaattcaatagctgttacctcgacgagtac600 atcgcaaggatggttcagatgatctgtctgctgtgcgtccggaccgcgtcctctgtggac660 atagaggtctccctgcaggtgctggacgccgtggtctgctacaactgcctgccggctgag720 agcctcccgctgttcatcgttaccctctgtcgcaccatcaacgtcaaggagctctgcgag780 ccttgctggaagctgatgcggaacctccttggcacccacctgggccacagcgccatctac840 aacatgtgccacctcatggaggacagagcctacatggaggacgcgcccctgctgagagga900 gccgtgttttttgtgggcatggctctctggggagcccaccggctctattctctcaggaac960 tcgccgacatctgtgtttccatcattttaccaggccatggcatgtccgaacgaggtggtg1020 tcctatgagatcgtcctgtccatcaccaggctcatcaagaagtataggaaggagctccag1080 gtggtggcgtgggacattctgctgaacatcatcgaacggctccttcaacagctccagacc1140 ttggacagcccggagctcaggaccatcgtccatgacctgttgaccacggtggaggagctg1200 tgtgaccagaacgagttccacgggtctcaggagagatactttgaactggtggagagatgt1260 gcggaccagaggcctgagtcctccctcctgaacctgatctcctatagagcgcagtccatc1320 cacccggccaaggacggctggattcagaacctgcaggcgctgatggagagattcttcagg1380 agcgagtcccgaggcgccgtgcgcatcaaggtgctggacgtgctgtcctttgtgctgctc1440 atcaacaggcagttctatgaggaggagctgattaactcagtggtcatctcgcagctctcc1500 cacatccccgaggataaagaccaccaggtccgaaagctggccacccagttgctggtggac1560 ctggcagagggctgccacacacaccacttcaacagcctgctggacatcatcgagaaggtg1620 atggcccgctccctctccccacccccggagctggaagaaagggatgtggccgcatactcg1680 gcctccttggaggatgtgaagacagccgtcctggggcttctggtcatccttcagaccaag1740 ctgtacaccctgcctgcaagccacgccacgcgtgtgtatgagatgctggtcagccacatt1800 cagctccactacaagcacagctacaccctgccaatcgcgagcagcatccggctgcaggcc1860 tttgacttcctgtttctgctgcgggccgactcactgcaccgcctgggcctgcccaacaag1920 gatggagtcgtgcggttcagcccctactgcgtctgcgactacatggagccagagagaggc1980 tctgagaagaagaccagcggccccctttctcctcccacagggcctcctggcccggcgcct2040 gcaggccccgccgtgcggctggggtccgtgccctactccctgctcttccgcgtcctgctg2100 cagtgcttgaagcaggagtctgactggaaggtgctgaagctggttctgggcaggctgcct2160 gagtccctgcgctataaagtgctcatctttacttccccttgcagtgtggaccagctgtgc2220 tctgctctctgctccatgctttcaggcccaaagacactggagcggctccgaggcgcccca2280 gaaggcttctccagaactgacttgcacctggccgtggttccagtgctgacagcattaatc2340 tcttaccataactacctggacaaaaccaaacagcgcgagatggtctactgcctggagcag2400 ggcctcatccaccgctgtgccagacagtgcgtcgtggccttgtccatctgcagcgtggag2460 atgcctgacatcatcatcaaggcgctgcctgttctggtggtgaagctcacgcacatctca2520 gccacagccagcatggccgtcccactgctggagttcctgtccactctggccaggctgccg2580 cacctctacaggaactttgccgcggagcagtatgccagtgtgttcgccatctccctgccg2640 tacaccaacccctccaagtttaatcagtacatcgtgtgtctggcccatcacgtcatagcc2700 atgtggttcatcaggtgccgcctgcccttccggaaggattttgtccctttcatcactaag2760 ggcctgcggtccaatgtcctcttgtcttttgatgacacccccgagaaggacagcttcagg2820 gcccggagtactagtctcaacgagagacccaagaggatacagacgtccctcaccagtgcc2880 agcttggggtctgcagatgagaactccgtggcccaggctgacgatagcctgaaaaacctc2940 cacctggagctcacggaaacctgtctggacatgatggctcgatacgtcttctccaacttc3000 acggctgtcccgaagaggtctcctgtgggcgagttcctcctagcgggtggcaggaccaaa3060 acctggctggttgggaacaagcttgtcactgtgacgacaagcgtgggaaccgggacccgg3120 tcgttactaggcctggactcgggggagctgcagtccggcccggagtcgagctccagcccc3180 ggggtgcatgtgagacagaccaaggaggcgccggccaagctggagtcccaggctgggcag3240 caggtgtcccgtggggcccgggatcgggtccgttccatgtcggggggccatggtcttcga3300 gttggcgccctggacgtgccggcctcccagttcctgggcagtgccacttctccaggacca3360 cggactgcaccagccgcgaaacctgagaaggcctcagctggcacccgggttcctgtgcag3420 gagaagacgaacctggcggcctatgtgcccctgctgacccagggctgggcggagatcctg3480 gtccggaggcccacagggaacaccagctggctgatgagcctggagaacccgctcagccct3540 ttctcctcggacatcaacaacatgcccctgcaggagctgtctaacgccctcatggcggct3600 gagcgcttcaaggagcaccgggacacagccctgtacaagtcactgtcggtgccggcagcc3660 agcacggccaaaccccctcctctgcctcgctccaacacagtggcctctttctcctccctg3720 taccagtccagctgccaaggacagctgcacaggagcgtttcctgggcaga~ctccgccgtg3780 g gtcatggaggagggaagtccgggcgaggttcctgtgctggtggagcccccagggttggag3840 gacgttgaggcagcgctaggcatggacaggcgcacggatgcctacagcaggtcgtcctca3900 gtctccagccaggaggagaagtcgctccacgcggaggagctggttggcaggggcatcccc3960 atcgagcgagtcgtctcctcggagggtggccggccctctgtggacctctccttccagccc4020 tcgcagcccctgagcaagtccagctcctctcccgagctgcagactctgcaggacatcctc4080 ggggaccctggggacaaggccgacgtgggccggctgagccctgaggttaaggcccggtca4140 cagtcagggaccctggacggggaaagtgctgcctggtcggcctcgggcgaagacagtcgg4200 ggccagcccgagggtcccttgccttccagctccccccgctcgcccagtggcctccggccc4260 cgaggttacaccatctccgactcggccccatcacgcaggggcaagagagtagagagggac4320 gccttaaagagcagagccacagcctccaatgcagagaaagtgccaggcatcaaccccagt4380 ttcgtgttcctgcagctctaccattcccccttctttggcgacgagtcaaacaagccaatc4440 ctgctgcccaatgagtcacagtcctttgagcggtcggtgcagctcctcgaccagatccca4500 tcatacgacacccacaagatcgccgtcctgtatgttggagaaggccagagcaacagcgag4560 ctcgccatcctgtccaatgagcatggctcctacaggtacacggagttcctgacgggcctg4620 ggccggctcatcgagctgaaggactgccagccggacaaggtgtacctgggaggcctggac4680' gtgtgtggtgaggacggccagttcacctactgctggcacgatgacatcatgcaagccgtc4740 ttccacatcgccaccctgatgcccaccaaggacgtggacaagcaccgctgcgacaagaag4800 cgccacctgggcaacgactttgtgtccattgtctacaatgactccggtgaggacttcaag4860 cttggcaccatcaagggccagttcaactttgtccacgtgatcgtcaccccgctggactac4920 gagtgcaacctggtgtccctgcagtgcaggaaagacatggagggccttgtggacaccagc4980 gtggccaagatcgtgtctgaccgcaacctgcccttcgtggcccgccagatggccctgcac5040 gcaaatatggcctcacaggtgcatcatagccgctccaaccccaccgatatctacccctcc5100 aagtggattgcccggctccgccacatcaagcggctccgccagcggatctgcgaggaagcc5160 gcctactccaaccccagcctacctctggtgcaccctccgtcccatagcaaagcccctgca5220 cagactccagccgagcccacacctggctatgaggtgggccagcggaagcgcctcatctcc5280 tcggtggaggacttcaccgagtttgtgtgaggccggggccctccctcctgcactggcctt5340 ggacggtattgcctgtcagtgaaataaataaagtcctgaccccagtgcacagacatagag5400 gcacagattgc 5411 <210> 6 <211> 5543 <212> DNA
<213> Homo Sapiens <400>

ggtgcgtcctggtccaccatggccaaaccaacaagcaaagattcaggcttgaaggagaag60 tttaagattctgttgggactgggaacaccgaggccaaatcccaggtctgcagagggtaaa120 cagacggagtttatcatcaccgcggaaatactgagagaactgagcatggaatgtggcctc180 aacaatcgcatccggatgatagggcagatttgtgaagtcgcaaaaaccaagaaatttgaa240 gagcacgcagtggaagcactctggaaggcggtcgcggatctgttgcagccggagcggccg300 ctggaggcccggcacgcggtgctggctctgctgaaggccatcgtgcaggggcagggcgag360 cgtttgggggtcctcagagccctcttctttaaggtcatcaaggattacccttccaacgaa420 gaccttcacgaaaggctggaggttttcaaggccctcacagacaatgggagacacatcacc480 tacttggaggaagagctggctgactttgtcctgcagtggatggatgttggcttgtcctcg540 gaattccttctggtgctggtgaacttggtcaaattcaatagctgttacctcgacgagtac600 atcgcaaggatggttcagatgatctgtctgctgtgcgtccggaccgcgtcctctgtggac660 atagaggtctccctgcaggtgctggacgccgtggtctgctacaactgcctgccggctgag720 agcctcccgctgttcatcgttaccctctgtcgcaccatcaacgtcaaggagctctgcgag780 ccttgctggaagctgatgcggaacctccttggcacccacctgggccacagcgccatctac840 aacatgtgccacctcatggaggacagagcctacatggaggacgcgcccctgctgagagga900 gccgtgttttttgtgggcatggctctctggggagcccaccggctctattctctcaggaac960 tcgccgacatctgtgttgccatcattttaccaggccatggcatgtccgaacgaggtggtg1020 tcctatgagatcgtcctgtccatcaccaggctcatcaagaagtataggaaggagctccag1080 gtggtggcgtgggacattctgctgaacatcatcgaacggctccttcaacagctccagacc1140 ttggacagcccggagctcaggaccatcgtccatgacctgttgaccacggtggaggagctg1200 tgtgaccagaacgagttccacgggtctcaggagagatactttgaactggtggagagatgt1260 gcggaccagaggcctgagtcctccctcctgaacctgatctcctatagagcgcagtccatc1320 cacccggccaaggacggctggattcagaacctgcaggcgctgatggagagattcttcagg1380 agcgagtcccgaggcgccgtgcgcatcaaggtgctggacgtgctgtcctttgtgctgctc1440 atcaacaggcagttctatgaggaggagctgattaactcagtggtcatctcgcagctctcc1500 cacatccccgaggataaagaccaccaggtccgaaagctggccacccagttgctggtggac1560 ctggcagagggctgccacacacaccacttcaacagcctgctggacatcatcgagaaggtg1620 atggcccgctccctctccccacccccggagctggaagaaagggatgtggccgcatactcg1680 gcctccttggaggatgtgaagacagccgtcctggggcttctggtcatccttcagaccaag1740 ctgtacaccctgcctgcaagccacgccacgcgtgtgtatgagatgctggtcagccacatt1800 cagctccactacaagcacagctacaccctgccaatcgcgagcagcatccggctgcaggcc1860 tttgacttcctgttgctgctgcgggccgactcactgcaccgcctgggcctgcccaacaag1920 gatggagtcgtgcggttcagcccctactgcgtctgcgactacatggagccagagagaggc1980 tctgagaagaagaccagcggccccctttctcctcccacagggcctcctggcccggcgcct2040 gcaggccccgccgtgcggctggggtccgtgccctactccctgctcttccgcgtcctgctg2100 cagtgcttgaagcaggagtctgactggaaggtgctgaagctggttctgggcaggctgcct2160 gagtccctgcgctataaagtgctcatctttacttccccttgcagtgtggaccagctgtgc2220 tctgctctctgctccatgctttcaggcccaaagacactggagcggctccgaggcgcccca2280 gaaggcttctccagaactgacttgcacctggccgtggttccagtgctgacagcattaatc2340 tcttaccataactacctggacaaaaccaaacagcgcgagatggtctactgcctggagcag2400 ggcctcatccaccgctgtgccagacagtgcgtcgtggccttgtccatctgcagcgtggag2460 atgcctgacatcatcatcaaggcgctgcctgttctggtggtgaagctcacgcacatctca2520 gccacagccagcatggccgtcccactgctggagttcctgtccactctggccaggctgccg2580 cacctctacaggaactttgccgcggagcagtatgccagtgtgttcgccatctccctgccg2640 tacaccaacccctccaagtttaatcagtacatcgtgtgtctggcccatcacgtcatagcc2700 atgtggttcatcaggtgccgcctgcccttccggaaggattttgtccctttcatcactaag2760 ggcctgcggtccaatgtcctcttgtcttttgatgacacccccgagaaggacagcttcagg2820 gcccggagtactagtctcaacgagagacccaagagtctgaggatagccagaccccccaaa2880 caaggcttgaataactctccacccgtgaaagaattcaaggagagctctgcagccgaggcc2940 ttccggtgccgcagcatcagtgtgtctgaacatgtggtccgcagcaggatacagacgtcc3000 ctcaccagtgccagcttggggtctgcagatgagaactccgtggcccaggctgacgatagc3060 ctgaaaaacctccacctggagctcacggaaacctgtctggacatgatggctcgatacgtc3120 ttctccaacttcacggctgtcccgaagaggtctcctgtgggcgagttcctcctagcgggt3180 ggcaggaccaaaacctggctggttgggaacaagcttgtcactgtgacgacaagcgtggga3240 accgggacccggtcgttactaggcctggactcgggggagctgcagtccggcccggagtcg3300 agctccagccccggggtgcatgtgagacagaccaaggaggcgccggccaagctggagtcc3360 caggctgggcagcaggtgtcccgtggggcccgggatcgggtccgttccatgtcggggggc3420 catggtcttcgagttggcgccctggacgtgccggcctcccagttcctgggcagtgccact3480 tctccaggaccacggactgcaccagccgcgaaacctgagaaggcctcagctggcacccgg3540 gttcctgtgcaggagaagacgaacctggcggcctatgtgcccctgctgacccagggctgg3600 gcggagatcctggtccggaggcccacagggaacaccagctggctgatgagcctggagaac3660 ccgctcagccctttctcctcggacatcaacaacatgcccctgcaggagctgtctaacgcc3720 ctcatggcggctgagcgcttcaaggagcaccgggacacagccctgtacaagtcactgtcg3780 gtgccggcagccagcacggccaaaccccctcctctgcctcgctccaacacagtggcctct3840 ttctcctccctgtaccagtccagctgccaaggacagctgcacaggagcgtttcctgggca3900 gactccgccgtggtcatggaggagggaagtccgggcgaggttcctgtgctggtggagccc3960 ccagggttggaggacgttgaggcagcgctaggcatggacaggcgcacggatgcctacagc4020 aggtcgtcctcagtctccagccaggaggagaagtcgctccacgcggaggagctggttggc4080 aggggcatccccatcgagcgagtcgtctcctcggagggtggccggccctctgtggacctc4140 tccttccagccctcgcagcccctgagcaagtccagctcctctcccgagctgcagactctg4200 caggacatcctcggggaccctggggacaaggccgacgtgggccggctgagccctgaggtt4260 aaggcccggtcacagtcagggaccctggacggggaaagtgctgcctggtcggcctcgggc4320 gaagacagtcggggccagcccgagggtcccttgccttccagctccccccgctcgcccagt4380 ggcctccggccccgaggttacaccatctccgactcggccccatcacgcaggggcaagaga4440 gtagagagggacgccttaaagagcagagccacagcctccaatgcagagaaagtgccaggc4500 atcaaccccagtttcgtgttcctgcagctctaccattcccccttctttggcgacgagtca4560 aacaagccaatcctgctgcccaatgagtcacagtcctttgagcggtcggtgcagctcctc4620 gaccagatcccatcatacgacacccacaagatcgccgtcctgtatgttggagaaggccag4680 agcaacagcgagctcgccatcctgtccaatgagcatggctcctacaggtacacggagttc4740 ctgacgggcctgggccggctcatcgagctgaaggactgccagccggacaaggtgtacctg4800 ggaggcctggacgtgtgtggtgaggacggccagttcacctactgctggcacgatgacatc4860 atgcaagccgtcttccacatcgccaccctgatgcccaccaaggacgtggacaagcaccgc4920 tgcgacaagaagcgccacctgggcaacgactttgtgtccattgtctacaatgactccggt4980 gaggacttcaagcttggcaccatcaagggccagttcaactttgtccacgtgatcgtcacc5040 ccgctggactacgagtgcaacctggtgtccctgcagtgcaggaaagacatggagggcctt5100 gtggacaccagcgtggccaagatcgtgtctgaccgcaacctgcccttcgtggcccgccag5160 atggccctgcacgcaaatatggcctcacaggtgcatcatagccgctccaaccccaccgat5220 atctacccctccaagtggattgcccggctccgccacatcaagcggctccgccagcggatc5280 tgcgaggaagccgcctactccaaccccagcctacctctggtgcaccctccgtcccatagc5340 aaagcccctgcacagactccagccgagcccacacctggctatgaggtgggccagcggaag5400 cgcctcatctcctcggtggaggacttcaccgagtttgtgtgaggccggggccctccctcc5460 tgcactggccttggacggtattgcctgtcagtgaaataaataaagtcctgaccccagtgc5520 acagacatag aggcacagat tgc 5543 <210>

<211>

<212>
DNA

<213>
Homo sapiens <400>

gcgcagttcgctgcgtgcagcgacgtggcggcggggccggcaccgggcagcggaagtggc60 tccggcggtgggacttgagtgtttgtgttttggttcgtgaaggagccggcggctggcctt120 aggggaggaggcagagggaggaggaggaggaagaattagtcggaactccagcgccggcgg180 cggcggcggcggcggaggaggagaaaggaaagaggaagggggagcggcgagaggcggaga240 cggagcccgacaggggcggcaccacggcacgagccccgcacagtccagtgtgaggggagc300 ggcgctaagagcaggcgacgccgccgccgccaccaccaccgccatagatacactctcatc360 ctacgggccacgcctgggccttgctgccaggaagcttcggccccgcagctcggcttgctg420 cggtctcaggtttctttacctccagaaagaagaatattggccccttgaattctggaagtt480 cattgaagagtctgaaattagggacttatttcaaatttggacatggctagtcgaggcgca540 acaagacccaacgggccaaatacgggaaataaaatatgccagttcaaactagtacttctg600 ggagagtccgctgttggcaaatcaagcctagtgcttcgttttgtgaaaggccaatttcat660 gaatttcaagagagtaccattggggctgcttttctaacccaaactgtatgtcttgatgac720 actacagtaaagtttgaaatatgggatacagctggtcaagaacgataccatagcctagca780 ccaatgtactacagaggagcacaagcagccatagttgtatatgatatcacaaatgaggag840 tcctttgcaagagcaaaaaattgggttaaagaacttcagaggcaagcaagtcctaacatt900 gtaatagctttatcgggaaacaaggccgacctagcaaataaaagagcagtagatttccag960 gaagcacagtcctatgcagatgacaatagtttattattcatggagacatccgctaaaaca1020 tcaatgaatgtaaatgaaatattcatggcaatagctaaaaaattgccaaagaatgaacca1080 caaaatccaggagcaaattctgccagaggaagaggagtagaccttaccgaacccacacaa1140 ccaaccaggaatcagtgttgtagtaactaaacctctagtttgaactagctggaatagtct1200 tctgcttcctaaatgttaataacaatggaattggagcatttaaccagcccagtatgactt1260 ccaaaagaagagacttatgatagagtcaagtttctaatacagaattattttaagtgtttt1320 gaacttaatttttaataacatgcatgggtccctctcactaatgtttcaacaatagggaaa1380 aatgagaactatgtggacacttgtttcattggaaggttagggggaataatttctcatcac1440 taggaatatagacaaatgactgtctgggcccacacagttaaccagcccatttctccacac1500 tggtacagtagtcacctgtggaaaaaaaaaattggaacttactaatttgggcttttcaaa1560 aacattctttgtttagaaggagattctaaagttatttatgatgcttagccatagtattca1620 ggcaaatgttcatttctcctggtacctgtatttaaaatgtacattccacattttaataaa1680 ttaaccacaagaaaataatcccacatatacaaggtcaggggtggggaagagtattaatgg1740 tatcttaattatacccagtctggttttttttttttaaatggggtaaaaatcaaatgcaac1800 cccatcttgttttaggaattttgagaactaataaatgcaccttaatggtcagtgttcctt1860 tcaaacatgtgagttctttaacaaaaatgaaataaaccaggtgtctgtgatttctaatta1920 atcaccgctggccattacacaggttttgttgtttggggtggggagggggcttttgttccc1980 ttttgacataatatagtcaatgcactaacaattatgtatattcaaacttgattattttaa2040 attcgatcttcagctgtactgtaaatagggtactgcattgtagtctccatatctgtatta2100 cttttctgtaatatttaagagttgcttaaaagcatacaaaatgtactgttactaaaacag2160 ctaattatttctctctccccctttgacaggaaggggcttcagttgttcctccatggctag2220 aaccataataaacaatgtacccgtaatttgtaacataaagtattggaatatgttagtaac2280 aatcttgcagccttcctttccaaagttcattttattttgatcagttcagtatattgcact2340 aattattttaggtattttcattatatgaaagctaccatgtgtcagagatgatttaatcta2400 tttaagtgttggactgctaggagaacttgtacatttatgataatgcagaattaggaaaac2460 ggttcaccagtgtttagttttatattgaggtgctcaggttggaataaagtggtataaaaa2520 gc 2522 <210>

<211>

<212>
DNA

<213> Sapiens Homo <400>

gcggctgagtcttcccagggtcagggtcaggcgctttgctgagtccctttgtggccgcca60 tggacaattccgggaaggaagcggaggcgatggcgctgttggccgaggcggagcgcaaag120 tgaagaactcgcagtccttcttctctggcctctttggaggctcatccaaaatagaggaag180 catgcgaaatctacgccagagcagcaaacatgttcaaaatggccaaaaactggagtgctg240 ctggaaacgcgttctgccaggctgcacagctgcacctgcagctccagagcaagcacgacg300 cagccacctgctttgtggacgctggcaacgcattcaagaaagccgacccccaagaggcca360 ttaactgtttgatgcgagcaatcgagatctacacagacatgggccgattcacgattgcgg420 ccaagcaccacatctccattgctgagatctatgagacagagttggtggacatcgagaagg480 ccattgcccactacgagcagtctgcagactactacaaaggcgaggagtccaacagctcag540 ccaacaagtgtctgctgaaggtggctggttacgctgcgctgctggagcagtatcagaagg600 ccattgacatctacgaacaggtggggaccaatgccatggacagccccctcctcaagtaca660 gcgccaaagactacttcttcaaggcggccctctgccacttctgcatcgacatgctcaacg720 ccaagctggctgtccaaaagtatgaggagctgttcccagctttctctgattcccgggaat780 gcaagttgatgaaaaaattgctagaggcccacgaggagcagaatgtggacagctacaccg840 agtcggtgaaggaatacgactccatctcccggctggaccagtggctcaccaccatgctgc900 tgcgcatcaagaagaccatccagggcgatgaggaggacctgcgctaagccccacccagcc960 ccccagtgcccgtcttcctgtcccatctgctcagagagagccaagctctaaagcacatgt1020 agccgctgagacctgctgtttctgctgggggcaggctcctcttcccccagccccgggagc1080 ctcccccagcttcctgcagccccgacctctcaggttagaccctgggccctggagcttagg1140 ggattctccccaccccagccccacacctgctccttccctaatgctttgaggttttcttgg1200 ttggaagctgcagctggcccaagaaagaaaataaaaaacaacacttttgc 1250 <210>

<211>

<212>
DNA

<213> sapiens Homo <400>

atctagcgcccccgtcaggacgtgcgaaaagcgacggcgcagcacggtgcggcgcagctc60 ctgctcgcctttcccttcgctgggcgagaggtgtctatggggcacccgctgccgccgccg120 ctaccgccaccgccaccgccaccgccgccgagtgctgtctctatggcgaggaggaggagg180 aggagcgcgagctcagcgatacaagtacataaataaaggataaaatattttatgaaacaa240 atcttcaatcaagtataacattttgatgcttggcatctagactcccttgtgccctcacta300 tgccagcggcaactgtagatcatagccaaagaatttgtgaagtttgggcttgcaacttgg360 atgaagagatgaagaaaattcgtcaagttatccgaaaatataattacgttgctatggaca420 ccgagtttccaggtgtggttgcaagacccattggagaattcaggagcaatgctgactatc480 aataccaactattgcggtgtaatgtagacttgttaaagataattcagctaggactgacat540 ttatgaatgagcaaggagaataccctccaggaacttcaacttggcagtttaattttaaat600 ttaatttgacggaggacatgtatgcccaggactctatagagctactaacaacatctggta660 tccagtttaaaaaacatgaggaggaaggaattgaaacccagtactttgcagaacttctta720 tgacttctggagtggtcctctgtgaaggggtcaaatggttgtcatttcatagcggttacg780 actttggctacttaatcaaaatcctaaccaactctaacttgcctgaagaagaacttgact840 tctttgagatccttcgattgttttttcctgtcatttatgatgtgaagtacctcatgaaga900 gctgcaaaaatctcaaaggtggattacaggaggtggcagaacagttagagctggaacgga960 taggaccacaacatcaggcaggatctgattcattgctcacaggaatggcctttttcaaaa1020 tgagagaagtatgaagacatcactgcctttttctcagttggttgttaggttgagaacatt1080 aaaaatcttgtggccaaagattttgggcaacaagtacctactaagtaaagatataattag1140 agataccatatgagtcacatccaccacacttaaaagtattcaaaaataagtcatcttgaa1200 atgtagttcagaaggaactgggagaacatgttcatcatagaaccaacaattttaaaacat1260 aaactacctgagaagtcatgtaggtccaaccatattatttctcaggtgagaaaacaggct1320 aataacttccatgattaaacacattactagtgggagaactccagagttcttttctgactc1380 ccattggtgctccttctacaaggcaaggaatctttatattaggtttattctagcatgacc1440 ccttttaaggtttaaactggtgataaatatattatttgctcatgtcattcttcagtgctt1500 tgaagattttatagagaagaactcaggctcttttactgcattgagtcttaaaaggggggt1560 tgaattccgaagggatcaaataaatccaacgtagtagttgcatcagaaaccatattagga1620 aaaccctccttaaacggcaaaaggcagagatcagttccttgagtataaagtgttagggat1680 ggaagaatgaaactaaatgaacccattatctactcctaagtaattaagtgatgtgcacag1740 atacacctagctataggtaaacaggaaaattggttgtgcaaaaaacaagtgaggtttttc1800 ttgactataagttttcccttttggaaaaattcgctgtggatttgagtatattttctctta1860 gacattaaattgagactgagaatttaaaactttttgtagcaatgtattgataatagaaag1920 cattaaagctgttttgctaagtaaaaaaaaaaaaaaaaa 1959 <210>

<211>

<212>
DNA

<213> sapiens Homo <400>

atggcgaacgacgagcagatcctggtcctcgatccgcccacagacctcaaattcaaaggc60 cccttcacagatgtagtcactacaaatcttaaattgcgaaatccatcggatagaaaagtg120 tgtttcaaagtgaagactacagcacctcgccggtactgtgtgaggcccaacagtggaatt180 attgacccagggtcaactgtgactgtttcagtaatgctacagccctttgactatgatccg240 aatgaaaagagtaaacacaagtttatggtacagacaatttttgctccaccaaacacttca300 gatatggaagctgtgtggaaagaggcaaaacctgatgaattaatggattccaaattgaga360 tgcgtatttgaaatgcccaatgaaaatgataaattgaatgatatggaacctagcaaagct420 gttccactgaatgcatctaagcaagatggacctatgccaaaaccacacagtgtttcactt480 aatgataccgaaacaaggaaactaatggaagagtgtaaaagacttcagggagaaatgatg540 aagctatcagaagaaaatcggcacctgagagatgaaggtttaaggctcagaaaggtagca600 cattcggataaacctggatcaacctcaactgcatccttcagagataatgtcaccagtcct660 cttccttcacttcttgttgtaattgcagccattttcattggattctttctagggaaattc720 atcttgtag <210>

<211>

<212>
DNA

<213> sapiens Homo <400>

gcgcgcccacccggtagaggacccccgcccgtgccccgaccggtccccgcctttttgtaa60 aacttaaagcgggcgcagcattaacgcttcccgccccggtgacctctcaggggtctcccc120 gccaaaggtgctccgccgctaaggaacatggcgaaggtggagcaggtcctgagcctcgag180 ccgcagcacgagctcaaattccgaggtcccttcaccgatgttgtcaccaccaacctaaag240 cttggcaacccgacagaccgaaatgtgtgttttaaggtgaagactacagcaccacgtagg300 tactgtgtgaggcccaacagcggaatcatcgatgcaggggcctcaattaatgtatctgtg360 atgttacagcctttcgattatgatcccaatgagaaaagtaaacacaagtttatggttcag420 tctatgtttgctccaactgacacttcagatatggaagcagtatggaaggaggcaaaaccg480 gaagaccttatggattcaaaacttagatgtgtgtttgaattgccagcagagaatgataaa540 ccacatgatgtagaaataaataaaattatatccacaactgcatcaaagacagaaacacca600 atagtgtctaagtctctgagttcttctttggatgacaccgaagttaagaaggttatggaa660 gaatgtaagaggctgcaaggtgaagttcagaggctacgggaggagaacaagcagttcaag720 gaagaagatggactgcggatgaggaagacagtgcagagcaacagccccatttcagcatta780 gccccaactgggaaggaagaaggccttagcacccggctcttggctctggtggttttgttc840 tttatcgttggtgtaattattgggaagattgccttgtagaggtagcatgcacaggatggt900 aaattggattggtggatccaccatatcatgggatttaaatttatcataaccatgtgtaaa960 aagaaattaatgtatgatgacatctcacaggtcttgcctttaaattacccctccctgcac1020 acacatacacagatacacacacacaaatataatgtaacgatcttttagaaagttaaaaat1080 gtatagtaactgattgagggggaaaagaatgatctttattaatgacaagggaaaccatga1140 gtaatgccacaatggcatattgtaaatgtcattttaaacattggtaggccttggtacatg1200 atgctggattacctctcttaaaatgacacccttcctcgcctgttggtgctggcccttggg1260 gagctggagcccagcatgctggggagtgcggtcagctccacacagtagtccccacgtggc1320 ccactcccggcccaggctgctttccgtgtcttcagttctgtccaagccatcagctccttg1380 ggactgatgaacagagtcagaagcccaaaggaattgcactgtggcagcatcagacgtact1440 cgtcataagtgagaggcgtgtgttgactgattgacccagcgctttggaaataaatggcag1500 tgctttgttcacttaaagggaccaagctaaatttgtattggttcatgtagtgaagtcaaa1560 ctgttattcagagatgtttaatgcatatttaacttatttaatgtatttcatctcatgttt1620 tcttattgtcacaagagtacagttaatgctgcgtgctgctgaactctgttgggtgaactg1680 gtattgctgctggagggctgtgggctcctctgtctctggagagtctggtcatgtggaggt1740 ggggtttattgggatgctggagaagagctgccaggaagtgttttttctgggtcagtaaat1800 aacaactgtcataggcagggaaattctcagtagtgacagtcaactctaggttaccttttt1860 taatgaagagtagtcagtcttctagattgttcttataccacctctcaaccattactcaca1920 cttccagcgcccaggtccaagtttgagcctgacctccccttggggacctagcctggagtc1980 aggacaaatggatcgggctgcaaagggttagaagcgagggcaccagcagttgtgggtggg2040 gagcaagggaagagagaaactcttcagcgaatccttctagtactagttgagagtttgact2100 gtgaattaattttatgccataaaagaccaacccagttctgtttgactatgtagcatcttg2160 aaaagaaaaattataataaagccccaaaattaaga 2195 <210>

<211>

<212>
DNA

<213> sapiens Homo <400>

ccgagccccagcccggccgccatggacgacaaggcgttcaccaaggagctggaccagtgg60 gtcgagcagctgaacgagtgtaagcagctgaacgagaaccaagtgcggacgctgtgcgag120 aaggcaaaggaaattttaacaaaagaatcaaatgtgcaagaggttcgttgccctgttact180 gtctgtggagatgtgcatggtcaatttcatgatcttatggaactctttagaattggtgga240 aaatcaccggatacaaactacttattcatgggtgactatgtagacagaggatattattca300 gtggagactgtgactcttcttgtagcattaaaggtgcgttatccagaacgcattacaata360 ttgagaggaaatcacgaaagccgacaaattacccaagtatatggcttttatgatgaatgt420 ctgcgaaagtatgggaatgccaacgtttggaaatattttacagatctctttgattatctt480 ccacttacagctttagtagatggacagatattctgcctccatggtggcctctctccatcc540 atagacacactggatcatataagagccctggatcgtttacaggaagttccacatgagggc600 ccaatgtgtgatctgttatggtcagatccagatgatcgtggtggatggggtatttcacca660 cgtggtgctggctacacatttggacaagacatttctgaaacctttaaccatgccaatggt720 ctcacactggtttctcgtgcccaccagcttgtaatggagggatacaattggtgtcatgat780 cggaatgtggttaccattttcagtgcacccaattactgttatcgttgtgggaaccaggct840 gctatcatggaattagatgacactttaaaatattccttccttcaatttgacccggcgcct900 cgtcgtggtgagcctcatgttacacggcgcaccccagactacttcctataaatttctcct960 gggaaacctgcctttgtatgtggaagtatacctggctttttaaaatatatgtatttaaaa1020 acaaaaagcaacagtaatctatgtgtttctgtaacaaattgggatctgtcttggcattaa1080 accacatcatggaccaaatgtgccatactaatgatgagcatttagcacaatttgagactg1140 aaatttagtacactatgttctagataggtcagtctaacagtttgcctgctgtatttatag1200 taaccattttcctttggactgttcaagcaaaaaaggtaactaactgcttcatctcctttt1260 gcgcttatttggaaattttagttatagtgtttaactggcatggattaatagagttggagt1320 tttatttttaagaaaaattcacaagctaacttccactaatccattatcctttattttatt1380 gaaatgtataattaacttaactgaagaaaaggttcttcttgggagtatgttgtcataaca1440 tttaaagagatttcccttcatttaaactaaattactgttttatgttgatctgcatatttc1500 tgtatatttgtcatgacagtgcttgcatcctatttggtgtg 1541 <210>

<211>

<212>
DNA

<213> Sapiens Homo <400>

agagagccgagctctggagcctcagcgagcggaggaggaggcgcagggccgacggccgag60 tactgcggtgagagccagcgggccagcgccagcctcaacagccgccagaagtacacgagg120 aaccggcggcggcgtgtgcgtgtaggcccgtgtgcgggcggcggcgcgggaggagcgcgg180 agcggcagccggctggggcgggtggcatcatggacgagaaggtgttcaccaaggagctgg240 accagtggatcgagcagctgaacgagtgcaagcagctgtccgagtcccaggtcaagagcc300 tctgcgagaaggctaaagaaatcctgacaaaagaatccaacgtgcaagaggttcgatgtc360 cagttactgtctgtggagatgtgcatgggcaatttcatgatctcatggaactgtttagaa420 ttggtggcaaatcaccagatacaaattacttgtttatgggagattatgttgacagaggat480 attattcagttgaaacagttacactgcttgtagctcttaaggttcgttaccgtgaacgca540 tcaccattcttcgagggaatcatgagagcagacagatcacacaagtttatggtttctatg600 atgaatgtttaagaaaatatggaaatgcaaatgtttggaaatattttacagatctttttg660 actatcttcctctcactgccttggtggatgggcagatcttctgtctacatggtggtctct720 cgccatctatagatacactggatcatatcagagcacttgatcgcctacaagaagttcccc780 atgagggtccaatgtgtgacttgctgtggtcagatccagatgaccgtggtggttggggta840 tatctcctcgaggagctggttacacctttgggcaagatatttctgagacatttaatcatg900 ccaatggcctcacgttggtgtctagagctcaccagctagtgatggagggatataactggt960 gccatgaccggaatgtagtaacgattttcagtgctccaaactattgttatcgttgtggta1020 accaagctgcaatcatggaacttgacgatactctaaaatactctttcttgcagtttgacc1080 cagcacctcgtagaggcgagccacatgttactcgtcgtaccccagactacttcctgtaat1140 gaaattttaaacttgtacagtattgccatgaaccatatatcgacctaatggaaatgggaa1200 gagcaacagtaactccaaagtgtcagaaaatagttaacattcaaaaaacttgttttcaca1260 tggaccaaaagatgtgccatataaaaatacaaagcctcttgtcatcaacagccgtgacca1320 ctttagaatgaaccagttcattgcatgctgaagcgacattgttggtcaagaaaccagttt1380 ctggcatagcgctatttgtagttacttttgctttctctgagagactgcagataataagat1440 gtaaacattaacacctcgtgaatacaatttaacttccatttagctatagctttactcagc1500 atgactgtagataaggatagcagcaaacaatcattggagcttaatgaacatttttaaaaa1560 taattaccaaggcctcccttctacttgtgagttttgaaattgttctttttattttcaggg1620 ataccgtttaatttaattatatgatttgtctgcactcagtttattccctactcaaatctc1680 agccccatgttgttctttgttattgtcagaacctggtgagttgttttgaacagaactgtt1740 ttttccccttcctgtaagacgatgtgactgcacaagagcactgcagtgtttttcataata1800 aacttgtgaactaagaactgagaaggtcaaattttaattgtatcaatgggcaagactggt1860 gctgtttattaaaaaagttaaatcaattgagtaaattttagaatttgtagacttgtaggt1920 aaaataaaaatcaagggcactacataacctctctggtaactccttgacattcttcagatt1980 aacttcaggatttatttgtatttcacatattacaatttgtcacattgttggtgtgcactt2040 tgtgggttcttcctgcatattaacttgtttgtaagaaaggaaatctgtgctgcttcagta2100 agacttaattgtaaaaccatataacttgagatttaagtctttgggttgtgttttaataaa2160 acagcatgttttcaggtagag 2181 <210>

<211>

<212>
DNA

<213>
Homo sapiens <400>

ggaagaaaacctgaaaaagaccccaaagaagaatatgaaaatggtaactggagccgtagc60 gtcggtgctggaagacgaggccacagacacttctgatagtgaaggaagctgtggatcgga120 aaaggaccacttttattctgatgatgacgcaatagaagctgacagtgagggtgatgctga180 gccctgtgacaaagaaaatgaaaatgatggagaatcaagtgttgggactaatatgggctg240 ggcagatgctatggctaaagtcctcaacaagaaaactcctgaaagtaaacctactattct300 ggtcaaaaataagaagctggaaaaggaaaaagaaaagttaaagcaagaaagactagagaa360 aataaaacagcgtgataagaggctggagtgggaaatgatgtgcagagtaaagccagatgt420 tgtccaagacaaagagacagagagaaatcttcagagaattgcaacaaggggtgtggtgca480 attatttaatgctgttcagaaacatcaaaagaatgttgatgaaaaggttaaggaagctgg540 aagttctatgagaaagcgtgctaagttgatatcaactgtttccaagaaagatttcatcag600 tgttttgagagggatggatggaagtacaaatgagactgcttcaagcaggaagaaaccaaa660 agccaaacagactgaagtgaaatcagaagaaggcccaggttggacgatcctacgtgatga720 tttcatgatgggagcatctatgaaagactgggacaaggaaagtgatgggccagatgacag780 cagaccagaatctgcaagtgactctgatacataaagcatcataggaaatacaattgcagt840 cgttttattttttctagaaaaatatgtcatcctctgatagttggggaattataaggatac900 catttgtaagaaagccaaaagacttttgccagatttcatatttccccttttcatgtacac960 tttatatatacttcattaaaattatattttaaacccttgtataattttaagcattgttcc1020 tcagaacatttgtaaaaggatatatttctgcttgaccagcgagatgtgcattttgccagg1080 atcatattggtcatgtctattggtgtattatttcagtatcaccaatgttttcagaaatac1140 agtactaattcatcattaaactctttgaagttaatatttttctgccttctaacttataga1200 ctcaactatgtatctgtagtttttgggaatggttggtgttttttgctttgtgttgggaag1260 ttattgagaaaacctatataataaaatttaaaattatagtttttcaaa 1308 <210>

<211>

<212>
DNA

<213> Sapiens Homo <400>

gaattcaaagtggagtaccgcaaacttgatatggaaaataaaaagaaagacaaggacaaa60 tcagatgatagaatggcacgacctagtggtcgatcgggacacaacactcgaggaactggg120 tcttcatcgtctggagttttaatggttggacctaactttagagttggaaaaaaaattgga180 tgtggcaattttggagaattacgattagggaaaaatttatacacaaatgaatatgtggca240 attaagttggagcccatgaaatcaagagcaccacagctacatttggaatacagattctat300 aagcagttaggatctggagatggtatacctcaagtttactatttcggcccttgtggtaaa360 tacaatgctatggtgctggaactgctgggacctagtttggaagacttgtttgacttgtgt420 gacagaacattttctcttaaaacagttctcatgatagctatacaactgatttctcgcatg480 gaatatgtccattcaaagaacttgatatacagagatgtaaaacctgagaacttcttaata540 ggacgaccaggaaacaaaacccagcaagttattcacattatagattttggtttggcaaag600 gaatatattgatccggagacaaagaaacacataccatacagagaacacaagagccttaca660 ggaacagctagatatatgagcataaacacacatttaggaaaagaacaaagtagaagagac720 gatttagaagctttaggtcatatgttcatgtattttctgagaggcagtcttccttggcaa780 ggcttaaaggctgacacattaaaggagaggtatcagaaaattggagatacaaaacgggct840 acaccaatagaagtgttatgtgaaaattttccagaaatggcaacatatcttcgttatgta900 agaaggctagatttttttgaaaaaccagactatgactacttaagaaagctttttactgac960 ttgtttgatcgaaaaggatatatgtttgattatgaatatgactggattggtaaacagttg1020 cctactccagtgggtgcagttcagcaagatcctgctttgtgatcaaacagagaagcacat1080 caacacagagataagatgcaacaatccaaaaaccagtcggcagaccacagggcagcttgg1140 gactcccagcaggcaaatccccaccatttgagagctcaccttgcagcagacagacatggt1200 ggctcggtacaggttgtaagttctacaaatggagagttaaacacagatgaccccaccgca1260 ggacgttcaaatgcacccatcacagcccctactgaagtagaagtgatggatgaaaccaag1320 tgctgctgttttttcaaacgaaggaaaaggaaaaccatacagcgccacaaatgactctgg1380 acacagacagatcctggggagttacttacatgttcatctgctgtcttgtgattaaaatca1440 tctctgtagtgaccacgtatattttcaaggactcactcttagaaacaaaaatgtcatact1500 ttcatacttcattttgtggttgtcttacattctttttctttttttttttttctctaattt1560 aacctttatggaagctttaaagttttgtcaaaacatgagtgctttgcccatcagtgaatg1620 gaatggaccaatgaggtggtatcaatgaatatagttccatagaacattttccagaagttc1680 ttctgttgtagaaagcagtacagtatcttaagtgtcaaccagttatatacctaatctggt1740 tttttataacttctgtaagagcataatcaaacaggaattttcttttctcagtggataata1800 caacagagaaaacagagttgcccaaatatttaaaagaagttattccttgagaagttcata1860 ttttgtgacatctgcattgatttcagtattactgatggtactgttattcataagtcatat1920 taacattctctccgtgaaatcatggtacagtcactgcccagaggtactgaggaaaaagca1980 atatgggttcggcagatggtggtggtaaaatgaatcttaaggagtgtggtaaatatgtgc2040 tccgcttttgttgcatcactatgtgaagtactgtgttgcagaagtggcaaaagcgcttat2100 ttttaaaaatgcaaaatatttgtacaatgtaactttatgcttccaaataataatgtatgt2160 tagacagcaagaaatgaatactttaaaaagtgatgtatgttggagttataaagaaataca2220 ctaaggagaggtagtaaatgtgaaccttgttgcagtgtataaggtggaagcctaaagaaa2280 tctcaccgaaacttactgctgaatgattacattctcccttaagcagaaaactttggatgt2340 gccatgcaatggtgtctgtgtaattattttgctctttgattaaaaaaaagacccccagca2400 ataaaaagtgggtcactctatgcc 2424 <210> 16 <211> 3653 <212> DNA
<213> Homo sapiens <400> 16 attgcaacac atgcagctgc ctggagagag ggagccggtg tcctacgtca gagccgccgc 60 cgccgcgagccgccgccggggaggagcagccgctgccgcccaggactgggcccttaggga120 ggaggaggcgagaagatggcggacgaccccagtgctgccgacaggaacgtggagatctgg180 aagatcaagaagctcattaagagcttggaggcggcccgcggcaatggcaccagcatgata240 tcattgatcattcctcccaaagaccagatttcacgagtggcaaaaatgttagcggatgag300 tttggaactgcatctaacattaagtcacgagtaaaccgcctttcagtcctgggagccatt360 acatctgtacaacaaagactcaaactttataacaaagtacctccaaatggtctggttgta420 tactgtggaacaattgtaacagaagaaggaaaggaaaagaaagtcaacattgactttgaa480 cctttcaaaccaattaatacgtcattgtatttgtgtgacaacaaattccatacagaggct540 cttacagcactactttcagatgatagcaagtttggattcattgtaatagatggtagtggt600 gcactttttggcacactccaaggaaacacaagagaagtcctgcacaaattcactgtggat660 ctcccaaagaaacacggtagaggaggtcagtcagccttgcgttttgcccgtttaagaatg720 gaaaagcgacataactatgttcggaaagtagcagagactgctgtgcagctgtttatttct780 ggggacaaagtgaatgtggctggtctagttttagctggatccgctgactttaaaactgaa840 ctaagtcaatctgatatgtttgatcagaggttacaatcaaaagttttaaaattagttgat900 atatcctatggtggtgaaaatggattcaaccaagctattgagttatctactgaagtcctc960 tccaacgtgaaattcattcaagagaagaaattaataggacgatactttgatgaaatcagc1020 caggacacgggcaagtactgttttggcgttgaagatacactaaaggctttggaaatggga1080 gctgtagaaattctaatagtctatgaaaatctggatataatgagatatgttcttcattgc1140 caaggcacagaagaggagaaaattctctatctaactccagagcaagaaaaggataaatct1200 catttcacagacaaagagaccggacaggaacatgagcttatcgagagcatgcccctgttg1260 gaatggtttgctaacaactataaaaaatttggagctacgttggaaattgtcacagataaa1320 tcacaagaagggtctcagtttgtgaaaggatttggtggaattggaggtatcttgcggtac1380 cgagtagatttccagggaatggaataccaaggaggagacgatgaattttttgaccttgat1440 gactactaggtagtcgacatgggtccggcaaaacgtgcctcaccctccagcatccaaccc1500 aaggagcatacccatggtggaatccaaacagatccctgccttacaattggaacatttcca1560 gaacttaatccatgagcattggatattgaaaagaaaaccgaaacaaaaccagacccagcc1620 ctacactttggtttgtcatggtgtcagcgcagcagcctacaactaagttcctaaacgcca1680 ctttggactaatttaaaaaagaatcccagtttttacttttactggatggtgaaattggtt1740 gctcttgtattttatgaaaaaaaatgatttttttaaccttcatacatagaagcaaaaata1800 ctttaactgctgtaaaccttcaaaagttaatagaagtgagatcatactggtttgtttctt1860 attttgattggagaaaaattaaattgctgcatttcgcagtgacccatttacatggcattc1920 tcagcttagactgcgtaagaagaaatatatgtggtgaaatgttggaaccatttctctctt1980 ggtctctgtttaatgttgaaagggtgagctaataggaggcactttcaacttcactccctc2040 acgctaccccgtccccctccagactggcagtttcaaggatgcaaattgcattgcaaaatc2100 aaactgactcatgaagcatttgggccagtgcactgtttacttccatctgtttgcagacac2160 atttgtgcccggcgtttgggagccctttgtatcaatgttctgacaagggtccctataacc2220 ttaacctactcgaaaccggtttgggatggatatgatggggcttctgtgctattgctggga2280 ttgggagaaataaaacatgcaatttaagtggaagcgaagaaatttaaagaggattttatt2340 ttgcttgggtcaatccttgttaaaagggaggtggatgtgtttccttgtgttggatggcat2400 gagattatgtgaatgttttgatttattaaaatgaactgcaaggtttttcacaggaacgac2460 agacatgtatgactgcatgtaattataaactcctgacctcctggtggggttggagcatct2520 gtttcaaatgtgggacttacaagcacttctcacatgagaaattaggggcgggtgggaagg2580 gatgggacacagcttctggcaccatggatttaagaccatgttggatccaaaagttggcct2640 gaaaccctgaagctgatgcttcacagctgggctgtaagtcagacttgaacccagctgata2700 tgcaaggtcatggcgtgccagggtggtgacagttgaacaaagtgtatagtacgtgcccag2760 tggtagcgatggaaaaaagtataccaaatggactttgaaggaccaaaggttttaaaagtc2820 aattggtatcacctccacactgactagggtagtggggtgcatttggttttcaaattgggt2880 acttttaacactttagtgcctgactgctgttctttactgacttgattcagtcactcgtag2940 ctttattggtctgaaccagctccttgttcccaggttacagacctgcctatcgttccaata3000 atcctgtttcacttgaatgaagggagtatgtcttaaatgtaaagtttctggttctcacac3060 tgtactctgaggtccaaatactgtctgtcaatgtgtaacctgatgtctcaaccccctgtg3120 agaagagtccattatttggtgttcaccaacgtgggagacttcaccggaacaggctttttt3180 gctttgggctctgctatttgtttgcagaacacccaagagcgagcaaacacgctctcttca3240 cagcagtaccttagggttttgccattgtaaatgggtctgatgtgatatgacaagaccaga3300 gaaattggatgtaaatttacatttttgaatatgcttgttgtttcacatgatacatttagg3360 gtatgcagctccttttgtagtttttatttttactatttaagtttggaaatgatgccaaat3420 ttttgtatttctttaatcaatgtgttctcttcggtgatatatattgcattatatattgat3480 gtgtgtatcaatatatattgatatgtattacacttacacatacaaacacatataagaggg3540 ggtgaaaaccgtagcctttgcattctctatagcctctgcagagagatactaagcagcaaa3600 atcttggtgttgtgatgtacagaaatggagaagagtattaaaccatatttaag 3653 <210> 17 <211> 540 <212> PRT

<213> Homo sapiens <400> 17 Met Val Arg Thr Asp Gly His Thr Leu Ser Glu Lys Arg Asn Tyr Gln Val Thr Asn Ser Met Phe Gly Ala Ser Arg Lys Lys Phe Val Glu Gly Val Asp Ser Asp Tyr His Asp Glu Asn Met Tyr Tyr Ser Gln Ser Ser Met Phe Pro His Arg Ser Glu Lys Asp Met Leu Ala Ser Pro Ser Thr Ser Gly Gln Leu Ser Gln Phe Gly Ala Ser Leu Tyr Gly Gln Gln Ser Ala Leu Gly Leu Pro Met Arg Gly Met Ser Asn Asn Thr Pro Gln Leu Asn Arg Ser Leu Ser Gln Gly Thr Gln Leu Pro Ser His Val Thr Pro Thr Thr Gly Val Pro Thr Met Ser Leu His Thr Pro Pro Ser Pro Ser Arg Gly Ile Leu Pro Met Asn Pro Arg Asn Met Met Asn His Ser Gln Val Gly Gln Gly Ile Gly Ile Pro Ser Arg Thr Asn Ser Met Ser Ser Ser Gly Leu Gly Ser Pro Asn Arg Ser Ser Pro Ser Ile Ile Cys Met Pro Lys Gln Gln Pro Ser Arg Gln Pro Phe Thr Val Asn Ser Met Ser Gly Phe Gly Met Asn Arg Asn Gln Ala Phe Gly Met Asn Asn Ser Leu Ser Ser Asn Ile Phe Asn Gly Thr Asp Gly Ser Glu Asn Val Thr Gly Leu Asp Leu Ser Asp Phe Pro Ala Leu Ala Asp Arg Asn Arg Arg Glu Gly Ser Gly Asn Pro Thr Pro Leu Ile Asn Pro Leu Ala Gly Arg Ala Pro Tyr Val Gly Met Val Thr Lys Pro Ala Asn Glu Gln Ser Gln Asp Phe Ser Ile His Asn Glu Asp Phe Pro Ala Leu Pro Gly Ser Ser Tyr Lys Asp Pro Thr Ser Ser Asn Asp Asp Ser Lys Ser Asn Leu Asn Thr Ser Gly Lys Thr Thr Ser Ser Thr Asp Gly Pro Lys Phe Pro Gly Asp Lys Ser Ser Thr Thr Gln Asn Asn Asn Gln Gln Lys Lys Gly Ile Gln Val Leu Pro Asp Gly Arg Val Thr Asn Ile Pro Gln Gly Met Val Thr Asp Gln Phe Gly Met Ile Gly Leu Leu Thr Phe Ile Arg Ala Ala Glu Thr Asp Pro Gly Met Val His Leu Ala Leu Gly Ser Asp Leu Thr Thr Leu Gly Leu Asn Leu Asn Ser Pro Glu Asn Leu Tyr Pro Lys Phe Ala Ser Pro Trp Ala Ser Ser Pro Cys Arg Pro Gln Asp Ile Asp Phe His Val Pro Ser Glu Tyr Leu Thr Asn Ile His Ile Arg Asp Lys Leu Ala Ala Ile Lys Leu Gly Arg Tyr Gly Glu Asp Leu Leu Phe Tyr Leu Tyr Tyr Met Asn Gly Gly Asp Val Leu Gln Leu Leu Ala Ala Val Glu Leu Phe Asn Arg Asp Trp Arg Tyr His Lys Glu Glu Arg Val Trp Ile Thr Arg Ala Pro Gly Met Glu Pro Thr Met Lys Thr Asn Thr Tyr Glu Arg Gly Thr Tyr Tyr Phe Phe Asp Cys Leu Asn Trp Arg Lys Val Ala Lys Glu Phe His Leu Glu Tyr Asp Lys Leu Glu Glu Arg Pro His Leu Pro Ser Thr Phe Asn Tyr Asn Pro Ala Gln Gln Ala Phe <210> 18 <211> 603 <212> PRT
<213> Homo Sapiens <400> 18 Met Ala Ser Ala Ser Thr Ser Lys Tyr Asn Ser His Ser Leu Glu Asn Glu Ser Ile Lys Arg Thr Ser Arg Asp Gly Val Asn Arg Asp Leu Thr Glu Ala Val Pro Arg Leu Pro Gly Glu Thr Leu Ile Thr Asp Lys Glu Val Ile Tyr Ile Cys Pro Phe Asn Gly Pro Ile Lys Gly Arg Val Tyr Ile Thr Asn Tyr Arg Leu Tyr Leu Arg Ser Leu Glu Thr Asp Ser Ser Leu Ile Leu Asp Val Pro Leu Gly Val Ile Ser Arg Ile Glu Lys Met Gly Gly Ala Thr Ser Arg Gly Glu Asn Ser Tyr Gly Leu Asp Ile Thr Cys Lys Asp Met Arg Asn Leu Arg Phe Ala Leu Lys Gln Glu Gly His Ser Arg Arg Asp Met Phe Glu Ile Leu Thr Arg Tyr Ala Phe Pro Leu Ala His Ser Leu Pro Leu Phe Ala Phe Leu Asn Glu Glu Lys Phe Asn Val Asp Gly Trp Thr Val Tyr Asn Pro Val Glu Glu Tyr Arg Arg Gln Gly Leu Pro Asn His His Trp Arg Ile Thr Phe Ile Asn Lys Cys Tyr Glu Leu Cys Asp Thr Tyr Pro Ala Leu Leu Val Val Pro Tyr Arg Ala Ser Asp Asp Asp Leu Arg Arg Val Ala Thr Phe Arg Ser Arg Asn Arg Ile Pro Val Leu Ser Trp Ile His Pro Glu Asn Lys Thr Val Ile Val Arg Cys Ser Gln Pro Leu Val Gly Met Ser Gly Lys Arg Asn Lys Asp Asp Glu Lys Tyr Leu Asp Val Ile Arg Glu Thr Asn Lys Gln Ile Ser Lys Leu Thr Ile Tyr Asp Ala Arg Pro Ser Val Asn Ala Val Ala Asn Lys Ala Thr Gly Gly Gly Tyr Glu Ser Asp Asp Ala Tyr His Asn Ala Glu Leu Phe Phe Leu Asp Ile His Asn Ile His Val Met Arg Glu Ser Leu Lys Lys Val Lys Asp Ile Val Tyr Pro Asn Val Glu Glu Ser His Trp Leu Ser Ser Leu Glu Ser Thr His Trp Leu Glu His Ile Lys Leu Val Leu Thr Gly Ala Ile Gln Val Ala Asp Lys Val Ser Ser Gly Lys Ser Ser Val Leu Val His Cys Ser Asp Gly Trp Asp Arg Thr Ala Gln Leu Thr Ser Leu Ala Met Leu Met Leu Asp Ser Phe Tyr Arg Ser Ile Glu Gly Phe Glu Ile Leu Val Gln Lys Glu Trp Ile Ser Phe Gly His Lys Phe Ala Ser Arg Ile Gly His Gly Asp Lys Asn His Thr Asp Ala Asp Arg Ser Pro Ile Phe Leu Gln Phe Ile Asp Cys Val Trp Gln Met Ser Lys Gln Phe Pro Thr Ala Phe Glu Phe Asn Glu Gln Phe Leu Ile Ile Ile Leu Asp His Leu Tyr Ser Cys Arg Phe Gly Thr Phe Leu Phe Asn Cys Glu Ser Ala Arg Glu Arg Gln Lys Val Thr Glu Arg Thr Val Ser Leu Trp Ser Leu Ile Asn Ser Asn Lys Glu Lys Phe Lys Asn Pro Phe Tyr Thr Lys Glu Ile Asn Arg Val Leu Tyr Pro Val Ala Ser Met Arg His Leu Glu Leu Trp Val Asn Tyr Tyr Ile Arg Trp Asn Pro Arg Ile Lys Gln Gln Gln Pro Asn Pro Val Glu Gln Arg Tyr Met Glu Leu Leu Ala Leu Arg Asp Glu Tyr Ile Lys Arg Leu Glu Glu Leu Gln Leu Ala Asn Ser Ala Lys Leu Ser Asp Pro Pro Thr Ser Pro Ser Ser Pro Ser Gln Met Met Pro His Val Gln Thr His Phe <210> 19 <211> 643 <212> PRT
<213> Homo Sapiens <400> 19 Met Glu Thr Ser Ser Ser Cys Glu Ser Leu Gly Ser Gln Pro Ala Ala Ala Arg Pro Pro Ser Val Asp Ser Leu Ser Ser Ala Ser Thr Ser His Ser Glu Asn Ser Val His Thr Lys Ser Ala Ser Val Val Ser Ser Asp Ser Ile Ser Thr Ser Ala Asp Asn Phe Ser Pro Asp Leu Arg Val Leu Arg Glu Ser Asn Lys Leu Ala Glu Met Glu Glu Pro Pro Leu Leu Pro Gly Glu Asn Ile Lys Asp Met Ala Lys Asp Val Thr Tyr Ile Cys Pro Phe Thr Gly Ala Val Arg Gly Thr Leu Thr Val Thr Asn Tyr Arg Leu Tyr Phe Lys Ser Met Glu Arg Asp Pro Pro Phe Val Leu Asp Ala Ser Leu Gly Val Ile Asn Arg Val Glu Lys Ile Gly Gly Ala Ser Ser Arg Gly Glu Asn Ser Tyr Gly Leu Glu Thr Val Cys Lys Asp Ile Arg Asn Leu Arg Phe Ala His Lys Pro Glu Gly Arg Thr Arg Arg Ser Ile Phe Glu Asn Leu Met Lys Tyr Ala Phe Pro Val Ser Asn Asn Leu Pro Leu Phe Ala Phe Glu Tyr Lys Glu Val Phe Pro Glu Asn Gly Trp Lys Leu Tyr Asp Pro Leu Leu Glu Tyr Arg Arg Gln Gly Ile Pro Asn Glu Ser Trp Arg Ile Thr Lys Ile Asn Glu Arg Tyr Glu Leu Cys Asp Thr Tyr Pro Ala Leu Leu Val Val Pro Ala Asn Ile Pro Asp Glu Glu Leu Lys Arg Val Ala Ser Phe Arg Ser Arg Gly Arg Ile Pro Val Leu Ser Trp Ile His Pro Glu Ser Gln Ala Thr Ile Thr Arg Cys Ser Gln Pro Met Val Gly Val Ser Gly Lys Arg Ser Lys Glu Asp Glu Lys Tyr Leu Gln Ala Ile Met Asp Ser Asn Ala Gln Ser His Lys Ile Phe Ile Phe Asp Ala Arg Pro Ser Val Asn Ala Val Ala Asn Lys Ala Lys Gly Gly Gly Tyr Glu Ser Glu Asp Ala Tyr Gln Asn Ala Glu Leu Val Phe Leu Asp Ile His Asn Ile His Val Met Arg Glu Ser Leu Arg Lys Leu Lys Glu Ile Val Tyr Pro Asn Ile Glu Glu Thr His Trp Leu Ser Asn Leu Glu Ser Thr His Trp Leu Glu His Ile Lys Leu Ile Leu Ala Gly Ala Leu Arg Ile Ala Asp Lys Val Glu Ser Gly Lys Thr Ser Val Val Val His Cys Ser Asp Gly Trp Asp Arg Thr Ala Gln Leu Thr Ser Leu Ala Met Leu Met Leu Asp Gly Tyr Tyr Arg Thr Ile Arg Gly Phe Glu Val Leu Val Glu Lys Glu Trp Leu Ser Phe Gly His Arg Phe Gln Leu Arg Val Gly His Gly Asp Lys Asn His Ala Asp Ala Asp Arg Ser Pro Val Phe Leu Gln Phe Ile Asp Cys Val Trp Gln Met Thr Arg Gln Phe Pro Thr Ala Phe Glu Phe Asn Glu Tyr Phe Leu Ile Thr Ile Leu Asp His Leu Tyr Ser Cys Leu Phe Gly Thr Phe Leu Cys Asn Ser Glu Gln Gln Arg Gly Lys Glu Asn Leu Pro Lys Arg Thr Val Ser Leu Trp Ser Tyr Ile Asn Ser Gln Leu Glu Asp Phe Thr Asn Pro Leu Tyr Gly Ser Tyr Ser Asn His Val Leu Tyr Pro Val Ala Ser Met Arg His Leu Glu Leu Trp Val Gly Tyr Tyr Ile Arg Trp Asn Pro Arg Met Lys Pro Gln Glu Pro Ile His Asn Arg Tyr Lys Glu Leu Leu Ala Lys Arg Ala Glu Leu Gln Lys Lys Val Glu Glu Leu Gln Arg Glu Ile Ser Asn Arg Ser Thr Ser Ser Ser Glu Arg Ala Ser Ser Pro Ala Gln Cys Val Thr Pro Val Gln Thr Val Val <210> 20 <211> 467 <212> PRT
<213> Homo Sapiens <400> 20 Met Ala Thr Gly Ala Asp Val Arg Asp Ile Leu Glu Leu Gly Gly Pro Glu Gly Asp Ala Ala Ser Gly Thr Ile Ser Lys Lys Asp Ile Ile Asn Pro Asp Lys Lys Lys Ser Lys Lys Ser Ser Glu Thr Leu Thr Phe Lys Arg Pro Glu Gly Met His Arg Glu Val Tyr Ala Leu Leu Tyr Ser Asp Lys Lys Asp Ala Pro Pro Leu Leu Pro Ser Asp Thr Gly Gln Gly Tyr Arg Thr Val Lys Ala Lys Leu Gly Ser Lys Lys Val Arg Pro Trp Lys Trp Met Pro Phe Thr Asn Pro Ala Arg Lys Asp Gly Ala Met Phe Phe 100 105 . 110 His Trp Arg Arg Ala Ala Glu Glu Gly Lys Asp Tyr Pro Phe Ala Arg Phe Asn Lys Thr Val Gln Val Pro Val Tyr Ser Glu Gln Glu Tyr Gln Leu Tyr Leu His Asp Asp Ala Trp Thr Lys Ala Glu Thr Asp His Leu Phe Asp Leu Ser Arg Arg Phe Asp Leu Arg Phe Val Val Ile His Asp Arg Tyr Asp His Gln Gln Phe Lys Lys Arg Ser Val Glu Asp Leu Lys Glu Arg Tyr Tyr His Ile Cys Ala Lys Leu Ala Asn Val Arg Ala Val Pro Gly Thr Asp Leu Lys Ile Pro Val Phe Asp Ala Gly His Glu Arg Arg Arg Lys Glu Gln Leu Glu Arg Leu Tyr Asn Arg Thr Pro Glu Gln Val Ala Glu Glu Glu Tyr Leu Leu Gln Glu Leu Arg Lys Ile Glu Ala Arg Lys Lys Glu Arg Glu Lys Arg Ser Gln Asp Leu Gln Lys Leu Ile Thr Ala Ala Asp Thr Thr Ala Glu Gln Arg Arg Thr Glu Arg Lys Ala Pro Lys Lys Lys Leu Pro Gln Lys Lys Glu Ala Glu Lys Pro Ala Val 290 ' 295 300 Pro Glu Thr Ala Gly Ile Lys Phe Pro Asp Phe Lys Ser Ala Gly Val Thr Leu Arg Ser Gln Arg Met Lys Leu Pro Ser Ser Val Gly Gln Lys Lys Ile Lys Ala Leu Glu Gln Met Leu Leu Glu Leu Gly Val Glu Leu Ser Pro Thr Pro Thr Glu Glu Leu Val His Met Phe Asn Glu Leu Arg Ser Asp Leu Val Leu Leu Tyr Glu Leu Lys Gln Ala Cys Ala Asn Cys Glu Tyr Glu Leu Gln Met Leu Arg His Arg His Glu Ala Leu Ala Arg Ala Gly Val Leu Gly Gly Pro Ala Thr Pro Ala Ser Gly Pro Gly Pro Ala Ser Ala Glu Pro Ala Val Thr Glu Pro Gly Leu Gly Pro Asp Pro Lys Asp Thr Ile Ile Asp Val Val Gly Ala Pro Leu Thr Pro Asn Ser Arg Lys Arg Arg Glu Ser Ala Ser Ser Ser Ser Ser Val Lys Lys Ala Lys Lys Pro <210> 21 <211> 1763 <212> PRT
<213> Homo Sapiens <400> 21 Met Ala Lys Pro Thr Ser Lys Asp Ser Gly Leu Lys Glu Lys Phe Lys Ile Leu Leu Gly Leu Gly Thr Pro Arg Pro Asn Pro Arg Ser Ala Glu Gly Lys Gln Thr Glu Phe Ile Ile Thr Ala Glu Ile Leu Arg Glu Leu Ser Met Glu Cys Gly Leu Asn Asn Arg Ile Arg Met Ile Gly Gln Ile Cys Glu Val Ala Lys Thr Lys Lys Phe Glu Glu His Ala Val Glu Ala Leu Trp Lys Ala Val Ala Asp Leu Leu Gln Pro Glu Arg Thr Leu Glu Ala Arg His Ala Val Leu Ala Leu Leu Lys Ala Ile Val Gln Gly Gln Gly Glu Arg Leu Gly Val Leu Arg Ala Leu Phe Phe Lys Val Ile Lys Asp Tyr Pro Ser Asn Glu Asp Leu His Glu Arg Leu Glu Val Phe Lys Ala Leu Thr Asp Asn Gly Arg His Ile Thr Tyr Leu Glu Glu Glu Leu Ala Asp Phe Val Leu Gln Trp Met Asp Val Gly Leu Ser Ser Glu Phe Leu Leu Val Leu Val Asn Leu Val Lys Phe Asn Ser Cys Tyr Leu Asp Glu Tyr Ile Ala Arg Met Val Gln Met Ile Cys Leu Leu Cys Val Arg Thr Ala Ser Ser Val Asp Ile Glu Val Ser Leu Gln Val Leu Asp Ala Val Val Cys Tyr Asn Cys Leu Pro Ala Glu Ser Leu Pro Leu Phe Ile Val Thr Leu Cys Arg Thr Ile Asn Val Lys Glu Leu Cys Glu Pro Cys ~Trp Lys Leu Met Arg Asn Leu Leu Gly Thr His Leu Gly His Ser Ala Ile Tyr Asn Met Cys His Leu Met Glu Asp Arg Ala Tyr Met Glu Asp Ala Pro Leu Leu Arg Gly Ala Val Phe Phe Val Gly Met Ala Leu Trp Gly Ala His Arg Leu Tyr Ser Leu Arg Asn Ser Pro Thr Ser Val Phe Pro Ser Phe Tyr Gln Ala Met Ala Cys Pro Asn Glu Val Val Ser Tyr Glu Ile Val Leu Ser Ile Thr Arg Leu Ile Lys Lys Tyr Arg Lys Glu Leu Gln Val Val Ala Trp Asp Ile Leu Leu Asn Ile Ile Glu Arg Leu Leu Gln Gln Leu Gln Thr Leu Asp Ser Pro Glu Leu Arg Thr Ile Val His Asp Leu Leu Thr Thr Val Glu Glu Leu Cys Asp Gln Asn Glu Phe His Gly Ser Gln Glu Arg Tyr Phe Glu Leu Val Glu Arg Cys Ala Asp Gln Arg Pro Glu Ser Ser Leu Leu Asn Leu Ile Ser Tyr Arg Ala Gln Ser Ile His Pro Ala Lys Asp Gly Trp Ile Gln Asn Leu Gln Ala Leu Met Glu Arg Phe Phe Arg Ser Glu Ser Arg Gly Ala Val Arg Ile Lys Val Leu Asp Val Leu Ser Phe Val Leu Leu Ile Asn Arg Gln Phe Tyr Glu Glu Glu Leu Ile Asn Ser Val Val Ile Ser Gln Leu Ser His Ile Pro Glu Asp Lys Asp His Gln Val Arg Lys Leu Ala Thr Gln Leu Leu Val Asp Leu Ala Glu Gly Cys His Thr His His Phe Asn Ser Leu Leu Asp Ile Ile Glu Lys Val Met Ala Arg Ser Leu Ser Pro Pro Pro Glu Leu Glu Glu Arg Asp Val Ala Ala Tyr Ser Ala Ser Leu Glu Asp Val Lys Thr Ala Val Leu Gly Leu Leu Val Ile Leu Gln Thr Lys Leu Tyr Thr Leu Pro Ala Ser His Ala Thr Arg Val Tyr Glu Met Leu Val Ser His Ile Gln Leu His Tyr Lys His Ser Tyr Thr Leu Pro Ile Ala Ser Ser Ile Arg Leu Gln Ala Phe Asp Phe Leu Phe Leu Leu Arg Ala Asp Ser Leu His Arg Leu Gly Leu Pro Asn Lys Asp Gly Val Val Arg Phe Ser Pro Tyr Cys Val Cys Asp Tyr Met Glu Pro Glu Arg Gly Ser Glu Lys Lys Thr Ser Gly Pro Leu Ser Pro Pro Thr Gly Pro Pro Gly Pro Ala Pro Ala Gly Pro Ala Val Arg Leu Gly Ser Val Pro Tyr Ser Leu Leu Phe Arg Val Leu Leu Gln Cys Leu Lys Gln Glu Ser Asp Trp Lys Val Leu Lys Leu Val Leu Gly Arg Leu Pro Glu Ser Leu Arg Tyr Lys Val Leu Ile Phe Thr Ser Pro Cys Ser Val Asp Gln Leu Cys Ser Ala Leu Cys Ser Met Leu Ser Gly Pro Lys Thr Leu Glu Arg Leu Arg Gly Ala Pro Glu Gly Phe Ser Arg Thr Asp Leu His Leu Ala Val Val Pro Val Leu Thr Ala Leu Ile Ser Tyr His Asn Tyr Leu Asp Lys Thr Lys Gln Arg Glu Met Val Tyr Cys Leu Glu Gln Gly Leu Ile His Arg Cys Ala Arg Gln Cys Val Val Ala Leu Ser Ile Cys Ser Val Glu Met Pro Asp Ile Ile Ile Lys Ala Leu Pro Val Leu Val Val Lys Leu Thr His Ile Ser Ala Thr Ala Ser Met Ala Val Pro Leu Leu Glu Phe Leu Ser Thr Leu Ala Arg Leu Pro His Leu Tyr Arg Asn Phe Ala Ala Glu Gln Tyr Ala Ser Val Phe Ala Ile Ser Leu Pro Tyr Thr Asn Pro Ser Lys Phe Asn Gln Tyr Ile Val Cys Leu Ala His His Val Ile Ala Met Trp Phe Ile Arg Cys Arg Leu Pro Phe Arg Lys Asp Phe Val Pro Phe Ile Thr Lys Gly Leu Arg Ser Asn Val Leu Leu Ser Phe Asp Asp Thr Pro Glu Lys Asp Ser Phe Arg Ala Arg Ser Thr Ser Leu Asn Glu Arg Pro Lys Arg Ile Gln Thr Ser Leu Thr Ser Ala Ser Leu Gly Ser Ala Asp Glu Asn Ser Val Ala Gln Ala Asp Asp Ser Leu Lys Asn Leu His Leu Glu Leu Thr Glu Thr Cys Leu Asp Met Met Ala Arg Tyr Val Phe Ser Asn Phe Thr Ala Val Pro Lys Arg 5er Pro Val Gly Glu Phe Leu Leu Ala Gly Gly Arg Thr Lys Thr Trp Leu Val Gly Asn Lys Leu Val Thr Val Thr Thr Ser Val Gly Thr Gly Thr Arg Ser Leu Leu Gly Leu Asp Ser Gly Glu Leu Gln Ser Gly Pro Glu Ser Ser Ser Ser Pro Gly Val His Val Arg Gln Thr Lys Glu Ala Pro Ala Lys Leu Glu Ser Gln Ala Gly Gln Gln Val Ser Arg Gly Ala Arg Asp Arg Val Arg Ser Met Ser Gly Gly His Gly Leu Arg Val Gly Ala Leu Asp Val Pro Ala Ser Gln Phe Leu Gly Ser Ala Thr Ser Pro Gly Pro Arg Thr Ala Pro Ala Ala Lys Pro Glu Lys Ala Ser Ala Gly Thr Arg Val Pro Val Gln Glu Lys Thr Asn Leu Ala Ala Tyr Val Pro Leu Leu Thr Gln Gly Trp Ala Glu Ile Leu Val Arg Arg Pro Thr Gly Asn Thr Ser Trp Leu Met Ser Leu Glu Asn Pro Leu Ser Pro Phe Ser Ser Asp Ile Asn Asn Met Pro Leu Gln Glu Leu Ser Asn Ala Leu Met Ala Ala Glu Arg Phe Lys Glu His Arg Asp Thr Ala Leu Tyr Lys Ser Leu Ser Val Pro Ala Ala Ser Thr Ala Lys Pro Pro Pro Leu Pro Arg Ser Asn Thr Val Ala Ser Phe Ser Ser Leu Tyr Gln Ser Ser Cys Gln Gly Gln Leu His Arg Ser Val Ser Trp Ala Asp Ser Ala Val Val Met Glu Glu Gly Ser Pro Gly Glu Val Pro Val Leu Val Glu Pro Pro Gly Leu Glu Asp Val Glu Ala Ala Leu Gly Met Asp Arg Arg Thr Asp Ala Tyr Ser Arg Ser Ser Ser Val Ser Ser Gln Glu Glu Lys Ser Leu His Ala Glu Glu Leu Val Gly Arg Gly Ile Pro Ile Glu Arg Val Val Ser Ser Glu Gly Gly Arg Pro Ser Val Asp Leu Ser Phe Gln Pro Ser Gln Pro Leu Ser Lys Ser Ser Ser Ser Pro Glu Leu Gln Thr Leu Gln Asp Ile Leu Gly Asp Pro Gly Asp Lys Ala Asp Val Gly Arg Leu Ser Pro Glu Val Lys Ala Arg Ser Gln Ser Gly Thr Leu Asp Gly Glu Ser Ala Ala Trp Ser Ala Ser Gly Glu Asp Ser Arg Gly Gln Pro Glu Gly Pro Leu Pro Ser Ser Ser Pro Arg Ser Pro Ser Gly Leu Arg Pro Arg Gly Tyr Thr Ile Ser Asp Ser Ala Pro Ser Arg Arg Gly Lys Arg Val Glu Arg Asp Ala Leu Lys Ser Arg Ala Thr Ala Ser Asn Ala Glu Lys Val Pro Gly Ile Asn Pro Ser Phe Val Phe Leu Gln Leu Tyr His Ser Pro Phe Phe Gly Asp Glu Ser Asn Lys Pro Ile Leu Leu Pro Asn Glu Ser Gln Ser Phe Glu Arg Ser Val Gln Leu Leu Asp Gln Ile Pro Ser Tyr Asp Thr His Lys Ile Ala Val Leu Tyr Val Gly Glu Gly Gln Ser Asn Ser Glu Leu Ala Ile Leu Ser Asn Glu His Gly Ser Tyr Arg Tyr Thr Glu Phe Leu Thr Gly Leu Gly Arg Leu Ile Glu Leu Lys Asp Cys Gln Pro Asp Lys Val Tyr Leu Gly Gly Leu Asp Val Cys Gly Glu Asp Gly Gln Phe Thr Tyr Cys Trp His Asp Asp Ile Met Gln Ala Val Phe His Ile Ala Thr Leu Met Pro Thr Lys Asp Val Asp Lys His Arg Cys Asp Lys Lys Arg His Leu Gly Asn Asp Phe Val Ser Ile Val Tyr Asn Asp Ser Gly Glu Asp Phe Lys Leu Gly Thr Ile Lys Gly Gln Phe Asn Phe Val His Val Ile Val Thr Pro Leu Asp Tyr Glu Cys Asn Leu Val Ser Leu Gln Cys Arg Lys Asp Met Glu Gly Leu Val Asp Thr Ser Val Ala Lys Ile Val Ser Asp Arg Asn Leu Pro Phe Val Ala Arg Gln Met Ala Leu His Ala Asn Met Ala Ser Gln Val His His Ser Arg Ser Asn Pro Thr Asp Ile Tyr Pro Ser Lys Trp Ile Ala Arg Leu Arg His Ile Lys Arg Leu Arg Gln Arg Ile Cys Glu Glu Ala Ala Tyr Ser Asn Pro Ser Leu Pro Leu Val His Pro Pro Ser His Ser Lys Ala Pro Ala Gln Thr Pro Ala Glu Pro Thr Pro Gly Tyr Glu Val Gly Gln Arg Lys Arg Leu Ile Ser Ser Val Glu Asp Phe Thr Glu Phe Val <210> 22 <211> 1807 <212> PRT
<213> Homo sapiens <400> 22 Met Ala Lys Pro Thr Ser Lys Asp Ser Gly Leu Lys Glu Lys Phe Lys Ile Leu Leu Gly Leu Gly Thr Pro Arg Pro Asn Pro Arg Ser Ala Glu Gly Lys Gln Thr Glu Phe Ile Ile Thr Ala Glu Ile Leu Arg Glu Leu Ser Met Glu Cys Gly Leu Asn Asn Arg Ile Arg Met Ile Gly Gln Ile Cys Glu Val Ala Lys Thr Lys Lys Phe Glu Glu His Ala.Val Glu Ala Leu Trp Lys Ala Val Ala Asp Leu Leu Gln Pro Glu Arg Pro Leu Glu Ala Arg His Ala Val Leu Ala Leu Leu Lys Ala Ile Val Gln Gly Gln Gly Glu Arg Leu Gly Val Leu Arg Ala Leu Phe Phe Lys Val Ile,Lys Asp Tyr Pro Ser Asn Glu Asp Leu His Glu Arg Leu Glu Val Phe Lys Ala Leu Thr Asp Asn Gly Arg His Ile Thr Tyr Leu Glu Glu Glu Leu Ala Asp Phe Val Leu Gln Trp Met Asp Val Gly Leu Ser Ser Glu Phe Leu Leu Val Leu Val Asn Leu Val Lys Phe Asn Ser Cys Tyr Leu Asp Glu Tyr Ile Ala Arg Met Val Gln Met Ile Cys Leu Leu Cys Val Arg Thr Ala Ser Ser Val Asp Ile Glu Val Ser Leu Gln Val Leu Asp Ala Val Val Cys Tyr Asn Cys Leu Pro Ala Glu Ser Leu Pro Leu Phe Ile Val Thr Leu Cys Arg Thr Ile Asn Val Lys Glu Leu Cys Glu Pro Cys Trp Lys Leu Met Arg Asn Leu Leu Gly Thr His Leu Gly His Ser Ala Ile Tyr Asn Met Cys His Leu Met Glu Asp Arg Ala Tyr Met Glu Asp Ala Pro Leu Leu Arg Gly Ala Val Phe Phe Val Gly Met Ala Leu Trp Gly Ala His Arg Leu Tyr Ser Leu Arg Asn Ser Pro Thr Ser Val Leu Pro Ser Phe Tyr Gln Ala Met Ala Cys Pro Asn Glu Val Val Ser Tyr Glu Ile Val Leu Ser Ile Thr Arg Leu Ile Lys Lys Tyr Arg Lys Glu Leu Gln Val Val Ala Trp Asp Ile Leu Leu Asn Ile Ile Glu Arg Leu Leu Gln Gln Leu Gln Thr Leu Asp Ser Pro Glu Leu Arg Thr Ile Val His Asp Leu Leu Thr Thr Val Glu Glu Leu Cys Asp Gln Asn Glu Phe His Gly Ser Gln Glu Arg Tyr Phe Glu Leu Val Glu Arg Cys Ala Asp Gln Arg Pro Glu Ser Ser Leu Leu Asn Leu Ile Ser Tyr Arg Ala Gln 420 ~ 425 430 Ser Ile His Pro Ala Lys Asp Gly Trp Ile Gln Asn Leu Gln Ala Leu Met Glu Arg Phe Phe Arg Ser Glu Ser Arg Gly Ala Val Arg Ile Lys Val Leu Asp Val Leu Ser Phe Val Leu Leu Ile Asn Arg Gln Phe Tyr Glu Glu Glu Leu Ile Asn Ser Val Val Ile Ser Gln Leu Ser His Ile Pro Glu Asp Lys Asp His Gln Val Arg Lys Leu Ala Thr Gln Leu Leu Val Asp Leu Ala Glu Gly Cys His Thr His His Phe Asn Ser Leu Leu Asp Ile Ile Glu Lys Val Met Ala Arg Ser Leu Ser Pro Pro Pro Glu Leu Glu Glu Arg Asp Val Ala Ala Tyr Ser Ala Ser Leu Glu Asp Val Lys Thr Ala Val Leu Gly Leu Leu Val Ile Leu Gln Thr Lys Leu Tyr Thr Leu Pro Ala Ser His Ala Thr Arg Val Tyr Glu Met Leu Val Ser His Ile Gln Leu His Tyr Lys His Ser Tyr Thr Leu Pro Ile Ala Ser Ser Ile Arg Leu Gln Ala Phe Asp Phe Leu Leu Leu Leu Arg Ala Asp Ser Leu His Arg Leu Gly Leu Pro Asn Lys Asp Gly Va1 Val Arg Phe Ser Pro Tyr Cys Val Cys Asp Tyr Met Glu Pro Glu Arg Gly Ser Glu Lys Lys Thr Ser Gly Pro Leu Ser Pro Pro Thr Gly Pro Pro Gly Pro Ala Pro Ala Gly Pro Ala Val Arg Leu Gly Ser Val Pro Tyr Ser Leu Leu Phe Arg Val Leu Leu Gln Cys Leu Lys Gln Glu Ser Asp Trp Lys Val Leu Lys Leu Val Leu Gly Arg Leu Pro Glu Ser Leu Arg Tyr Lys Val Leu Ile Phe Thr Ser Pro Cys Ser Val Asp Gln Leu Cys Ser Ala Leu Cys Ser Met Leu Ser Gly Pro Lys Thr Leu Glu Arg Leu Arg Gly Ala Pro Glu Gly Phe Ser Arg Thr Asp Leu His Leu Ala Val Val Pro Val Leu Thr Ala Leu Ile Ser Tyr His Asn Tyr Leu Asp Lys Thr Lys Gln Arg Glu Met Val Tyr Cys Leu Glu Gln Gly Leu Ile His Arg Cys Ala Arg Gln Cys Val Val Ala Leu Ser Ile Cys Ser Val Glu Met Pro Asp Ile Ile Ile Lys Ala Leu Pro Val Leu Val Val Lys Leu Thr His Ile Ser Ala Thr Ala Ser Met Ala Val Pro Leu Leu Glu Phe Leu Ser Thr Leu Ala Arg Leu Pro His Leu Tyr Arg Asn Phe Ala Ala Glu Gln Tyr Ala Ser Val Phe Ala Ile Ser Leu Pro Tyr Thr Asn Pro Ser Lys Phe Asn Gln Tyr Ile Val Cys Leu Ala His His Val Ile Ala Met Trp Phe Ile Arg Cys Arg Leu Pro Phe Arg Lys Asp Phe Val Pro Phe Ile Thr Lys Gly Leu Arg Ser Asn Val Leu Leu Ser Phe Asp Asp Thr Pro Glu Lys Asp Ser Phe Arg Ala Arg Ser Thr Ser Leu Asn Glu Arg Pro Lys Ser Leu Arg Ile Ala Arg Pro Pro Lys Gln Gly Leu Asn Asn Ser Pro Pro Val Lys Glu Phe Lys Glu Ser Ser Ala Ala Glu Ala Phe Arg Cys Arg Ser Ile Ser Val Ser Glu His Val Val Arg Ser Arg Ile Gln Thr Ser Leu Thr Ser Ala Ser Leu Gly Ser Ala Asp Glu Asn Ser Val Ala Gln Ala Asp Asp Ser Leu Lys Asn Leu His Leu Glu Leu Thr Glu Thr Cys Leu Asp Met Met Ala Arg Tyr Val Phe Ser Asn Phe Thr Ala Val Pro Lys Arg Ser Pro Val Gly Glu Phe Leu Leu Ala Gly Gly Arg Thr Lys Thr Trp Leu Val Gly Asn Lys Leu Val Thr Val Thr Thr Ser Val Gly Thr Gly Thr Arg Ser Leu Leu Gly Leu Asp Ser Gly Glu Leu Gln Ser Gly Pro Glu Ser Ser Ser Ser Pro Gly Val His Val Arg Gln Thr Lys Glu Ala Pro Ala Lys Leu Glu Ser Gln Ala Gly Gln Gln Val Ser Arg Gly Ala Arg Asp Arg Val Arg Ser Met Ser Gly Gly His Gly Leu Arg Val Gly Ala Leu Asp Val Pro Ala Ser Gln Phe Leu Gly Ser Ala Thr Ser Pro Gly Pro Arg Thr Ala Pro Ala Ala Lys Pro Glu Lys Ala Ser Ala Gly Thr Arg Val Pro Val Gln Glu Lys Thr Asn Leu Ala Ala Tyr Val Pro Leu Leu Thr Gln Gly Trp Ala Glu Ile Leu Val Arg Arg Pro Thr Gly Asn Thr Ser Trp Leu Met Ser Leu Glu Asn Pro Leu Ser Pro Phe Ser Ser Asp Ile Asn Asn Met Pro Leu Gln Glu Leu Ser Asn Ala Leu Met Ala Ala Glu Arg Phe Lys Glu His Arg Asp Thr Ala Leu Tyr Lys Ser Leu Ser Val Pro Ala Ala Ser Thr Ala Lys Pro Pro Pro Leu Pro Arg Ser Asn Thr Val Ala Ser Phe Ser Ser Leu Tyr Gln Ser Ser Cys Gln Gly Gln Leu His Arg Ser Val Ser Trp Ala Asp Ser Ala Val Val Met Glu Glu Gly Ser Pro Gly Glu Val Pro Val Leu Val Glu Pro Pro Gly Leu Glu Asp Val Glu Ala Ala Leu Gly Met Asp Arg Arg Thr Asp Ala Tyr Ser Arg Ser Ser Ser Val Ser Ser Gln Glu Glu Lys Ser Leu His Ala Glu Glu Leu Val Gly Arg Gly Ile Pro Ile Glu Arg Val Val Ser Ser Glu Gly Gly Arg Pro Ser Val Asp Leu Ser Phe Gln Pro Ser Gln Pro Leu Ser Lys Ser Ser Ser Ser Pro Glu Leu Gln Thr Leu Gln Asp Ile Leu Gly Asp Pro Gly Asp Lys Ala Asp Val Gly Arg Leu Ser Pro Glu Val Lys Ala Arg Ser Gln Ser Gly Thr Leu Asp Gly Glu Ser Ala Ala Trp Ser Ala Ser Gly Glu Asp Ser Arg Gly Gln Pro Glu Gly Pro Leu Pro Ser Ser Ser Pro Arg Ser Pro Ser Gly Leu Arg Pro Arg Gly Tyr Thr Ile Ser Asp Ser Ala Pro Ser Arg Arg Gly Lys Arg Val Glu Arg Asp Ala Leu Lys Ser Arg Ala Thr Ala Ser Asn Ala Glu Lys Val Pro Gly Ile Asn Pro Ser Phe Val Phe Leu Gln Leu Tyr His Ser Pro Phe Phe Gly Asp Glu Ser Asn Lys Pro Ile Leu Leu Pro Asn Glu Ser Gln Ser Phe Glu Arg Ser Val Gln Leu Leu Asp Gln Ile Pro Ser Tyr Asp Thr His Lys Ile Ala Val Leu Tyr Val Gly Glu Gly Gln Ser Asn Ser Glu Leu Ala Ile Leu Ser Asn Glu His Gly Ser Tyr Arg Tyr Thr Glu Phe Leu Thr Gly Leu Gly Arg Leu Ile Glu Leu Lys Asp Cys Gln Pro Asp Lys Val Tyr Leu Gly Gly Leu Asp Val Cys Gly Glu Asp Gly Gln Phe Thr Tyr Cys Trp His Asp Asp Ile Met Gln Ala Val Phe His Ile Ala Thr Leu Met Pro Thr Lys Asp Val Asp Lys His Arg Cys Asp Lys Lys Arg His Leu Gly Asn Asp Phe Val Ser Ile Val Tyr Asn Asp Ser Gly Glu Asp Phe Lys Leu Gly Thr Ile Lys Gly Gln Phe Asn Phe Val His Val Ile Val Thr Pro Leu Asp Tyr Glu Cys Asn Leu Val Ser Leu Gln Cys Arg Lys Asp Met Glu Gly Leu Val Asp Thr Ser Val Ala Lys Ile Val Ser Asp Arg Asn Leu Pro Phe Val Ala Arg Gln Met Ala Leu His Ala Asn Met Ala Ser Gln Val His His Ser Arg Ser Asn Pro Thr Asp Ile Tyr Pro Ser Lys Trp Ile Ala Arg Leu Arg His Ile Lys Arg Leu Arg Gln Arg Ile Cys Glu Glu Ala Ala Tyr Ser Asn Pro Ser Leu Pro Leu Val His Pro Pro Ser His Ser Lys Ala Pro Ala Gln Thr Pro Ala Glu Pro Thr Pro Gly Tyr Glu Val Gly Gln Arg Lys Arg Leu Ile Ser Ser Val Glu Asp Phe Thr Glu Phe Val <210> 23 <211> 215 <212> PRT
<213> Homo Sapiens <400> 23 Met Ala Ser Arg Gly Ala Thr Arg Pro Asn Gly Pro Asn Thr Gly Asn Lys Ile Cys Gln Phe Lys Leu Val Leu Leu Gly Glu Ser Ala Val Gly Lys Ser Ser Leu Val Leu Arg Phe Val Lys Gly Gln Phe His Glu Phe Gln Glu Ser Thr Ile Gly Ala Ala Phe Leu Thr Gln Thr Val Cys Leu Asp Asp Thr Thr Val Lys Phe Glu Ile Trp Asp Thr Ala Gly Gln Glu Arg Tyr His Ser Leu Ala Pro Met Tyr Tyr Arg Gly Ala Gln Ala Ala Ile Val Val Tyr Asp Ile Thr Asn Glu Glu Ser Phe Ala Arg Ala Lys Asn Trp Val Lys Glu Leu Gln Arg Gln Ala Ser Pro Asn Ile Val Ile Ala Leu Ser Gly Asn Lys Ala Asp Leu Ala Asn Lys Arg Ala Val Asp Phe Gln Glu Ala Gln Ser Tyr Ala Asp Asp Asn Ser Leu Leu Phe Met Glu Thr Ser Ala Lys Thr Ser Met Asn Val Asn Glu Ile Phe Met Ala Ile Ala Lys Lys Leu Pro Lys Asn Glu Pro Gln Asn Pro Gly Ala Asn Ser Ala Arg Gly Arg Gly Val Asp Leu Thr Glu Pro Thr Gln Pro Thr Arg Asn Gln Cys Cys Ser Asn <210> 24 <211> 295 <212> PRT
<213> Homo sapiens <400> 24 Met Asp Asn Ser Gly Lys Glu Ala Glu Ala Met Ala Leu Leu Ala Glu Ala Glu Arg Lys Val Lys Asn Ser Gln Ser Phe Phe Ser Gly Leu Phe Gly Gly Ser Ser Lys Ile Glu Glu Ala Cys Glu Ile Tyr Ala Arg Ala Ala Asn Met Phe Lys Met Ala Lys Asn Trp Ser Ala Ala Gly Asn Ala Phe Cys Gln Ala Ala Gln Leu His Leu Gln Leu Gln Ser Lys His Asp Ala Ala Thr Cys Phe Val Asp Ala Gly Asn Ala Phe Lys Lys Ala Asp Pro Gln Glu Ala Ile Asn Cys Leu Met Arg Ala Ile Glu Ile Tyr Thr Asp Met Gly Arg Phe Thr Ile Ala Ala Lys His His Ile Ser Ile Ala Glu Ile Tyr Glu Thr Glu Leu Val Asp Ile Glu Lys Ala Ile Ala His Tyr Glu Gln Ser Ala Asp Tyr Tyr Lys Gly Glu Glu Ser Asn Ser Ser Ala Asn Lys Cys Leu Leu Lys Val Ala Gly Tyr Ala Ala Leu Leu Glu Gln Tyr Gln Lys Ala Ile Asp Ile Tyr Glu Gln Val Gly Thr Asn Ala Met Asp Ser Pro Leu Leu Lys Tyr Ser Ala Lys Asp Tyr Phe Phe Lys Ala Ala Leu Cys His Phe Cys Ile Asp Met Leu Asn Ala Lys Leu Ala Val Gln Lys Tyr Glu Glu Leu Phe Pro Ala Phe Ser Asp Ser Arg Glu Cys Lys Leu Met Lys Lys Leu Leu Glu Ala His Glu Glu Gln Asn Val Asp Ser Tyr Thr Glu Ser Val Lys Glu Tyr Asp Ser Ile Ser Arg Leu Asp Gln Trp Leu Thr Thr Met Leu Leu Arg Ile Lys Lys Thr Ile Gln Sl 275 280 . 285 Gly Asp Glu Glu Asp Leu Arg <210> 25 <211> 221 <212> PRT
<213> Homo sapiens <400> 25 Met Lys Lys Ile Arg Gln Val Ile Arg Lys Tyr Asn Tyr Val Ala Met Asp Thr Glu Phe Pro Gly Val Val Ala Arg Pro Ile Gly Glu Phe Arg Ser Asn Ala Asp Tyr Gln Tyr Gln Leu Leu Arg Cys Asn Val Asp Leu Leu Lys Ile Ile Gln Leu Gly Leu Thr Phe Met Asn Glu Gln Gly Glu Tyr Pro Pro Gly Thr Ser Thr Trp Gln Phe Asn Phe Lys Phe Asn Leu Thr Glu Asp Met Tyr Ala Gln Asp Ser Ile Glu Leu Leu Thr Thr Ser Gly Ile Gln Phe Lys Lys His Glu Glu Glu Gly Ile Glu Thr Gln Tyr Phe Ala Glu Leu Leu Met Thr Ser Gly Val Val Leu Cys Glu Gly Val Lys Trp Leu Ser Phe His Ser Gly Tyr Asp Phe Gly Tyr Leu Ile Lys Ile Leu Thr Asn Ser Asn Leu Pro Glu Glu Glu Leu Asp Phe Phe Glu Ile Leu Arg Leu Phe Phe Pro Val Ile Tyr Asp Val Lys Tyr Leu Met Lys Ser Cys Lys Asn Leu Lys Gly Gly Leu Gln Glu Val Ala Glu Gln Leu Glu Leu Glu Arg Ile Gly Pro Gln His Gln Ala Gly Ser Asp Ser Leu Leu Thr Gly Met Ala Phe Phe Lys Met Arg Glu Val <210> 26 <211> 242 <212> PRT
<213> Homo Sapiens <400> 26 Met Ala Asn Asp Glu Gln Ile Leu Val Leu Asp Pro Pro Thr Asp Leu Lys Phe Lys Gly Pro Phe Thr Asp Val Val Thr Thr Asn Leu Lys Leu Arg Asn Pro Ser Asp Arg Lys Val Cys Phe Lys Val Lys Thr Thr Ala Pro Arg Arg Tyr Cys Val Arg Pro Asn Ser Gly Ile Ile Asp Pro Gly Ser Thr Val Thr Val Ser Val Met Leu Gln Pro Phe Asp Tyr Asp Pro Asn Glu Lys Ser Lys His Lys Phe Met Val Gln Thr Ile Phe Ala Pro Pro Asn Thr Ser Asp Met Glu Ala Val Trp Lys Glu Ala Lys Pro Asp Glu Leu Met Asp Ser Lys Leu Arg Cys Val Phe Glu Met Pro Asn Glu Asn Asp Lys Leu Asn Asp Met Glu Pro Ser Lys Ala Val Pro Leu Asn Ala Ser Lys Gln Asp Gly Pro Met Pro Lys Pro His Ser Val Ser Leu Asn Asp Thr Glu Thr Arg Lys Leu Met Glu Glu Cys Lys Arg Leu Gln Gly Glu Met Met Lys Leu Ser Glu Glu Asn Arg His Leu Arg Asp Glu Gly Leu Arg Leu Arg Lys Val Ala His Ser Asp Lys Pro Gly Ser Thr Ser Thr Ala Ser Phe Arg Asp Asn Val Thr Ser Pro Leu Pro Ser Leu Leu Val Val Ile Ala Ala Ile Phe Ile Gly Phe Phe Leu Gly Lys Phe Ile Leu <210> 27 <211> 243 <212> PRT
<213> Homo Sapiens <400> 27 Met Ala Lys Val Glu Gln Val Leu Ser Leu Glu Pro Gln His Glu Leu Lys Phe Arg Gly Pro Phe Thr Asp Val Val Thr Thr Asn Leu Lys Leu Gly Asn Pro Thr Asp Arg Asn Val Cys Phe Lys Val Lys Thr Thr Ala Pro Arg Arg Tyr Cys Val Arg Pro Asn Ser Gly Ile Ile Asp Ala Gly Ala Ser Ile Asn Val Ser Val Met Leu Gln Pro Phe Asp Tyr Asp Pro Asn Glu Lys Ser Lys His Lys Phe Met Val Gln Ser Met Phe Ala Pro Thr Asp Thr Ser Asp Met Glu Ala Val Trp Lys Glu Ala Lys Pro Glu Asp Leu Met Asp Ser Lys Leu Arg Cys Val Phe Glu Leu Pro Ala Glu Asn Asp Lys Pro His Asp Val Glu Ile Asn Lys Ile Ile Ser Thr Thr Ala Ser Lys Thr Glu Thr Pro Ile Val Ser Lys Ser Leu Ser Ser Ser Leu Asp Asp Thr Glu Val Lys Lys Val Met Glu Glu Cys Lys Arg Leu Gln Gly Glu Val Gln Arg Leu Arg Glu Glu Asn Lys Gln Phe Lys Glu Glu Asp Gly Leu Arg Met Arg Lys Thr Val Gln Ser Asn Ser Pro Ile Ser Ala Leu Ala Pro Thr Gly Lys Glu Glu Gly Leu Ser Thr Arg Leu Leu Ala Leu Val Val Leu Phe Phe Ile Val Gly Val Ile Ile Gly Lys Ile Ala Leu <210> 28 <211> 309 <212> PRT
<213> Homo sapiens <400> 28 Met Asp Asp Lys Ala Phe Thr Lys Glu Leu Asp Gln Trp Val Glu Gln Leu Asn Glu Cys Lys Gln Leu Asn Glu Asn Gln Val Arg Thr Leu Cys Glu Lys Ala Lys Glu Ile Leu Thr Lys Glu Ser Asn Val Gln Glu Val Arg Cys Pro Val Thr Val Cys Gly Asp Val His Gly Gln Phe His Asp Leu Met Glu Leu Phe Arg Ile Gly Gly Lys Ser Pro Asp Thr Asn Tyr Leu Phe Met Gly Asp Tyr Val Asp Arg Gly Tyr Tyr Ser Val Glu Thr Val Thr Leu Leu Val Ala Leu Lys Val Arg Tyr Pro Glu Arg Ile Thr Ile Leu Arg Gly Asn His Glu Ser Arg Gln Ile Thr Gln Val Tyr Gly Phe Tyr Asp Glu Cys Leu Arg Lys Tyr Gly Asn Ala Asn Val Trp Lys Tyr Phe Thr Asp Leu Phe Asp Tyr Leu Pro Leu Thr Ala Leu Val Asp Gly Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro Ser Ile Asp Thr Leu Asp His Ile Arg Ala Leu Asp Arg Leu Gln Glu Val Pro His Glu Gly Pro Met Cys Asp Leu Leu Trp Ser Asp Pro Asp Asp Arg Gly Gly Trp Gly Ile Ser Pro Arg Gly Ala Gly Tyr Thr Phe Gly Gln Asp Ile Ser Glu Thr Phe Asn His Ala Asn Gly Leu Thr Leu Val Ser Arg Ala His Gln Leu Val Met Glu Gly Tyr Asn Trp Cys His Asp Arg Asn Val Val Thr Ile Phe Ser Ala Pro Asn Tyr Cys Tyr Arg Cys Gly Asn Gln Ala Ala Ile Met Glu Leu Asp Asp Thr Leu Lys Tyr Ser Phe Leu Gln Phe Asp Pro Ala Pro Arg Arg Gly Glu Pro His Val Thr Arg Arg Thr Pro Asp Tyr Phe Leu <210> 29 <211> 309 <212> PRT
<213> Homo sapiens <400> 29 Met Asp Glu Lys Val Phe Thr Lys Glu Leu Asp Gln Trp Ile Glu Gln Leu Asn Glu Cys Lys Gln Leu Ser Glu Ser Gln Val Lys Ser Leu Cys Glu Lys Ala Lys Glu Ile Leu Thr Lys Glu Ser Asn Val Gln Glu Val Arg Cys Pro Val Thr Val Cys Gly Asp Val His Gly Gln Phe His Asp Leu Met Glu Leu Phe Arg Ile Gly Gly Lys Ser Pro Asp Thr Asn Tyr Leu Phe Met Gly Asp Tyr Val Asp Arg Gly Tyr Tyr Ser Val Glu Thr Val Thr Leu Leu Val Ala Leu Lys Val Arg Tyr Arg Glu Arg Ile Thr Ile Leu Arg Gly Asn His Glu Ser Arg Gln Ile Thr Gln Val Tyr Gly Phe Tyr Asp Glu Cys Leu Arg Lys Tyr Gly Asn Ala Asn Val Trp Lys Tyr Phe Thr Asp Leu Phe Asp Tyr Leu Pro Leu Thr Ala Leu Val Asp Gly Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro Ser Ile Asp Thr Leu Asp His Ile Arg Ala Leu Asp Arg Leu Gln Glu Val Pro His Glu Gly Pro Met Cys Asp Leu Leu Trp Ser Asp Pro Asp Asp Arg Gly Gly Trp Gly Ile Ser Pro Arg Gly Ala Gly Tyr Thr Phe Gly Gln Asp Ile Ser Glu Thr Phe Asn His Ala Asn Gly Leu Thr Leu Val Ser Arg Ala His Gln Leu Val Met Glu Gly Tyr Asn Trp Cys His Asp Arg Asn Val Val Thr Ile Phe Ser Ala Pro Asn Tyr Cys Tyr Arg Cys Gly Asn Gln Ala Ala Ile Met Glu Leu Asp Asp Thr Leu Lys Tyr Ser Phe Leu Gln Phe Asp Pro Ala Pro Arg Arg Gly Glu Pro His Val Thr Arg Arg Thr Pro Asp Tyr Phe Leu <210> 30 <211> 259 <212> PRT
<213> Homo sapiens <400> 30 Met Lys Met Val Thr Gly Ala Val Ala Ser Val Leu Glu Asp Glu Ala Thr Asp Thr Ser Asp Ser Glu Gly Ser Cys Gly Ser Glu Lys Asp His Phe Tyr Ser Asp Asp Asp Ala Ile Glu Ala Asp Ser Glu Gly Asp Ala Glu Pro Cys Asp Lys Glu Asn Glu Asn Asp Gly Glu Ser Ser Val Gly Thr Asn Met Gly Trp Ala Asp Ala Met Ala Lys Val Leu Asn Lys Lys Thr Pro Glu Ser Lys Pro Thr Ile Leu Val Lys Asn Lys Lys Leu Glu Lys Glu Lys Glu Lys Leu Lys Gln Glu Arg Leu Glu Lys Ile Lys Gln Arg Asp Lys Arg Leu Glu Trp Glu Met Met Cys Arg Val Lys Pro Asp Val Val Gln Asp Lys Glu Thr Glu Arg Asn Leu Gln Arg Ile Ala Thr Arg Gly Val Val Gln Leu Phe Asn Ala Val Gln Lys His Gln Lys Asn Val Asp Glu Lys Val Lys Glu Ala Gly Ser Ser Met Arg Lys Arg Ala 165 ' 170 175 Lys Leu Ile Ser Thr Val Ser Lys Lys Asp Phe Ile Ser Val Leu Arg Gly Met Asp Gly Ser Thr Asn Glu Thr Ala Ser Ser Arg Lys Lys Pro Lys Ala Lys Gln Thr Glu Val Lys Ser Glu Glu Gly Pro Gly Trp Thr Ile Leu Arg Asp Asp Phe Met Met Gly Ala Ser Met Lys Asp Trp Asp Lys Glu Ser Asp Gly Pro Asp Asp Ser Arg Pro Glu Ser Ala Ser Asp Ser Asp Thr <210> 31 <211> 447 <212> PRT
<213> Homo sapiens <400> 31 Met Glu Asn Lys Lys Lys Asp Lys Asp Lys Ser Asp Asp Arg Met Ala Arg Pro Ser Gly Arg Ser Gly His Asn Thr Arg Gly Thr Gly Ser Ser Ser Ser Gly Val Leu Met Val Gly Pro Asn Phe Arg Val Gly Lys Lys Ile Gly Cys Gly Asn Phe Gly Glu Leu Arg Leu Gly Lys Asn Leu Tyr Thr Asn Glu Tyr Val Ala Ile Lys Leu Glu Pro Met Lys Ser Arg Ala Pro Gln Leu His Leu Glu Tyr Arg Phe Tyr Lys Gln Leu Gly Ser Gly Asp Gly Ile Pro Gln Val Tyr Tyr Phe Gly Pro Cys Gly Lys Tyr Asn Ala Met Val Leu Glu Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asp Leu Cys Asp Arg Thr Phe Ser Leu Lys Thr Val Leu Met Ile Ala Ile Gln Leu Ile Ser Arg Met Glu Tyr Val His Ser Lys Asn Leu Ile Tyr Arg Asp Val Lys Pro Glu Asn Phe Leu Ile Gly Arg Pro Gly Asn Lys Thr Gln Gln Val Ile His Ile Ile Asp Phe Gly Leu Ala Lys Glu Tyr Ile Asp Pro Glu Thr Lys Lys His Ile Pro Tyr Arg Glu His Lys Ser Leu Thr Gly Thr Ala Arg Tyr Met Ser Ile Asn Thr His Leu Gly Lys Glu Gln Ser Arg Arg Asp Asp Leu Glu Ala Leu Gly His Met Phe Met Tyr Phe Leu Arg Gly Ser Leu Pro Trp Gln Gly Leu Lys Ala Asp Thr Leu Lys Glu Arg Tyr Gln Lys Ile Gly Asp Thr Lys Arg Ala Thr Pro Ile Glu Val Leu Cys Glu Asn Phe.Pro Glu Met Ala Thr Tyr Leu Arg Tyr Val Arg Arg Leu Asp Phe Phe Glu Lys Pro Asp Tyr Asp Tyr Leu Arg Lys Leu Phe Thr Asp Leu Phe Asp Arg Lys Gly Tyr Met Phe Asp Tyr Glu Tyr Asp Trp Ile Gly Lys Gln Leu Pro Thr Pro Val Gly Ala Val Gln Gln Asp Pro Ala Leu Ser Ser Asn Arg Glu Ala His Gln His Arg Asp Lys Met Gln Gln Ser Lys Asn Gln Ser Ala Asp His Arg Ala Ala Trp Asp Ser Gln Gln Ala Asn Pro His His Leu Arg Ala His Leu Ala Ala Asp Arg His Gly Gly Ser Val Gln Val Val Ser Ser Thr Asn Gly Glu Leu Asn Thr Asp Asp Pro Thr Ala Gly Arg Ser Asn Ala Pro Ile Thr Ala Pro Thr Glu Val Glu Val Met Asp Glu Thr Lys Cys Cys Cys Phe Phe Lys Arg Arg Lys Arg Lys Thr Ile Gln Arg His Lys <210> 32 <211> 437 <212> PRT
<213> Homo Sapiens <400> 32 Met Ala Asp Asp Pro Ser Ala Ala Asp Arg Asn Val Glu Ile Trp Lys Ile Lys Lys Leu Ile Lys Ser Leu Glu Ala Ala Arg Gly Asn Gly Thr Ser Met Ile Ser Leu Ile Ile Pro Pro Lys Asp Gln Ile Ser Arg Val Ala Lys Met Leu Ala Asp Glu Phe Gly Thr Ala Ser Asn Ile Lys Ser Arg Val Asn Arg Leu Ser Val Leu Gly Ala Ile Thr Ser Val Gln Gln Arg Leu Lys Leu Tyr Asn Lys Val Pro Pro Asn Gly Leu Val Val Tyr Cys Gly Thr Ile Val Thr Glu Glu Gly Lys Glu Lys Lys Val Asn Ile Asp Phe Glu Pro Phe Lys Pro Ile Asn Thr Ser Leu Tyr Leu Cys Asp Asn Lys Phe His Thr Glu Ala Leu Thr Ala Leu Leu Ser Asp Asp Ser Lys Phe Gly Phe Ile Val Ile Asp Gly Ser Gly Ala Leu Phe Gly Thr Leu Gln Gly Asn Thr Arg Glu Val Leu His Lys Phe Thr Val Asp Leu Pro Lys Lys His Gly Arg Gly Gly Gln Ser Ala Leu Arg Phe Ala Arg Leu Arg Met Glu Lys Arg His Asn Tyr Val Arg Lys Val Ala Glu Thr Ala Val Gln Leu Phe Ile Ser Gly Asp Lys Val Asn Val Ala Gly Leu Val Leu Ala Gly Ser Ala Asp Phe Lys Thr Glu Leu Ser Gln Ser Asp Met Phe Asp Gln Arg Leu Gln Ser Lys Val Leu Lys Leu Val Asp Ile Ser Tyr Gly Gly Glu Asn Gly Phe Asn Gln Ala Ile Glu Leu Ser Thr Glu Val Leu Ser Asn Val Lys Phe Ile Gln Glu Lys Lys Leu Ile Gly Arg Tyr Phe Asp Glu Ile Ser Gln Asp Thr Gly Lys Tyr Cys Phe Gly Val Glu Asp Thr Leu Lys Ala Leu Glu Met Gly Ala Val Glu Ile Leu Ile Val Tyr Glu Asn Leu Asp Ile Met Arg Tyr Val Leu His Cys Gln Gly Thr Glu Glu Glu Lys Ile Leu Tyr Leu Thr Pro Glu Gln Glu Lys Asp Lys Ser His Phe Thr Asp Lys Glu Thr Gly Gln Glu His Glu Leu Ile Glu Ser Met Pro Leu Leu Glu Trp Phe Ala Asn Asn Tyr Lys Lys Phe Gly Ala Thr Leu Glu Ile Val Thr Asp Lys Ser Gln Glu Gly Ser Gln Phe Val Lys Gly Phe Gly Gly Ile Gly Gly Ile Leu Arg Tyr Arg Val Asp Phe Gln Gly Met Glu Tyr Gln Gly Gly Asp Asp Glu Phe Phe Asp Leu Asp Asp Tyr

Claims (22)

WHAT IS CLAIMED IS:

1. A method of identifying a candidate INR signaling modulating agent, said method comprising the steps of:
(a) providing an assay system comprising a MINK polypeptide or nucleic acid;
(b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity;
and (c) detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate INR signaling modulating agent.

2. The method of Claim 1 wherein the assay system includes a screening assay comprising a MINR polypeptide, and the candidate test agent is a small molecule modulator.

3. The method of Claim 2 wherein the screening assay is a binding assay.

4. The method of Claim 1 wherein the assay system includes a binding assay comprising a MINR polypeptide and the candidate test agent is an antibody.

5. The method of Claim 1 wherein the assay system includes an expression assay comprising a MINR nucleic acid and the candidate test agent is a nucleic acid modulator.

6. The method of Claim 5 wherein the nucleic acid modulator is an antisense oligomer.

7. The method of Claim 6 wherein the nucleic acid modulator is a PMO.

8. The method of Claim 1 wherein the assay system comprises cultured cells or a non-human animal expressing MINR, and wherein the assay system includes an assay that detects an agent-biased change in INR signaling or an output of INR signaling.

9. The method of Claim 8 wherein the assay system comprises cultured cells.

10. The method of Claim 9 wherein the assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an INR
signaling pathway component, kinase activity of an INR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.

11. The method of Claim 8 wherein the assay system comprises a non-human animal.

12. The method of Claim 11 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.

13. The method of Claim 12 wherein the assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.

14. The method of Claim 1, comprising the additional steps of:
(d) providing a second assay system comprising cultured cells or a non-human animal expressing MINR , (e) contacting the second 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 INR signaling modulating agent, and wherein the second assay system includes a second assay that detects an agent-biased change in an activity associated with INR signaling or an output of INR
signaling.

15. The method of Claim 14 wherein the second assay system comprises cultured cells.

16. The method of Claim 15 wherein the second assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an INR
signaling pathway component, kinase activity of an INR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.

17. The method of Claim 14 wherein the second assay system comprises a non-human animal.

18. The method of Claim 17 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.

19. The method of Claim 18 wherein the second assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.

20. A method of modulating INR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds a MINK 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 INR signaling.

22. The method of Claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody.