WO2002031152A2 - Intracellular signaling molecules - Google Patents

Abstract

The invention provides human intracellular signaling molecules (INTSIG) and polynucleotides which identify and encode INTSIG. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of INTSIG.

Description

INTRACELLULAR SIGNALING MOLECULES

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences of intracellular signaling molecules and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, and developmental disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of intracellular signaling molecules.

BACKGROUND OF THE INVENTION

Cell-cell communication is essential for the growth, development, and survival of multicellular organisms. Cells communicate by sending and receiving molecular signals. An example of a molecular signal is a growth factor, which binds and activates a specific transmembrane receptor on the surface of a target cell. The activated receptor transduces the signal intracellularly, thus initiating a cascade of biochemical reactions that ultimately affect gene transcription and cell cycle progression in the target cell.

Intracellular signaling is the process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule. Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered. The intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules such as cyclic nucleotides, calcium-calmodulin, inositol, and various mitogens that regulate protein phosphorylation.

Cells also respond to changing conditions by switching off signals. Many signal transduction proteins are short-lived and rapidly targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Cells also maintain mechanisms to monitor changes in the concentration of denatured or unfolded proteins in membrane-bound extracytoplasmic compartments, including a transmembrane receptor that monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones in the endoplasmic reticulum. Certain proteins in intracellular signaling pathways serve to link or cluster other proteins involved in the signaling cascade. These proteins are referred to as scaffold, anchoring, or adaptor proteins. (For review, see Pawson, T. and J.D. Scott (1997) Science 278:2075-2080.) As many intracellular signaling proteins such as protem kinases and phosphatases have relatively broad substrate specificities, the adaptors help to organize the component signaling proteins into specific biochemical pathways. Many of the above signaling molecules are characterized by the presence of particular domains that promote protein-protein interactions. A sampling of these domains is discussed below, along with other important intracellular messengers.

Intracellular Signaling Second Messenger Molecules

Protem Phosphorylation

Protein kinases and phosphatases play a key role in the intracellular signaling process by controlling the phosphorylation and activation of various signaling proteins. The high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule (ATP) to a particular protein by a protein kinase and removed from that protein by a protein phosphatase. Protem kinases are roughly divided into two groups: those that phosphorylate serine or threonine residues (serine/threonine kinases, STK) and those that phosphorylate tyrosine residues (protem tyrosine kinases, PTK). A few protein kinases have dual specificity for serine/threonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. Hanks (1995) The Protem Kinase Facts Books. Vol 1:7-20, Academic Press, San Diego, CA).

STKs include the second messenger dependent protein kinases such as the cyclic -AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smootii muscle contraction, glycogen breakdown, and neurotransmission; and the mitogen-activated protein kinases (MAP) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, KJ. et al. (1994) Harrison's Principles of Internal Medicine. McGraw-Hill, New York, NY, pp. 416-431, 1887). PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor

PTKs. Transmembrane PTKs are receptors for most growth factors. Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Charbonneau H. and N.K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-493).

An additional family of protein kinases previously thought to exist only in prokaryotes is the histidine protein kinase family (HPK). HPKs bear little homology with mammalian STKs or PTKs but have distinctive sequence motifs of their own (Davie, J.R. et al. (1995) J. Biol. Chem. 270:19861-19867). A histidine residue in the N-terminal half of the molecule (region I) is an autophosphorylation site. Three additional motifs located in the C-terminal half of the molecule include an invariant asparagine residue in region π and two glycine-rich loops characteristic of nucleotide binding domains in regions Ul and IV. Recently a branched chain alpha-ketoacid dehydrogenase kinase has been found with characteristics of HPK in rat (Davie et al., supra).

Protein phosphatases regulate the effects of protein kinases by removing phosphate groups from molecules previously activated by kinases. The two principal categories of protein phosphatases are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine phosphatases (PTPs). PPs dephosphorylate phosphoserine/threonine residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508). PTPs reverse the effects of protem tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonneau and Tonks, supra). As previously noted, many PTKs are encoded by oncogenes, and oncogenesis is often accompanied by increased tyrosine phosphorylation activity. It is therefore possible that PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific inhibition of PTPs can enhance cell transformation (Charbonneau and Tonks, supra). Phospholipid and Inositol-phosphate Signaling

Inositol phospholipids (phosphoinositides) are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane. This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma membrane to the biphosphate state (PIP₂) by inositol kinases. Simultaneously, the G-protein linked receptor binding stimulates a trimeric G-protein which in turn activates a phosphoinositide-specific phospholipase C-β. Phospholipase C-β then cleaves PIP₂ into two products, inositol triphosphate (IP₃) and diacylglycerol. These two products act as mediators for separate signaling events. IP₃ diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), while diaacylglycerol remains in the membrane and helps activate protein kinase C, a serine-threonine kinase that phosphorylates selected proteins in the target cell. The calcium response initiated by IP₃ is terminated by the dephosphorylation of IP₃ by specific inositol phosphatases. Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation.

Oxysterols are oxygenated derivatives of cholesterol and have a wide range of biological activities. Oxysterols mediate cholesterol homeostasis, steroid biosynthesis and sphingolipid metabolism within the cell, but can also diffuse through the plasma membrane and act as extracellular messengers, affecting such processes as platelet aggregation, cell growth and apoptosis. Oxysterols interact with a number of receptors, including the oxysterol binding protein (OSBP), the sterol regulatory element binding protein, the cellular nucleic acid binding protein, the LXR nuclear hormone receptors, and the LDL receptor (for a review, see Schroepfer, G.J. (2000) Physiol. Rev. 80:361-554). OSBP is a high-affinity intracellular receptor for a variety of oxysterols that down-regulate cholesterol synthesis and stimulate cholesterol esterification. Upon ligand binding, OSBP translocates from the cytoplasm to the Golgi. This movement seems to be dependent on the presence of a pleckstrin homology domain (Lagace, T.A. et al. (1997) Biochem. J. 326:205-213). The oxysterol-induced apoptosis of leukemic T-cells seems to be mediated by OSBP occupancy (Bakos, J.T. et al. (1993) J. Steroid Biochem. Mol. Biol. 46:415-426). Cyclic Nucleotide Signaling Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. In particular, cyclic-AMP dependent protein kinases (PKA) are thought to account for all of the effects of cAMP in most mammalian cells, including various hormone-induced cellular responses. Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca²⁺-specific channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter. Thus adenylyl cyclase, which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to β-adrenergic receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the Ca²⁺-specific channels and recovery of the dark state in the eye. In contrast, hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the opposite of these and other effects mediated by increased cyclic nucleotide levels. PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins. At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, J.A. (1995) Physiol. Rev. 75:725-748). PDE inhibitors have been found to be particularly useful in treating various clinical disorders. Rolipram, a specific inhibitor of PDE4, has been used in the treatment of depression, and similar inhibitors are undergoing evaluation as anti-inflammatory agents. Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K.H. and CP. Page (1995) Eur. Respir. J. 8:996-1000). G-Protein Signaling

Guanine nucleotide binding proteins (G-proteins) are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCRs), and intracellular second messengers such as cAMP and Ca²⁺. G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to GTP, inducing an "active" state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca²⁺ into the cytosol from the ER, which, in turn, regulate phosphorylation and activation of other intracellular proteins. Recycling of the G-protein to the inactive state involves hydrolysis of the bound GTP to GDP by a GTPase activity in the G-protein. (See Alberts, B. et al. (1994) Molecular Biology of the Cell Garland Publishing, Inc. New York, NY, pp.734-759.) Two structurally distinct classes of G- proteins are recognized: heterotrimeric G-proteins, consisting of three different subunits, and monomeric, low molecular weight (LMW), G-proteins consisting of a single polypeptide chain. The three polypeptide subunits of heterotrimeric G-proteins are the α, β, and γ subunits. The subunit binds and hydrolyzes GTP. The β and γ subunits form a tight complex that anchors the protein to the inner side of the plasma membrane. The β subunits, also known as G-β proteins or β transducins, contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. Mutations and variant expression of β transducfn proteins are linked with various disorders (Neer, E. J. et al. (1994) Nature 371 :297-300; Margottin, F. et al. (1998) Mol. Cell. 1:565-574).

LMW GTP-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the α subunit of the heterotrimeric G-proteins, are able to bind and hydrolyze GTP, thus cycling between an inactive and an active state. At least sixty members of the LMW G-protein superfamily have been identified and are currently grouped into the six subfamilies of ras, rho, arf, sari, ran, and rab. Activated ras genes were initially found in human cancers, and subsequent studies confirmed that ras function is critical in determining whether cells continue to grow or become differentiated. Other members of the LMW G-protein superfamily have roles in signal transduction that vary with the function of the activated genes and the locations of the G-proteins.

Guanine nucleotide exchange factors regulate the activities of LMW G-proteins by determining whether GTP or GDP is bound. GTPase-activating protein (GAP) binds to GTP-ras and induces it to hydrolyze GTP to GDP. In contrast, guanine nucleotide releasing protein (GNRP) binds to GDP-ras and induces the release of GDP and the binding of GTP.

Other regulators of G-protein signaling (RGS) also exist that act primarily by negatively regulating the G-protein pathway by an unknown mechanism (Druey, K.M. et al. (1996) Nature 379:742-746). Some 15 members of the RGS family have been identified. RGS family members are related structurally through similarities in an approximately 120 amino acid region termed the RGS domain and functionally by their ability to inhibit the interleukin (cytokine) induction of MAP kinase in cultured mammalian 293T cells (Druey et al., supra). Calcium Signaling Molecules

Ca²⁺ is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP. Ca²⁺ can enter the cytosol by two pathways, in response to extracellular signals. One pathway acts primarily in nerve signal transduction where Ca²⁺ enters a nerve terminal through a voltage-gated Ca²⁺ channel. The second is a more ubiquitous pathway in which Ca²⁺ is released from the ER into the cytosol in response to binding of an extracellular signaling molecule to a receptor. Ca²⁺ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways. Ca²⁺ also binds to specific Ca ⁺-binding proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels. CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M.R. et al. (1996) Guidebook to Calcium-binding Proteins. Oxford University Press, Oxford, UK, pp. 15-20). Some Ca²⁺ binding proteins are characterized by the presence of one or more EF-hand Ca²⁺ binding motifs, which are comprised of 12 amino acids flanked by α-helices (Celio, supra). The regulation of CBPs has implications for the control of a variety of disorders. Calcineurin, a CaM-regulated protein phosphatase, is a target for inhibition by the immunosuppressive agents cyclosporin and FK506. This indicates the importance of calcineurin and CaM in the immune response and immune disorders (Schwaninger M. et al. (1993) J. Biol Chem. 268:23111-23115). The level of CaM is increased several-fold in tumors and tumor-derived cell lines for various types of cancer (Rasmussen, CD. and A.R. Means (1989) Trends Neurosci. 12:433-438). The annexins are a family of calcium-binding proteins that associate with the cell membrane (Towle, CA. and B.V. Treadwell (1992) J. Biol. Chem. 267:5416-5423). Annexins reversibly bind to negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium dependent manner. Annexins participate in various processes pertaining to signal transduction at the plasma membrane, including membrane-cytoskeleton interactions, phospholipase inhibition, anticoagulation, and membrane fusion. Annexins contain four to eight repeated segments of about 60 residues. Each repeat folds into five alpha helices wound into a right-handed superhelix. Signaling Complex Protein Domains

PDZ domains were named for three proteins in which this domain was initially discovered. These proteins include PSD-95 (postsynaptic density 95), Dig (Drosophila lethal(l)discs large-1), and ZO-1 (zonula occludens-1). These proteins play important roles in neuronal synaptic transmission, tumor suppression, and cell junction formation, respectively. Since the discovery of these proteins, over sixty additional PDZ-containing proteins have been identified in diverse prokaryotic and eukaryotic organisms. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For a review of PDZ domain-containing proteins, see Ponting, CP. et al. (1997) Bioessays 19:469-479.) A large proportion of PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels. However, PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serine/threonine kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS). Generally, about one to three PDZ domains are found in a given protein, although up to nine PDZ domains have been identified in a single protein. The glutamate receptor interacting protein (GRIP) contains seven PDZ domains. GRIP is an adaptor that links certain glutamate receptors to other proteins and may be responsible for the clustering of these receptors at excitatory synapses in the brain (Dong, H. et al. (1997) Nature 386:279-284). The Drosophila scribble (SCRIB) protein contains both multiple PDZ domains and leucine-rich repeats. SCRIB is located at the epithelial septate junction, which is analogous to the vertebrate tight junction, at the boundary of the apical and basolateral cell surface. SCRIB is involved in the distribution of apical proteins and correct placement of adherens junctions to the basolateral cell surface (Bilder, D. and N. Perrimon (2000) Nature 403:676-680). The PX domain is an example of a domain specialized for promoting protein-protein interactions. The PX domain is found in sorting nexins and in a variety of other proteins, including the PhoX components of NADPH oxidase and the Cpk class of phosphatidylinositol 3-kinase. Most PX domains contain a polyproline motif which is characteristic of SH3 domain-binding proteins (Ponting, CP. (1996) Protein Sci. 5:2353-2357). SH3 domain-mediated interactions involving the PhoX components of NADPH oxidase play a role in the formation of the NADPH oxidase multi-protein complex (Leto, T.L. et al. (1994) Proc. Natl. Acad. Sci. USA 91:10650-10654; Wilson, L. et al. (1997) Inflamm. Res. 46:265-271).

The SH3 domain is defined by homology to a region of the proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase. SH3 is a small domain of 50 to 60 amino acids that interacts with proline-rich ligands. SH3 domains are found in a variety of eukaryotic proteins involved in signal transduction, cell polarization, and membrane-cytoskeleton interactions. In some cases, SH3 domain- containing proteins interact directly with receptor tyrosine kinases. For example, the SLAP-130 protein is a substrate of the T-cell receptor (TCR) stimulated protein kinase. SLAP-130 interacts via its SH3 domain with the protein SLP-76 to affect the TCR-induced expression of interleukin-2 (Musci, M. A. et al. (1997) J. Biol. Chem. 272: 11674-11677). Another recently identified SH3 domain protein is macrophage actin-associated tyrosine-phosphorylated protein (MAYP) which is phosphorylated during the response of macrophages to colony stimulating factor- 1 (CSF-1) and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton (Yeung, Y.-G. et al. (1998) J. Biol. Chem. 273:30638-30642). The structure of the SH3 domain is characterized by two antiparallel beta sheets packed against each other at right angles. This packing forms a hydrophobic pocket lined with residues that are highly conserved between different SH3 domains. This pocket makes critical hydrophobic contacts with proline residues in the ligand (Feng, S. et al. (1994) Science 266:1241- 1247). A novel domain, called the WW domain, resembles the SH3 domain in its ability to bind proline-rich ligands. This domain was originally discovered in dystrophin, a cytoskeletal protein with direct involvement in Duchenne muscular dystrophy (Bork, P. and M. Sudol (1994) Trends Biochem. Sci. 19:531-533). WW domains have since been discovered in a variety of intracellular signaling molecules involved in development, cell differentiation, and cell proliferation. The structure of the WW domain is composed of beta strands grouped around four conserved aromatic residues, generally tryptophan.

Like SH3, the SH2 domain is defined by homology to a region of c-Src. SH2 domains interact directly with phospho-tyrosine residues, thus providing an immediate mechanism for the regulation and transduction of receptor tyrosine kinase-mediated signaling pathways. For example, as many as ten distinct SH2 domains are capable of binding to phosphorylated tyrosine residues in the activated PDGF receptor, thereby providing a highly coordinated and finely tuned response to ligand-mediated receptor activation. (Reviewed in Schaffhausen, B. (1995) Biochim. Biophys. Acta. 1242:61-75.)

The GSG domain (GRP33, Sam68, GLD-1) and the KH domain (an RNA binding domain), are found within Sam68, a 68-kDa Src substrate associated during mitosis protein, which is an RNA- binding protein with signaling properties. It is known to be a substrate for Src-family tyrosine kinases during mitosis and associates with various SH3 and SH2 domain-containing signaling molecules. SLM-1 and SLM-2 (Sam68-like mammalian) proteins have sequence identity with Sam68, also contain the GSG domain, have proline-rich motifs, arginine-gylcine repeats, and a C-terminal tyrosine-rich region. SLM-1 is a Src substrate during mitosis, suggesting a possible involvement in the steps of mitosis. It has been suggested by Di Fruscio et al. that Sam68/SLM defines a family in which the members have the potential to link tyrosine kinase signaling cascades with some aspects of RNA metabolism, possibly as multifunctional adapter proteins during mitosis (Di Fruscio, M. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2710-2715.) The pleckstrin homology (PH) domain was originally identified in pleckstrin, the predominant substrate for protein kinase C in platelets. Since its discovery, this domain has been identified in over 90 proteins involved in intracellular signaling or cytoskeletal organization. Proteins containing the pleckstrin homology domain include a variety of kinases, phospholipase-C isoforms, guanine nucleotide release factors, and GTPase activating proteins. For example, members of the FGD1 family contain both Rho-guanine nucleotide exchange factor (GEF) and PH domains, as well as a FYVE zinc finger domain. FGD1 is the gene responsible for faciogenital dysplasia, an inherited skeletal dysplasia (Pasteris, N.G. and J.L. Gorski (1999) Genomics 60:57-66). Many PH domain proteins function in association with the plasma membrane, and this association appears to be mediated by the PH domain itself. PH domains share a common structure composed of two antiparallel beta sheets flanked by an amphipathic alpha helix. Variable loops connecting the component beta strands generally occur within a positively charged environment and may function as ligand binding sites (Lemmon, M.A. et al. (1996) Cell 85:621-624). Ankyrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular signaling functions. For example, ANK repeats are found in proteins involved in cell proliferation such as kinases, kinase inhibitors, tumor suppressors, and cell cycle control proteins. (See, for example, Kalus, W. et al. (1997) FEBS Lett. 401 : 127-132; Ferrante, A.W. et al. (1995) Proc. Natl. Acad. Sci. USA 92:1911-1915.) These proteins generally contain multiple ANK repeats, each composed of about 33 amino acids. Myotrophin is an ANK repeat protein that plays a key role in the development of cardiac hypertrophy, a contributing factor to many heart diseases. Structural studies show that the myotrophin ANK repeats, like other ANK repeats, each form a helix-turn-helix core preceded by a protruding "tip." These tips are of variable sequence and may play a role in protein- protein interactions. The helix-turn-helix region of the ANK repeats stack on top of one another and are stabilized by hydrophobic interactions (Yang, Y. et al. (1998) Structure 6:619-626).

The tetratricopeptide repeat (TPR) is a 34 amino acid repeated motif found in organisms from bacteria to humans. TPRs are predicted to form ampipathic helices, and appear to mediate protein- protein interactions. TPR domains are found in CDC16, CDC23, and CDC27, members of the the anaphase promoting complex which targets proteins for degradation at the onset of anaphase. Other processes involving TPR proteins include cell cycle control, transcription repression, stress response, and protein kinase inhibition (Lamb, J.R. et al. (1995) Trends Biochem. Sci. 20:257-259). The armadillo/beta-catenin repeat is a 42 amino acid motif which forms a superhelix of alpha helices when tandemly repeated. The structure of the armadillo repeat region from beta-catenin revealed a shallow groove of positive charge on one face of the superhelix, which is a potential binding surface. The armadillo repeats of beta-catenin, plakoglobin, and pl20^cas bind the cytoplasmic domains of cadherins. Beta-catenin/cadherin complexes are targets of regulatory signals that govern cell adhesion and mobility (Huber, A.H. et al. (1997) Cell 90:871-882).

Eight tandem repeats of about 40 residues (WD-40 repeats), each containing a central Trp-Asp motif, make up beta-transducin (G-beta), which is one of the three subunits (alpha, beta, and gamma) of the guanine nucleotide-binding proteins (G proteins). In higher eukaryotes G-beta exists as a small multigene family of highly conserved proteins of about 340 amino acid residues. The discovery of new intracellular signaling molecules, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, and developmental disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of intracellular signaling molecules.

SUMMARY OF THE INVENTION

The invention features purified polypeptides, intracellular signaling molecules, referred to collectively as "INTSIG" and individually as "INTSIG-1," 'TNTSIG-2," 'TNTSIG-3," 'TNTSIG-4," "ΓNTSIG-5," "ΓNTSIG-6," "ΓNTSIG-7," "ΓNTSIG-8," "ΓNTSIG-9," "ΓNTSIG-IO," "ΓNTSIG-Π," 'TNTSIG-12," "INTSIG-13," "INTSIG-14," "INTSIG-15," 'TNTSIG-16," "INTSIG-17," 'TNTSIG-

18," "INTSIG-19," and 'TNTSIG-20." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-20.

The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D3 NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ DD NO: 1-20. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:21-40. Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-20. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.

Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.

Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide_. having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides. The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition.

The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition. Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional INTSIG, comprising administering to a patient in need of such treatment the composition.

The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ DD NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.

The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ DD NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ DD NO: 1-20. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.

The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ DD NO:21-40, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.

The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ DD NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ DD NO:21-40, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ DD NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ DD NO:21-40, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.

Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is also shown.

Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.

Table 5 shows the representative cDNA library for polynucleotides of the invention.

Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5. Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.

DESCRIPTION OF THE INVENTION Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DEFINITIONS

"INTSIG" refers to the amino acid sequences of substantially purified INTSIG obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

The term "agonist" refers to a molecule which intensifies or mimics the biological activity of INTSIG. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of INTSIG either by directly interacting with INTSIG or by acting on components of the biological pathway in which INTSIG participates.

An "allelic variant" is an alternative form of the gene encoding INTSIG. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding INTSIG include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as INTSIG or a polypeptide with at least one functional characteristic of INTSIG. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding INTSIG, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding INTSIG. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent INTSIG. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of INTSIG is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may ^' include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine. The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule. "Amplification" relates to the production of additional copies of a nucleic acid sequence.

Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.

The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of INTSIG. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of rNTSIG either by directly interacting with INTSIG or by acting on components of the biological pathway in which INTSIG participates.

The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind INTSIG polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH₂), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)

The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610). The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.

The term "antisense" refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule. The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic" refers to the capability of the natural, recombinant, or synthetic INTSIG, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. "Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.

A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.

Compositions comprising polynucleotide sequences encoding INTSIG or fragments of INTSIG may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.). "Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.

"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions. Original Residue Conservative Substitution Ala Gly, Ser

Arg His, Lys

Asn Asp, Gin, His

Asp Asn, Glu

Cys Ala, Ser Gin Asn, Glu, His

Glu Asp, Gin, His

Gly Ala

His Asn, Arg, Gin, Glu

He Leu, Val Leu He, Val

Lys Arg, Gin, Glu

Met Leu, He

Phe His, Met, Leu, Trp, Tyr

Ser Cys, Thr Thr Ser, Val

Trp Phe, Tyr

Tyr His, Phe, Trp VaT lie, Leu, Thr

Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

The term "derivative" refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.

"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.

"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures,°thus allowing acceleration of the evolution of new protein functions.

A "fragment" is a unique portion of INTSIG or the polynucleotide encoding INTSIG which is identical in sequence to but shorter in length than the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

A fragment of SEQ DD NO :21-40 comprises a region of unique polynucleotide sequence that specifically identifies SEQ DD NO:21-40, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ DD NO:21-40 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ DD NO:21-40 from related polynucleotide sequences. The precise length of a fragment of SEQ DD NO:21-40 and the region of SEQ DD NO:21-40 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. A fragment of SEQ DD NO: 1-20 is encoded by a fragment of SEQ DD NO:21-40. A fragment of SEQ DD NO: 1-20 comprises a region of unique amino acid sequence that specifically identifies SEQ DD NO: 1-20. For example, a fragment of SEQ DD NO: 1-20 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ DD NO: 1-20. The precise length of a fragment of SEQ DD NO: 1-20 and the region of SEQ DD NO: 1-20 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

A "full length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence. "Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS 8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.

Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: -2

Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50 Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ DD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and_hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.

Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=l, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.

Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap x drop-off: 50 Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ DD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

"Human artificial chromosomes" (HACs) are linear rmcrocrrromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.

The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 μg/ml sheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thennal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating T_m and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. 2^nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.

The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C₀t or R₀t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed). The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively. "Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.

An "immunogenic fragment" is a polypeptide or oligopeptide fragment of INTSIG which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of INTSIG which is useful in any of the antibody production methods disclosed herein or known in the art.

The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.

The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray. The term "modulate" refers to a change in the activity of INTSIG. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of INTSIG.

The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.

"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.

"Post-translational modification" of an INTSIG may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of INTSIG.

"Probe" refers to nucleic acid sequences encoding INTSIG, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR). Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.

Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. 2^nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biology. Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA). Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "misprinting library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarray s. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.

A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell. Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.

A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.

"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.

An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

The term "sample" is used in its broadest sense. A sample suspected of containing INTSIG, nucleic acids encoding INTSIG, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc. The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.

"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

A "transcript image" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time. "Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.

A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at^'least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state. A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.

THE INVENTION

The invention is based on the discovery of new human intracellular signaling molecules (INTSIG), the polynucleotides encoding INTSIG, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, and developmental disorders.

Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project DD). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ DD NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide DD) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ DD NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide DD) as shown. Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ DD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide DD) for polypeptides of the invention. Column 3 shows the GenBank identification number (Genbank DD NO:) of the nearest GenBank homolog. Column 4 shows the probability score for the match between each polypeptide and its GenBank homolog. Column 5 shows the annotation of the GenBank homolog along with relevant citations where applicable, all of which are expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ DD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide DD) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIF'S program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are intracellular signaling molecules. For example, SEQ DD NO:2 is 37% identical to Schizosaccharomyces pombe beta transducin (GenBank DD g3451308) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.1e-146, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ DD NO:2 also contains a G-beta repeat WD40 domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from MOTIFS analysis provides further corroborative evidence that SEQ DD NO:2 is a transducin.

In an alternative example, SEQ DD NO:6 is 85% identical to murine nedd-1 protein (GenBank DD g286103) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ DD NO:6 also contains a WD domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTTFS analyses provide further corroborative evidence that SEQ DD NO: 6 is a protein involved in signal transduction. In an alternative example, SEQ ID NO: 10 is 51% identical to the human rho GTPase activating protein pi 15 (GenBank DD g840786) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.2e-211, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ DD NO: 10 contains a rhoGAP domain, an SH3 domain, and a Fes/CIP4 actin cytoskeleton regulatory protein domain, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) The presence of these domains is confirmed by BLIMPS and MOTTFS analyses, providing further corroborative evidence that SEQ DD NO: 10 is a GTPase activating protein. In an alternative example, SEQ DD NO: 16 is 49% identical to the human ras-related tumor suppressor NOEY2 (GenBank DD g4100355) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.6e-45, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ DD NO: 16 also contains a ras family domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ DD NO: 16 is a signaling protein of the ras family.

In an alternative example, SEQ DD NO:20 is 95% identical to murine SLM-1 protein (GenBank DD g4426613) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.1e-183, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ DD NO:20 also contains a KH domain (E- value is 0.11) as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) SEQ DD NO:l, SEQ DD NO:3-5, SEQ DD NO:7-9, SEQ DD NO: 11-13, SEQ DD NO: 14-15, and SEQ DD NO:17-19 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ DD NO:1-20 are described in Table 7.

As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ DD NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide DD) for each polynucleotide of the invention. Column 3 shows the length of each polynucleotide sequence in basepairs. Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ DD NO:21-40 or that distinguish between SEQ DD NO:21-40 and related polynucleotide sequences. Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.

The identification numbers in Column 5 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding cDNA libraries. For example, 105283R6 is the identification number of an Incyte cDNA sequence, and BMARNOT02 is the cDNA library from which it is derived. Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 71206562V1). Alternatively, the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g3034305) which contributed to the assembly of the full length polynucleotide sequences. In addition, the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the identification numbers in column 5 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, ¥L_XXXXXXJ!₁_N₂_YYYYY_N₃_N₄ represents a "stitched" sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N_1A3__„, if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the identification numbers in column 5 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example,

FLXXXXXX_gAAAAA_gBBBBB_l_N is the identification number of a "stretched" sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "ΝM," "ΝP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.

Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6. The invention also encompasses INTSIG variants. A preferred INTSIG variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the INTSIG amino acid sequence, and which contains at least one functional or structural characteristic of INTSIG.

The invention also encompasses polynucleotides which encode INTSIG. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ DD NO:21-40, which encodes INTSIG. The polynucleotide sequences of SEQ DD NO:21-40, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

The invention also encompasses a variant of a polynucleotide sequence encoding INTSIG. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding INTSIG. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ DD NO:21- 40 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ DD NO:21-40. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of INTSIG.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding INTSIG, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring INTSIG, and all such variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode INTSIG and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring INTSIG under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding INTSIG or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding INTSIG and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encode INTSIG and INTSIG derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding INTSIG or any fragment thereof.

Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ DD NO:21-40 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507- 511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."

Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology. John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)

The nucleic acid sequences encoding INTSIG may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)

Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C

When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate^' software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode INTSIG may be cloned in recombinant DNA molecules that direct expression of INTSIG, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express INTSIG.

The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter INTSIG-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.

5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of INTSIG, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner. In another embodiment, sequences encoding INTSIG may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.) Alternatively, INTSIG itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties. WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of INTSIG, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active INTSIG, the nucleotide sequences encoding INTSIG or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding INTSIG. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding INTSIG. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding INTSIG and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding INTSIG and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to contain and express sequences encoding INTSIG. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, I.M. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding INTSIG. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding INTSIG can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding INTSIG into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of INTSIG are needed, e.g. for the production of antibodies, vectors which direct high level expression of INTSIG may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used. Yeast expression systems may be used for production of INTSIG. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, CA. et al. (1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of INTSIG. Transcription of sequences encoding INTSIG may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding INTSIG may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses INTSIG in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV- based vectors may also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355.) For long term production of recombinant proteins in mammalian systems, stable expression of

INTSIG in cell lines is preferred. For example, sequences encoding INTSIG can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a ι selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in fk and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C and R.C Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding INTSIG is inserted within a marker gene sequence, transformed cells containing sequences encoding INTSIG can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding INTSIG under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encoding INTSIG and that express INTSIG may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of INTSIG using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on INTSIG is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.)

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding INTSIG include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Alternatively, the sequences encoding INTSIG, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding INTSIG may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode INTSIG may be designed to contain signal sequences which direct secretion of INTSIG through a prokaryotic or eukaryotic cell membrane. In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein. In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding INTSIG may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric INTSIG protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of INTSIG activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transf erase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the INTSIG encoding sequence and the heterologous protein sequence, so that INTSIG may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeled INTSIG may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, ³⁵S-methionine.

INTSIG of the present invention or fragments thereof may be used to screen for compounds that specifically bind to INTSIG. At least one and up to a plurality of test compounds may be screened for specific binding to INTSIG. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

In one embodiment, the compound thus identified is closely related to the natural ligand of INTSIG, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a ^■ natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunology 1(2): Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which INTSIG binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express INTSIG, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or K coli. Cells expressing INTSIG or cell membrane fractions which contain INTSIG are then contacted with a test compound and binding, stimulation, or inhibition of activity of either INTSIG or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with INTSIG, either in solution or affixed to a solid support, and detecting the binding of INTSIG to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support. INTSIG of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of INTSIG. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for INTSIG activity, wherein INTSIG is combined with at least one test compound, and the activity of INTSIG in the presence of a test compound is compared with the activity of INTSIG in the absence of the test compound. A change in the activity of INTSIG in the presence of the test compound is indicative of a compound that modulates the activity of INTSIG. Alternatively, a test compound is combined with an in vitro or cell-free system comprising INTSIG under conditions suitable for INTSIG activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of INTSIG may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.

In another embodiment, polynucleotides encoding INTSIG or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.

Polynucleotides encoding INTSIG may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding INTSIG can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding INTSIG is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress INTSIG, e.g., by secreting INTSIG in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS

Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of INTSIG and intracellular signaling molecules. In addition, the expression of INTSIG is closely associated with brain and neurological tissues including thoracic dorsal root ganglion tissue, dermal tissue, reproductive tissue, digestive and hemic/immune tissue, diseased prostate tissue, and tumorous tissues including bladder, tongue, and testicular. Therefore, INTSIG appears to play a role in cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, and developmental disorders. In the treatment of disorders associated with increased INTSIG expression or activity, it is desirable to decrease the expression or activity of INTSIG. In the treatment of disorders associated with decreased INTSIG expression or activity, it is desirable to increase the expression or activity of INTSIG.

Therefore, in one embodiment, INTSIG or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of INTSIG. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (ADDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia,

Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and

Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (ADDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphaj-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno- occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, ectopic pregnancy, teratogenesis; cancer of the breast, fibrocystic breast disease, galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, gynecomastia, hypergonadotropic and hypogonadotropic hypogonadism, pseudohermaphroditism, azoospermia, premature ovarian failure, acrosin deficiency, delayed puperty, retrograde ejaculation and anejaculation, haemangioblastomas, cystsphaeochromocytomas, paraganglioma, cystadenomas of the epididymis, and endolymphatic sac tumours; and a developmental disorder such as renal tubular acidosis, anemia, Gushing' s syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss. In another embodiment, a vector capable of expressing INTSIG or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of INTSIG including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantially purified INTSIG in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of INTSIG including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity of INTSIG may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of INTSIG including, but not limited to, those listed above. In a further embodiment, an antagonist of INTSIG may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of INTSIG. Examples of such disorders include, but are not limited to, those cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, and developmental disorders described above. In one aspect, an antibody which specifically binds INTSIG may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express INTSIG.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding INTSIG may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of INTSIG including, but not limited to, those described above. In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

An antagonist of INTSIG may be produced using methods which are generally known in the art. In particular, purified INTSIG may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind INTSIG. Antibodies to INTSIG may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with INTSIG or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to INTSIG have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of INTSIG amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.

Monoclonal antibodies to INTSIG may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimeric antibodies," such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce INTSIG-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for INTSIG may also be generated. For example, such fragments include, but are not limited to, F(ab')₂ fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between INTSIG and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering INTSIG epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for INTSIG. Affinity is expressed as an association constant, K_a, which is defined as the molar concentration of INTSIG-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K, determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple INTSIG epitopes, represents the average affinity, or avidity, of the antibodies for INTSIG. The K_a determined for a preparation of monoclonal antibodies, which are monospecific for a particular INTSIG epitope, represents a true measure of affinity. High-affinity antibody preparations with K, ranging from about 10⁹ to 10¹² L/mole are preferred for use in immunoassays in which the INTSIG- antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_a ranging from about 10⁶ to 10⁷ L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of INTSIG, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach. IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).

The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of INTSIG-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encoding INTSIG, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding INTSIG. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding INTSIG. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.)

In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13): 1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull. 51(l):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.)

In another embodiment of the invention, polynucleotides encoding INTSIG may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCDD)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene

Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VDI or Factor IX deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, I.M. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trvpanosoma cruzi). In the case where a genetic deficiency in INTSIG expression or regulation causes disease, the expression of INTSIG from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders caused by deficiencies in INTSIG are treated by constructing mammalian expression vectors encoding INTSIG and introducing these vectors by mechanical means into INTSIG-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of INTSIG include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). INTSIG may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding INTSIG from a normal individual. Commercially available liposome transformation kits (e.g., the PERFECT LIPDD

TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to INTSIG expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding INTSIG under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cz^'s-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines, and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4⁺ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding INTSIG to cells which have one or more genetic abnormalities with respect to the expression of INTSIG. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding INTSIG to target cells which have one or more genetic abnormalities with respect to the expression of INTSIG. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing INTSIG to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.

In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding INTSIG to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for INTSIG into the alphavirus genome in place of the capsid-coding region results in the production of a large number of INTSIG-coding RNAs and the synthesis of high levels of INTSIG in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of INTSIG into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.

Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Furura Publishing, Mt. Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding INTSIG.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding INTSIG. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability and half -life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases. An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding INTSIG. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased INTSIG expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding INTSIG may be therapeutically useful, and in the treatment of disorders associated with decreased INTSIG expression or activity, a compound which specifically promotes expression of the polynucleotide encoding INTSIG may be therapeutically useful.

At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding INTSIG is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding INTSIG are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding INTSIG. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomvces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, ML. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of INTSIG, antibodies to INTSIG, and mimetics, agonists, antagonists, or inhibitors of INTSIG.

The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast- acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.

Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising INTSIG or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, INTSIG or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572). For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient, for example

INTSIG or fragments thereof, antibodies of INTSIG, and agonists, antagonists or inhibitors of ENTTSIG, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₃₀ (the dose therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS

In another embodiment, antibodies which specifically bind INTSIG may be used for the diagnosis of disorders characterized by expression of INTSIG, or in assays to monitor patients being treated with INTSIG or agonists, antagonists, or inhibitors of INTSIG. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for INTSIG include methods which utilize the antibody and a label to detect INTSIG in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used. A variety of protocols for measuring INTSIG, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of INTSIG expression. Normal or standard values for INTSIG expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to INTSIG under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of INTSIG expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding INTSIG may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of INTSIG may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of INTSIG, and to monitor regulation of INTSIG levels during therapeutic intervention. In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding INTSIG or closely related molecules may be used to identify nucleic acid sequences which encode INTSIG. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding INTSIG, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the INTSIG encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ DD NO:21-40 or from genomic sequences including promoters, enhancers, and introns of the INTSIG gene.

Means for producing specific hybridization probes for DNAs encoding ESfTSIG include the cloning of polynucleotide sequences encoding INTSIG or INTSIG derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding INTSIG may be used for the diagnosis of disorders associated with expression of INTSIG. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (ADDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjδgren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt- Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory- Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (ADDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphaj-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, ectopic pregnancy, teratogenesis; cancer of the breast, fibrocystic breast disease, galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, gynecomastia, hypergonadotropic and hypogonadotropic hypogonadism, pseudohermaphroditism, azoospermia, premature ovarian failure, acrosin deficiency, delayed puperty, retrograde ejaculation and anejaculation, haemangioblastomas, cystsphaeochromocytomas, paraganglioma, cystadenomas of the epididymis, and endolymphatic sac tumours; and a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss. The polynucleotide sequences encoding INTSIG may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiforrnat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered INTSIG expression. Such qualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding INTSIG may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding INTSIG may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding INTSIG in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associated with expression of INTSIG, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding INTSIG, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer. Additional diagnostic uses for oligonucleotides designed from the sequences encoding INTSIG may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding INTSIG, or a fragment of a polynucleotide complementary to the polynucleotide encoding INTSIG, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding INTSIG may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding INTSIG are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer- based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).

Methods which may also be used to quantify the expression of INTSIG include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile. In another embodiment, INTSIG, fragments of INTSIG, or antibodies specific for INTSIG may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity. Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.

Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.

In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample. Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for , example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.

A proteomic profile may also be generated using antibodies specific for INTSIG to quantify the levels of INTSIG expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element. Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases. In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.

In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and Heller, MJ. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference. In another embodiment of the invention, nucleic acid sequences encoding INTSIG may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMDV1) World Wide Web site. Correlation between the location of the gene encoding INTSIG on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts. In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, INTSIG, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between INTSIG and the agent being tested may be measured.

Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with INTSIG, or fragments thereof, and washed. Bound INTSIG is then detected by methods well known in the art. Purified INTSIG can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding INTSIG specifically compete with a test compound for binding INTSIG. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with INTSIG.

In additional embodiments, the nucleotide sequences which encode INTSIG may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The disclosures of all patents, applications and publications, mentioned above and below, are expressly incorporated by reference herein: U.S. Ser. No. 60/240,871, U.S. Ser. No. 60/244,723, U.S. Ser. No. 60/249,402, U.S. Ser. No. 60/252,622, and U.S. Ser. No. 60/255,622.

EXAMPLES I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LJ-FESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods. Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).

In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or pTNCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C

Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VDI.

The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM. (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples TV and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).

The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ DD NO:21-40. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 4.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative intracellular signaling molecules were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode intracellular signaling molecules, the encoded polypeptides were analyzed by querying against PFAM models for intracellular signaling molecules. Potential intracellular signaling molecules were also identified by homology to Incyte cDNA sequences that had been annotated as intracellular signaling molecules. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan- predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences

Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary. "Stretched" Sequences

Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example πi were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of INTSIG Encoding Polynucleotides The sequences which were used to assemble SEQ DD NO:21-40 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ DD NO:21-40 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ DD NO:, to that map location. Map locations are represented by ranges, or intervals, of human chromosomes. The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above. In this manner, SEQ DD NO:38 was mapped to chromosome 7 within the interval from 112.90 to 113.40 centiMorgans. VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as: BLAST Score x Percent Identity 5 x minimum {length(Seq. 1), length(Seq. 2)}

The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.

Alternatively, polynucleotide sequences encoding INTSIG are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example ID). Each cDNA sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding INTSIG. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). In particular, SEQ DD NO:30 shows a strong association with neurological tissues. 1292 libraries present in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) isolated from 20 tissue types were examined. SEQ ID NO:30 was found in 73 libraries, 43 (59%) of which were isolated from neurological tissues. Of 113 incidences of SEQ DD NO:30 in all libraries, 75 were in nervous system libraries. SEQ IN NO:30 is useful for distinguishing between nervous tissues and, for example, cardiovascular or endocrine tissues. VIII. Extension of INTSIG Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂S0₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384- well plates in LB/2x carb liquid media.

The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above. Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).

In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5 'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library. IX. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ DD NO:21-40 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 p ol of each oligomer, 250 Ci of [γ-³²P] adenosine rriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10⁷ counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl D, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dόdecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. X. Microarrays

The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection. After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element. Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below. Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl 5X SSC/0.2% SDS. Microarray Preparation

Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.

Array elements are applied to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference. 1 μl of the array element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C followed by washes in 0.2% SDS and distilled water as before. Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm² coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection

Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477,

Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously. The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.

The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). XL Complementary Polynucleotides

Sequences complementary to the INTSIG-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring INTSIG. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of INTSIG. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the INTSIG-encoding transcript. XII. Expression of INTSIG

Expression and purification of INTSIG is achieved using bacterial or virus-based expression systems. For expression of INTSIG in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express INTSIG upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG). Expression of INTSIG in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica calif ornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding INTSIG by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)

In most expression systems, INTSIG is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from INTSIG at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6- His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified INTSIG obtained by these methods can be used directly in the assays shown in Examples XVI, XVϋ, and XVDI, where applicable. XIII. Functional Assays

INTSIG function is assessed by expressing the sequences encoding INTSIG at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or elecrroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics- based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.

The influence of INTSIG on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding INTSIG and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding INTSIG and other genes of interest can be analyzed by northern analysis or microarray techniques.

XIV. Production of INTSIG Specific Antibodies

INTSIG substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.

Alternatively, the INTSIG amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)

Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-INTSIG activity by, for example, binding the peptide or INTSIG to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

XV. Purification of Naturally Occurring INTSIG Using Specific Antibodies Naturally occurring or recombinant INTSIG is substantially purified by immunoaffinity chromatography using antibodies specific for INTSIG. An immunoaffinity column is constructed by covalently coupling anti-INTSIG antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions. Media containing INTSIG are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of INTSIG (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/TNTSIG binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and INTSIG is collected. XVI. Identification of Molecules Which Interact with INTSIG

INTSIG, or biologically active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled INTSIG, washed, and any wells with labeled INTSIG complex are assayed. Data obtained using different concentrations of INTSIG are used to calculate values for the number, affinity, and association of INTSIG with the candidate molecules.

Alternatively, molecules interacting with INTSIG are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).

INTSIG may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101). XVII. Demonstration of INTSIG Activity

INTSIG activity is associated with its ability to form protein-protein complexes and is measured by its ability to regulate growth characteristics of NDT3T3 mouse fibroblast cells. A cDNA encoding INTSIG is subcloned into an appropriate eukaryotic expression vector. This vector is transfected into NIH3T3 cells using methods known in the art. Transfected cells are compared with non-transfected cells for the following quantifiable properties: growth in culture to high density, reduced attachment of cells to the substrate, altered cell morphology, and ability to induce tumors when injected into immunodeficient mice. The activity of INTSIG is proportional to the extent of increased growth or frequency of altered cell morphology in NIH3T3 cells transfected with INTSIG. Alternatively, INTSIG activity is measured by binding of INTSIG to radiolabeled for in polypeptides containing the proline-rich region that specifically binds to SH3 containing proteins (Chan, D.C et al. (1996) EMBO J. 15:1045-1054). Samples of INTSIG are run on SDS-PAGE gels, and transferred onto nitrocellulose by electroblotting. The blots are blocked for 1 hr at room temperature in TBST (137 mM NaCl, 2.7 mM KC1, 25 mM Tris (pH 8.0) and 0.1% Tween-20) containing non-fat dry milk. Blots are then incubated with TBST containing the radioactive formin polypeptide for 4 hrs to overnight. After washing the blots four times with TBST, the blots are exposed to autoradiographic film. Radioactivity is quantitated by cutting out the radioactive spots and counting them in a radioisotope counter. The amount of radioactivity recovered is proportional to the activity of INTSIG in the assay.

Alternatively, INTSIG protein kinase activity is measured by quantifying the phosphorylation of an appropriate substrate in the presence of gamma-labeled ³²P-ATP. INTSIG is incubated with the substrate, ³²P-ATP, and an appropriate kinase buffer. The ³²P incorporated into the product is separated from free ³²P-ATP by electrophoresis, and the incorporated ³²P is quantified using a beta radioisotope counter. The amount of incorporated ³ P is proportional to the protein kinase activity of INTSIG in the assay. A determination of the specific amino acid residue phosphorylated by protein kinase activity is made by phosphoamino acid analysis of the hydrolyzed protein.

Alternatively, an assay for INTSIG protein phosphatase activity measures the hydrolysis of para-nitrophenyl phosphate (PNPP). INTSIG is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1% β-mercaptoethanol at 37°C for 60 min. The reaction is stopped by the addition of 6 ml of 10 N NaOH, and the increase in light absorbance of the reaction mixture at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in light absorbance is proportional to the activity of INTSIG in the assay (Diamond, R.H. et al. (1994) Mol. Cell Biol. 14:3752-3762).

An alternative assay measures INTSIG-mediated G-protein signaling activity by monitoring the mobilization of Ca²⁺ as an indicator of the signal transduction pathway stimulation. (See, e.g., Grynkiewicz, G. et al. (1985) J. Biol. Chem. 260:3440; McColl, S. et al. (1993) J. Immunol. 150:4550-4555; and Aussel, C et al. (1988) J. Immunol. 140:215-220). The assay requires preloading neutrophils or T cells with a fluorescent dye such as FURA-2 or BCECF (Universal Imaging Corp, Westchester PA) whose emission characteristics are altered by Ca⁺⁺ binding. When the cells are exposed to one or more activating stimuli artificially (e.g., anti-CD3 antibody ligation of the T cell receptor) or physiologically (e.g., by allogeneic stimulation), Ca⁺⁺ flux takes place. This flux can be observed and quantified by assaying the cells in a fluorometer or fluorescent activated cell sorter. Measurements of Ca** flux are compared between cells in their normal state and those transfected with INTSIG. Increased Ca⁺⁺ mobilization attributable to increased INTSIG concentration is proportional to INTSIG activity.

Alternatively, INTSIG activity is measured by binding of INTSIG to a substrate which recognizes WD-40 repeats, such as ElonginB, by coimmunoprecipitation (Kamura, T. et al. (1998) Genes Dev. 12:3872-3881). Briefly, epitope tagged substrate and INTSIG are mixed and immunoprecipitated with commercial antibody against the substrate tag. The reaction solution is run on SDS-PAGE and the presence of INTSIG visualized using an antibody to the INTSIG tag. Substrate binding is proportional to INTSIG activity.

Alternatively, INTSIG activity is measured by measuring oxysterol binding. Epitope-tagged INTSIG is incubated with a radio-labeled oxysterol ligand, such as ³H-25-hydroxycholesterol. INTSIG is collected by immunoprecipitation with a commercial antibody against the epitope, and bound hydroxycholesterol quantitated by scintillation counting. INTSIG activity is proportional to the amount of ligand bound.

XVIII. Assay to Detect INTSIG Binding to RNA

The binding of INTSIG to RNA can be assayed using a solid phase RNA binding assay. Hemagglutinin- (HA) tagged wild type and mutant INTSIG in pcDNA3 are transiently transfected into COS cells using LipofectAMINE reagent (Life Technologies, Inc.) for expression and analysis of RNA binding to multiple, simutaneously purified INTSIG proteins. Anti-HA immunoprecipitated INTSIG bound to protein G-Sepharose is incubated with 30 ng of ³²P-labeled G8-5 RNA in 30 μl of RNA binding buffer containing 1 μg/μl poly(C) at room temperature for 20 min. with occasional shaking. The beads are then washed twice with 700 μl of RNA binding buffer and resuspended in 20 μl of SDS-polyacrylamide gel electrophoresis sample buffer. The protein and RNA were separated by 10% SDS-polyacrylamdie gel electrophoresis. The RNA bands ran with a mobility equivalent to 25-35 kDa, and this part of the gel is cut out and dried for autoradiography. The upper part of the gel is transferred to a polyvinylidene difluoride membrane and blotted with anti-HA antibody to detect HA-INTSIG (Lin, et al. (1997) J. Biol. Chem. 272:27274-27280).

Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Table 1

Table 2

Table 2 (cont.)

Table 3

Table 3 (cont.)

Table 3 (cont.)

Table 3 (cont.)

Table 3 (cont.)

Table 3 (cont.)

Table 3 (cont.)

Table 3 (cont.)

Table 4

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Table 4 (cont.)

Table 4 (cont.)

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Table 4 (cont.)

Table 4 (cont.)

Table 5

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Table 6

Table 6 (cont.)

Table 6 (cont.)

Table 6 (cont.)

Table 7

Program Description Reference Parameter Threshold

ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.

ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch <50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.

ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.

BLAST A Basic Local Alignment Search Tool useful in Altschul, S.F. et al. (1990) J. Mol. Biol. ESTs: Probability value= 1.0E-8 sequence similarity search for amino acid and 215:403-410; Altschul, S.F. et al. (1997) or less nucleic acid sequences. BLAST includes five Nucleic Acids Res. 25:3389-3402. Full Length sequences: Probabilit functions: blastp, blastn, blasts, tblastn, and tblastx. valuer l.OE-10 or less

FASTA A Pearson and Lipman algorithm that searches for Pearson, W.R. and D J. Lipman (1988) Proc. ESTs: fasta E value=1.06E-6

O oo similarity between a query sequence and a group of Natl. Acad Sci. USA 85:2444-2448; Pearson, Assembled ESTs: fasta Identity= sequences of the same type. FASTA comprises as W.R. (1990) Methods Enzymol. 183:63-98; 95% or greater and least five functions: fasta, tfasta, fastx, tfastx, and and Smith, T.F. and M.S. Waterman (1981) Match length=200 bases or greate ssearch. Adv. Appl. Math. 2:482-489. fastx E value=1.0E-8 or less

Full Length sequences: fastx score=100 or greater

BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S. and J.G. Henikoff (1991) Nucleic Probability value= 1.0E-3 or less sequence against those in BLOCKS, PRINTS, Acids Res. 19:6565-6572; Henikoff, J.G. and DOMO, PRODOM, and PFAM databases to search S. Henikoff (1996) Methods Enzymol. for gene families, sequence homology, and 266:88-105; and Attwood, T.K. et al. (1997) J. structural fingerprint regions. Chem. Inf. Comput. Sci. 37:417-424.

HMMER An algorithm for searching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol. PFAM hits: Probability value= hidden Markov model (HMM)-based databases of 235:1501-1531; Sonnhammer, E.L.L. et al. 1.0E-3 or less protein family consensus sequences, such as PFAM. (1988) Nucleic Acids Res. 26:320-322; Signal peptide hits: Score= 0 or Durbin, R. et al. (1998) Our World View, in a greater Nutshell, Cambridge Univ. Press, pp. 1-350.

Table 7 (cont.)

Program Description Reference Parameter Threshold

ProfileScan An algorithm that searches for structural and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66; Normalized quality score≥GCG motifs in protein sequences that match sequence patterns Gribskov, M. et al. (1989) Methods Enzymol. specified "HIGH" value for that defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) particular Prosite motif. Nucleic Acids Res. 25:217-221. Generally, score=l.4-2.1.

Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.

Phrap A Phils Revised Assembly Program including SWAT and Smith, T.F. and M.S. Waterman (1981) Adv. Score= 120 or greater; CrossMatch, programs based on efficient implementation Appl. Math. 2:482-489; Smith, T.F. and M.S. Match length= 56 or greater of the Smith-Waterman algorithm, useful in searching Waterman (1981) J. Mol. Biol. 147:195-197; sequence homology and assembling DNA sequences. and Green, P., University of Washington, Seattle, WA.

Consed A graphical tool for viewing and editing Phrap Gordon, D. et al. (1998) Genome Res. 8:195-202. assemblies. SPScan A weight matrix analysis program that scans protein Nielson, H. et al. (1997) Protein Engineering Score=3.5 or greater sequences for the presence of secretory signal peptides. 10:1-6; Claverie, J.M. and S. Audic (1997) CABIOS 12:431-439.

TMAP A program that uses weight matrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol. transmembrane segments on protein sequences and 237:182-192; Persson, B. and P. Argos (1996) determine orientation. Protein Sci. 5:363-371.

TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.

Motifs A program that searches amino acid sequences for patterns Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221; that matched those defined in Prosite. Wisconsin Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.

Claims

What is claimed is:

1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO.21-40.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. A method of claim 9, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.

11. An isolated antibody which specifically binds to a polypeptide of claim 1.

12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

90% identical to a polynucleotide sequence selected from the group consisting of SEQ

ID NO:21-40, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).

13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.

19. A method for treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition of claim 17.

20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.

22. A method for treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment a composition of claim 21.

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional INTSIG, comprising administering to a patient in need of such treatment a composition of claim 24.

26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

30. A diagnostic test for a condition or disease associated with the expression of INTSIG in a biological sample, the method comprising: a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11, wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')₂ fragment, or e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of INTSIG in a subject, comprising administering to said subject an effective amount of the composition of claim

32.

34. A composition of claim 32, wherein the antibody is labeled.

35. A method of diagnosing a condition or disease associated with the expression of INTSIG in a subject, comprising administering to said subject an effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim

11, the method comprising: a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11 , wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20 in a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20 in the sample.

45. A method of purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20 from a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim

13.

47. A method of generating a transcript image of a sample which contains polynucleotides, the method comprising: a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An array of claim 48, which is a microarray.

53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.

56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:l.

57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ED NO:2.

58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:3.

59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:4.

60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:5.

61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 8.

64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:10.

66. A polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO: 11.

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:12.

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 13.

69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:14.

70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:15.

71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:16.

72. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 17.

73. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:18.

74. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 19.

75. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:20.

76. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:21.

77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:24.

80. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:25.

81. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:26.

82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:27.

83. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:28.

84. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:29.

85. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:30.

86. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:31.

87. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:32.

88. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:33.

89. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:34.

90. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:35.

91. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:36.

92. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:37.

93. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:38.

94. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:39.

95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:40.

<110> INCYTE GENOMICS, INC. BAUGHN, Mariah R. DING, Li

ELLIOTT, Vicki S. GANDHI, Ameena R. Gietzen, Kimberly J. GRIFFIN, Jennifer A. GURURAJAN, Ra agopal HAFALIA, April J.A. KEARNEY, Liam KHAN, Farrah A. LAL, Preeti LEE, Ernestine A. LU, Dyung Aina M. LU, Yan

NGUYEN, Danniel B. ARVIZU, Chandra RAMKU AR, Jaya TANG, Y. Tom THANGAVELU, Kavitha THORNTON, Michael WALIA, Narinder K. WARREN, Bridget A. XU, Yuming YAO, Monique G. YUE, Henry

<120> INSTRACELLULAR SIGNALING MOLECULES

<130> PF-0827 PCT

<140> To Be Assigned <141> Herewith

<150> 60/240,871; 60/244,723; 60/249,402; 60/252,622; 60/255,622 <151> 2000-10-13; 2000-10-30; 2000-11-15; 2000-11-22; 2000-12-13

<160> 40

<170> PERL Program

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Phe Leu Ser Ser Arg Gin Val Arg Ser Gin Lys He Leu Thr His

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Arg Leu Gly Pro Glu Ala Ser Phe Ser Ala Glu Asp Leu Ser Leu

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Val Gin His Phe His Lys Pro Ala Leu Leu Pro Leu Leu Gin Arg

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Ala Phe His Pro Pro His Arg Val Val Arg Leu Leu Cys Gly Val

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Arg Asp Ser Glu Glu Phe Leu Asp Phe Phe Pro Asp Trp Ala His

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Trp Gin Glu Leu Thr Cys Asp Asp Glu Pro Glu Thr Tyr Val Ala

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Thr Asp Thr Glu Pro Glu Asp Glu Lys Val Val Ser Tyr Ser Lys

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Gin Gin Asn Leu Pro Thr Val Thr Ser Pro Gly Asn Leu Met Val

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Val Gin Pro Asp Arg He Arg Cys Gly Ala Glu Thr Thr Val Tyr

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Glu Phe Ser Pro Glu Asp Ser Pro Ser Val Arg Met Glu Ala Lys

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Val Glu Asn Glu Tyr Thr He Ser Val Lys Ala Pro Asn Leu Ser

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Ser Gly Asn Val Ser Leu Lys He Tyr Ser Gly Asp Leu Val Val

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Asn Leu Leu Ser Asn Ala Ala Asn Pro Val Glu Phe Met Cys Gin

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Ala Phe Lys He Val Pro Tyr Asn Thr Glu Thr Leu Asp Lys Leu

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Leu Thr Glu Ser Leu Lys Asn Asn He Pro Ala Ser Gly Leu His

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Leu Phe Gly He Asn Gin Leu Glu Glu Glu Asp Met Met Thr Asn

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Asn Thr He Ala Glu Lys His Gly Phe Arg Asp Leu Arg Gin Phe

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He Asp Glu Tyr Val Glu Thr Val Asp Met Leu Lys Ser His He

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Asn Pro Gly Cys Asp Glu Asp Leu Tyr Glu Ser Met Ala Ala Phe

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Val Pro Ala Ala Thr Glu Asp Leu Tyr Val Glu Met Leu Gin Ala

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Ser Thr Ser Asn Pro He Pro Gly Asp Gly Phe Ser Arg Ala Thr

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Lys Asp Ser Met He Arg Lys Phe Leu Glu Gly Asn Ser Met Gly

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Asp Val Tyr His Thr Val Asp Asp Asp Glu Ala Phe Ser Val Asp

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Leu Ala Ser Arg Pro Pro Val Pro Val Pro Arg Pro Glu Thr Thr

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Ala Pro Gly Ala His Gin Leu Pro Asp Asn Glu Pro Tyr He Phe

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Lys Val Phe Ala Glu Lys Ser Gin Glu Arg Pro Gly Asn Phe Tyr

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Val Ser Ser Glu Ser He Arg Lys Gly Pro Pro Val Arg Pro Trp

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Arg Asp Arg Pro Gin Ser Ser He Tyr Asp Pro Phe Ala Gly Met

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Lys Thr Pro Gly Gin Arg Gin Leu He Thr Leu Gin Glu Gin Val

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Lys Leu Gly He Val Asn Val Asp Glu Ala Val Leu His Phe Lys

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Glu Trp Gin Leu Asn Gin Lys Arg Arg Ser Glu Ser Phe Arg Phe

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Gin Gin Glu Asn Leu Lys Arg Leu Arg Asp Ser He Thr Arg Arg

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Gin Arg Glu Lys Gin Lys Ser Gly Lys Gin Thr Asp Leu Glu He

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Thr Val Pro He Arg His Ser Gin His Leu Pro Ala Lys Val Glu

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Phe Gly Val Tyr Glu Ser Gly Pro Arg Lys Ser Val He Pro Pro

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Leu Ser Val Ser Ser Gly Met Glu Gly Asp Asn Glu Asp Asn Glu

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Asp Gly Thr Pro Thr Met Ser Leu Glu Arg Pro Pro Arg Val Pro

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Pro Arg Ala Ala Ser Gin Arg Pro Pro Thr Arg Glu Thr Phe His

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Pro Pro Pro Pro Val Pro Pro Arg Gly Arg

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Pro Leu Asp Thr Leu Lys Gly Leu Gly Thr Cys Phe Pro Ser Gly 35 40 45

Pro Glu Leu Arg Gly Ala Gly He Ala Ala Ala Met Glu Arg Ala

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Ser Glu Arg Arg Thr Ala Ser Ala Leu Phe Ala Gly Phe Arg Ala

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Leu Gly Leu Phe Ser Asn Asp He Pro His Val Val Arg Phe Ser

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Ala Leu Lys Arg Arg Phe Tyr Val Thr Thr Cys Val Gly Lys Ser

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Phe His Thr Tyr Asp Val Gin Lys Leu Ser Leu Val Ala Val Ser

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Asn Lys Glu He Val His Thr Phe Lys Gly His Lys Ala Glu He

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His Phe Leu Gin Pro Phe Gly Asp His He He Ser Val Asp Thr

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Asp Gly He Leu He He Trp His He Tyr Ser Glu Glu Glu Tyr

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Leu His Pro Ser Thr Tyr Leu Asn Lys He Leu Leu Gly Ser Glu

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Pro He Arg Gin Thr Phe He Pro Ala Gly Tyr Leu Leu Gly Ala

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His Gly Leu Lys Thr Gin Ala Pro Ala Val Asp Val Val Ala He

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Gly Leu Met Ser Gly Gin Val He He His Asn He Lys Phe Asn

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Glu Thr Leu Met Lys Phe Arg Gin Asp Trp Gly Pro He Thr Ser

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He Arg Tyr Tyr Gly Gin Asn Gly Gin Gin He Leu Ser Ala Ser

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Gin Asp Gly Thr Leu Gin Ser Phe Ser Thr Val His Glu Lys Phe

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Asn Lys Ser Leu Gly His Gly Leu He Asn Lys Lys Arg Val Lys

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Arg Lys Gly Leu Gin Asn Thr Met Ser Val Arg Leu Pro Pro He

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Leu Lys Lys Asp Asp He Thr Ala Thr Ala Val Asp He Thr Ser

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Cys Gly Asn Phe Ala Val He Gly Leu Ser Ser Gly Thr Val Asp

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Val Tyr Asn Met Gin Ser Gly He His Arg Gly Ser Phe Gly Lys

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Asp Gin Ala His Lys Gly Ser Val Arg Gly Val Ala Val Asp Gly

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Leu Asn Gin Leu Thr Val Thr Thr Gly Ser Glu Gly Leu Leu Lys

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Phe Trp Asn Phe Lys Asn Lys He Leu He His Ser Val Ser Leu

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Ser Ser Ser Pro Asn He Met Leu Leu His Arg Asp Ser Gly He

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Leu Gly Leu Ala Leu Asp Asp Phe Ser He Ser Val Leu Asp He

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' He Asn Asp Met Ala Phe Ser Pro Asp Gly Arg Trp Leu He Ser

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Ala Ala Met Asp Cys Ser He Arg Thr Trp Asp Leu Pro Ser Gly

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Cys Leu He Asp Cys Phe Leu Leu Asp Ser Ala Pro Leu Asn Val

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Ser Met Ser Pro Thr Gly Asp Phe Leu Ala Thr Ser His Val Asp

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His Leu Gly He Tyr Leu Trp Ser Asn He Ser Leu Tyr Ser Val

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Met Leu Pro Gly Thr Cys Gin Thr Gin Asp Val Glu Val Ser Glu

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Glu Thr Val Glu Pro Ser Asp Glu Leu He Glu Tyr Asp Ser Pro

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Glu Gin Leu Asn Glu Gin Leu Val Thr Leu Ser Leu Leu Pro Glu

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Ser Arg Trp Lys Asn Leu Leu Asn Leu Asp Val He Lys Lys Lys

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Asn Lys Pro Lys Glu Pro Pro Lys Val Pro Lys Ser Ala Pro Phe

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Phe He Pro Thr He Pro Gly Leu Val Pro Arg Tyr Ala Ala Pro

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Val Leu Ala Gin Lys Ser Asp Phe Cys Leu Lys Leu Glu Glu Gly

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Leu Val Asn Asn Lys Tyr Asp Thr Ala Leu Asn Leu Leu Lys Glu

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Ser Gly Pro Ser Gly He Glu Thr Glu Leu Arg Ser Leu Ser Pro

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Asp Cys Gly Gly Ser He Glu Val Met Gin Ser Phe Leu Lys Met

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Val Glu Glu Asn Trp Thr His Leu Gin Ser Leu Phe Asn Gin Ser

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Met Cys He Leu Asn Tyr Leu Lys Ser Ala Leu Leu

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Cys He Ser Gin He Thr Asn Leu Asn Tyr Ala Thr Asn Leu Thr

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His Leu Tyr Leu Gin Asn Asn Cys He Ser Cys He Glu Asn Leu

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Arg Ser Leu Lys Lys Leu Glu Lys Leu Tyr Leu Gly Gly Asn Tyr

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He Ala Val He Glu Gly Leu Glu Gly Leu Gly Glu Leu Arg Glu

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Leu His Val Glu Asn Gin Arg Leu Pro Leu Gly Glu Lys Leu Leu

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Phe Asp Pro Arg Thr Leu His Ser Leu Ala Lys Ser Leu Cys He

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Leu Asn He Ser Asn Asn Asn He Asp Asp He Thr Asp Leu Glu

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Leu Leu Glu Asn Leu Asn Gin Leu He Ala Val Asp Asn Gin Leu

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Leu His Val Lys Asp Leu Glu Phe Leu Leu Asn Lys Leu Met Lys

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Leu Trp Lys He Asp Leu Asn Gly Asn Pro Val Cys Leu Lys Pro

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Lys Tyr Arg Asp Arg Leu He Leu Val Ser Lys Ser Leu Glu Phe

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Leu Asp Gly Lys Glu He Lys Asn He Glu Arg Gin Phe Leu Met

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Asn Trp Lys Ala Ser Lys Asp Ala Lys Lys He Ser Lys Lys Arg

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Ser Ser Lys Asn Glu Asp Ala Ser Asn Ser Leu He Ser Lys His

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Lys Lys Leu Lys Lys Lys Leu Val Arg Ser Glu Glu Asn He Ser

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Pro Asp Thr He Arg Ser Asn Leu His Tyr Met Lys Glu Thr Thr

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Ser Asp Asp Pro Asp Thr He Arg Ser Asn Leu Pro His He Lys

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Glu Thr Thr Ser Asp Asp Val Ser Ala Ala Asn Thr Asn Asn Leu

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Lys Lys Ser Thr Arg Val Thr Lys Asn Lys Leu Arg Asn Thr Gin

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Leu Ala Thr Glu Asn Pro Asn Gly Asp Ala Ser Val Glu Glu Asp

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Lys Gin Gly Lys Pro Asn Lys Lys Val He Lys Thr Val Pro Gin

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Leu Thr Thr Gin Asp Leu Lys Pro Glu Thr Pro Glu Asn Lys Val

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Asp His Gin Lys Ser Glu Lys Ala Asn Glu Gly Arg Glu Glu Thr

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Asp Leu Glu Glu Asp Glu Glu Leu Met Gin Ala Tyr Gin Cys His

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Val Thr Glu Glu Met Ala Lys Glu He Lys Arg Lys He Arg Lys

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Lys Leu Lys Glu Gin Leu Thr Tyr Phe Pro Ser Asp Thr Leu Phe

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His Asp Asp Lys Leu Ser Ser Glu Lys Arg Lys Lys Lys Lys Glu

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Val Pro Val Phe Ser Lys Ala Glu Thr Ser Thr Leu Thr He Ser

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Gly Asp Thr Val Glu Gly Glu Gin Lys Lys Glu Ser Ser Val Arg

260 265 270

Ser Val Ser Ser Asp Ser His Gin Asp Asp Glu He Ser Ser Met

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Glu Gin Ser Thr Glu Asp Ser Met Gin Asp Asp Thr Lys Pro Lys

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Pro Lys Lys Thr Lys Lys Lys Thr Lys Ala Val Ala Asp Asn Asn

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Glu Asp Val Asp Gly Asp Gly Val His Glu He Thr Ser Arg Asp

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Ser Pro Val Tyr Pro Lys Cys Leu Leu Asp Asp Asp Leu Val Leu

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Gly Val Tyr He His Arg Thr Asp Arg Leu Lys Ser Asp Phe Met

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He Ser His Pro Met Val Lys He His Val Val Asp Glu His Thr 365 370 375

Gly Gin Tyr Val Lys Lys Asp Asp Ser Gly Arg Pro Val Ser Ser

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Tyr Tyr Glu Lys Glu Asn Val Asp Tyr He Leu Pro He Met Thr

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Gin Pro Tyr Asp Phe Lys Gin Leu Lys Ser Arg Leu Pro Glu Trp

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Glu Glu Gin He Val Phe Asn Glu Asn Phe Pro Tyr Leu Leu Arg

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Gly Ser Asp Glu Ser Pro Lys Val He Leu Phe Phe Glu He Leu

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Asp Phe Leu Ser Val Asp Glu He Lys Asn Asn Ser Glu Val Gin

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Asn Gin Glu Cys Gly Phe Arg Lys He Ala Trp Ala Phe Leu Lys

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Leu Leu Gly Ala Asn Gly Asn Ala Asn He Asn Ser Lys Leu Arg

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Leu Gin Leu Tyr Tyr Pro Pro Thr Lys Pro Arg Ser Pro Leu Ser

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Val Val Glu Ala Phe Glu Trp Trp Ser Lys Cys Pro Arg Asn His

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Tyr Pro Ser Thr Leu Tyr Val Thr Val Arg Gly Leu Lys Val Pro

530 535 540

Asp Cys He Lys Pro Ser Tyr Arg Ser Met Met Ala Leu Gin Glu

545 550 555

Glu Lys Gly Lys Pro Val His Cys Glu Arg His His Glu Ser Ser

560 565 570

Ser Val Asp Thr Glu Pro Gly Leu Glu Glu Ser Lys Glu Val He

575 580 585

Lys Trp Lys Arg Leu Pro Gly Gin Ala Cys Arg He Pro Asn Lys

590 595 600

His Leu Phe Ser Leu Asn Ala Gly Glu Arg Gly Cys Phe Cys Leu

605 610 615

Asp Phe Ser His Asn Gly Arg He Leu Ala Ala Ala Cys Ala Ser

620 625 630

Arg Asp Gly Tyr Pro He He Leu Tyr Glu He Pro Ser Gly Arg

635 640 645

Phe Met Arg Glu Leu Cys Gly His Leu Asn He He Tyr Asp Leu

650 655 660

Ser Trp Ser Lys Asp Asp His Tyr He Leu Thr Ser Ser Ser Asp

665 670 675

Gly Thr Ala Arg He Trp Lys Asn Glu He Asn Asn Thr Asn Thr

680 685 690 Phe Arg Val Leu Pro His Pro Ser Phe Val Tyr Thr Ala Lys Phe

695 700 705

His Pro Ala Val Arg Glu Leu Val Val Thr Gly Cys Tyr Asp Ser

710 715 720 Met He Arg He Trp Lys Val Glu Met Arg Glu Asp Ser Ala He

725 730 735

Leu Val Arg Gin Phe Asp Val His Lys Ser Phe He Asn Ser Leu

740 745 750 Cys Phe Asp Thr Glu Gly His His Met Tyr Ser Gly Asp Cys Thr

755 760 765

Gly Val He Val Val Trp Asn Thr Tyr Val Lys He Asn Asp Leu

770 775 780

Glu His Ser Val His His Trp Thr He Asn Lys Glu He Lys Glu

785 790 795 Thr Glu Phe Lys Gly He Pro He Ser Tyr Leu Glu He His Pro 800 805 810

Asn Gly Lys Arg Leu Leu He His Thr Lys Asp Ser Thr Leu Arg

815 ^' 820 825

He Met Asp Leu Arg He Leu Val Ala Arg Lys Phe Val Gly Ala

830 835 840

Ala Asn Tyr Arg Glu Lys He His Ser Thr Leu Thr Pro Cys Gly

845 850 855

Thr Phe Leu Phe Ala Gly Ser Glu Asp Gly He Val Tyr Val Trp

860 865 870

Asn Pro Glu Thr Gly Glu Gin Val Ala Met Tyr Ser Asp Leu Pro

875 880 885

Phe Lys Ser Pro He Arg Asp He Ser Tyr His Pro Phe Glu Asn

890 895 900

Met Val Ala Phe Cys Ala Phe Gly Gin Asn Glu Pro He Leu Leu

905 910 915

Tyr He Tyr Asp Phe His Val Ala Gin Gin Glu Ala Glu Met Phe

920 925 930

Lys Arg Tyr Asn Gly Thr Phe Pro Leu Pro Gly He His Gin Ser

935 940 945

Gin Asp Ala Leu Cys Thr Cys Pro Lys Leu Pro His Gin Gly Ser

950 955 960

Phe Gin He Asp Glu Phe Val His Thr Glu Ser Ser Ser Thr Lys

965 970 975

Met Gin Leu Val Lys Gin Arg Leu Glu Thr Val Thr Glu Val He

980 985 990

Arg Ser Cys Ala Ala Lys Val Asn Lys Asn Leu Ser Phe Thr Ser

995 1000 1005

Pro Pro Ala Val Ser Ser, Gin Gin Ser Lys Leu Lys Gin Ser Asn

1010 1015 1020

Met Leu Thr Ala Gin Glu He Leu His Gin Phe Gly Phe Thr Gin

1025 1030 1035

Thr Gly He He Ser He Glu Arg Lys Pro Cys Asn His Gin Val

1040 1045 1050

Asp Thr Ala Pro Thr Val Val Ala Leu Tyr Asp Tyr Thr Ala Asn

1055 1060 1065

Arg Ser Asp Glu Leu Thr He His Arg Gly Asp He He Arg Val

1070 1075 1080

Phe Phe Lys Asp Asn Glu Asp Trp Trp Tyr Gly Ser He Gly Lys

1085 1090 1095

Gly Gin Glu Gly Tyr Phe Pro Ala Asn His Val Ala Ser Glu Thr

1100 1105 1110

Leu Tyr Gin Glu Leu Pro Pro Glu He Lys Glu Arg Ser Pro Pro

1115 1120 1125

Leu Ser Pro Glu Glu Lys Thr Lys He Glu Lys Ser Pro Ala Pro

1130 1135 1140

Gin Lys Val Lys

<210> 5

<211> 513

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No : 4596874CD1

<400> 5 Met Ala Tyr Gin Val Val Glu Lys Gly Ala Ala Leu Gly Thr Leu

1 5 10 15

Glu Ser Glu Leu Gin Gin Arg Gin Ser Arg Leu Ala Ala Leu Glu

20 25 30

Ala Arg Val Ala Gin Leu Arg Glu Ala Arg Ala Gin Gin Ala Gin

35 40 45

Gin Val Glu Glu Trp Arg Ala Gin Asn Ala Val Gin Arg Ala Ala

50 55 60

Tyr Glu Ala Leu Arg Ala His Val Gly Leu Arg Glu Ala Ala Leu

65 70 75

Arg Arg Leu Gin Glu Glu Ala Arg Asp Leu Leu Glu Arg Leu Val

80 85 90

Gin Arg Lys Ala Arg Ala Ala Ala Glu Arg Asn Leu Arg Asn Glu

95 100 105

Arg Arg Glu Arg Ala Lys Gin Ala Arg Val Ser Gin Glu Leu Lys

110 115 120

Lys Ala Ala Lys Arg Thr Val Ser He Ser Glu Gly Pro Asp Thr

125 130 135

Leu Gly Asp Gly Met Arg Glu Arg Arg Glu Thr Leu Ala Leu Ala

140 145 150

Pro Glu Pro Glu Pro Leu Glu Lys Glu Ala Cys Glu Lys Trp Lys

155 160 165

Arg Pro Phe Arg Ser Ala Ser Ala Thr Ser Leu Thr Leu Ser His

170 175 180

Cys Val Asp Val Val Lys Gly Leu Leu Asp Phe Lys Lys Arg Arg

185 190 195

Gly His Ser He Gly Gly Ala Pro Glu Gin Arg Tyr Gin He He

200 205 210

Pro Val Cys Val Ala Ala Arg Leu Pro Thr Arg Ala Gin Asp Val

215 220 225

Leu Asp Ala His Leu Ser Glu Val Asn Ala Val Arg Phe Gly Pro

230 235 240

Asn Ser Ser Leu Leu Ala Thr Gly Gly Ala Asp Arg Leu He His

245 250 255

Leu Trp Asn Val Val Gly Ser Arg Leu Glu Ala Asn Gin Thr Leu

260 255 270

Glu Gly Ala Gly Gly Ser He Thr Ser Val Asp Phe Asp Pro Ser

275 280 285

Gly Tyr Gin Val Leu Ala Ala Thr Tyr Asn Gin Ala Ala Gin Leu

290 295 300

Trp Lys Val Gly Glu Ala Gin Ser Lys Glu Thr Leu Ser Gly His

305 310 315

Lys Asp Lys Val Thr Ala Ala Lys Phe Lys Leu Thr Arg His Gin

320 325 330

Ala Val Thr Gly Ser Arg Asp Arg Thr Val Lys Glu Trp Asp Leu

335 340 ^• 345

Gly Arg Ala Tyr Cys Ser Arg Thr He Asn Val Leu Ser Tyr Cys

350 355 360

Asn Asp Val Val Cys Gly Asp His He He He Ser Gly His Asn

365 370 375

Asp Gin Lys He Arg Phe Trp Asp Ser Arg Gly Pro His Cys Thr

380 385 390

Gin Val He Pro Val Gin Gly Arg Val Thr Ser Leu Ser Leu Ser

395 400 405

His Asp Gin Leu His Leu Leu Ser Cys Ser Arg Asp Asn Thr Leu

410 415 420

Lys Val He Asp Leu Arg Val Ser Asn He Arg Gin Val Phe Arg

425 430 435 Ala Asp Gly Phe Lys Cys Gly Ser Asp Trp Thr Lys Ala Val Phe

440 445 450

Ser Pro Asp Arg Ser Tyr Ala Leu Ala Gly Ser Cys Asp Gly Ala

455 460 465

Leu Tyr He Trp Asp Val Asp Thr Gly Lys Leu Glu Ser Arg Leu

470 475 480

Gin Gly Pro His Cys Ala Ala Val Asn Ala Val Ala Trp Cys Tyr

485 490 495

Ser Gly Ser His Met Val Ser Val Asp Gin Gly Arg Lys Val Val

500 505 510 Leu Trp Gin

<210> 6

<211> 667

<212> PRT

<213> Homo sapiens

<220>

<221> misσ_feature

<223> Incyte ID No: 3594012CD1

<400> 6

Met His Phe Thr Gly Ala Val Met Gin Glu Asn Leu Arg Phe Ala

1 5 10 15

Ser Ser Gly Asp Asp He Lys He Trp Asp Ala Ser Ser Met Thr

20 25 30

Leu Val Asp Lys Phe Asn Pro His Thr Ser Pro His Gly He Ser

35 ■ 40 45

Ser He Cys Trp Ser Ser Asn Asn Asn Phe Leu Val Thr Ala Ser

50 55 60

Ser Ser Gly Asp Lys He Val Val Ser Ser Cys Lys Cys Lys Pro

65 70 75

Val Pro Leu Leu Glu Leu Ala Glu Gly Gin Lys Gin Thr Cys Val

80 . 85 90

Asn Leu Asn Ser Thr Ser Met Tyr Leu Val Ser Gly Gly Leu Asn

95 100 105

Asn Thr Val Asn He Trp Asp Leu Lys Ser Lys Arg Val His Arg

110 115 120

Ser Leu Lys Asp His Lys Asp Gin Val Thr Cys Val Thr Tyr Asn

125 130 135

Trp Asn Asp Cys Tyr He Ala Ser Gly Ser Leu Ser Gly Glu He

140 145 150

He Leu His Ser Val Thr Thr Asn Leu Ser Ser Thr Pro Phe Gly

155 160 165

His Gly Ser Asn Gin Ser Val Arg His Leu Lys Tyr Ser Leu Phe

170 175 180

Lys Lys Ser Leu Leu Gly Ser Val Ser Asp Asn Gly He Val Thr

185 190 195

Leu Trp Asp Val Asn Ser Gin Ser Pro Tyr His Asn Phe Asp Ser

200 205 210

Val His Lys Ala Pro Ala Ser Gly He Cys Phe Ser Pro Val Asn

215 220 225

Glu Leu Leu Phe Val Thr He Gly Leu Asp Lys Arg He He Leu

230 235 240

Tyr Asp Thr Ser Ser Lys Lys Leu Val Lys Thr Leu Val Ala Asp

245 250 255

Thr Pro Leu Thr Ala Val Asp Phe Met Pro Asp Gly Ala Thr Leu 260 265 270

Ala He Gly Ser Ser Arg Gly Lys He Tyr Gin Tyr Asp Leu Arg

275 280 285

Met Leu Lys Ser Pro Val Lys Thr He Ser Ala His Lys Thr Ser

290 295 300

Val Gin Cys He Ala Phe Gin Tyr Ser Thr Val Leu Thr Lys Ser

305 310 315

Ser Leu Asn Lys Gly Cys Ser Asn Lys Pro Thr Thr Val Asn Lys

320 325 330

Arg Ser Val Asn Val Asn Ala Ala Ser Gly Gly Val Gin Asn Ser

335 340 345

Gly He Val Arg Glu Ala Pro Ala Thr Ser He Ala Thr Val Leu

350 355 360

Pro Gin Pro Met Thr Ser Ala Met Gly Lys Gly Thr Val Ala Val

365 370 375

Gin Glu Lys Ala Gly Leu Pro Arg Ser He Asn Thr Asp Thr Leu

380 385 390

Ser Lys Glu Thr Asp Ser Gly Lys Asn Gin Asp Phe Ser Ser Phe

395 400 405

Asp Asp Thr Gly Lys Ser Ser Leu Gly Asp Met Phe Ser Pro He

410 415 420

Arg Asp Asp Ala Val Val Asn Lys Gly Ser Asp Glu Ser He Gly

425 430 435

Lys Gly Asp Gly Phe Asp Phe Leu Pro Gin Leu Asn Ser Val Phe

440 445 450

Pro Pro Arg Lys Asn Pro Val Thr Ser Ser Thr Ser Val Leu His

455 460 465

Ser Ser Pro Leu Asn Val Phe Met Gly Ser Pro Gly Lys Glu Glu

470 475 480

Asn Glu Asn Arg Asp Leu Thr Ala Glu Ser Lys Lys He Tyr Met

485 490 495

Gly Lys Gin Glu Ser Lys Asp Ser Phe Lys Gin Leu Ala Lys Leu

500 505 510

Val Thr Ser Gly Ala Glu Ser Gly Asn Leu Asn Thr Ser Pro Ser

515 520' 525

Ser Asn Gin Thr Arg Asn Ser Glu Lys Phe Glu Lys Pro Glu Asn

530 535 540

Glu He Glu Ala Gin Leu He Cys Glu Pro Pro He Asn Gly Ser

545 550 555

Ser Thr Pro Asn Pro Lys He Ala Ser Ser Val Thr Ala Gly Val

560 565 570

Ala Ser Ser Leu Ser Glu Lys He Ala Asp Ser He Gly Asn Asn

575 580 585

Arg Gin Asn Ala Pro Leu Thr Ser He Gin He Arg Phe He Gin

590 595 600

Asn Met He Gin Glu Thr Leu Asp Asp Phe Arg Glu Ala Cys His

605 610 615

Arg Asp He Val Asn Leu Gin Val Glu Met He Lys Gin Phe His

620 625 630

Met Gin Leu Asn Glu Met His Ser Leu Leu Glu Arg Tyr Ser Val

635 640 645

Asn Glu Gly Leu Val Ala Glu He Glu Arg Leu Arg Glu Glu Asn

650 655 660

Lys Arg Leu Arg Ala His Phe

665

<210> 7 <211> 897 <212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 7482435CD1

<400> 7

Met Ala Glu He Pro Leu Tyr Phe Val Asp Leu Gin Asp Asp Leu

1 5 10 15

Asp Asp Tyr Gly Phe Glu Asp Tyr Gly Thr Asp Cys Asp Asn Met

20 25 30

Arg Val Thr Ala Phe Leu Asp He Pro Gly Gin Asp Asn Leu Pro

35 40 45

Pro Leu Thr Arg Leu Glu Lys Tyr Ala Phe Ser Glu Asn Thr Phe

50 55 60

Asn Arg Gin He He Ala Arg Gly Leu Leu Asp He Phe Arg Asp

65 70 75

Phe Gly Asn Asn Glu Glu Asp Phe Leu Thr Val Met Glu He Val

80 85 90

Val Arg Leu Ser Glu Asp Ala Glu Pro Thr Val Arg Thr Glu Leu

95 100 105

Met Glu Gin He Pro Pro He Ala He Phe Leu Gin Glu Asn Arg

110 115 120

Ser Asn Phe Pro Val Val Leu Ser Glu Tyr Leu He Pro He Val

125 130 135

Met Arg Tyr Leu Thr Asp Pro Asn Asn Gin He He Cys Lys Met

140 145 150

Ala Ser Met Leu Ser Lys Ser Thr Val Glu Arg Leu Leu Leu Pro

155 160 165

Arg Phe Cys Glu Leu Cys Gly Asp Arg Lys Leu Phe Gin Val Arg

170 175 180

Lys Val Cys Ala Ala Asn Phe Gly Asp He Cys His Ala Val Gly

185 190 195

Gin Glu Ala Thr Glu Lys Phe Leu He Pro Lys Phe Phe Glu Leu

200 205 210

Cys Ser Asp Ala Val Trp Gly Met Arg Lys Ala Cys Ala Glu Cys

215 220 225

Phe Thr Ala Val Ser His Ser Ser Ser Pro Gly Val Arg Arg Thr

230 235 240

Gin Leu Phe Pro Leu Phe He Arg Leu Val Ser Asp Pro Cys Arg

245 250 255

Trp Val His Gin Ala Ala Phe Gin Ser Leu Gly Pro Phe He Ser

260 265 270

Thr Phe Ala Asn Pro Ser Arg Ala Gly Leu Tyr Leu Arg Glu Asp

275 280 285

Gly Ala Leu Ser He Trp Pro Leu Thr Gin Asp Leu Asp Ser Gly

290 295 300

Phe Ala Ser Gly Ser Pro Ala Pro Ser Ser Gly Gly Asn He Ser

305 310 315

Pro Ala Ser Leu He Arg Ser Ala Lys Pro Val Arg Ser Glu Pro

320 325 330

Glu Leu Pro Val Glu Gly Thr Ser Ala Lys Thr Ser Asp Cys Pro

335 340 345

His Ser Ser Ser Ser Ser Asp Gly Pro Ala Glu Ser Pro Val Glu

350 355 360

Ser Cys Val Ser Ala Gly Ala Glu Trp Thr Arg Val Ser Pro Glu

365 370 375 Thr Ser Ala Arg Ser Lys Leu Ser Asp Met Asn Asp Leu Pro He

380 385 390

Ser Ser Tyr Pro Gly Ser Asp Ser Trp Ala Cys Pro Gly Asn Thr

395 400 405

Glu Asp Val Phe Ser His Phe Leu Tyr Cys Lys Asp Leu Glu Leu

410 415 420

Leu Leu Ser Glu Ala Gly Pro Gin Glu Asp Asp Cys Ser Arg Pro

425 430 435

Gly Val Val His Asn Ser Cys Val Ala Arg Ser Glu He Gin Lys

440 445 450

Val Leu Asp Ser Leu Gin Glu His Leu Met Asn Asp Pro Asp Val

455 460 465

Gin Ala Gin Val Gin Val Leu Ser Ala Ala Leu Arg Ala Ala Gin

470 475 480

Leu Asp Cys Val Asn Glu Ala Glu Ser Lys Pro Thr Ala Gly Leu

485 490 495

Lys Glu Val Ser lie Ser His Pro Ser Ser Ala Ser Asp Asn Gin

500 505 510

He Ala Leu Ala Ala Ser Ser Ser Gin Asp Glu Leu Phe Val Ala

515 520 525

Arg He Leu Gin Ser Pro Asp Pro Gly Gly Pro Arg Asn Gly Thr

530 535 540

Ser Asp His Leu Glu Thr Asp Gin Arg Gin Asp Pro Thr Pro Leu

545 550 555

Glu Glu Asn Lys Ser Lys Leu Gin Asp Val He Pro Gin Pro Leu

560 565 570

Leu Asp Gin Tyr Val Ser Met Thr Asp Pro Ala Arg Ala Gin Thr

575 580 585

Val Asp Thr Asp He Ala Lys His Cys Ala Tyr Ser Leu Pro Gly

590 595 600

Val Ala Leu Thr Leu Gly Arg Gin Asn Trp His Cys Leu Lys Asp

605 610 615

Thr Tyr Glu Thr Leu Ala Ser Asp Val Gin Trp Lys Val Arg Arg

620 625 630

' Ala Leu Ala Phe Ser He His Glu Leu Ala Val He Leu Gly Asp

635 640 645

Gin Leu Thr Ala Ala Asp Leu Val Pro He Phe Asn Gly Phe Leu

650 655 660

Lys Asp Leu Asp Glu Val Arg He Gly Val Leu Arg His Leu Tyr

665 670 675

Asp Phe Leu Lys Leu Leu His Glu Asp Lys Arg Arg Asp Tyr Leu

680 685 690

Tyr Gin Leu Gin Glu Phe Val Val Thr Asp Asn Ser Arg Asn Trp

695 700 705

Arg Phe Arg Tyr Glu Leu Ala Glu Gin Leu He Leu He Leu Glu

710 715 720

Leu Tyr Ser Pro Asn Asp Val Tyr Asp Tyr Leu Met His He Ala

725 730 735

Leu Lys Leu Cys Ala Asp Gin Val Ser Glu Val Arg Trp He Ser

740 745 750

Phe Lys Leu Val Val Ala He Leu Gin Lys Phe Tyr Ser Asn Ser

755 760 765

Glu Ser Ala Leu Gly Leu Asn Phe He Asn Glu Leu He He Arg

770 775 780

Phe Arg His Cys Ser Lys Trp Val Gly Arg Gin Ala Phe Ala Phe

785 790 795

He Cys Gin Ala Val Val Ser Lys Glu Cys Val Pro Val Asp Gin

800 805 810 Phe Met Glu His Leu Leu Pro Ser Leu Leu Ser Leu Ala Ser Asp

815 820 825

Pro Val Pro Asn Val Arg Val Leu Leu Ala Lys Ala Leu Arg Gin

830 835 840

Met Leu Leu Glu Lys Ala Tyr Phe Arg Asn Ala Gly Asn Pro His

845 850 855

Leu Glu Val He Glu Glu Thr He Leu Ala Leu Gin Ser Asp Arg

860 865 870

Asp Gin Asp Val Ser Phe Phe Ala Ala Leu Glu Pro Lys Arg Arg

875 880 885

Asn He He Asp Thr Ala Val Leu Glu Lys Gin Asn

890 895

<210> 8

<211> 454

<212> PRT

<213> Homo sapiens

<220>

<221> misc_£eature

<223> Incyte ID No : 3882333CD1

<400> 8

Met Leu Lys Gin He Leu Ser Glu Met Tyr He Asp Pro Asp Leu

1 5 10 15

Leu Ala Glu Leu Ser Glu Glu Gin Lys Gin He Leu Phe Phe Lys

20 25 30

Met Arg Glu Glu Gin He Arg Arg Trp Lys Glu Arg Glu Ala Ala

35 40 45

Met Glu Arg Lys Glu Ser Leu Pro Val Lys Pro Arg Pro Lys Lys

50 55 60

Glu Asn Gly Lys Ser Val His Trp Lys Leu Gly Ala Asp Lys Glu

65 70 75

Val Trp Val Trp Val Met Gly Glu His His Leu Asp Lys Pro Tyr

80 85 90

Asp Val Leu Cys Asn Glu He He Ala Glu Arg Ala Arg Leu Lys

95 100 iθ5

Ala Glu Gin Glu Ala Glu Glu Pro Arg Lys Thr His Ser Glu Glu

110 115 120

Phe Thr Asn Ser Leu Lys Thr Lys Ser Gin Tyr His Asp Leu Gin

125 130 135

Ala Pro Asp Asn Gin Gin Thr Lys Asp He Trp Lys Lys Val Ala

140 145 150

Glu Lys Glu Glu Leu Glu Gin Gly Ser Arg Pro Ala Pro Thr Leu

155 160 165

Glu Glu Glu Lys He Arg Ser Leu Ser Ser Ser Ser Arg Asn He

170 175 180

Gin Gin Met Leu Ala Asp Ser He Asn Arg Met Lys Ala Tyr Ala

185 190 195

Phe His Gin Lys Lys Glu Ser Met Lys Lys Lys Gin Asp Gly Glu

200 205 210

He Asn Gin He Glu Gly Glu Arg Thr Lys Gin He Cys Lys Ser

215 220 225

Trp Lys Glu Asp Ser Glu Trp Gin Ala Ser Leu Arg Lys Ser Lys

230 235 240

Ala Ala Asp Glu Lys Arg Arg Ser Leu Ala Lys Gin Ala Arg Glu

245 250 255

Asp Tyr Lys Arg Leu Ser Leu Ala Ala Gin Lys Gly Arg Gly Gly 260 265 270

Glu Arg Leu Gin Ser Pro Leu Arg Val Pro Gin Lys Pro Glu Arg

275 280 285

Pro Pro Leu Pro Pro Lys Pro Gin Phe Leu Asn Ser Gly Ala Tyr

290 295 300

Pro Gin Lys Pro Leu Arg Asn Gin Gly Val Val Arg Thr Leu Ser

305 310 315

Ser Ser Ala Gin Glu Asp He He Arg Trp Phe Lys Glu Glu Gin

320 325 330

Leu Pro Leu Arg Ala Gly Tyr Gin Lys Thr Ser Asp Thr He Ala

335 340 345

Pro Trp Phe His Gly He Leu Thr Leu Lys Lys Ala Asn Glu Leu

350 355 360

Leu Leu Ser Thr Gly Met Pro Gly Ser Phe Leu He Arg Val Ser

365 370 375

Glu Arg He Lys Gly Tyr Ala Leu Ser Tyr Leu Ser Glu Asp Gly

380 385 390

Cys Lys His Phe Leu He Asp Ala Ser Ala Asp Ala Tyr Ser Phe

395 400 405

Leu Gly Val Asp Gin Leu Gin His Ala Thr Leu Ala Asp Leu Val

410 415 420

Glu Tyr His Lys Glu Glu Pro He Thr Ser Leu Gly Lys Glu Leu

425 430 435

Leu Leu Tyr Pro Cys Gly Gin Gin Asp Gin Leu Pro Asp Tyr Leu

440 , 445 450 Glu Leu Phe Glu

<210> 9

<211> 344

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> incyte ID No: 7482809CD1

<400> 9 i

Met Ala Thr Pro Tyr Val Pro Val Pro Met Pro He Gly Asn' Ser

1 5 10 15

Ala Ser Ser Phe Thr Thr Asn Arg Asn Gin Arg Ser Ser Ser Phe

20 25 30

Gly Ser Val Ser Thr Ser Ser Asn Ser Ser Lys Gly Gin Leu Glu

35 40 45

Asp Ser Asn Met Gly Asn Phe Lys Gin Thr Ser Val Pro Asp Gin

50 55 60

Met Asp Asn Thr Ser Ser Val Cys Ser Ser Pro Leu He Arg Thr

65 70 75

Lys Phe Thr Gly Thr Ala Ser Ser He Glu Tyr- Ser Thr Arg Pro

80 85 90

Arg Asp Thr Glu Glu Gin Asn Pro Glu Thr Val Asn Trp Glu Asp

95 100 105

Arg Pro Ser Thr Pro Thr He Leu Gly Tyr Glu Val Met Glu Glu

110 115 120

Arg Ala Lys Phe Thr Val Tyr Lys He Leu Val Lys Lys Thr Pro

125 130 135

Glu Glu Ser Trp Val Val Phe Arg Arg Tyr Thr Asp Phe Ser Arg

140 145 150 Leu Asn Asp Lys Leu Lys Glu Met Phe Pro Gly Phe Arg Leu Ala

155 160 165

Leu Pro Pro Lys Arg Trp Phe Lys Asp Asn Tyr Asn Ala Asp Phe

170 175 180

Leu Glu Asp Arg Gin Leu Gly Leu Gin Ala Phe Leu Gin Asn Leu

185 190 195

Val Ala His Lys Asp He Ala Asn Cys Leu Ala Val Arg Glu Phe

200 205 210

Leu Cys Leu Asp Asp Pro Pro Gly Pro Phe Asp Ser Leu Glu Glu

215 220 225

Ser Arg Ala Phe Cys Glu Thr Leu Glu Glu Thr Asn Tyr Arg Leu

230 235 240

Gin Lys Glu Leu Leu Glu Lys Gin Lys Glu Met Glu Ser Leu Lys

245 250 255

Lys Leu Leu Ser Glu Lys Gin Leu His He Asp Thr Leu Glu Asn

260 265 270

Arg He Arg Thr Leu Ser Leu Glu Pro Glu Glu Ser Leu Asp Val

275 280 285

Ser Glu Thr Glu Gly Glu Gin He Leu Lys Val Glu Ser Ser Ala

290 295 300

Leu Glu Val, Asp Gin Asp Val Leu Asp Glu Glu Ser Arg Ala Asp

305 310 315

Asn Lys Pro Cys Leu Ser Phe Ser Glu Pro Glu Asn Ala Val Ser

320 325 330

Glu He Glu Val Ala Glu Val Ala Tyr Asp Ala Glu Glu Asp

335 340

<210> 10

<211> 1115

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1739178CD1 *

<400> 10

Met Ala Lys He Val Val Val Thr Cys Ser Asp Ser Ser Phe Gly

1 5 10 15

Asn Phe Trp Leu Asp Gin Trp Gin Lys Arg Ala Arg Glu Lys Ser

20 25 30

Leu Cys Gin Cys Ser Ala Lys Gin Glu He Arg Thr Gin Leu Val

35 40 45 ^■

Glu Gin Phe Lys Cys Leu Glu Gin Gin Ser Glu Ser Arg Leu Gin

50 55 60

Leu Leu Gin Asp Leu Gin Glu Phe Phe Arg Arg Lys Ala Glu He

65 70 75

Glu Leu Glu Tyr Ser Arg Ser Leu Glu Lys Leu Ala Glu Arg Phe

80 85 90

Ser Ser Lys He Arg Ser Ser Arg Glu His Gin Phe Lys Lys Asp

95 100 105

Gin Tyr Leu Leu Ser Pro Val Asn Cys Trp Tyr Leu Val Leu His

110 115 120

Gin Thr Arg Arg Glu Ser Arg Asp His Ala Thr Leu Asn Asp He

125 130 135

Phe Met Asn Asn Val He Val Arg Leu Ser Gin He Ser Glu Asp

140 145 150

Val He Arg Leu Phe Lys Lys Ser Lys Glu He Gly Leu Gin Met 155 160 165

His Glu Glu Leu Leu Lys Val Thr Asn Glu Leu Tyr Thr Val Met

170 175 180

Lys Thr Tyr His Met Tyr His Ala Glu Ser He Ser Ala Glu Ser

185 190 195

Lys Leu Lys Glu Ala Glu Lys Gin Glu Glu Lys Gin Phe Asn Lys

200 205 210

Ser Gly Asp Leu Ser Met Asn Leu Leu Arg His Glu Asp Arg Pro

215 220 225

Gin Arg Arg Ser Ser Val Lys Lys He Glu Lys Met Lys Glu Lys

230 235 240

Arg Gin Ala Lys Tyr Ser Glu Asn Lys Leu Lys Cys Thr Lys Ala

245 250 255

Arg Asn Asp Tyr Leu Leu Asn Leu Ala Ala Thr Asn Ala Ala He

260 265 • 270

Ser Lys Tyr Tyr He His Asp Val Ser Asp Leu He Asp Cys Cys

275 280 285

Asp Leu Gly Phe His Ala Ser Leu Ala Arg Thr Phe Arg Thr Tyr

290 295 300

Leu Ser Ala Glu Tyr Asn Leu Glu Thr Ser Arg His Glu Gly Leu

305 310 315

Asp Val He Glu Asn Ala Val Asp Asn Leu Asp Ser Arg Ser Asp

320 325 330

Lys His Thr Val Met Asp Met Cys Asn Gin Val Phe Cys Pro Pro

335 340 345

Leu Lys Phe Glu Phe Gin Pro His Met Gly Asp Glu Val Cys Gin

350 355 360

Val Ser Ala Gin Gin Pro Val Gin Thr Glu Leu Leu Met Arg Tyr

365 370 375

His Gin Leu Gin Ser Arg Leu Ala Thr Leu Lys He Glu Asn Glu

380 385 390

Glu Val Arg Lys Thr Leu Asp Ala Thr Met Gin Thr Leu Gin Asp

395 400 405

Met Leu Thr Val Glu Asp Phe Asp Val Ser Asp Ala Phe Gin His

410 415 420

Ser Arg Ser Thr Glu Ser Val Lys Ser Ala Ala Ser Glu Thr Tyr

425 430 435

Met Ser Lys He Asn He Ala Lys Arg Arg Ala Asn Gin Gin Glu

440 445 450

Thr Glu Met Phe Tyr Phe Thr Lys Phe Lys Glu Tyr Val Asn Gly

455 460 465

Ser Asn Leu He Thr Lys Leu Gin Ala Lys His Asp Leu Leu Lys

470 475 480

Gin Thr Leu Gly Glu Gly Glu Arg Ala Glu Cys Gly Thr Thr Arg

485 490 495

Pro Pro Cys Leu Pro Pro Lys Pro Gin Lys Met Arg Arg Pro Arg

500 505 510

Pro Leu Ser Val Tyr Ser His Lys Leu Phe Asn Gly Ser Met Glu

515 520 525

Ala Phe He Lys Asp Ser Gly Gin Ala He Pro Leu Val Val Glu

530 535 540

Ser Cys He Arg Tyr He Asn Leu Tyr Gly Leu Gin Gin Gin Gly

545 550 555

He Phe Arg Val Pro Gly Ser Gin Val Glu Val Asn Asp He Lys

560 565 570

Asn Ser Phe Glu Arg Gly Glu Asp Pro Leu Val Asp Asp Gin Asn

575 580 585

Glu Arg Asp He Asn Ser Val Ala Gly Val Leu Lys Leu Tyr Phe 590 595 600

Arg Gly Leu Glu Asn Pro Leu Phe Pro Lys Glu Arg Phe Gin Asp

605 610 615

Leu He Ser Thr He Lys Leu Glu Asn Pro Ala Glu Arg Val His

620 625 630

Gin He Gin Gin He Leu Val Thr Leu Pro Arg Val Val He Val

635 640 645

Val Met Arg Tyr Leu Phe Ala Phe Leu Asn His Leu Ser Gin Tyr

650 655 660

Ser Asp Glu Asn Met Met Asp Pro Tyr Asn Leu Ala He Cys Phe

665 670 675

Gly Pro Thr Leu Met His He Pro Asp Gly Gin Asp Pro Val Ser

680 685 690

Cys Gin Ala His He Asn Glu Val He Lys Thr He He He His

695 700 705

His Glu Ala He Phe Pro Ser Pro Arg Glu Leu Glu Gly Pro Val

710 715 720

Tyr Glu Lys Cys Met Ala Gly Gly Glu Glu Tyr Cys Asp Ser Pro

725 730 735

His Ser Glu Pro Gly Ala He Asp Glu Val Asp His Asp Asn Gly

740 745 750

Thr Glu Pro His Thr Ser Asp Glu Glu Val Glu Gin He Glu Ala

755 760 765

He Ala Lys Phe Asp Tyr Met Gly Arg Ser Pro Arg Glu Leu Ser

770 775 780

Phe Lys Lys Gly Ala Ser Leu Leu Leu Tyr His Arg Ala Ser Glu

785 790 795

Asp Trp Trp Glu Gly Arg His Asn Gly Val Asp Gly Leu He Pro

800 805 810

His Gin Tyr He Val Val Gin Asp Met Asp Asp Ala Phe Ser Asp

815 820 825

Ser Leu Ser Gin Lys Ala Asp Ser Glu Ala Ser Ser Gly Pro Leu

830 835 840

Leu Asp Asp Lys Ala Ser Ser Lys Asn Asp Leu Gin Ser Pro Thr

845 850 855

Glu His He Ser Asp Tyr Gly Phe Gly Gly Val Met Gly Arg Val

860 865 870

Arg Leu Arg Ser Asp Gly Ala Ala He Pro Arg Arg Arg Ser Gly

875 880 885

Gly Asp Thr His Ser Pro Pro Arg Gly Leu Gly Pro Ser He Asp

890 895 900

Thr Pro Pro Arg Ala Ala Ala Cys Pro Ser Ser Pro His Lys He

905 910 915

Pro Leu Thr Arg Gly Arg He Glu Ser Pro Glu Lys Arg Arg Met

920 925 930

Ala Thr Phe Gly Ser Ala Gly Ser He Asn Tyr Pro Asp Lys Lys

935 940 945

Ala Leu Ser Glu Gly His Ser Met Arg Ser Thr Cys Gly Ser Thr

950 955 960

Arg His Ser Ser Leu Gly Asp His Lys Ser Leu Glu Ala Glu Ala

965 970 975

Leu Ala Glu Asp He Glu Lys Thr Met Ser Thr Ala Leu His Glu

980 985 990

Leu Arg Glu Leu Glu Arg Gin Asn Thr Val Lys Gin Ala Pro Asp

995 1000 1005

Val Val Leu Asp Thr Leu Glu Pro Leu Lys Asn Pro Pro Gly Pro

1010 1015 1020

Val Ser Ser Glu Pro Ala Ser Pro Leu His Thr He Val He Arg 1025 1030 1035

Asp Pro Asp Ala Ala Met Arg Arg Ser Ser Ser Ser Ser Thr Glu

1040 1045 1050

Met Met Thr Thr Phe Lys Pro Ala Leu Ser Ala Arg Leu Ala Gly

1055 1060 1065

Ala Gin Leu Arg Pro Pro Pro Met Arg Pro Val Arg Pro Val Val

1070 1075 1080

Gin His Arg Ser Ser Ser Ser Ser Ser Ser Gly Val Gly Ser Pro

1085 1090 1095

Ala Val Thr Pro Thr Glu Lys Met Phe Pro Asn Ser Ser Ala Asp

1100 1105 1110 Lys Ser Gly Thr Met

1115

<210> 11

<211> 839

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 7473630CD1 •

<400> 11

Met Ala Pro Asn Leu Lys Lys Gly Thr Ser Ser Cys Pro Gly Leu

1 5 10 15

Thr Asn Gin Glu Thr His Ser Asp Ser Lys Gly Glu Gly Ala Gly

20 25 30

Pro Asp Gly Lys He Tyr Asp Gly Lys Asp Lys Thr Thr His Leu

35 40 45

Leu Gly Ala Phe Thr Gly Ala Ser Met Arg Gly Leu Thr Leu Ser

50 55 60

Ser Thr Ser Asn Gin Leu Trp Leu Glu Phe Asn Ser Asp Thr Glu

65 70 75

Gly Thr Asp Glu Gly Phe Gin Leu Val Tyr Thr Lys Arg He He

80 85 90

Gly He Ala Glu Glu Val Thr Val Leu Thr Leu Thr Glu Ser Glu

95 100 105

Gin Glu Arg Glu His Leu Ser Arg Glu Asp Gin Val Leu Asn Ser

110 115 120

His Thr Val Lys He Leu Ala Phe His Asn Leu Asp Thr Arg Ser

125 130 135

Val Thr Lys Ala Thr Leu Leu Val Ala Pro Ser Phe Met Asp Ala

140 145 150 lie Gin Ala Thr Leu Ser Thr Glu Val Ala Phe Ser Thr Glu Cys

155 160 165

Gly Gly Arg Phe Lys Gly Glu Ser Ser Gly Arg He Leu Ser Pro

170 175 180

Gly Tyr Pro Phe Pro Tyr Asp Asn Asn Leu Arg Cys Met Trp Met

185 190 195

He Glu Val Asp Pro Gly Asn He Val Ser Leu Gin Phe Leu Ala

200 205 210

Phe Asp Thr Glu Ala Ser His Asp He Leu Arg Val Trp Asp Gly

215 220 225

Pro Pro Glu Asn Asp Met Leu Leu Lys Glu He Ser Gly Ser Leu

230 235 240

He Pro Glu Gly He His Ser Thr Leu Asn He Val Thr He Gin

245 250 255 Phe Asp Thr Asp Phe Tyr He Ser Lys Ser Gly Phe Ala He Gin

260 265 270

Phe Ser Ser Ser Val Ala Thr Ala Cys Arg Asp Pro Gly Val Pro

275 280 285

Met Asn Gly Thr Arg Asn Gly Asp Gly Arg Glu Pro Gly Asp Thr

290 295 300

Val Val Phe Gin Cys Asp Pro Gly Tyr Glu Leu Gin Gly Glu Glu

305 310 315

Arg He Thr Cys He Gin Val Glu Asn Arg Tyr Phe Trp Gin Pro

320 325 330

Ser Pro Pro Val Cys He Ala Pro Cys Gly Gly Asn Leu Thr Gly

335 340 345

Ser Ser Gly Phe He Leu Ser Pro Asn Phe Pro His Pro Tyr Pro

350 355 360

His Ser Arg Asp Cys Asp Trp Thr He Thr Val Asn Ala Asp Tyr

365 370 375

Val He Ser Leu Ala Phe He Ser Phe Ser He Glu Pro Asn Tyr

380 385 390

Asp Phe Leu Tyr He Tyr Asp Gly Pro Asp Ser Asn Ser Pro Leu

395 400 405

He Gly Ser Phe Gin Asp Ser Lys Leu Pro Glu Arg He Glu Ser

410 415 420

Ser Ser Asn Thr Met His Leu Ala Phe Arg Ser Asp Gly Ser Val

425 430 435

Ser Tyr Thr Gly Phe His Leu Glu Tyr Lys Ala Lys Leu Arg Glu

440 445 450

Ser Cys Phe Asp Pro Gly Asn He Met Asn Gly Thr Arg Leu Gly

455 460 465

Met Asp Tyr Lys Leu Gly Ser Thr Val Thr Tyr Tyr Cys Asp Ala

470 475 480

Gly Tyr Val Leu Gin Gly Tyr Ser Thr Leu Thr Cys Phe Met Gly

485 490 495

Asp Asp Gly Arg Pro Gly Trp Asn Arg Ala Leu Pro Ser Cys His

500 505 510

Ala Pro Cys Gly Ser Arg Ser Thr Gly Ser Glu Gly Thr Val Leu

515 520 525

Ser Pro Asn Tyr Pro Lys Asn Tyr Ser Val Gly His Asn Cys Val

530 535 540

Tyr Ser He Ala Val Pro Lys Glu Leu Trp Cys Trp Pro Val Val

545 550 555

Phe Phe Gin Thr Ser Leu His Asp Val Val Glu Val Tyr Asp Gly

560 565 570

Pro Thr Gin Gin Ser Ser Leu Leu Ser Ser Leu Ser Gly Ser His

575 580 585

Ser Gly Glu Ser Leu Pro Leu Ser Ser Gly Asn Gin He Thr He

590 595 600

Arg Phe Thr Ser Val Gly Pro He Thr Ala Lys Gly Phe His Phe

605 610 615

Val Tyr Gin Ala Val Pro Arg Thr Ser Ser Thr Gin Cys Ser Ser

620 625 630

Val Pro Glu Pro Arg Phe Gly Arg Arg He Gly Asn Glu Phe Ala

635 640 645

Val Gly Ser Ser Val Leu Phe Asp Cys Asn Pro Gly Tyr He Leu

650 655 660

His Gly Ser He Ala He Arg Cys Glu Thr Val Pro Asn Ser Leu

665 670 675

Ala Gin Trp Asn Asp Ser Leu Pro Thr Cys He Val Pro Cys Gly

680 685 690 Gly He Leu Thr Lys Arg Lys Gly Thr He Leu Ser Pro Gly Tyr

695 700 705

Pro Glu Pro Tyr Asp Asn Asn Leu Asn Cys Val Trp Lys He Thr

710 715 720

Val Pro Glu Gly Ala Gly He Gin Val Gin Val Val Ser Phe Ala

725 730 735

Thr Glu His Asn Trp Asp Ser Leu Asp Phe Tyr Asp Gly Gly Asp

740 745 750

Asn Asn Ala Pro Arg Leu Gly Ser Tyr Ser Gly Thr Thr He Pro

755 760 765

His Leu Leu Asn Ser Thr Ser Asn Asn Leu Tyr Leu Asn Phe Gin

770 775 780

Ser Asp He Ser Val Ser Ala Ala Gly Phe His Leu Glu Tyr Thr

785 790 795

Ala He Gly Leu Asp Ser Cys Pro Glu Pro Gin Thr Pro Ser Ser

800 805 810

Gly He Lys He Gly Asp Arg Tyr Met Val Gly Asp Val Val Ser

815 820 825

Phe Gin Cys Asp Gin Gly Tyr Ser Leu Gin Val Ser Leu Phe

830 835

<210> 12

<211> 304

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1431520CD1

<400> 12

Met Ser Ser He Lys His Leu Val Tyr Ala Val He Arg Phe Leu

1 5 10 15

Arg Glu Gin Ser Gin Met Asp Thr Tyr Thr Ser Asp Glu Gin Glu

20 25 30

Ser Leu Glu Val Ala He Gin Cys Leu Glu Thr Val Phe Lys He

35 40 45

Ser Pro Glu Asp Thr His Leu Ala Val Ser Gin Pro Leu Thr Glu

50 55 60

Met Phe Thr Ser Ser Phe Cys Lys Asn Asp Val Leu Pro Leu Ser

65 70 75

Asn Ser Val Pro Glu Asp Val Gly Lys Ala Asp Gin Leu Lys Asp

80 85 90

Glu Gly Asn Asn His Met Lys Glu Glu Asn Tyr Ala Ala Ala Val

95 100 105

Asp Cys Tyr Thr Gin Ala He Glu Leu Asp Pro Asn Asn Ala Val

110 115 120

Tyr Tyr Cys Asn Arg Ala Ala Ala Gin Ser Lys Leu Gly His Tyr

125 130 135

Thr Asp Ala He Lys Asp Cys Glu Lys Ala He Ala He Asp Ser

140 145 150

Lys Tyr Ser Lys Ala Tyr Gly Arg Met Gly Leu Ala Leu Thr Ala

155 160 165

Leu Asn Lys Phe Glu Glu Ala Val Thr Ser Tyr Gin Lys Ala Leu

170 175 180

Asp .Leu Asp Pro Glu Asn Asp Ser Tyr Lys Ser Asn Leu Lys He

185 190 195

Ala Glu Gin Lys Leu Arg Glu Val Ser Ser Pro Thr Gly Thr Gly 200 205 210

Leu Ser Phe Asp Met Ala Ser Leu He Asn Asn Pro Ala Phe He

215 220 225

Ser Met Ala Ala Ser Leu Met Gin Asn Pro Gin Val Gin Gin Leu

230 235 240 Met Ser Gly Met Met Thr Asn Ala He Gly Gly Pro Ala Ala Gly

245 250 255

Val Gly Gly Leu Thr Asp Leu Ser Ser Leu He Gin Ala Gly Gin

260 265 270

Gin Phe Ala Gin Gin He Gin Gin Gin Asn Pro Glu Leu He Glu

275 280 285

Gin Leu Arg Asn His He Arg Ser Arg Ser Phe Ser Ser Ser Ala

290 295 300 Glu Glu His Ser

<210> 13

<211> 440

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1916304CD1

<400> 13

Met Pro He Pro Pro Pro Pro Pro Pro Pro Pro Gly Pro Pro Pro

1 5 10 15

Pro Pro Thr Phe His Gin Ala Asn Thr Glu Gin Pro Lys Leu Ser

20 25 30

Arg Asp Glu Gin Arg Gly Arg Gly Ala Leu Leu Gin Asp He Cys

35 40 45

Lys Gly Thr Lys Leu Lys Lys Val Thr Asn He Asn Asp Arg Ser

50 55 60

Ala Pro He Leu Glu Lys Pro Lys Gly Ser Ser Gly Gly Tyr Gly

65 70 75

Ser Gly Gly Ala Ala Leu Gin Pro Lys Gly Gly Leu Phe Gin Gly

80 85 90

Gly Val Leu Lys Leu Arg Pro Val Gly Ala Lys Asp Gly Ser Glu

95 100 105

Asn Leu Ala Gly Lys Pro Ala Leu Gin He Pro Ser Ser Arg Ala

110 115 120

Ala Ala Pro Arg Pro Pro Val Ser Ala Ala Ser Gly Arg Pro Gin

125 ^' 130 135

Asp Asp Thr Asp Ser Ser Arg Ala Ser Leu Pro Glu Leu Pro Arg

140 145 150

Met Gin Arg Pro Ser Leu Pro Asp Leu Ser Arg Pro Asn Thr Thr

155 160 165

Ser Ser Thr Gly Met Lys His Ser Ser Ser Ala Pro Pro Pro Pro

170 175 180

Pro Pro Gly Arg Arg Ala Asn Ala Pro Pro Thr Pro Leu Pro Met

185 190 195

His Ser Ser Lys Ala Pro Ala Tyr Asn Arg Glu Lys Pro Leu Pro

200 205 210

Pro Thr Pro Gly Gin Arg Leu His Pro Gly Arg Glu Gly Pro Pro

215 220 225

Ala Pro Pro Pro Val Lys Pro Pro Pro Ser Pro Val Asn He Arg

230 235 240 Thr Gly Pro Ser Gly Gin Ser Leu Ala Pro Pro Pro Pro Pro Tyr

245 250 255

Arg Gin Pro Pro Gly Val Pro Asn Gly Pro Ser Ser Pro Thr Asn

260 265 270

Glu Ser Ala Pro Glu Leu Pro Gin Arg His Asn Ser Leu His Arg

275 280 285

Lys Thr Pro Gly Pro Val Arg Gly Leu Ala Pro Pro Pro Pro Thr

290 295 300

Ser Ala Ser Pro Ser Leu Leu Ser Asn Arg Pro Pro Pro Pro Ala

305 310 315

Arg Asp Pro Pro Ser Arg Gly Ala Ala Pro Pro Pro Pro Pro Pro

320 325 330

Val He Arg Asn Gly Ala Arg Asp Ala Pro Pro Pro Pro Pro Pro

335 340 345

Tyr Arg Met His Gly Ser Glu Pro Pro Ser Arg Gly Lys Pro Pro

350 355 360

Pro Pro Pro Ser Arg Thr Pro Ala Gly Pro Pro Pro Pro Pro Pro

365 370 375

Pro Pro Leu Arg Asn Gly His Arg Asp Ser He Thr Thr Val Arg

380 385 390

Ser Phe Leu Asp Asp Phe Glu Ser Lys Tyr Ser Phe His Pro Val

395 400 405

Glu Asp Phe Pro Ala Pro Glu Glu Tyr Lys His Phe Gin Arg He

410 415 420

Tyr Pro Ser Lys Thr Asn Arg Ala Ala Arg Gly Ala Pro Pro Leu

425 430 435

Pro Pro He Leu Arg

440

<210> 14

<211> 747

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 378504CD1

<400> 14

Met Gin Gly Gly Glu Pro Val Ser Thr Met Lys Val Ser Glu Ser

1 5 10 15

Glu Gly Lys Leu Glu Gly Gin Ala Thr Ala Val Thr Pro Asn Lys

20 25 30

Asn Ser Ser Cys Gly Gly Gly He Ser Ser Ser Ser Ser Ser Arg

35 40 45

Gly Gly Ser Ala Lys Gly Trp Gin Tyr Ser Asp His Met Glu Asn

50 55 60

Val Tyr Gly Tyr Leu Met Lys Tyr Thr Asn Leu Val Thr Gly Trp

65 70 75

Gin Tyr Arg Phe Phe Val Leu Asn Asn Glu Ala Gly Leu Leu Glu

80 85 90

Tyr Phe Val Asn Glu Gin Ser Arg Asn Gin Lys Pro Arg Gly Thr

95 100 105

Leu Gin Leu Ala Gly Ala Val He Ser Pro Ser Asp Glu Asp Ser

110 115 120

His Thr Phe Thr Val Asn Ala Ala Ser Gly Glu Gin Tyr Lys Leu

125 130 135

Arg Ala Thr Asp Ala Lys Glu Arg Gin His Trp Val Ser Arg Leu 140 145 150

Gin He Cys Thr Gin His His Thr Glu Ala He Gly Lys Asn Asn

155 160 165

Pro Pro Leu Lys Ser Arg Ser Phe Ser Leu Ala Ser Ser Ser Asn

170 175 180

Ser Pro He Ser Gin Arg Arg Pro Ser Gin Asn Ala He Ser Phe

185 190 195

Phe Asn Val Gly His Ser Lys Leu Gin Ser Leu Ser Lys Arg Thr

200 205 210

Asn Leu Pro Pro Asp His Leu Val Glu Val Arg Glu Met Met Ser

215 220 225

His Ala Glu Gly GlnOln Arg Asp Leu He Arg Arg He Glu Cys

230 235 240

Leu Pro Thr Ser Gly His Leu ^• Ser Ser Leu Asp Gin Asp Leu Leu

245 250 255

Met Leu Lys Ala Thr Ser Met Ala Thr Met Asn Cys Leu Asn Asp

250 265 270

Cys Phe His He Leu Gin Leu Gin His Ala Ser His Gin Lys Gly

275 280 285

Ser Leu Pro Ser Gly Thr Thr He Glu Trp Leu Glu Pro Lys He

290 295 300

Ser Leu Ser Asn His Tyr Lys Asn Gly Ala Asp Gin Pro Phe Ala

305 310 315

Thr Asp Gin Ser Lys Pro Val Ala Val Pro Glu Glu Gin Pro Val

320 325 330

Ala Glu Ser Gly Leu Leu Ala Arg Glu Pro Glu Glu He Asn Ala

335 340 345

Asp Asp Glu He Glu Asp Thr Cys Asp His Lys Glu Asp Asp Leu

350 355 360

Gly Ala Val Glu Glu Gin Arg Ser Val He Leu His Leu Leu Ser

365 370 375

Gin Leu Lys Leu Gly Met Asp Leu Thr Arg Val Val Leu Pro Thr

380 385 390

Phe He Leu Glu Lys Arg Ser Leu Leu Glu Met Tyr Ala Asp Phe

395 400 405

Met Ser His Pro Asp Leu Phe He Ala He Thr Asn Gly Ala Thr

410 415 420

Ala Glu Asp Arg Met He Arg Phe Phe Glu Tyr Tyr Leu Thr Ser

425 430 435

Phe His Glu Gly Arg Lys Gly Ala He Ala Lys Lys Pro Tyr Asn

440 445 450

Pro He He Gly Glu Thr Phe His Cys Ser Trp Lys Met Pro Lys

455 460 465

Ser Glu Val Ala Ser Ser Val Phe Ser Ser Ser Ser Thr Gin Gly

470 475 480

Val Thr Asn His Ala Pro Leu Ser Gly Glu Ser Leu Thr Gin Val

485 490 495

Gly Ser Asp Cys Tyr Thr Val Arg Phe Val Ala Glu Gin Val Ser

500 505 510

His His Pro Pro Val Ser Gly Phe Tyr Ala Glu Cys Thr Glu Arg

515 520 525

Lys Met Cys Val Asn Ala His Val Trp Thr Lys Ser Lys Phe Leu

530 535 540

Gly Met Ser He Gly Val Thr Met Val Gly Glu Gly He Leu Ser

545 550 555

Leu Leu Glu His Gly Glu Glu Tyr Thr Phe Ser Leu Pro Cys Ala

560 565 570

Tyr Ala Arg Ser He Leu Thr Val Pro Trp Val Glu Leu Gly Gly 575 580 585

Lys Val Ser Val Asn Cys Ala Lys Thr Gly Tyr Ser Ala Ser He

590 595 600

Thr Phe His Thr Lys Pro Phe Tyr Gly Gly Lys Leu His Arg Val

605 610 615

Thr Ala Glu Val Lys His Asn He Thr Asn Thr Val Val Cys Arg

620 625 630

Val Gin Gly Glu Trp Asn Ser Val Leu Glu Phe Thr Tyr Ser Asn

635 640 645

Gly Glu Thr Lys Tyr Val Asp Leu Thr Lys Leu Ala Val Thr Lys

650 655 660

Lys Arg Val Arg Pro Leu Glu Lys Gin Asp Pro Phe Glu Ser Arg

665 670 675

Arg Leu Trp Lys Asn Val Thr Asp Ser Leu Arg Glu Ser Glu He

680 685 590

Asp Lys Ala Thr Glu His Lys His Thr Leu Glu Glu Arg Gin Arg

695 700 705

Thr Glu Glu Arg His Arg Thr Glu Thr Gly Thr Pro Trp Lys Thr

710 715 720

Lys Tyr Phe He Lys Glu Gly Asp Gly Trp Val Tyr His Lys Pro ' 725 730 735

Leu Trp Lys He He Pro Thr Thr Gin Pro Ala Glu

740 745

<210> 15

<211> 770

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 5275371CD1

<400> 15

Met Pro Leu Leu Glu Lys Asn Glu Pro Lys Met Ser Glu Ala Lys

1 5 10 15

Asn Tyr Leu Ser Ser He Leu Asn His Gly Arg Leu Ser Pro Gin

20 25 30

Tyr Met Cys Glu Ala Met Leu He Leu Gly Lys Leu His Tyr Val

35 40 45

Glu Gly Ser Tyr Arg Asp Ala He Ser Met Tyr Ala Arg Ala Gly

50 55 60

He Asp Asp Met Ser Met Glu Asn Lys Pro Leu Tyr Gin Met Arg

65 70 75

Leu Leu Ser Glu Ala Phe Val He Lys Gly Leu Ser Leu Glu Arg

80 85 90

Leu Pro Asn Ser He Ala Ser Arg Phe Arg Leu Thr Glu Arg Glu

95 100 105

Glu Glu Val He Thr Cys Phe Glu Arg Ala Ser Trp He Ala Gin

110 115 120

Val Phe Leu Gin Glu Leu Glu Lys Thr Thr Asn Asn Ser Thr Ser

125 130 135

Arg His Leu Lys Gly Cys His Pro Leu Asp Tyr Glu Leu Thr Tyr

140 145 ■ 150

Phe Leu Glu Ala Ala Leu Gin Ser Ala Tyr Val Lys Asn Leu Lys

155 160 165

Lys Gly Asn He Val Lys Gly Met Arg Glu Leu Arg Glu Val Leu

170 175 180 Arg Thr Val Glu Thr Lys Ala Thr Gin Asn Phe Lys Val Met Ala

185 190 195

Ala Lys His Leu Ala Gly Val Leu Leu His Ser Leu Ser Glu Glu

200 205 210

Cys Tyr Trp Ser Pro Leu Ser His Pro Leu Pro Glu Phe Met Gly

215 220 225

Lys Glu Glu Ser Ser Phe Ala Thr Gin Ala Leu Arg Lys Pro His

230 ^' 235 240

Leu Tyr Glu Gly Asp Asn Leu Tyr Cys Pro Lys Asp Asn He Glu

245 250 255

Glu Ala Leu Leu Leu Leu Leu He Ser Glu Ser Met Ala Thr Arg

260 265 270

Asp Val Val Leu Ser Arg Val Pro Glu Gin Glu Glu Asp Arg Thr

275 280 285

Val Ser Leu Gin Asn Ala Ala Ala He Tyr Asp Leu Leu Ser He

290 295 300

Thr Leu Gly Arg Arg Gly Gin Tyr Val Met Leu Ser Glu Cys Leu

305 310 315

Glu Arg Ala Met Lys Phe Ala Phe Gly Glu Phe His Leu Trp Tyr

320 325 330

Gin Val Ala Leu Ser Met Val Ala Cys Gly Lys Ser Ala Tyr Ala

335 340 345

Val Ser Leu Leu Arg Glu Cys Val Lys Leu Arg Pro Ser Asp Pro

350 355 360

Thr Val Pro Leu Met Ala Ala Lys Val Cys He Gly Ser Leu Arg

365 370 375

Trp Leu Glu Glu Ala Glu His Phe Ala Met Met Val He Ser Leu

380 • 385 390

Gly Glu Glu Ala Gly Glu Phe Leu Pro Lys Gly Tyr Leu Ala Leu

395 400 405

Gly Leu Thr Tyr Ser Leu Gin Ala Thr Asp Ala Thr Leu Lys Ser

410 415 420

Lys Gin Asp Glu Leu His Arg Lys Ala Leu Gin Thr Leu Glu Arg

425 430 435

Ala Gin Gin Leu Ala Pro Ser Asp Pro Gin Val He Leu Tyr Val

440 445 450

Ser Leu Gin Leu Ala Leu Val Arg Gin He Ser Ser Ala Met Glu

455 460 465

Gin Leu Gin Glu Ala Leu Lys Val Arg Lys Asp Asp Ala His Ala

470 475 480

Leu His Leu Leu Ala Leu Leu Phe Ser Ala Gin Lys His His Gin

485 490 495

His Ala Leu Asp Val Val Asn Met Ala He Thr Glu His Pro Glu

500 505 510

Asn Phe Asn Leu Met Phe Thr Lys Val Lys Leu Glu Gin Val Leu

515 520 525

Lys Gly Pro Glu Glu Ala Leu Val Thr Cys Arg Gin Val Leu Arg

530 535 540

Leu Trp Gin Thr Leu Tyr Ser Phe Ser Gin Leu Gly Gly Leu Glu

545 550 555

Lys Asp Gly Ser Phe Gly Glu Gly Leu Thr Met Lys Lys Gin Ser

560 565 570

Gly Met His Leu Thr Leu Pro Asp Ala His Asp Ala Asp Ser Gly

575 580 585

Ser Arg Arg Ala Ser Ser He Ala Ala Ser Arg Leu Glu Glu Ala

590 595 600

Met Ser Glu Leu Thr Met Pro Ser Ser Val Leu Lys Gin Gly Pro

605 610 615 Met Gin Leu Trp Thr Thr Leu Glu Gin He Trp Leu Gin Ala Ala

620 625 630

Glu Leu Phe Met Glu Gin Gin His Leu Lys Glu Ala Gly Phe Cys

635 640 645

He Gin Glu Ala Ala Gly Leu Phe Pro Thr Ser His Ser Val Leu

650 655 660

Tyr Met Arg Gly Arg Leu Ala Glu Val Lys Gly Asn Leu Glu Glu

665 670 675

Ala Lys Gin Leu Tyr Lys Glu Ala Leu Thr Val Asn Pro Asp Gly

680 685 690

Val Arg He Met His Ser Leu Gly Leu Met Leu Ser Arg Leu Gly

695 700 705

His Lys Ser Leu Ala Gin Lys Val Leu Arg Asp Ala Val Glu Arg

710 715 720

Gin Ser Thr Cys His Glu Ala Trp Gin Gly Leu Gly Glu Val Leu

725 730 735

Gin Ala Gin Gly Gin Asn Glu Ala Ala Val Asp Cys Phe Leu Thr

740 745 750 Ala Leu Glu Leu Glu Ala Ser Ser Pro Val Leu Pro Phe Ser He

755 760 765 lie Pro Arg Glu Leu

770

<210> 16

<211> 199

<212> PRT

<213> Homo sapiens

<220>

<221> ιrtisc_f eature

<223> Incyte ID No : 490576CD1

<400> 16

Met Pro Glu Gin Ser Asn Asp Tyr Arg Val Ala Val Phe Gly Ala

1 5 10 15

Gly Gly Val Gly Lys Ser Ser Leu Val Leu Arg Phe Val Lys Gly

20 25 30

Thr Phe Arg Glu Ser Tyr He Pro Thr Val Glu Asp Thr Tyr Arg

35 40 45

Gin Val He Ser Cys Asp Lys Ser He Cys Thr Leu Gin He Thr

50 55 60

Asp Thr Thr Gly Ser His Gin Phe Pro Ala Met Gin Arg Leu Ser

65 70 75

He Ser Lys Gly His Ala Phe He Leu Val Tyr Ser He Thr Ser

80 85 90

Arg Gin Ser Leu Glu Glu Leu Lys Pro He Tyr Glu Gin He Cys

95 100 105

Glu He Lys Gly Asp Val Glu Ser He Pro He Met Leu Val Gly

110 115 120

Asn Lys Cys Asp Glu Ser Pro Ser Arg Glu Val Gin Ser Ser Glu

125 130 135

Ala Glu Ala Leu Ala Arg Thr Trp Lys Cys Ala Phe Met Glu Thr

140 145 150

Ser Ala Lys Leu Asn His Asn Val Lys Glu Leu Phe Gin Glu Leu

155 160 165

Leu Asn Leu Glu Lys Arg Arg Thr Val Ser Leu Gin lie Asp Gly

170 175 180

Lys Lys Ser Lys Gin Gin Lys Arg Lys Glu Lys Leu Lys Gly Lys 185 190 195

Cys Val He Met

<210> 17

<211> 790

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1417657CD1

<400> 17

Met Glu Lys Met Ser Arg Val Thr Thr Ala Leu Gly Gly Ser Val

1 5 10 15

Leu Thr Gly Arg Thr Met His Cys His Leu Asp Ala Pro Ala Asn

20 25 30

Ala He Ser Val Cys Arg Asp Ala Ala Gin Val Val Val Ala Gly

35 40 45

Arg Ser He Phe Lys He Tyr Ala He Glu Glu Glu Gin Phe Val

50 55 60

Glu Lys Leu Asn Leu Arg Val Gly Arg Lys Pro Ser Leu Asn Leu

65 70 75

Ser Cys Ala Asp Val Val Trp His Gin Met Asp Glu Asn Leu Leu

80 85 90

Ala Thr Ala Ala Thr Asn Gly Val Val Val Thr Trp Asn Leu Gly

95 100 105

Arg Pro Ser Arg Asn Lys Gin Asp Gin Leu Phe Thr Glu His Lys

110 115 120

Arg Thr Val Asn Lys Val Cys Phe His Pro Thr Glu Ala His Val

125 130 135

Leu Leu Ser Gly Ser Gin Asp Gly Phe Met Lys Cys Phe Asp Leu

140 145 150

Arg Arg Lys Asp Ser Val Ser Thr Phe Ser Gly Gin Ser Glu Ser

155 160 165

Val Arg Asp Val Gin Phe Ser He Arg Asp Tyr Phe Thr Phe Ala

170 175 180

Ser Thr Phe Glu Asn Gly Asn Val Gin Leu Trp Asp He Arg Arg

185 190 195

Pro Asp Arg Cys Glu Arg Met Phe Thr Ala His Asn Gly Pro Val

200 205 210

Phe Cys Cys Asp Trp His Pro Glu Asp Arg Gly Trp Leu Ala Thr

215 220 225

Gly Gly Arg Asp Lys Met Val Lys Val Trp Asp Met Thr Thr His

230 " 235 240

Arg Ala Lys Glu Met His Cys Val Gin Thr He Ala Ser Val Ala

245 250 255

Arg Val Lys Trp Arg Pro Glu Cys Arg His His Leu Ala Thr Cys

260 265 270

Ser Met Met Val Asp His Asn He Tyr Val Trp Asp Val Arg Arg

275 ^• 280 285

Pro Phe Val Pro Ala Ala Met Phe Glu Glu His Arg Asp Val Thr

290 295 300

Thr Gly He Ala Trp Arg His Pro His Asp Pro Ser Phe Leu Leu

305 310 315

Ser Gly Ser Lys Asp Ser Ser Leu Cys Gin His Leu Phe Arg Asp

320 325 330 Ala Ser Gin Pro Val Glu Arg Ala Asn Pro Glu Gly Leu Cys Tyr

335 340 345

Gly Leu Phe Gly Asp Leu Ala Phe Ala Ala Lys Glu Ser Leu Val

350 355 360

Ala Ala Glu Ser Gly Arg Lys Pro Tyr Thr Gly Asp Arg Arg His

365 370 375

Pro He Phe Phe Lys Arg Lys Leu Asp Pro Ala Glu Pro Phe Ala

380 385 390

Gly Leu Ala Ser Ser Ala Leu Ser Val Phe Glu Thr Glu Pro Gly

395 400 405

Gly Gly Gly Met Arg Trp Phe Val Asp Thr Ala Glu Arg Tyr Ala

410 415 420

Leu Ala Gly Arg Pro Leu Ala Glu Leu Cys Asp His Asn Ala Lys

425 430 435

Val Ala Arg Glu Leu Gly Arg Asn Gin Val Ala Gin Thr Trp Thr

440 445 450

Met Leu Arg He He Tyr Cys Ser Pro Gly Leu Val Pro Thr Ala

455 460 - 465

Asn Leu Asn His Ser Val Gly Lys Gly Gly Ser Cys Gly Leu Pro

470 475 480

Leu Met Asn Ser Phe Asn Leu Lys Asp Met Ala Pro Gly Leu Gly

485 490 495

Ser Glu Thr Arg Leu Asp Arg Ser Lys Gly Asp Ala Arg Ser Asp

500 505 510

Thr Val Leu Leu Asp Ser Ser Ala Thr Leu He Thr Asn Glu Asp

515 520 525

Asn Glu Glu Thr Glu Gly Ser Asp Val Pro Ala Asp Tyr Leu Leu

530 535 540

Gly Asp Val Glu Gly Glu Glu Asp Glu Leu Tyr Leu Leu Asp Pro

545 550 555

Glu His Ala His Pro Glu Asp Pro Glu Cys Val Leu Pro Gin Glu

560 565 570

Ala Phe Pro Leu Arg His Glu He Val Asp Thr Pro Pro Gly Pro

575 580 585

Glu His Leu Gin Asp Lys Ala Asp Ser Pro His Val Ser Gly Ser

590 595 600

Glu Ala Asp Val Ala Ser Leu Ala Pro Val Asp Ser Ser Phe Ser

605 610 615

Leu Leu Ser Val Ser His Ala Leu Tyr Asp Ser Arg Leu Pro Pro

620 625 630

Asp Phe Phe Gly Val Leu Val Arg Asp Met Leu His Phe Tyr Ala

635 640 645

Glu Gin Gly Asp Val Gin Met Ala Val Ser Val Leu He Val Leu

650 655 660

Gly Glu Arg Val Arg Lys Asp He Asp Glu Gin Thr Gin Glu His

665 670 675

Trp Tyr Thr Ser Tyr He Asp Leu Leu Gin Arg Phe Arg Leu Trp

680 685 690

Asn Val Ser Asn Glu Val Val Lys Leu Ser Thr Ser Arg Ala Val

695 700 705

Ser Cys Leu Asn Gin Ala Ser Thr Thr Leu His Val Asn Cys Ser

710 715 720

His Cys Lys Arg Pro Met Ser Ser Arg Gly Trp Val Cys Asp Arg

725 730 735

Cys His Arg Cys Ala Ser Met Cys Ala Val Cys His His Val Val

740 745 750

Lys Gly Leu Phe Val Trp Cys Gin Gly Cys Ser His Gly Gly His

755 760 765 Leu Gin His He Met Lys Trp Leu Glu Gly Ser Ser His Cys Pro 770 775 780

Ala Gly Cys Gly His Leu Cys Glu Tyr Ser 785 790

<210> 18

<211> 490

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1773215CD1

<400> 18

Met Glu Glu Glu Gly Val Lys Glu Ala Gly Glu Lys Pro Arg Gly

1 5 10 15

Ala Gin Met Val Asp Lys Ala Gly Trp He Lys Lys Ser Ser Gly

20 25 30

Gly Leu Leu Gly Phe Trp Lys Asp Arg Tyr Leu Leu Leu Cys Gin

35 40 45

Ala Gin Leu Leu Val Tyr Glu Asn Glu Asp Asp Gin Lys Cys Val

50 55 60

Glu Thr Val Glu Leu Gly Ser Tyr Glu Lys Cys Gin Asp Leu Arg

65 70 75

Ala Leu Leu Lys Arg Lys His Arg Phe He Leu Leu Arg Ser Pro

80 85 90

Gly Asn Lys Val Ser Asp He Lys Phe Gin Ala Pro Thr Gly Glu

95 100 105

Glu Lys Glu Ser Trp He Lys Ala Leu Asn Glu Gly He Asn Arg

110 115 120

Gly Lys Asn Lys Ala Phe Asp Glu Val Lys Val Asp Lys Ser Cys

125 130 135

Ala Leu Glu His Val Thr Arg Asp Arg Val Arg Gly Gly Gin Arg

140 145 150

Arg Arg Pro Pro Thr Arg Val His Leu Lys Glu Val Ala Ser Ala

155 160 165

Ala Ser Asp Gly Leu Leu Arg Leu Asp Leu Asp Val Pro Asp Ser

170 175 180

Gly Pro Pro Val Phe Ala Pro Ser Asn His Val Ser Glu Ala Gin

185 190 195

Pro Arg Glu Thr Pro Arg Pro Leu Met Pro Pro Thr Lys Pro Phe

200 205 210

Leu Ala Pro Glu Thr Thr Ser Pro Gly Asp Arg Val Glu Thr Pro

215 220 225

Val Gly Glu Arg Ala Pro Thr Pro Val Ser Ala Ser Ser Glu Val

230 235 240

Ser Pro Glu Ser Gin Glu Asp Ser Glu Thr Pro Ala Glu Glu Asp

245 250 255

Ser Gly Ser Glu Gin Pro Pro Asn Ser Val Leu Pro Asp Lys Leu

260 265 270

Lys Val Ser Trp Glu Asn Pro Ser Pro Gin Glu Ala Pro Ala Ala

275 280 285

Glu Ser Ala Glu Pro Ser Gin Ala Pro Cys Ser Glu Thr Ser Glu

290 295 300

Ala Ala Pro Arg Glu Gly Gly Lys Pro Pro Thr Pro Pro Pro Lys

305 310 315

He Leu Ser Glu Lys Leu Lys Ala Ser Met Gly Glu Met Gin Ala 320 325 330

Ser Gly Pro Pro Ala Pro Gly Thr Val Gin Val Ser Val Asn Gly

335 340 345

Met Asp Asp Ser Pro Glu Pro Ala Lys Pro Ser Gin Ala Glu Gly

350 355 360

Thr Pro Gly Thr Pro Pro Lys Asp Ala Thr Thr Ser Thr Ala Leu

365 370 375

Pro Pro Trp Asp Leu Pro Pro Gin Phe His Pro Arg Cys Ser Ser

380 385 390

Leu Gly Asp Leu Leu Gly Glu Gly Pro Arg His Pro Leu Gin Pro

395 400 405

Arg Glu Arg Leu Tyr Arg Ala Gin Leu Glu Val Lys Val Ala Ser

410 415 420

Glu Gin Thr Glu Lys Leu Leu Asn Lys Val Leu Gly Ser Glu Pro

425 430 435

Ala Pro Val Ser Ala Glu Thr Leu Leu Ser Gin Ala Val Glu Gin

440 445 450

Leu Arg Gin Ala Thr Gin Val Leu Gin Glu Met Arg Asp Leu Gly

455 460 465

Glu Leu Ser Gin Glu Ala Pro Gly Leu Arg Glu Lys Arg Lys Glu

470 475 480

Leu Val Thr Leu Tyr Arg Arg Ser Ala Pro

485 490

<210> 19

<211> 914

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 3036986CD1

<400> 19

Met Ala Asn He Asn Leu Lys Glu He Thr 'Leu He Val Gly Val

1 5 10 15

Val Thr Ala Cys Tyr Trp Asn Ser Leu Phe Cys Gly Phe Val Phe

20 25 30

Asp Asp Val Ser Ala He Leu Asp Asn Lys Asp Leu His Pro Ser

35 40 45

Thr Pro Leu Lys Thr Leu Phe Gin Asn Asp Phe Trp Gly Thr Pro

50 55 60

Met Ser Glu Glu Arg Ser His Lys Ser Tyr Arg Pro Leu Thr Val

65 70 75

Leu Thr Phe Arg Leu Asn Tyr Leu Leu Ser Glu Leu Lys Pro Met

80 85 90

Ser Tyr His Leu Leu Asn Met He Phe His Ala Val Val Ser Val

95 100 105

He Phe Leu Lys Val Cys Lys Leu Phe Leu Asp Asn Lys Ser Ser

110 115 120

Val He Ala Ser Leu Leu Phe Ala Val His Pro He His Thr Glu

125 130 135

Ala Val Thr Gly Val Val Gly Arg Ala Glu Leu Leu Ser Ser He

140 145 150

Phe Phe Leu Ala Ala Phe Leu Ser Tyr Thr Arg Ser Lys Gly Pro

155 160 165

Asp Asn Ser He He Trp Thr Pro He Ala Leu Thr Val Phe Leu

170 175 180 Val Ala Val Ala Thr Leu Cys Lys Glu Gin Gly He Thr Val Val

185 190 195

Gly He Cys Cys Val Tyr Glu Val Phe He Ala Gin Gly Tyr Thr

200 205 210

Leu Pro Leu Leu Cys Thr Thr Ala Gly Gin Phe Leu Arg Gly Lys

215 220 225

Gly Ser He Pro Phe Ser Met Leu Gin Thr Leu Val Lys Leu He

230 235 240

Val Leu Met Phe Ser Thr Leu Leu Leu Val Val He Arg Val Gin

245 250 255

Val He Gin Ser Gin Leu Pro Val Phe Thr Arg Phe Asp Asn Pro

260 265 270 Ala Ala Val Ser Pro Thr Pro Thr Arg Gin Leu Thr Phe Asn Tyr

275 280 285

Leu Leu Pro Val Asn Ala Trp Leu Leu Leu Asn Pro Ser Glu Leu

290 295 300

Cys Cys Asp Trp Thr Met Gly Thr He Pro Leu He Glu Ser Leu

305 310 315

Leu Asp He Arg Asn Leu Ala Thr Phe Thr Phe Phe Cys Phe Leu

320 325 330

Gly Met Leu Gly Val Phe Ser He Arg Tyr Ser Gly Asp Ser Ser

335 340 345

Lys Thr Val Leu Met Ala Leu Cys Leu Met Ala Leu Pro Phe He

350 355 ^' 360

Pro Ala Ser Asn Leu Phe Phe Pro Val Gly Phe Val Val Ala Glu

365 370 375

Arg Val Leu Tyr Val Pro Ser Met Gly Phe Cys He Leu Val Ala

380 385 390

His Gly Trp Gin Lys He Ser Thr Lys Ser Val Phe Lys Lys Leu

395 400 405

Ser Trp He Cys Leu Ser Met Val He Leu Thr His Ser Leu Lys

410 415 420 Thr Phe His Arg Asn Trp Asp Trp Glu Ser Glu Tyr Thr Leu Phe

425 430 435

Met Ser Ala Leu Lys Val Asn Lys Asn Asn Ala Lys Leu Trp Asn

440 445 450

Asn Val Gly His Ala Leu Glu Asn Glu Lys Asn Phe Glu Arg Ala

455 460 465

Leu Lys Tyr Phe Leu Gin Ala Thr His Val Gin Pro Asp Asp He

470 475 480

Gly Ala His Met Asn Val Gly Arg Thr Tyr Lys Asn Leu Asn Arg

485 490 495

Thr Lys Glu Ala Glu Glu Ser Tyr Met Met Ala Lys Ser Leu Met

500 505 510

Pro Gin He He Pro Gly Lys Lys Tyr Ala Ala Arg He Ala Pro

515 520 525

Asn His Leu Asn Val Tyr He Asn Leu Ala Asn Leu He Arg Ala

530 535 540 Asn Glu Ser Arg Leu Glu Glu Ala Asp Gin Leu Tyr Arg Gin Ala

545 550 555

He Ser Met Arg Pro Asp Phe Lys Gin Ala Tyr He Ser Arg Gly

560 565 570

Glu Leu Leu Leu Lys Met Asn Lys Pro Leu Lys Ala Lys Glu Ala

575 580 585

Tyr Leu Lys Ala Leu Glu Leu Asp Arg Asn Asn Ala Asp Leu Trp

590 595 600

Tyr Asn Leu Ala He Val His He Glu Leu Lys Glu Pro Asn Glu

605 610 615 Ala Leu Lys Asn Phe Asn Arg Ala Leu Glu Leu Asn Pro Lys His

620 625 630

Lys Leu Ala Leu Phe Asn Ser Ala He Val Met Gin Glu Ser Gly

635 640 645

Glu Val Lys Leu Arg Pro Glu Ala Arg Lys Arg Leu Leu Ser Tyr

650 655 660

He Asn Glu Glu Pro Leu Asp Ala Asn Gly Tyr Phe Asn Leu Gly

665 670 675

Met Leu Ala Met Asp Asp Lys Lys Asp Asn Glu Ala Glu He Trp

680 685 690

Met Lys Lys Ala He Lys Leu Gin Ala Asp Phe Arg Ser Ala Leu

695 700 705

Phe Asn Leu Ala Leu Leu Tyr Ser Gin Thr Ala Lys Glu Leu Lys

710 715 720

Ala Leu Pro He Leu Glu Glu Leu Leu Arg Tyr Tyr Pro Asp His

725 730 735

He Lys Gly Leu He Leu Lys Gly Asp He Leu Met Asn Gin Lys

740 745 750

Lys Asp He Leu Gly Ala Lys Lys Cys Phe Glu Arg He Leu Glu

755 760 765

Met Asp Pro Ser Asn Val Gin Gly Lys His Asn Leu Cys Val Val

770 775 780

Tyr Phe Glu Glu Lys Asp Leu Leu Lys Ala Glu Arg Cys Leu Leu

785 790 795

Glu Thr Leu Ala Leu Ala Pro His Glu Glu Tyr He Gin Arg His

800 805 810

Leu Asn He .Val Arg Asp Lys He Ser Ser Ser Ser Phe He Glu

815 820 825

Pro He Phe Pro Thr Ser Lys He Ser Ser Val Glu Gly Lys Lys

830 835 840

He Pro Thr Glu Ser Val Lys Glu He Arg Gly Glu Ser Arg Gin

845 850 855

Thr Gin He Val Lys Thr Ser Asp Asn Lys Ser Gin Ser Lys Ser

860 865 870

Asn Lys Gin Leu Gly Lys Asn Gly Asp Glu Glu Thr Pro His Lys

875 880 885

Thr Thr Lys Asp He Lys Glu He Glu Lys Lys Arg Val Ala Ala

, 890 895 900 Leu Lys Arg Leu Glu Glu He Glu Arg He Leu Asn Gly Glu

905 910

<210> 20

<211> 349

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 2041080CD1

<400> 20

Met Glu Glu Glu Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp

1 5 10 15

Ser Leu Asp Pro Ser Phe Val His Ala Ser Arg Leu Leu Ala Glu

20 25 30

Glu He Glu Lys Phe Gin Gly Ser Asp Gly Lys Lys Glu Asp Glu

35 40 45

Glu Lys Lys Tyr Leu Asp Val He Ser Asn Lys Asn He Lys Leu 50 55 60

Ser Glu Arg Val Leu He Pro Val Lys Gin Tyr Pro Lys Phe Asn

65 70 75

Phe Val Gly Lys Leu Leu Gly Pro Arg Gly Asn Ser Leu Lys Arg

80 85 90

Leu Gin Glu Glu Thr Gly Ala Lys Met Ser He Leu Gly Lys Gly

95 100 105

Ser Met Arg Asp Lys Ala Lys Glu Glu Glu Leu Arg Lys Ser Gly

110 115 120

Glu Ala Lys Tyr Ala His Leu Ser Asp Glu Leu His Val Leu He

125 130 135

Glu Val Phe Ala Pro Pro Gly Glu Ala Tyr Ser Arg Met Ser His

140 145 150

Ala Leu Glu Glu He Lys Lys Phe Leu Val Pro Asp Tyr Asn Asp

155 160 165

Glu He Arg Gin Glu Gin Leu Arg Glu Leu Ser Tyr Leu Asn Gly

170 175 180

Ser Glu Asp Ser Gly Arg Gly Arg Gly He Arg Gly Arg Gly He

185 190 195

Arg He Ala Pro Thr Ala Pro Ser Arg Gly Arg Gly Gly Ala He

200 205 210

Pro Pro Pro Pro Pro Pro Gly Arg Gly Val Leu Thr Pro Arg Gly

215 220 225

Ser Thr Val Thr Arg Gly Ala Leu Pro Val Pro Pro Val Ala Arg

230 235 240

Gly Val Pro Thr Pro Arg Ala Arg Gly Ala Pro Thr Val Pro Gly

245 250 255

Tyr Arg Ala Pro Pro Pro Pro Ala His Glu Ala Tyr Glu Glu Tyr

260 265 270

Gly Tyr Asp Asp Gly Tyr Gly Gly Glu Tyr Asp Asp Gin Thr Tyr

275 280 285

Glu Thr Tyr Asp Asn Ser Tyr Ala Thr Gin Thr Gin Ser Val Pro

290 295 300

Glu Tyr Tyr Asp Tyr Gly His Gly Val Ser Glu Asp Ala Tyr Asp

305 310 315

Ser Tyr Ala Pro Glu Glu Trp Ala Thr Thr Arg Ser Ser Leu Lys

320 325 330

Ala Pro Pro Gin Arg Ser Ala Arg Gly Gly Tyr Arg Glu His Pro

335 340 345 Tyr Gly Arg Tyr

<210> 21

<211> 2860

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 105283CB1

<400> 21 gccagagcgg cggccggtcc cgcgcggagc ccggcgcccc tccagcccga gccaggacgc 60 cgccggcccc ggtcccggcc ccgggcacgc agcgagccag ggatgtgagc ggcgσcccgσ 120 ggcatggcag cctcaggggt gcccagagga tgcgacatcc tcatcgtcta cagcccggat 180 gccgaggaat ggtgccagta cctgcagacc ctgttcctgt ccagtcggca ggtccgcagc 240 cagaagatac tgactcacag gctgggcccc gaggcctcct tctcggcaga ggacctaagc 300 cttttcctca gcaccσgctg tgtcgtggtg ctgctgtcσg cggagctggt gσagcaσttc 360 cacaagσccg ccttgctgcc cctgctgcag agagctttcc atcctcσgca ccgcgtggtc 420 aggctgctct gcggcgtgσg ggacagcgag gagttcctag acttctttcc agattgggcc 480 cattggcagg agctcacctg tgacgatgag ccagagacct acgtggcagc tgtgaaaaaa 540 gccatttccg aagattσtgg ctgtgactσa gtcactgaca ctgagcctga ggacgagaag 600 gttgtttcct actcgaagca gcagaacctg ccgacggtga cttcacσtgg gaacσtgatg 660 gtggtgσagσ cggaσσgσat tcgctgtggg gσagaaacca ctgtctatgt tattgtgaga 720 tgtaagctgg atgacagggt ggcgacagaa gcagagtttt ctcctgagga ttctccctct 780 gtaaggatgg aagcσaaggt ggagaatgag taσaσσattt σagtgaaggc tcccaacσtt 840 tcatctggga acgtttctct gaagatatat tctggagact tagtggtgtg tgaaaccgtt 900 atcagctatt atactgacat ggaagaaatt gggaatttat tgtccaatgc cgcgaatcct 960 gtggaattca tgtgtcaggc ctttaaaatt gtgccctaca acacagagac ccttgataaa 1020 ctgctaaccg aatcσσtgaa gaacaatatσ cσtgσaagcg gactgcacct σtttggaatc 1080 aaccagctgg aagaagaaga tatgatgaca aatcagaggg atgaagagct gcccaccctg 1140 ttgcattttg ctgσgaagta tggactgaag aacctcactg cσttgttgct cacctgccca 1200 ggagccctgc aggcgtacag cgtggccaac aagcatggcc actaccccaa caccatcgct 1260 gagaaacacg gcttcaggga cctgcggcag ttcatcgacg agtatgtgga aacggtggac 1320 atgctcaaga gtcacattaa agaggaactg atgcacgggg aggaggctga tgctgtgtac 1380 gagtcσatgg cσσaσσtttσ cacagacctg cttatgaaat gctcgctcaa cσcσggσtgt 1440 gacgaggatc tctatgagtc catggctgcc tttgtcccag ctgccactga agacctctat 1500 gttgaaatgc ttσaggσσag taσatσtaaσ σσaatccctg gagatggttt ctctcgggcσ 1560 actaaggact ctatgatccg caagttttta gaaggcaaca gcatgggaat gaccaatctg 1620 gagagagatc agtgccatct tggtσaggaa gaagatgttt atcaσaσggt ggatgaσgat 1680 gaggcctttt ctgtggactt ggccagcagg ccccctgtcc cagtgcccag accagagacc 1740 actgctσσtg gtgσtσaσσa gσtgσctgac aaσgaaσσat acatttttaa agtttttgca 1800 gaaaaaagtc aagagcggcc tgggaatttc tacgtttcct cagagagcat caggaaaggg 1860 cσgcccgtca gaσcatggag ggacaggσσσ σagtcgagta tatatgaccσ ttttgσggga 1920 atgaaaacgc caggccagcg gcagcttatc accctccagg agcaggtgaa gctgggcatt 1980 gtcaacgtgg atgaggctgt gctcσacttc aaagagtggc agctcaacca gaagagaσga 2040 tcggagtcct ttcgtttcca gcaggaaaat cttaaacggc taagagacag catcaσccga 2100 agacagagag agaagcaaaa atcaggaaag cagacagact tggagatcac ggtcccaatt 2160 cggcactcac agcacctgcc tgcaaaagtg gagtttggag tctatgagag tggccccagg 2220 aaaagtgtca ttccccctag gacggagσtg agaσgaggag actggaaaac agacagcacc 2280 tccagcacag caagtagcac aagtaaccgc tccagcaccc ggagcctcct cagtgtgagc 2340 agcgggatgg aaggggacaa cgaggataat gaagtccctg aggttaccag aagtcgcagt 2400 ccaggccccc cacaagtgga tgggacaccc accatgtccc tcgagagacc ccccagggtg 2460 cσtccgagag σtgσσtσaσa gaggcctccg accagggaga ccttccatcc tσctcσaσσt 2520 gttccacσca gaggacgctg attccacctc ctaaaacctg cctacttcag gactttaaga 2580 ctσaσagtct tσagσσtgtt aatgatgtσt tσatgttgag ttttatagca tgactgttga 2640 ccttaagatc cattctcatt gctgataatg ctgcagccct gctggtttgg gcttgcctcg 2700 aagattttat taaggcaσga agaagtgaaa aactaagggc ttcattcaσσ atcaccaagt 2760 atatcgaacc atatacttgt ttgccaaaag gatgaagact taatcgaaat acttacctct 2820 aatttgccat atcagaagσσ taaaaagaat gatσataaat 2860

<210> 22

<211> 3542

<212> DNA

<213> Homo sapiens

<220>

<221> misσ_feature

<223> Incyte ID No : 3350821CB1

<400> 22 atgtgttgca σtgagggσag tctcσggaaa cgσgattσgσ agcgggcgcσ ggaagcggtg 60 ttgtgtctgc agctctggca gaggactgtt ccactagaca cgctgaaggg actgggtacg 120 tgttttcσtt caggaσcaga gctgagagga gctgggatσg σggσggσaat ggaaσgggσσ 180 tcagaaaggc gcacggccag cgcgcttttt gcggggttcc gggccttggg acttttcagc 240 aacgacattc cacaσgtggt gcggttcagσ gσgctcaagc gccggttcta tgtaacaacc 300 tgcgtgggca agagtttcca cacctatgac gttcagaaac ttagtctggt tgcagtaagt 360 aattctgttc cacaggatat ctgctgtatg gcagσtgatg gσagattagt ctttgctgct 420 tatggaaatg ttttctctgc atttgcccgt aataaagaga tagtaσatac ctttaagggt 480 cataaggcag aaatcσattt cttgcaaccc tttggagacc acattatctc tgttgatact 540 gatggcattc ttattatttg gcacatatat tcagaagaag aatacctgca gttgactttt 600 gataaatcag tatttaaaat ttσtgcaatt ttgcatσσaa gtacctactt gaataaaata 660 cttctgggca gtgaacaagg aagcctgcag ttgtggaatg taaaatccaa ccagaaatac 720 ccaatcaggc agacatttat ccctgcgggc tatttgttgg gtgcaσatgg gttgaagaσt 780 caggcaccag ccgtggatgt tgttgctatt ggtcttatgt caggtcaagt tatcattcac 840 aacattaaat ttaatgaaaσ attaatgaag tttσgtσaag actggggacσ σattaσttσa 900 atttcatttc gcacagatgg tcatccagta atggcagctg gaagcccatg tggccatatt 960 ggactctggg atσtagaaga caaaaaatta atcaaccaaa tgagaaatgc acaσtctaca 1020 gcaattgccg gactgacatt tctcσataga gagccacttσ ttgtcacaaa tggcgctgac 1080 aatgctctta ggatatggat atttgatggt cctacaggtg aaggccgact tttgagattc 1140 agaafcgggtc atagtgctcc tcttaccaat atcagatatt atggacagaa tggacagcag 1200 attσtaagtg σaagtcaaga tggaaσtσtt cagtσatttt ccacggtaca tgaaaaattσ 1260 aataagagσt tgggacatgg attaataaat aaaaagagag ttaaaσgtaa aggacttcag 1320 aataccatgt cagtgagact tσσaσσcatσ acaaagtttg σagσagagga agctcgtgaa 1380 agtgactggg atggtatcat tgcttgccat caaggtaagc tatcttgctc aacσtggaat 1440 tatσagaaat σtaσaatagg cgcttacttt ctcaagccaa aagagttgaa gaaagatgac 1500 ataactgcaa cagcagtgga tataacttct tgtggaaact ttgctgtaat tggcctctca 1560 tcaggaactg tagatgtata taacatgcag tσtggσatac atcgaggaag ttttggσaag 1620 gatcaagctc acaagggatc tgttagaggt gtcgcagtgg atggattaaa ccagttgaca 1680 gttaσaactg gtagtgaagg attactσaaa ttctggaact ttaaaaacaa aattttaatc 1740 cattctgtga gcctσagttc atctcσaaat atcatgttgc tacataggga cagtggσatt 1800 ctgggactσg σσttggatga cttctccatt agtgttctgg acatagaagc taggaagatt 1860 gtcagagagt tttctggaca ccaaggccaa ataaatgaca tggcttttag tcctgatggt 1920 cgttggttaa taagtgctgc gatggattgσ tσtattagga cttgggaσσt tccttctggg 1980 tgccttatag actgcttttt gttggactcg gctcctctca atgtttσtat gtctcctact 2040 ggagactttc tggcaacttc ccatgtggac cacσttggaa tttatσtatg gtccaatatt 2100 tccctgtatt cagttgtttc attacggcca cttcctgσag attatgtσσσ ttcaatagtσ 2160 atgσfctσσtg gtacttgtca aacccaagat gtagaagttt cagaagaaac agtagaacca 2220 agtgatgaat tgatagaata tgattcgcca gaacagttga atgagcaatt ggtgactctt 2280 tcaσttcttc ctgaatcacg atggaaaaac cttσttaaσc ttgatgttat taagaaaaag 2340 aataaaccaa aggaaccacc caaagtacσσ aaatcagσac catttttcat tccaacaatt 2400 cσtggccttg tacccagata tgctgσaσσt gaacaaaata atgatσσσσa gcagtctaaa 2460 gtggtaaatc ttggagtttt ggctσaaaaa tcagatttct gcttgaaact tgaagaagga 2520 ctggtaaata ataagtatga σaσtgctctc aaccttctga aagaatcagg cσcatcagga 2580 attgaaacag agctgcgaag cttgtσtσσt gattgtggtg ggtccataga agttatgσag 2640 agσttcttga aaatgattgg gatgatgctg gacagaaagc gtgattttga gttagcσcag 2700 gcataccttg cattgtttct aaagttacac cttaaaatgc ttcσttσaga gσσagtactc 2760 ctagaagaaa taacaaattt gtcatcσσag gtggaagaaa actggaccca tttgcaatσa 2820 ctσttcaatc aaagcatgtg tattttaaat tatctσaaaa gtgctttgtt gtaaaaataa 2880 atttgtgaσt aaaσaaagac tttcatatta aatgggttca attgaactca tttcttattt 2940 tccaagtgtc aatgtgaaaa gaaaataaat gctagcacta ctgactagtc agtatatctc 3000 cactttaaat gctaaatact ttcttgaaat aaaatcgcac ctσccggcca ggcatgatgg 3060 ataatgcctg taattccagc actttcagag gccaaagcgg gaggattgct tgaagccagg 3120^' aatttgagcc cagcctgggc aacatagcaa gcaagatσσσ atctctacaa aaagttaaaa 3180 aaataaatta cacctgcσtt tgttttgaag acttaaagaa tctgctcaaa tgatttattt 3240 tctgagtctc ttcaσattaa cttttatatg atttttaaaa aattattacc tgcttatatt 3300 tttgagtatc tgtttcatat aaagagatgc tgσatgttσa σtgtgaagta ttgctgaatt 3360 aaattgaaaa tgtcagataa aaσtgataσa tgatgtagtt ataaaaatgσ tgttatgaag 3420 aaaaaaggaa tgcttttttσ ctcggtggcc tttgataaaa atatgaatga ttgactctca 3480 gagtggagga taaactaatc atcttctagσ aσagccσtgt gtgtgcagga gaggaatcag 3540 gg 3542

<210> 23 <211> 1014 <212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 5876846CB1

<400> 23 ggacccggcg ccgatgaaac cgaccaaact ggagttagct accggactgt cccgcgagcg 60 aaagttggaa ataatacσta aacctacttσ ttgaσatggt gtσaaaaσag agσttttgσt 120 gagagtttga tttataaaga aattatggtt cgactgacct tggatctaat tgccagaaac 180 agcaatctta aaσσσσgaaa agaagaaacc atttcacagt gcctgaagaa aataactcat 240 ataaattttt cagacaaaaa tatagatgca attgaagacc tctctctttg caaaaatctt 300 agtgttttat atttatatga taattgtatt agtcaaatca ctaacctgaa ttatgccaca 360 aatctgaccc acttgtacct acaaaacaat tgtatttcat gtatagagaa cctcaggtca 420 ttaaagaaaσ tggaaaaact gtatσtggga ggσaattaσa ttgσtgtσat agaaggttta 480 gaaggattag gagaactaag agagcttcat gttgagaatc agaggcttcc ccttggggaa 540 aagctfcσtgt ttgatcσaag aactσttσat tσtctggσaa aatccctctg tatattgaat 600 atcagcaata ataatattga tgacattaca gacttagaac tactagagaa tcttaatcag 660 ctcatagσσg ttgaσaacca acttctgσat gtgaaggatt tggagttttt actgaaσaag 720 ttgatgaagc tgtggaaaat tgatctaaat ggaaatcctg tttgtctcaa accaaaatac 780 agggaσagac tgatattggt gtσσaaatσa σtggaatttσ ttgatggaaa agaaattaaa 840 aatatagaaa gacaattcct aatgaattgg aaagcatcca aagatgccaa gaaaatcagc 900 aaaaaaagga gcagtaaaaa tgaggatgσa agcaattσaσ tcataagtaa gσactctgtc 960 actcattagg tgactgctct tctagcaaag gaaatattct aggcaacaaa aaaa 1014

<210> 24

<211> 4040

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 3560269CB1

<400> 24 gcactgcggc acagagccgc ggcttgcttt caggacacgg gtcgctggcg ggttaggtgg 60 ctgσgtggcc gcctσaσttc gcgcaσgcgc cgctaggσtg ggggagttga tttgcactgc 120 tccgggtgtc ggctccggcg ggacgggcgg ttctgcgtac ccctctccag tgtcaacctg 180 gggctgaatc ttcaacctgt σaaagσσttc ttaattatgt ctgagaaaag taσtaaaata 240 ggtgccattt taaattccca gacaaggaaa aaccaaggat tagaaattaa gtagcttgcc 300 , caaggcσaσa σatcagatσσ tgattgagtσ aatgctttgσ attctcagtc ctctgcataa 360 agctgagaga tgcctacagc tgagagtgaa gcaaaagtaa aaaccaaagt tcgctttgaa 420 gaattgctta agacccacag tgatctaatg σgtgaaaaga aaaaaσtgaa gaaaaaaσtt 480 gtcaggtctg aagaaaacat ctcacctgac actattagaa gcaatcttca ctatatgaaa 540 gaaaσtaσaa gtgatgatcσ σgaσaσtatt agaagσaatσ ttσσσσatat taaagaaact 600 acaagtgatg atgtaagtgc tgctaacact aacaacctga agaagagcac gagagtcact 660 aaaaacaaat tgaggaacac aσagttagσa actgaaaatc ctaatggtga tgσtagtgta 720 gaggaagaca aacaaggaaa gccaaataaa aaggtgataa agacggtgcc ccagttgact 780 acacaagacσ tgaaaccgga aaσtσσtgag aataaggttg attσtacaca σσagaaaaca 840 catacaaagc cacagccagg cgttgatcat cagaaaagtg agaaggcaaa tgagggaaga 900 gaagagactg atttagaaga ggatgaagaa ttgatgcaag catatσagtg σσatgtaact 960 gaagaaatgg caaaggagat taagaggaaa ataagaaaga aactgaaaga acagttgact 1020 tactttcσσt σagataσttt attcσatgat gacaaactaa gcagtgaaaa aaggaaaaag 1080 aaaaaggaag ttccagtctt ctctaaagct gaaacaagta cattgaccat ctctggtgac 1140 acagttgaag gtgaacaaaa gaaagaatct tcagttagat cagtttcttσ agattctcat 1200 caagatgatg aaataagctc aatggaacaa agcacagaag acagcatgca agatgataca 1260 aaacctaaac σaaaaaaaaσ aaaaaagaag actaaagcag ttgcagataa taatgaagat 1320 gttgatggtg atggtgttca tgaaataaca agcσgagata gcccggttta tcccaaatgt 1380 ttgcttgatg atgaccttgt cttgggagtt tacattcacc gaactgatag acttaagtca 1440 gattttatga tttctcaccc aatggtaaaa attcatgtgg ttgatgagca taσtggtσaa 1500 tatgtcaaga aagatgatag tggacggcσt gtttcatctt actatgaaaa agagaatgtg 1560 gattatattc ttcctattat gacccagcσa tatgatttta aacagttaaa atcaagactt 1620 ccagagtggg aagaacaaat tgtatttaat gaaaattttc cσtatttgct tcgaggctct 1680 gatgagagtc ctaaagtcat σctgttcttt gagattcttg atttσttaag cgtggatgaa 1740 attaagaata attσtgaggt tcaaaaσcaa gaatgtggσt ttcggaaaat tgcσtgggca 1800 tttσttaagσ ttctgggagc caatggaaat gcaaacatca actcaaaact tcgcttgcag 1860 ctatattacc cacctaσtaa gcctσgatσσ σcattaagtg ttgttgaggσ atttgaatgg 1920 tggtcaaaat gtccaagaaa tcattaccca tcaacactgt acgtaactgt aagaggactg 1980 aaagttccag actgtataaa gccatcttac cgσtctatga tggctcttca ggaggaaaaa 2040 ggtaaaccag tgcattgtga acgtcaccat gagtcaagct cagtagacac agaacctgga 2100 ttagaagagt caaaggaagt aataaagtgg aaacgactσc ctgggσaggσ ttgccgtatc 2160 ccaaacaaac acctcttctc actaaatgca ggagaacgag gatgtttttg tcttgatttc 2220 tcccaσaatg gaagaatatt agσagσagσt tgtgσcagσσ gggatggata tccaattatt 2280 ttatatgaaa ttccttctgg acgtttcatg agagaattgt gtggccacct caatatcatt 2340 tatgatcttt cσtggtσaaa agatgatcac taσatσσtta cttσatcatσ tgatggcact 2400 gccaggatat ggaaaaatga aataaacaat acaaatactt tcagagtttt acctcatcct 2460 tcttttgttt acacggctaa attcσatσσa gctgtaagag agσtagtagt tacaggatgc 2520 tatgattcca tgatacggat atggaaagtt gagatgagag aagattctgc catattggtc 2580 σgacagtttg atgttσaσaa aagttttatc aaσtσacttt gttttgataσ tgaaggtσat 2640 catatgtatt caggagattg tacaggggtg attgttgttt ggaataccta tgtcaagatt 2700 aatgatttgg aacattcagt gσaccactgg aσtataaata aggaaattaa agaaactgag 2760 tttaagggaa ttccaataag ttatttggag attcatccca atggaaaacg tttgttaatc 2820 cataccaaag acagtacttt gagaattatg gatctccgga tattagtagc aaggaagttt 2880 gtaggagcag caaattatcg ggagaagatt catagtactt tgactccafcg tgggactttt 2940 ctgtttgσtg gaagtgagga tggtatagtg tatgtttgga acσσagaaac aggagaacaa 3000 gtagccatgt attctgactt gccattcaag tcacccattc gagacatttc ttatcatcca 3060 tttgaaaata tggttgcatt σtgtgcattt gggcaaaatg agσσaattct tσtgtatatt 3120 tacgatttcc atgttgccca gcaggaggct gaaatgttca aacgctacaa tggaacattt 3180 cσattacctg gaatacaσσa aagtσaagat gccctatgta σσtgtσσaaa actaσσccat 3240 caaggctctt ttcagattga tgaatttgtc cacactgaaa gttσttcaac gaagatgcag 3300 σtagtaaaac agaggcttga aaσtgtσaσa gaggtgataσ gttcctgtgσ tgcaaaagtc 3360 aacaaaaatc tctcatttac ttcaccacca gcagtttcct cacaacagtc taagttaaag 3420 cagtcaaaca tgσtgaσcgσ tσaagagatt ctacatσagt ttggtttcac tσagaσσggg 3480 attatcagca tagaaagaaa gccttgtaac catcaggtag atacagcacc aacggtagtg 3540 gσtσtttatg actacacagc gaatcgatca gatgaaσtaa ccatccatcg cggagaσatt 3600 atccgagtgt ttttcaaaga taatgaagac tggtggtatg gcagcatagg aaagggacag 3660 gaaggttatt ttσσagσtaa tcatgtggct agtgaaacac tgtatcaaga aσtgcctcσt 3720 gagataaagg agcgatcccc tcctttaagσ cctgaggaaa aaaσtaaaat agaaaaatct 3780 ccagσtσctc aaaaggtaaa ataaaacaaa acagσtσaca gagacaσσtc σttσttcσcc 3840 ctgcttctgc ctccatgagt aactacctat atgacgtgct gctgctgaga ccaaggaatg 3900 agtgagtaaa ggtgtttgga agtcaaatafc tggtcctagt taagtaagtg tccttttcaa 3960 gtaagtggta aactttgtta tgtggacccc cttctaactt aggattcata taatttgaac 4020 acaggσtaaa gσtgσtσtgg 4040

<210> 25

<211> 2006

<212> DNA

<213> Homo sapiens

<220>

<221> misσ_feature

<223> Incyte ID No: 4596874CB1

<400> 25 tcσgaσtggc cgccataσat ctctagagaa gctgaσttct ggaσagttct caaagaaσcg 60 cagcσgagcσ aaacaaggca actσcσaσσa ccccccaggc ccctgggagg agtcagagσt 120 tgactccaga cccagtgccc atcactggtc gcactgaggg tgaagtggca cggaggagga 180 ggaggggctc cggctggtct gtggtgagat ggcσtacσag gtggtggaga agggcgcggc 240 cctgggcacg ctggagtcgg agctgcagca gaggcaaagc aggctggcag ccctggaggc 300 ccgcgtggcg cagctgcgag aggcgcgggσ gcagσaggcc cagcaggtgg aggagtggcg 360 ggcgcagaat gcggtgcagc gggcagccta cgaggcgctg cgcgcgcacg tcgggctccg 420 ggaggcggσa ctgcgcaggc tσσaggaaga ggσgcgcgac ctgσtggaga ggctcgtgca 480 gcgcaaggcg cgcgccgcgg ccgagcgcaa cctgcgcaac gagcgccggg agcgggccaa 540 gcaggcgcgg gtgtcccagg agσtgaagaa ggctgccaag cggaccgtga gcatcagσga 600 gggcccggac accctaggcg atgggatgag ggagagaagg gagactctgg ctctggcccc 660 tgagcσagag cσcctggaga aggaagcttg tgagaagtgg aagaggccct tσaggtctgσ 720 ctcagccacc tccctgacgc tgtcccactg tgtggatgtg gtgaaggggc ttctggattt 780 taagaagagg agaggtcact caattggggg agσσcctgag cagcgatacc agatcatσcσ 840 tgtgtgtgtg gctgcccgac ttcctacccg ggctcaggat gtgctggatg cccacctctc 900 tgaggtcaat gσtgttcgtt ttggccccaa cagσagσσtc ctggcσactg gaggggctga 960 ccgcctgatc cacctctgga atgttgtggg aagtcgcctg gaggccaacc agaccctgga 1020 gggagctggt ggcagσatca cσagtgtgga ctttgaσσσσ tσgggctacσ aggttttagσ 1080 agcaacttac aaccaggctg cccagctctg gaaggtgggg gaggcacagt ccaaggagac 1140 actgtctgga cacaaggata aggtgacagc tgcσaaattc aagσtaaσga ggcaccaggc 1200 agtgactggg agccgcgacc ggacagtgaa ggagtgggac ctcggccgtg cctattgctc 1260 caggaccatc aatgtσcttt cctaσtgtaa tgacgtggtg tgtggggaσσ atatσatcat 1320 tagtggccac aatgaccaga agatccggtt ctgggacagc agggggcccc actgcaccca 1380 ggtcatcσσt gtgcagggσσ gggtσaσctc cctgagcσtσ agccacgaσc aactgcacct 1440 gctcagctgt tcccgagaca acacactcaa ggtcatcgac ctgcgtgtca gcaacatccg 1500 ccaggtgttc agggcσgatg gσttcaagtg tggttσtgac tggacσaaag ctgtgttcag 1560 cccggacaga agctatgcac tggcaggctc ctgtgatggg gccctttaca tcfcgggatgt 1620 ggaσaccggg aaactggaga gcagactaca gggaccccat tgσgctgccg tcaaσgσcgt 1680 ggcctggtgc tactccggga gccacatggt gagcgtggac cagggcagga aggttgtgct 1740 ctggσagtag ggσcacgacc tgcctgcσtg ggctggagct σttgcccgaa gcσtgaagct 1800 tccttcggcg ccatgcaggg gttggggttg ggactggagc tggccttggg atttaatggg 1860 gaagaaggcc tggcaggacc tggcctgttt gtttaaaaat gaagtatggg ttgggggatt 1920 acgctagttt ttctttgtat ttttatctct atctatctcc tcactttttc tcσcaaagta 1980 gaaaaaaatg atatctgaaa aaaaaa 2006

<210> 26

<211> 3643

<212> DNA

<213> Homo sapiens

<220>

<221> misσ_feature

<223> Incyte ID No: 3594012CB1

<400> 26 gcggagaggt acttggatgc attttacagg σgcagtcatg caggaaaacc tcagatttgc SO ttcatcagga gatgatatta aaatatggga tgcttcatct atgacattgg tggataaatt 120 caacccacac acatcaccac atggaatcag ctcaatatgt tggagcagca ataataactt 180 tttagtaaca gcatcttcca gtggσgacaa aatagttgtc tcaagttgca aatgtaaacc 240 tgttccactt ttagagcttg ctgaagggca aaagcagaσa tgtgtσaatt taaattσtac 300 atctatgtat ttggtaagcg gaggcctaaa taacactgtt aatatttggg atttaaaatc 360 aaaaagagtt catcgatctc ttaaggatca taaagatσaa gtaacttgtg taacataσaa 420 ttggaatgat tgctacattg cttctggatc tcttagtggt gaaattattt tacacagtgt 480 aaccactaat ttatctagta ctccttttgg σσatggtagt aaccagtσtg ttcggcactt 540 gaagtactcc ttgtttaaga aatcactact gggcagtgtt tcggataatg gaatagtaac 600 tctcfcgggat gtaaatagtσ agagtcσata σcataacttt gaσagtgtac acaaagctcc 660 agcgtcaggc atσtgttttt ctcctgtcaa tgaattgctc tttgtaacσa taggcttgga 720 taaaagaatc atcctctatg acacttcaag taagaagcta gtgaaaaσtt tagtggctga 780 cactcctcta actgcggtag atttcatgcc tgatggagcc actttggcta ttggatcttc 840 ccgggggaaa atatatcaat atgatttaag aatgttgaaa tcaccagtta agaccatcag 900 tgσtcaσaag aσatctgtgc agtgtatagc atttσagtac tcσactgttc ttactaagtc 960 aagtttaaat aaaggctgtt caaataagcc cacaacagtg aacaaacgaa gtgttaatgt 1020 gaatgctgct agtggaggag ttcagaattc cggaattgtc agagaagσaσ σtgccaσgtσ 1080 cattgccaca gttctaccac aacctatgac atcagctatg gggaaaggaa cagttgctgt 1140 tcaagaaaaa gσaggtttgσ σtσgaagcat aaacacagac actttatσta aggaaaσaga 1200 cagtggaaaa aatcaggatt tctccagctt tgatgatact gggaaaagta gtttaggtga 1260 σatgttctca cctatσagag atgatgσtgt agttaaσaag ggaagtgatg agtccatagg 1320 caaaggagat ggctttgact ttctaccgca gttgaactca gtgtttcctc caagaaaaaa 1380 tccagtaact tcaagtactt cagtattgca ttctagtσct cttaatgttt ttatgggatc 1440 tccagggaaa gaggaaaatg aaaaccgtga tctaacagct gagtctaaga aaatatatat 1500 gggaaaacag gaatctaaag aσtccttcaa aσagttagca aagttggtca catσtggtgc 1560 tgaaagtgga aatctaaata cctctccatc atctaaccaa acaagaaatt ctgagaaatt 1620 tgaaaagσσa gagaatgaaa ttgaagccca gttgatatgt gaacσcccaa tcaatggatc 1680 σtcaactcca aatccaaaga tagcatcttc tgtcactgct ggagttgcca gttcactctc 1740 agaaaaaata gccgacagca ttggaaataa ccggcaaaat gcaccattga cttσσattσa 1800 aattcgtttt attcagaaca tgatacagga aacgttggat gactttagag aagcatgσca 1860 tagggacatt gtgaatttgc aagtggagat gattaaaσag tttcatatgσ aactgaatga 1920 aatgcattct ttgctggaaa gatactcagt gaatgaaggt ttagtggctg aaattgaaag 1980 actaσgagaa gaaaacaaaa gattacgggc ccacttttga aatttcagtg aataσcttaa 2040 tgttctgtaa tttgggaagt ttctggcaac acagaactac atagaatcag tattgttfctc 2100 atggcctcσa gggaaaaaat gtttttσaag taagagtaaa agggtgatgg gattttataσ 2160 caacaactgt ttcatcttaa aaatatgtat atttttatat taaaaattgt acagtatgtc 2220 atctaσccaa taggaaagtσ aaσaggatσt ttattttttg aaagctttag ccatσcacta 2280 agtgcccttt ttcataagag aagaaaattg tgcataaaaa ttggttatgt ttgtttttta 2340 gtcatctttt ttaaσatata tttttgattg aσaaattgcc tttcaaattt ttggggσtag 2400 ttgagattta aagagtttga tatgσcttct atttttatgg agaaagtaat tttaaaatgg 2460 caattggtgfc ttctaagcσa ttgaσtaata aaaσataggg ttggctagta attattttgt 2520 fcaacttgatg aagtcaagta tgactattat ttattgtaca tttgataaga caatttttgg 2580 aattttgaat tgcaσaaatt acatgatatc ttttgcattt atgttaσtat attgtaσttσ 2640 tgacaaatct ttattσσtgg gtggtatttt taagatatct ttaσσtataa aaaatgttta 2700 aggttσatag gactcgacaa gagctatctg gtgattttct cattagtaac atgcaaσgtt 2760 gtactgcaaa atttcaatca acatgacaac ttataatgag tggagatttc atattaggta 2820 ctaaatatta tagtattatt tctattttct ttttccaaat aagaagσttg gattatttta 2880 ttttgtggtc tttatcatta actttaattc tttσtgtact gtgtataata tttttatatt 2940 attggcctta ccataaaatt atttagaaag gttgtcaaaa taagttatac ctctttggca 3000 atagatagat gtatacatct acctactatg atctacaatt ttaggttaag tgaagcttgg 3060 gggggctact gacttggtta cσttcttgtc tcttgtccca aagatttaaa σtatgtacct 3120 ttgtatagct cttctgcσcc attttgactt ctgagatgaa agtatttact aaaattaaaa 3180 aaaaaaaaac aaaaaacaaa cctttagctc actaacttta tgggtttctg aagtgatgga 3240 aatttttaag gatatattta ataagcataa acttactaat aattacttcc aaaaataaaa 3300 acaggaatat tacttttaσc cagtgtggtt tatagcatac atttgtactg aagcatatag 3360 ggatgttaat gtgatctttt σctgacagat tatgaaagca ttatgacttg taacaagttt 3420 σcttgtatat caσtaacagg tttagaagaσ ataaatatta gtgtgttttg σctacatggt 3480 gtatttaaat ctattaatat tttσctgttg cttttttaaa aaaataaata caσataatgt 3540 atattaaaag aggtgggatg aaataatttt agtaattatg tgtaσagatg aaacattttt 3600 gtσatggaat ttaaaagcta agtaagtata aaaaataaaa tgt 3643

<210> 27

<211> 2694

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 7482435CB1

<400> 27 atggcggaga tccccσtgta σtttgtggac ttgcaggatg acttagacga σtatggattt 60 gaagactatg gtacagattg cgacaacatg agagtaacgg ccttcttgga cattcσaggc 120 caggataacσ tgcctcσact cactcgσσtg gagaagtatg σtttσagσga gaaσaccttc 180 aaccggcaga ttattgccag agggctgctt gatatcttcc gggacttcgg taacaatgaa 240 gaagaσttcc tcacggtaat ggagattgta gtcagattgt cagaagatgc agagcccaca 300 gtgcggactg agctgatgga acagattcct σσtattgcca tttttttaca agaaaacaga 360 tcaaattttσ cagtggtgct ctctgaatat ctcataccta ttgtaatgag gtacctcaca 420 gatccaaaca atcagataat σtgcaaaatg gcttcaatgσ tgagtaagag tacσgttgaa 480 σgcctgctac tcccccgatt ctgtgaactg tgtggtgaca ggaagctctt tcaagttcgg 540 aaggtttgtg ctgcaaattt tggtgatatt tgtσatgctg ttggaσaaga agccactgaa 600 aaatttttga tcccaaagtt ctttgagctc tgctctgatg ctgtttgggg catgcggaaa 660 gcσtgtgσag aatgcttcac ggctgtgtcg σaσagctcct cccctggggt ccgcagaaσσ 720 cagctcttcc cgctcttcat σagacttgtc agcgacccct gccgatgggt gcaccaggct 780 gccttccagt cσcttggσcc σttcatttσc acσtttgσaa accσctccag ggctggσσtt 840 tatcttcgag aggatggcgc cctgagcatc tggccactca cccaggattt ggactctggt 900 tttgcσtctg gσtcccσtgσ tccσagcagt ggtggtaaca tttccσctgσ σagtttaatt 960 cgttcagcaa agcctgtgcg gagcgagcca gagctacctg tggaagggac ctcagcgaaa 1020 accagtgatt gσσcaσaσag σagtagσtσσ tσtgaσggσc cagσggaaag cσσtgtggaa 1080 agctgtgtgt cggctggagc tgagtggacc agggtttccc cagagaccag cgcccgctca 1140 aagσtttctg aσatgaatga cctσσσcatσ agcagctacc ctggatcaga ttcctgggσσ 1200 tgcccaggga acactgagga tgtgttcagt cattttcttt attgcaagga cttagagctg 1260 cttctgagtg aggctgggcσ tσaggaggaσ gactgσagσa gacσtggggt tgtgσacaac 1320 agctgtgtgg cccggagtga gattcagaaa gtccttgata gcttgcagga gcatctgatg 1380 aatgatccag atgttcaagc tσaagttcag gtattatccg σtgcaσtgag agctgcacag 1440 ctcgactgcg tgaatgaagc tgagagcaag ccaacagcag gcctaaagga agtgtccatt 1500 tcacatσcca gctσtgcσtc tgaσaatcag atσgσtσtgg σggσσtcatσ atctcaggat 1560 gagctctttg tggccaggat attacaaagc ccagatccag gtggacccag aaatggaacc 1620 agtgaσσatc tggagactga ccagaggcag gatσσσaσcσ caσttgaaga gaataaatσt 1680 aaattacagg atgtaatacc tcagccgctg ctagatcagt atgtgtccat gactgaccca 1740 gctcgagccc agactgtcga tactgacata gccaaaσact gtgcσtacag σσtσccaggg 1800 gtggcactga ccctgggcag gcaaaattgg cactgcctga aagatacata tgaaacactg 1860 gcttσtgatg tacagtggaa ggtaσggσga gccσtagcct tctccattca σgagctggct 1920 gtgattcttg gggatcagtt aacagcagct gacctggtgc ctatcttcaa tggattttta 1980 aaggatctgg atgaagtgσg aataggagtt cttagaσacσ tgtatgattt tctaaagttg 2040 cttcatgaag acaagaggag agactatctt tatcagcttc aagaatttgt agtgactgat 2100 aacagcagga attggaggtt tσgatatgaa ctagcagaaσ agctgatact gattctggaa 2160 ctctatagtc ccaatgatgt ttatgattac ctaatgcaca ttgccttaaa gttgtgtgca 2220 gatcaagttt ctgaagttσg gtggatctcσ ttσaaaσtag tcgtggcaat tσtgσagaag 2280 ttctattcca acagtgaaag tgcattgggg ttaaatttca tcaatgagct catcataagg 2340 ttccggcact gttctaagtg ggttggaagg caagσtttcg σtttσatttg tcaggσagtg 2400 gtgagcaagg agtgtgtccc cgtggaccag ttcatggagc acctgcttcc cagσctcctg 2460 agcctσgcat cagatσσtgt gcσσaaσgtg agggttσtgc tagσcaaggσ cσtaaggcag 2520 atgctgttgg aaaaggcgta ttttagaaat gctggtaacc ctcatcttga agtcattgaa 2580 gagaccatct tagσattgca gtcagaσσgg gaσσaagatg tttσσttttt tgcagcσσta 2640 gaaccaaagc ggcggaatat catagacact gctgtactag aaaaacagaa ttaa 2694

<210> 28

<211> 2349

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 3882333CB1

<400> 28 cgccgcttgg gacgcctctg cctttccctc cctcccttcc ccgacggctt ctggcggcca 60 agtggatgtg gcgggtgatσ gagσcaccσt gσσσaggggc gcccagcaσt ggttcagtga 120 aσagσatttt ggaσaggaca tttggtgcca ggtctgagta gccagtttgc tgaattattg 180 tcccagtcag ccaggattgt gagctgtttg ggaagtttσg tggaaacgcc caagtgccag 240 σaσaggtgga gggacacctg gaggccagtt tcaggaactt ttgccacaag tataaaagac 300 ttcagaagtg caaagatgct gaaacagata ctgtcggaga tgtacataga tcctgatcta 360 ctggcagagc tcagcgaaga acagaaacag atcctgttct tcaagatgag agaggaacag 420 atccgaσgat ggaaagaaag agaagσagct atggaaagaa aggagtcσct gccagtgaaa 480 cccagaccaa agaaagagaa tggcaaatcg gttcattgga aacttggagc tgataaggaa 540 gtctgggtat gggtgatggg cgaacaccat σtagataaac cctatgatgt gctctgtaat 600 gaaattattg ctgagagggc ccggctgaaa gcagaacagg aggcagaaga gcccagaaaa 660 actcaσtctg aagaattcac σaatagcttg aaaacaaaat caσagtacca tgatctgcag 720 gctccggata accagcagac taaagacatc tggaagaaag tggcagaaaa ggaggaactg 780 gagcaaggat ctaggccagc aσσaaccctg gaagaagaga aaatccgatc actctccagt 840 tcttcaagaa atattcaaca aatgttggca gattcaatca atcgtatgaa ggcatatgca 900 tttcaccaga agaaagaatc tatgaagaaa aaacaagatg gagaaataaa tσaaatagaa 960 ggagagagaa cgaagcagat ttgtaagagc tggaaagaag actcggaatg gcaggcatct 1020 ctgσgaaaat ccaaagcagσ tgatgagaag agacgctcct tggctaaaca agcaσgagaa 1080 gactacaaga ggttatccct cgcggcccag aaaggaagag gcggtgagag gctgcaaagc 1140 ccσttgσgtg tfcccgcagaa acσagaaaga σσtσσσσttc cacσcaagσσ tcagttσσta 1200 aactcagggg catatcctca aaaaσctctt agaaatcagg gagtggtgag gacactgtcc 1260 agctctgσσσ aagaggaσat catccggtgg tttaaagagg agcagctacc acttcgagcg 1320 ggσtaccaga aaacσtcaga caccatagcc ccctggttcc atggaattct cacactcaag 1380 aaagσaaatg aacttcttct gagcacaggc atgσccggσa gttttctσat ccgagtcagt 1440 gaaaggatca aaggctatgc cctgtcctat ctgtcggagg acggctgtaa acatttcctc 1500 atcgatgσσt σtgσagacgc ctacagcttc ctgggσgtgg accagctaσa gcatgcσacσ 1560 ttggcggatt tggtggaata tcacaaggag gaacccatσa cttccctggg gaaggagσtc 1620 cttctctatσ cctgtggtσa gcaggacσag ctgσctgact acctggagct gtttgagtga 1680 cagcctccat cagggtcatc ctacagcctc caagcgggct ttcccctgga caaatgccac 1740 tgcaaσattt atgtgtgaag σσaaaatσaσ σctgcagcag agcσaatact gatcaactga 1800 aagtaaagta tccatggagt cctcattgac acctcttttc tgcacaaata ctggaattca 1860 atgtcaagag aaaatgaσσt σtgctσaaaa gggagaagag tctσaatttc agcaagtacc 1920 tgtcatgaag ggtatgacct taatgatgta cataaaataa aacaaatgaa gaaatggaaa 1980 acttttagaa attaaggtgt acttgaaaac gaatatσtat σatatgaccc ctgσaσtccc 2040 tctgtatcat ctcaggaggt ttcaggggcc tgttgacatg aagtttcgaa gtttcatgtt 2100 ggctttggaa tggtagσaaa agcσtttσct ggctgagatg atgcttaaaa cacacctcac 2160 ttattgtaca tgttggaacc aggacatgag agacatagaa aaacagaagt catgaatgta 2220 aattgaatga gaggcttaaσ atgcatgaaa ataσagatgg aσσtgcagga aagtgagσaa 2280 acatcgctga gtttgttttc ttgttcggga gaatggggcc ggggctggcc tggcctccct 2340 ggatataσt 2349

<210> 29

<211> 1213

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 7482809CB1

<400> 29 aggcgaaaag ctgσggttaa ggagagtccg gtttaaccgt caσσgggaag σgσgσtcgtt 60 cgggatcgcc gagtgggctg agatagtgaa ttcctaagaa gaaaataatg gattgcatat 120 tagttgttct σtaagtggaσ tcaacagtgt gcaagcttgt tggaaaagcc aaaagaagat 180 ggcaactcct tatgtcccag ttcctatgcc cataggaaac tctgcttcca gttttacaac 240 aaacagaaat caaagaagtt σttσttttgg σagtgtσtσa aσaagσtcaa attcttctaa 300 gggccagtta gaagactcaa atatgggtaa ttttaaacag acaagtgttc ctgatcaaat 360 ggataatact tcatctgtσt gtagcagtcσ σσtσattagg aσtaaattta caggtacagc 420 ttcttccatt gagtattcta ctagaccaag agacactgaa gaacaaaatc cggaaaσagt 480 gaattgggaa gatagaccat ctacacσtaσ tatactgggt tatgaagtga tggaagaaag 540 agctaaattt actgtatata aaatactagt aaagaaaacc ccagaagaaa gctgggtagt 600 tttcagaaga tacactgact tctctaggct taatgaσaaa ttaaaagaga tgtttσcagg 660 ttttcgacfca gcacttcctc caaaacgctg gtttaaagat aattacaatg ctgacttttt 720 agaagaσaga caattaggat tacaagcgtt tcttcaaaat ttagtagctc acaaggacat 780 tgctaactgσ σttgcagtga gagaatttct ttgtttggat gatccaσσgg gtccatttga 840 tagcctagaa gaaagcaggg cattctgtga aactttagaa gagacaaaσt accgcttaca 900 gaaagaacta cttgaaaaac aaaaggagat ggaatcacta aagaaactgc tcagtgagaa 960 gσaacttcat atagacaσtt tagagaacag aatcagaaca ttgtctttag aaσctgaaga 1020 atσactggat gtgtcagaaa cagaaggtga acagatccta aaggtggagt cctctgcact 1080 tgaggttgat σaagatgtcc tggatgaaga atctagagσt gataataaaσ σatgσttaag 1140 ttttagtgaa cctgaaaatg ctgtatcaga gatagaagta gcagaagtgg catatgatgc 1200 tgaagaagac taa 1213

<210> 30

<211> 3465

<212> DNA

<213> Homo sapiens

<220>

<221> misσ_feature

<223> Incyte ID No: 1739178CB1

<400> 30 atggcaaaaa tcgtggtggt gacatgcagt gattcaagtt ttggaaaσtt ttggttagat 60 caatggcaaa aacgagcaag agagaaatct ttgtgccaat gctctgccaa gcaagagatc 120 cgcacgcagc tggtggagca gttcaaatgt ctggagcagσ aatσagagtσ gσgaσtgσag 180 ctgcttcaag acctccagga gtttttccgc cggaaagctg agattgagct cgagtactcc 240 cgcagcctgg agaagctggσ tgagcgcttσ tσσtσcaaaa tσcgcagσtσ σσgggagσaσ 300 cagttcaaga aggaccagta cctcctctcg cctgtgaact gttggtatct ggttctgcat 360 cagacccggc gggagagccg agaccatgcσ aσσσtσaatg aσatσttσat gaaσaatgtσ 420 atcgtccgcc tctcccagat cagtgaggat gtcatcagac tcttcaaaaa gagcaaggag 480 attggcσtgσ agatgcacga ggagctσctg aaggtgacσa atgagσtσta caσagtcatg 540 aaaacctacc acatgtacca tgcagagagc atcagtgcgg aaagcaagct gaaggaggct 600 gagaagcagg aggagaagca gttσaataag tσaggagaσσ tσagσatgaa σctgctσcgg 660 cacgaggacc ggccccagcg ccgcagσtct gtgaagaaga ttgagaagat gaaggagaag 720 aggcaggcca agtaσtσtga gaacaagctg aaatgcacaa aggcσσggaa tgaσtacttg 780 ctcaatctgg cagccaccaa cgcagctata agcaaatact acatccatga tgtctctgat 840 σtgatσgatt gctgtgattt gggσttccat gcσagσσtgg ccσgcacctt ccggaσσtat 900 ctctcagctg aatacaacct ggagacctct cgccacgaag ggctggatgt cattgagaat 960 gcagtggaca aσσtggattσ ccgaagtgac aagcacacag tcatggaσat gtgcaatcaa 1020 gtcttctgcc ctccactcaa gttcgagttc cagccccaca tgggggatga ggtctgσcag 1080 gtcagcgσtσ agσagcccgt cσagacagaa ctgctcatgc gttatcacca gσtgcagtσσ 1140 agactggcca ccctcaagat agagaatgag gaggttagga aaaccctgga tgccaccatg 1200 cagacattac aggaσatgct gactgtggag gaσtttgatg tσtccgatgσ σttσσaaσaσ 1260 agtcgatcga cagagtccgt caagtcggct gcctctgaga cctacatgag caagatσaac 1320 attgccaaga ggagagσσaa σcagσaggaa aσagaaatgt tttattttaσ aaaatttaaa 1380 gagtatgtga atggcagtaa cctcatcacc aagctgcagg ccaagcacga tttactcaag 1440 cagaccctgg gcgaagggga aagagcagaa tgcggcacca ccaggσσσσσ σtgtσttσcc 1500 cctaaaccac agaaaatgag gagacctagg cctctctcag tgtatagcca taaactcttt 1560 aacggσagta tggaagcatt σattaaggat tcaggacaag ctatacσgct tgtagtcgag 1620 agctgcatcc gttacatcaa tttatatgga ctccagcagc aggggatctt cagagtgcca 1680 ggatctcagg tggaagtcaa tgacatcaaa aattcctttg agagaggtga agacccσctt 1740 gtggacgatc aaaatgaacg agatatcaat tcagtcgctg gtgttttaaa actgtatttc 1800 cgaggaσtgg aaaacσσaσt σtttcσtaag gaaaggtttc aagatttgat atctactatt 1860 aaactggaga acccagccga gagggtgcac cagatccaac aaatcctcgt cacccttccc 1920 cgσgtggtca ttgtggtcat gagatacctc ttcgctttσc tcaaσcaσct ctccσagtat 1980 agcgacgaga acatgatgga tccctacaac ctggccatct gcttcgggcc taccctcatg 2040 cacatccctg atgggcagga σσctgtgtcc tgccaggσaσ acatcaatga agtcatcaaa 2100 aσcatcatca tσcatcatga agccatσttσ cccagccccc gggagσtaga gggacctgtg 2160 tatgaaaaat gcatggσtgg aggggaagaa tattgtgaσa gσσcacacag cgagcσaggg 2220 gσσatσgatg aagttgacca tgacaatggc actgagcctc ataccagcga tgaagaagtg 2280 gagcagatcg aggctattgσ σaagtttgac tacatggggc ggtccccgcg tgagσtatσσ 2340 ttσaagaagg gggcctcgct gctcctgtac caccgcgcσt cggaggactg gtgggagggc 2400 cggcacaacg gcgtggatgg actcatσσcc catcagtaca tagttgtaca ggacatggat 2460 gatgccttct cσgacagcct gagccagaag gctgacagcg aggccagcag tgggccattg 2520 ctggatgaca aggcctcttc caaaaacgaσ ctcσagtσcc ccacggagca catctcggat 2580 tacggctttg ggggggtgat gggccgagtg cggttacgat ctgatggagc agccatcccc 2640 agacgcσgaa gσgggggcga σacacacagc ccgccccggg gcσtgggccc cagσatagaσ 2700 acaccacccc gggctgσtgc ctgccccagc agcccccaca aaatccccct cacccggggg 2760 aggatcgaga gσcctgagaa gcggaggatg gσgaσgttcg ggagcgctgg σagσatcaac 2820 taccctgaca agaaggcgct ctccgaaggg cactcgatga ggtcgacctg cggttccacc 2880 aggcacagca gcctagggga ccacaagtσσ ctggaggcσg aggσcctggσ agaagacatσ 2940 gagaagacca tgagcacggc tctgcacgag ttgcgggaac tcgagaggca gaacacggtc 3000 aagσaggσgσ cagatgtggt gctggacacc ctggagcσσσ tgaagaaσσσ gσσaggσσσσ 3060 gtcagctcgg agcccgccag tccccttcac accatcgtca tccgcgaccc cgatgccgcc 3120 atgcgcσgσa gσagcagσtc ctcσaσσgag atgatgaσσa σσttcaagcc agccctgtcσ 3180 gcccgcctgg ctggcgccca gctccgcccg ccccccatgc ggcccgtgcg gccggtggtc 3240 cagcacσggt ccagcagcag cagcagctcg ggσgtgggσa gσσσggcσgt gaσgσσσaσc 3300 gagaagatgt tccccaacag ctcagcggac aagtcgggca ccatgtgacc tgcaggatgg 3360 gccgcgcσgσ cgtggcccgc tgtggσtσaσ σacggcccag ggtggccttg gtggcttσσa 3420 cgtgcttccc agtgaccctg gccttggagg gcagaggtcc gtctg 3465

<210> 31

<211> 2609

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 7473630CB1

<400> 31 gtcggcaaca ggctcagccc taccctcaga agcgccctaa aaaacctaca gtgtagaaga 60 gcaσaccaga cagtgtgtct gσaagaagga tggctcσtaa tctgaaaaaa ggtaσaagtt 120 cctgtccagg gctcaccaat caggaaactc attctgactc caagggtgaa ggagcagggc 180 cagatggcaa gatttatgat ggaaaagata aaaσgaσtσa tσtaσtaggt gcttttactg 240 gtgcatctat gcgcggactg acacttagta gtacttcaaa tcaactctgg ctagaattta 300 attcσgatac tgaagggaca gatgaaggct ttcaacttgt gtataσσaag agaatσatag 360 gtattgcaga ggaggtcaca gtactcacat tgacagaatc agaacaggag cgagaacatt 420 tgtctaggga ggatcaggtt ttgaaσtσtσ aσaσtgtgaa gatσctggca ttσcacaatt 480 tggatacaag atcagtgacc aaggccactt tgctggtagc acσatcattt atggatgcaa 540 tσσaggσtaσ aσtσtccacg gaagtagcct tctσaactga atgtggaggt cgttttaaag 600 gagaatcatc aggaagaatc ttatctcctg gctatccttt tccatatgac aataacctgc 660 gttgcatgtg gatgattgag gtagatσctg gaaatattgt cagcttgcag tttσttgσtt 720 ttgatacgga agcatcacat gatatactcc gagtctggga cggtccacca gaaaatgata 780 tgcttttaaa ggaaattagt ggatσtctta ttcctgaagg aattσatagc acσσtσaata 840 tagtaaccat ccagtttgac acggattttt atattagcaa atctggattt gcaattcagt 900 tttcaagttc tgttgccact gσgtgtcgtg aσccaggggt σcσcatgaat gggactcgaa 960 atggggatgg aagagaacct ggggacactg ttgtttttca atgtgaccca ggafcatgaac 1020 ttcaaggaga ggaaagaata aσσtgcattc aggtagaaaa tσggtaσttc tggcagcσca 1080 gcccaccagt ctgtatagca ccσtgtggag gcaatttaac aggatcttσa ggσtttattc 1140 tttcaccaaa σttσcctcat σσatatccgc atagcagaga ctgtgactgg actatσaσcg 1200 tcaatgcaga ctatgttatc tccttggcgt tcatcagttt tagcatagaa ccaaactatg 1260 acttcctσta tatσtatgat ggaσcagaca gtaatagcσσ aσtgattgga agttttσaag 1320 acagcaagtt accagagaga atagaaagca gctcaaatac aatgcatttg gcttttcgga 1380 gtgatggatc tgttagttac actggatttc atσtagaata caaagcaaaa ctgcgagagt 1440 cctgctttga tcσaggσaat ataatgaatg gcacσagact tggaatggat tataaattag 1500 ggtσaacagt σaσσtattaσ tgtgatgctg gttatgttct tcaaggttat tcaacactσa 1560 cctgtttcat gggagatgat ggaagacctg gatggaatag agcσttgcca agttgtσatg 1620 σgccctgtgg aagtcgttca aσaggttcag aaggcactgt tctatcacca aactatcσaa 1680 aaaattacag tgtgggacat aattgtgttt attctatagc agttccaaag gagttgtggt 1740 gttggσcagt tgtatttttc cagacatcac tccacgatgt tgttgaggtg tatgatgggc 1800 caactcagca atcttctctg ttatcttccc tctcaggatc ccattcagga gaatcacttc 1860 cactgagttσ aggtaatσag atcaσaattσ gatttacttc agttggacσa ataacagcta 1920 agggatttca ctttgtttac caagctgttc ctagaacaag ttctacacaa tgcagttctg 1980 tgcctgaacσ aagattcgga agaagaattg gcaatgaatt tgcagtσggt tcatcggttc 2040 tttttgattg taatccagga tatattctcc atggatccat agcaattagg tgtgaaacag 2100 tgccσaattc tttggcσσag tggaatgatt ccttaσσtac ttgtattgtg ccσtgtggtg 2160 gaattttaaσ taagcgcaaa gggactattt tgtcacctgg ataccctgag ccttatgaca 2220 acaatσtgaa ttgtgtgtgg aagatσacag tgccagaggg agctggcatt caagtgcaag 2280 ttgttagctt tgctacagaa cataattggg attctctgga cttttatgat gggggagaca 2340 acaatgctσσ aagacttgga agctattcag gaacaacaat acσσσatσtt ttgaatagta 2400 cgtctaataa tctgtatcta aattttcaat cagacatcag tgtttctgct gcaggatttc 2460 atcttgaata σacagcaatt ggtttggatt cctgtcσtga aσσaσaaact σσtagcagtg 2520 gaattaaaat tggagacaga tatatggttg gagatgtagt atcctttcag tgtgatcaag 2580 gatattctσt tcaggtaagt σtattttaa 2609

<210> 32

<211> 2580

<212> DNA

<213> Homo sapiens

<220>

<221> misσ_feafcure

<223> Incyte ID No : 1431520CB1

<400> 32 gagagagagσ acagcctggt gggttctggg gtctacggσc taggggσcgg ggaagtttgc 60 gccgccgcga ccagtgctgc gatcccgagc cgggctccag ccccgaggac caggggtcgg 120 gcgggcctgc σtacggaacσ σσgcgggσca gσagcagtcg tctσgσgtcc tcσtgcttgg 180 aaaagtgttt aagcttctaa aatgtcatct atcaagcacc tggtttatgc agttattcgt 240 ttcttaσggg aaσaaagtca gatggaσaσt tacaσσtcgg atgaaσaaga aagtttggaa 300 gttgcaattc agtgcttgga gacagttttt aagatcagcc cagaagatac acacctagca 360 gtttσaσagσ σtttgaσaga aatgtttacc agttσσttσt gtaagaatga σgttctgσcc 420 ctttcaaact cagtgcctga agatgtggga aaagctgacc aattaaaaga tgaaggcaat 480 aaccacatga aagaagaaaa ttatgctgσt gσagtggatt gttacacaca ggcaatagaa 540 ttggatccca ataatgcagt ttactattgc aacagggctg ctgctcagag caaattaggt 600 cactaσacag atgcgataaa ggattgtgaa aaagcaatag caattgattc aaagtacagc 660 aaggcctatg ggagaatggg gctggccctc actgccttga ataaatttga agaagcagtt 720 acaagttatc aaaaggcatt agatcttgaσ σctgaaaatg attcσtataa gtcaaatctg 780 aaaatagcag aacagaagtt aagagaggta tccagtccta caggaactgg actgagcttt 840 gacatggcta gσttgataaa taatccagcσ ttσattagta tggcggcaag tttaatgcag 900 aaccctcaag ttcaacagct aatgtcagga atgatgacaa atgccattgg gggacctgct 960 gctggagttg ggggcσtaaσ tgaσσtgtσa agσctcatσc aagcgggaca gcagtttgct 1020 cagcagatac agcaacaaaa tcctgaactt atagagcaac tgagaaatca catccggagc 1080 agatcattca gcagcagcgc tgaagagcat tσσtgattta aσcaggggct caagσσσaag 1140 atacaaatgg tttatggcta tgaatgaagt atttgttgta gatagtaccc cctccctcct 1200 tcaaaaaacc aaacaσcata tctgatgtgt gaaaataatg gaggaaaaσc tσttgtgtat 1260 attcttaaag aataaatctg tttagaσaat aggtttatca caaacaaact caaaacataa 1320 catgaσtcct tttgaaatga tcaσtagcca tgttacgtgc σaatatagca ctgggataag 1380 gttccatttt caaacttcta tcatatttgt atattagact taatagcaaa gcatatttat 1440

-tgaactctag tagggctatt tgttggtaag ctσtttagσt σσaatttσcc ttaagcaaag 1500 ttttttgaga agggaaatga cccaaaggaa aggtttccta tgctggttga gaagaagtgt 1560 acttgccaσt gagσagtatg tcaggaaaga aggaatatta tσtaggttta gσtttgataa 1620 gtgctattag taatgaatat ataacatggg aaccaatgtt atctttaatg ttgcttgttc 1680 tggtgaaσag agaatcttaa gagctgttag aaagtagcac ttgatgcaag ggatgttttg 1740 aaaagaaaaa attggtaatg cgaatgtata gaaagtaaag gtaggatgct caggttttct 1800- gcatagttσt taaσtaatct tgtσtgσagt ttggtattga taatattagσ atggccactt 1860 atgctaaata cacaataaga tacatttaga aatccttaat gtactggtta ggtcagtggt 1920 acaactgttt gacttaatta tcaσaatttc cccaatggta accttacσtt ggaaactatc 1980 aaatataaat atactttaac taagccagtg acctggtcct aagttaagga ttttctaaat 2040 ggtatttaat tgtgtgccσa taaacactga ttttttttaa aaagaaaact tagtaattat 2100 ttgatggata acaaaaagca tctatcacta acattgcttg ttcatgtaat ctgtgataat 2160 ttggcttggc atgσcagtct tataattaac ataatttaaa tttgatgtaa tttaaatata 2220 acaggagact taaaatattt tattataaat tagatgccat attagttagt ggagaaaagg 2280 ggcattaggt attaattgca tgggtctaac tacattgatg atgagagttt aattccaact 2340 agagtatatt ttttgtttat taataatgaa attggtgctg acatgtaatg aσttcaσaag 2400 ttttacgagg gaaaactgaa aaaggaagta atataaattt tgtggggttt ttttgagatg 2460 gagtcttgct cttttgccag gctggagtgg tgttgcaatg tcagctcact gcaacctcσa 2520 cctcctggat tcaagtgatt ctcgagcσtt aagσtccσaa gtagctggga ttacgggcgc 2580

<210> 33

<211> 2181

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1916304CB1

<400> 33 cggσggσggc gacggcgaga aagagcttgc cggggggcga gcaggacagg acgaagccgg 60 agtgtaggcg gcagaggatt cgctcccaga gcagctgcgg ccaggtcgga aagaggccgg 120 ggcggσtggg tatatgaatg acctaaaggt acaaataaag acggagagag aacagtgcca 180 actgggagca gggcaagaat gccaattcct σσtcccccgc caσσσσσaσσ tggtcctcσt 240 σcacctccca catttcatσa ggcaaacaca gagcagccca agctgagtag agatgagcag 300 cggggtcgag gcgccctctt acaggacatt tgσaaaggga σσaagσtgaa gaaggtgaσσ 360 aacattaatg atcggagtgc tcccatcctσ gagaagccga aaggaagcag tggtggctat 420 ggctctggag gagctgccct gcagcccaag ggaggtctct tccaaggagg agtgctgaag 480 cttcgacσtg tgggagccaa ggatggttca gagaacctag σtggtaagcc agccctgcaa 540 atccccagtt ctcgagctgc tgcσccaagg cσtσσagtat σtgccgcσag σgggσgtσct 600 caggatgata cagacagσag ccgggcσtσa ctcccagaac tgccccggat gcagagaccc 660 tctttaccgg acctctctcg gcctaatacc accagcagta cgggcatgaa gσaσagσtσσ 720 tσtgσσσσtc ccccaσcacc cσσagggσgg σgtgccaacg caccccccac acctctgcct 780 atgcacagca gcaaagcccc cgcσtacaac agagagaaac ccttgcσaσσ gaσgσctgga 840 caaaggcttσ aσcctggtcg agagggaσct cctgctcσac σcccagtcaa accacctcct 900 tcccctgtga atatcagaaσ aggaccaagt ggcσagtσtc tggctcctcc tσctσσgσσt 960 taccgccagc ctcctggggt cccσaatgga ccσtσtagcc ccactaatga gtcagcccct 1020 gagctgccac agagacacaa ttctttgcat aggaagacac cagggcσtgt cagaggccta 1080 gcacctcctc caσccacctc ggσσtcccca tctttactga gtaataggcc acctccccca 1140 gcccgagacc ctcccagtcg gggagcagct cctccaccσc cacσacσtgt gatσcgaaat 1200 ggtgccaggg atgσtσcccc tccσccacca ccataccgaa tgcatgggtc agaacccccg 1260 agccgaggaa agσccccacσ tccaccctca aggacgσcag ctgggccacc σσσtcctcct 1320 ccaccgccσc tgaggaatgg ccacagagat tctatcacca ctgtccggtc tttcttggat 1380 gattttgagt caaagtattc cttccatcca gtagaagact ttcσtgctcc agaagaatat 1440 aaacactttc agaggatata tccσagcaaa aσaaaccgag ctgcccgtgg agccccacct 1500 ctgccaccσa ttσtcaggtg aagσσtggct tggtcccgtt cctσaggaaa aggatggacc 1560 ttctcttctt σtσagatggt cccttccatt cccctgaaac ctgcatgaga gctcctaaca 1620 tgtttctcca atgcaatcaa gcσσtagaσt ccaaatgtcσ tccσagctσa σctσσatσta 1680 tgcatσtσat σtσtggattt ggtgatσaga ctctatattg acagtaggat ctcaaaccct 1740 gcatccatcc ttcctccagc aagccctgct agσcacatga ggaacaagtt tσcgtgtctt 1800 ctgccttcct σttggggaaa ggtgccttgt tgtgatgaat taactcactg ttagggcagg 1860 gtggagaatg gtactccttc cttctcctgt ccactgtggg ggaagcttgg caggtatatt 1920 atatttcatσ atttaggagg ctggcatgac caggaσttat gggtgggagg ggagcatttt 1980 tagtgaagca agaaaggagt ttgccaagaa gtgatctgtt ttaaaggtca tatttggaga 2040 aagggcaagg aattgggtct gctttatttt tgggggtatt ttgtttttgt tctcacctgc 2100 tgccccccca ccσcaccacc ccagggataa attggatata aacactaaat actaatcagt 2160 tgaacttaaσ atttaataaa a 2181

<210> 34

<211> 4149

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 378504CB1

<400> 34 atttgacagt σccgσσccag cσgσσgtgtg atttagσaaσ cggtatcata ttcagatcaa 60 atcttaaaaa aaaaaagfcca cctacagaac tacagaatgg ttctctcccg ctcaagaagg 120 gσaagtggac ttgggcgtta agatgcaggg gggtgaacca gtgtcσaσaa tgaaagtctc 180 ggagagcgaa ggaaagctgg agggccaggc cacagcggtg accccgaaca agaacagcag 240 ctgtggaggt ggaatcagta gσagcagcag cagccgcggt ggcagtgcaa aaggctggσa 300 gtacagtgat cacatggaaa atgtgtatgg ctatttaatg aagtatacca accttgtcac 360 tgggtggσag tacaggtttt ttgttttaaa caatgaagσt gggctgttgg agtactttgt 420 gaatgaacag tctagaaatc agaaacctag aggaactttg cagcttgcag gagctgtaat 480 atcacccagt gatgaggatt ctσacacctt caσtgtaaac gctgσσagtg gggaacaata 540 taaactcaga gctacagatg caaaagagcg acagcactgg gttagcagac ttcagatatg 600 tacaσagσat catactgaag ctattggaaa gaataatcct cctctgaagt cacggagctt 660 ctcacttgca tctagtagta attctcctat atcgcagagg agaccaagtc aaaatgccat 720 ttcttttttt aatgttggac attccaaact gcaatcactg agcaaaagaa ctaatttacc 780 tccagaccat cttgtggaag tcagagaaat gatgtctcat gctgaaggac aacaaagaga 840 cttaattaga cgaattgaat gccttcctac ttctggσcat cttagttσσt tggaccagga 900 tctcttaatg ctcaaagcta cttccatggσ aactatgaaσ tgcttaaatg actgctttca 960 tattctccag ttacagcatg catcacatca gaagggctca ttgccttcag gaacgacaat 1020 cgagtggtta gaaccaaaga tatctttatc aaaccactat aaaaatggag ctgaccagcc 1080 ctttgσaaσt gatσagagta agccggtggc agtcccagaa gagcagcctg ttgcagaatc 1140 tggactatta gcgagggagσ ctgaagaaat aaatgcagat gatgagatag aggatacatg 1200 tgaccacaaa gaggatgacc tgggagctgt agaagaaσaa σgtagtgtca tcctacatσt 1260 cttgtcacag cttaagctgg gcatggattt aacaagagtg gtgσttccta cafcttafccct 1320 agagaagcgt tσσttgctgg aaatgtatgσ agaσtttatg tctcatσσag acctatttat 1380 agccatcact aatggagcca cagctgagga cagaatgatfc cgcttttttg agtactacct 1440 tacctcattt catgaaggcc gtaagggagc σattgctaaa aaaccataca atcctatσat 1500 tggagaaaca tttcactgtt cctggaagat gccaaaaagc gaggtagcat ccagtgtttt 1560 tagcagttσt tcσaccσagg gagtcacaaa tcatgσtσσt ttatcggggg agtctttgac 1620 ccaggtggga tcagactgtt acacagtcag atttgttgct gagcaggttt ctcatcatcc 1680 tcσagtctca ggattttatg cagaatgtac agagaggaag atgtgtgtaa atgcgcatgt 1740 ctggactaag agcaagttct taggcatgtc aataggcgtg acaatggttg gagaaggtat 1800 ccttagtctg ttggagcatg gagaagagta cacattttσt ctaccctgtg catatgctcg 1860 gtcaattttg actgttcσtt gggtagaact gggtggcaaa gtcagtgtca actgtgcaaa 1920 aactggatat tσagcσagca tcaσttttca taccaagσσa ttttatggtg gcaaactgca 1980 tcgggttaσa gctgaagtaa agcaσaaσat caccaacact gtggtatgca gagtgcaagg 2040 ggaatggaat agtgttcttg agttσaσata tagσaatgga gagaσaaagt atgtggaσtt 2100 gactaaattg gcagtgacga agaaaagagt gagacctctg gagaagcagg atccatttga 2160 atσcaggcga ttgtggaaaa atgtgacaga ctσgctgaga gaatctgaaa ttgataaggc 2220 cacagagcat aagcataccc tggaagaacg tcagaggact gaagaaaggc atσgtactga 2280 aacaggcaca cσttggaaaa ccaaatattt tattaaagag ggagatggct gggtttatca 2340 taaaccaσtt tggaaaataa ttccaacaac acaaccagca gagtgacaca tactatctaa 2400 aactσgaσσa aatgaggttc ttctctgttt aσσctaaatc ctσσσagaat ggagtcattg 2460 cactgagtga cctgcttcct gattgcgcag actgaaacta gctaaacctg aatgtaccta 2520 ctagggcaσc ataatactgc agcaagacca aagtggtaaa gaaacaσagt ggacctttta 2580 ccaacctgtt catgtgatgt gagcaatacc atcttaaaac ttgttacctg aatcagtaga 2640 tgaatctttt atcσagttσt tgσtσσtaaa gttaagtttg aatσccctat ttttgσaσag 2700 gggcagcaga tacacacaaσ aatgagaact cagtgacttt gatttctttg tagtgaaaag 2760 tgaagtctσσ gtttσagagt ttgtgtσttt σtttσtgtcc ataactgaag tattcaσtaσ 2820 tcttgtaaac caaccaagag gaggagaaag atgacccaga agtggattca gccattgtgc 2880 ctgaaatcag tgtttaaaaa aaaaaatcaa cσaggttgtg gtaaσaaggσ attσtatttc 2940 ttcaaaaaga ctgtatgcct gtgtctgagg aacttaccta ttatccacct σtgttggaac 3000 tctcttttaa aaagtacatt tatagattga tcagaattat aaσcatggag aattttttct 3060 tctgagcatt ttaatatact tgaaaacaac attgacttga aaaatttcag aacatttttc 3120 agtacctagt tttattaaat attacaσttg agagacaσtt tttaaaaatg tgttaatgtc 3180 aatatgatga gattttagcc tttctccaga actaaggcat taaagaaaat agcaaatatt 3240 aaaaaataaa actgttactt ttttccttct ttσttttcac ctttaggtta atatccagta 3300 ttatgtgtta tccctttgga taagtatgct ttattttacc tctgttaaaa attaaaataa 3360 atgattσtat tcatatttgt cagtaattca aaacttatat gtgtaactga acgcgcatgt 3420 aaggtatggt tttatttatt tttttttttt ttgaggaaat ttaaatgcta aagaaacaac 3480 gaaatgaaaa ggtatcagga aaaaaagatc aggaagttgt attcaggtac aaatσttttt 3540 ttaaataagt attttgttga ggttgaagaa ttgctggcaa ttaaaagaat agagctaatt 3600 atggctttσa tσattσattσ atgtatttat tgagcaccta cttattatgg tgctσaaσaσ 3660 ttgttactgc aagctaσctt aatttcccaa gagtggtgcc ttactctgtt ttttctgata 3720 tggfccttcca atcagtgtgt gtaaσatacσ tgttgtttat cagσσattgt aggtggσtgt 3780 gtctgttgca tcatcataag aagtttaagc tttgtgctct gataaattgt gttctgttaa 3840 agaggttagt aggatgaaaa cagσaaaaσa ataatttttt σaaσaaattg taaattataa 3900 gaaaaagagt tggtttgtgt acaacaattt taatgattcc cttgttcatt tttgctgtga 3960 aatgcactga aaaaaatcct caaaatgagt tatagttcσt gtgttgggaa aattgaσaaa 4020 taataaaact agagaacaac catataacaa aaaacaaaaa acaacaaaac aaaaaaaaca 4080 acacaacaca aacaaaaaca aaaaaacaaa caacσacaaa caaaaaaaaa σagagσσσσσ 4140 catagacca 4149

<210> 35

<211> 3080

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 5275371CB1

<400> 35 gctagaaatg gggaaaacca attatggtta catagaaatt aggagcatgt aaaagtacat 60 tattaaacca cttcatgtat tttttctctt σaatcatttg gggagaattσ ttσttggttt 120 aagaacttat tatccacagc acagcatatg tatgactttg tcccttattt taaattctga 180 aattctggga atttgtcccσ gagtσσtggg aactctgtct tccσaaσcat taaactaσag 240 cagaaaataa atccaagggg caatttgtat tgtgaccata gtagaatgcc tgtgtctttt 300 ctttaaaggc tgttgaaatt tatgaaaaat gtgσctttat tttσtσagac ctgagσσatt 360 catgtactgg gtatgggtgg ttgggtgggt ccaggagtgt gcaactccct ccattatccg 420 ccttgttcgg ggtttgσtgσ tctgaccσσt aσtttgσttt tcagatgact ttgggaaatt 480 gctgctggct gaggccctcc tggagcagtg tttgaaggag aaccatgcca aaataaaaga 540 ctccatgσct ttgctggaga agaatgagσc gaagatgagc gaagccaaaa attatctaag 600 cagtatcctt aaccatggga ggctctcgcc acagtacatg tgtgaggcca tgctgatcct 660 gggcaaactg cattacgtgg agggctcata ccgagatgcc atcagcatgt acgcaσgggσ 720 cgggattgat gacatgtcca tggagaacaa gcccctgtat cagatgcggc tgctgtcgga 780 ggcttttgtc atcaaaggσσ tσtσtctgga acgcctaccc aactccatcg cctcσσgσtt 840 ccgcctgaca gagagggagg aggaagtgat cacσtgtttt gagagggcct cctggatcgc 900 tcaggtgttc ctgσaggaat tggagaagac caσaaataac agcacgtcga ggσatctgaa 960 aggctgtcac ccgcttgact atgagctcac ctacttσσtg gaagσtgσσσ tccagagcgc 1020 ctatgtgaaa aaσctgaaga aggggaacat σgtgaagggσ atgagagagσ tccgggaagt 1080 gctgcggact gtggagacca aagcaactca gaacttcaaa gtgatggcgg ccaagcacct 1140 ggσgggggtσ σtgσtgσaσt σcctgagtga ggagtgctaσ tggagcσσcσ tgtcccacσσ 1200 tσtgcctgag ttcatgggca aggaggagag ttctttcgcc actcaggccc tgcggaaacc 1260 tσaσctσtat gaaggagaσa aσσtσtaσtg σσσcaaggaσ aacatσgagg aagσσσtcσt 1320 gσtcctcctc atcagcgaat ccatggcaac tcgagatgtg gtgctgagcc gggtgccgga 1380 gcaggaggag gaccggacag tgagσttgσa gaatgccgca gccatctatg acσtσσtgag 1440 catcacgttg ggcagaaggg gacagtacgt catgctctcg gagtgcctgg agcgagccat 1500 gaagtttgcg tttggagaat ttcaσctttg gtacσaggtg gσσσtσtcca tggtggcttg 1560 tgggaagtca gcctacgctg tgtccctgct gcgggagtgt gtgaagttgc ggccctcgga 1620 cccσaσσgtg cσσσtgatgg ccgσgaaggt σtgcatcggg tcσcttcgct ggctagagga 1680 agcagagcac tttgccatga tggtgatcag cctcggagag gaagccgggg agttcctccc 1740 caagggctaσ ctggσtσtgg gtσtcaσσta tagcctgcag gccacσgaσg σcacσctgaa 1800 gtccaagcaa gatgaattgc accggaaggc actgcagacg ctggagaggg ctcagcagct 1860 ggcgσσσagt gacccσσagg tσatcσtσta tgtσtcgctg cagctggσσc tcgtccgaca 1920- gatctccagt gccatggagc agctgcagga ggccctgaag gtacgcaagg atgatgccca 1980 cgccctσcac ctgctggcaσ tgctcttctc tgcσσagaag σaσσacσagσ atgσσctgga 2040 tgttgtcaac atggccatca ccgagcaccc tgagaacttσ aacctgatgt tcaccaaggt 2100 gaagctggag caggtgctga aaggcccaga ggaagccctc gtgacctgca gacaagtgct 2160 gaggctgtgg cagaccctgt acagcttctc ccagctggga ggcctagaaa aggatggcag 2220 cttσggtgag ggcctcacca tgaagaagσa gagtggcatg σacctgaσtt tgσσtgatgσ 2280 ccatgatgca gactctggct cccggcgggc ttcgtccatc gccgcctccc ggctggagga 2340 ggcσatgtσa gagσtgacta tgccσtcttσ ggtcσtgaag σagggccσca tgcagσtgtg 2400 gaccacgctg gaacagatct ggctgcaggc tgctgagctg ttcatggagc agcagcacct 2460 caaggaagca ggtttctgσa tσσaggaggc ggcgggcσtc ttccσσaσtt σtcactcagt 2520 actctatatg cggggccggc tggctgaggt gaagggcaac ctggaggagg ccaagcagct 2580 gtaσaaggag gcgctcaσgg tgaaσccaga tggσgtgcgc atcatgcata gcσtgggtσt 2640 gatgctgagt cggctgggcc acaagagctt ggcccagaag gtgcttcgtg atgccgtgga 2700 gaggcagagt acgtgccacg aggcgtggca gggσσtgggc gaggtgctgc aggcσσaggg 2760 ccagaacgag gctgccgttg actgcttcct caccgccctt gagctggagg ccagcagccc 2820 tgtactgσσσ ttσtσcatca tcccσagaga gctσtgaσga cgctgcagcc gcagggaggg 2880 aggggctggc cagagggaga ggcagcaggg aacgtgggtc agggtggggc aacagtggca 2940 tcaggtgcgg ggσctcaggg aaataσatct ttagtgaacg cctctgcagc tgcagccσtσ 3000 gttctcttgg ctgggccaag aggccttcct ggatttcttt gttgtcaatt gggaacaggc 3060 tgσtgagσaσ σgaσtgσcag 3080

<210> 36

<211> 4167

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 490576CB1

<400> 36 ggσggσggga gggagtgcgc tgσgcσtgcc tσσgggagga gccgcatcca caσaσσσtgσ 60 gctgccctgt cctgcgcgag tggagctctg aagaagctct gagcggagtt gtgttcttcc 120 ccaggtgcgt σσtggctgag agttggagct ctσσagσaaσ atgσctgagc agagtaacga 180 ttaccgggtg gccgtgtttg gggctggcgg tgttggcaag agctccctgg tgttgaggtt 240 tgtgaaaggc aσattσcggg agagσtacat cσσgacggtg gaagacaσct accggσaagt 300 gatcagctgt gacaagagca tatgcacatt gcagatcacc gacacgacgg ggagccacca 360 gttcccggcc atgcagcggc tgtcσatctσ σaaagggσac gσcttcatcc tggtgtactσ 420 cattaccagc cgacagtcct tggaggagct caagσccatc tacgaacaaa tctgcgagat 480 caaaggggac gtggagagca tσcccatσat gσtggtgggg aacaagtgtg atgagagσσσ 540 cagccgcgag gtgcagagca gcgaggcgga ggccttggcc cgcacatgga agtgtgσσtt 600 catggagaσc tcagccaagσ tσaaσcataa cgtgaaggag σttttσσagg agσtgctcaa 660 cctggagaag cgcaggaccg tgagtctcca gatcgacggg aaaaagagca agcagcagaa 720 aaggaaagag aagctcaaag gcaagtgσgt gatcatgtga aggcσcttcc tgcgggagga 780 gcagctgtgt gtccccggca cctcactccc ccaaaatgac acccaccgtc gtcagggtag 840 catgtataat gσσcaσgtgt taaaσattgc atttaatcga gatgcgtcct attgtcctta 900 agagggcgtt tcacaccacc aacagtaagc cacccactct ggagtcacag aatctgccag 960 gσggttσaag tgaaaaccaa σaσaσtcagc atσσσtggga aσtgagaggt gccagcaatt 1020 gctgaaggtg gcgatgaaca cccgaaggtg ggagggagga ctggtaccca caaagcaaca 1080 tgtaccgaga ggactaaatg tcatσtaσgt gσatgtgaga gσgtgttaaσ ctagagttaσ 1140 ctgcaccaac cccagacaga agccaatcac atctttgggg gaggggaggg gcaggaagag 1200 gtgagaagat cagatggtcc aaagtggacc aσacttggtσ σattttacac ttttttaaag 1260 gggattaaaa aacacagcct ctcccccaaa gggtgtccgt tcttaattcc cacctggcct 1320 gttaggagσc ttgσtaσcσt gaggggatgt gttσacctta cctagacσta gttaggaagt 1380 atcattttaa gctattagag tatttatctt catgtgcagg gataagtgca ctaacagtgt 1440 gctgctctgt cggaagttct tcagttttta agtgaggata tcgtgacagt attaaaacat 1500 cgcaataatg ttcctgtgtg ttatacatcg agggttttag aaatgtgatt ttcttctttt 1560 gacctgtgag gagtataact tctttσagσc ctcagatttt aaatacaagc aaataaactc 1620 actattttta gacgtttttt tcctccaagg tggttttctt ctcttaaata actcgatctg 1680 tacccagctg ggtagcagσσ agσaaaggcc atcagacaac cagaagcaσa tccatttttg 1740 tagtgtcaσa aacatgtata tgccacactt tgcaccttaa tgaaatactt tgaaacagaa 1800 gttattcact gtgtttttga tgatσtatct gtattggaaa tatgttcσtg gaaaatgcat 1860 ttaaataata gtaaattctc ttgcatgttc cattatacgt gtcttσtaag agctgttcaa 1920 taσagtattσ aσtctagaaa σaattatσtt tttσtcttaa tgattttgtg tgcatcttta 1980 atctttcaag ccaaattaca gctatttcag gtttcctgtg ttagcttggg gataggatgg 2040 tggctggaga σaggσaggσt tctctgσcct gggaagagσσ σactcagctt aattgctσtg 2100 ccatcgtaga gcctggttgg acttggcttc ctgaaaactc ccactgatag tgcctgttag 2160 atctcctgtt tgtttσagtt ggcagaacat ttactggccc caaσtgtggc atcatcσtct 2220 cagcagtctt cctgtcaccc gcctggcagg cagaaggagc tgcagtccca cgtgggcctg 2280 cσtggggggg tgggggctgc atggctgttg ggtggcagtg tcagcacagg gagggcttaa 2340 gttggggatg tttgaccagg ccacσtcctg caactgctgt ttctcctgtc cctcctatgc 2400 agggσttgca gσagσagcag tgtggσcatc tcσatσσσσc aaagcacact tgσtctctca 2460 atatgtccta gttttcttca gccttttctg gttcagttcc cttgtcctga tctcatcctc 2520 tctggtσtcc caataaσtca σccttgggat gtgtttagag cgtgggaggt gcσtttgaga 2580 actgcttgac tccatgatct σctagaacaa aaccgccctg actttacagg gggaacactc 2640 atgctgagct gagaaagcag agaagtggcg tgggagccag ctgggggtga agagcatttg 2700 ggccagtccc gtggccccct tcagattσσt caagcaggat tgttctgttc taaaaagctg 2760 ttgcacagca ttcgσaatga gatσtttagt tggσggattt tctggaacat ttgtttttca 2820 acttgtcccg acattttttt ttctgtttct attctgagag agagatgatc aagttttaat 2880 ttgggtatag gttaaatgga agaagaaaca gaacttσatg gccaaagtag acσtatagat 2940 tttgattggg ttσtttgtta acagtagaat gcgatctttg ccactgactg tagtattaat 3000 aaggttttaa tgtgagatat tcctgcaaac catcccattt σtactgattg taagtcagaa 3060 tttcttttat ccctttcaaa tcagtttcta catgtttaag tgttcagggc ttcatcagca 3120 tgagaagttt gtaattactg aaagtctgat ttcattcagg acacattttt ttccttcata 3180 ttttttctgt gaatttatag gctaggaagg ctattgaagc ctcaattatg ggtcttcatt 3240 ttgagatcgt tttctatgag ctgaaσtgag gatatcaatg gttatctcaa aatσgtσttt 3300 taggagatcc ccaattgact σagagtttga ggagttagta tcacagaatt agattttttt 3360 aaagcatttg taσgtttcca ttcccaaata tgtagctgtg gttcttgaaa acacatccta 3420 cattgcatat gggcatagca gtttttgacc caggcagaat aagttaatat ttaattaaat 3480 attgctttga agatggcgct ctgggcatga gcatggggσt σσatgacttc ccttctatcc 3540 ccatgagccc ctcctccatc cagcgacaag ccatgggcat gcatacaatg cagcaagacc 3600 aacacaagag caatattgaa ttgttcattc tatctaaaat tacatgtata taaaatatat 3660 aatttatctt cctgcatttt tgaagtataa agtcataaat tgtaσatatc tgtaagctag 3720 tatatttgtt tcactgtttg taatatttaa gaaatgctca ttctttgtag aacaaaaatg 3780 tattaaatat tttaaaaatt gctctgtgat acttaatttt tttcccσaaa atttgtaatg 3840 tgttgcttct acataagttc tctggaaata tctaσaaσta ataggacaca tgtaaatcct 3900 tgaagacaca tcσtggaatt cataccccac aaggacagtg tgtataσaaa gtatttgcag 3960 agcatgactt ttatatgtgt gggatatσaa tgtgtatatt tatatttaaa gtgtatttat 4020 tgttacaagt ctattσtcta ttatatttta tttactctgc ggttataaaa atcacccttg 4080

- catacaagtt tctagttgσc agtgatgttc tggaaataat gggagatatt acaataaagc 4140 tacagttatg aσaaaaaaaa aaaaaaa 4167 <210> 37

<211> 3591

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No : 1417657CB1

<400> 37 gggcccctcc tggcctctgσ σaaσgσagct cσσtgacccg gaactttcgt ggtgtcgggc 60 gagagtttgt gcσcacttcc tgagagggat gggtaagaag aggaccactt ccggcgaggg 120 acgggaaaga cagcggcttc cggσgcggcg gttccggaσa aσcgtgcgct tttagtaaaa 180 gattggggtt cgσgcggggg agaagggctg ccccgggccc tctggttctc gtcccgcagc 240 gtccgctcσσ σcgcgccact gcgccgσtσc caggaaccct gtactccggg gtcgecggct 300 tctctcctgc ctccggtccc gccagacacc tcgagctcct taagtagctc ggtccttgac 360 gtcσσtσtgg gσσcttcccg cgtctatσgσ σtgagtcσcc gggcccctct agcσctctgt 420 tccctcccct cttttgttcc tccctagagc cccgccgccc tcagggctga cagtgtggac 480 ggcgggagtc tcctcgσtcc cctgctggga ttgactgacc gagcgtttag tgactgcσσa 540 gatctggctg atgggggtac cgagaggtgg cctgggccgg gaatgtccag ctagagtctt 600 σcgtggaagt σagacatgaa aσtgacaggc ctaagggaag ctaggaagtc ccctσaccgc 660 tcagccaggg tgatgggctg gactgacaga ctccagtgaa tttgagcttg cctgtcaggc 720 tgattggctg atagaσagcc ctggattggc tcaσtaagac tgaccagσσσ gggaσσaagc 780 agttctgggg tcccaacctg ggtggaaggt ctgaactgat gacccaccca ggctgaccag 840 gσσagcccac σtcaσtgaσσ tcctgaσσσc tgaσσtσatc acσtgtgcag σcatggagaa 900 gatgtcccgt gtgaccacag ccctgggtgg cagcgtgctg acaggccgca ccatgcactg 960 ccacσtggat gσtσccgcca atgccatcag tgtgtgσσgc gacgσagccc aggtggtcgt 1020 ggcaggccgt agcatcttca agatctatgc catcgaggag gaacagttcg tggaaaagct 1080 gaacctgcgt gtggggcgca agccttcgct taacctgagc tgtgctgacg tggtσtggca 1140 ccagatggat gagaacctgc tggccacagc agccaccaat ggcgtggtgg tcacgtggaa 1200 cctgggccgg cσatcccgca aσaagσagga ccagσtgttc acagaacaca agσgcacggt 1260 aaacaaagtc tgcttccacc ccaccgaagc ccaσgtgctg ctcagtggct cccaggatgg 1320 cttcatgaag tgctttgacc tccgcagaaa ggaσtσtgtc agσaccttct σgggccagtσ 1380 ggagagcgtg cgggacgtgc agttcagtat ccgggactac ttcaccttcg cctccacctt 1440 tgagaacggc aatgtgcagc tctgggaσat ccggσgtσcc gacσggtgcg agaggatgtt 1500 cacagcccac aaσggacccg tcttctgctg cgactggcac cccgaggaca ggggctggtt 1560 ggccactgga gggcgcgaca agatggtgaa ggtctgggac atgaccacgc accgtgcσaa 1620 ggagatgcaσ tgtgtgσaga ccatcgcctc ggtggcccgt gtgaagtggc ggccagagtg 1680 cσgσσaσcac ctggσσaσgt gσtccatgat ggtggaccac aacatctatg tttgggaσgt 1740 gcgccggccc ttcgtgccag ctgccatgtt tgaggaacac cgagaσgfcca ccacgggaat 1800 tgcctggcgc σaσccccacg acccctcσtt σctgctgtct ggσtσσaagg aσagctcgct 1860 gtgccagcac ctgttccgcg acgccagcca gcccgtcgag cgcgccaacc ctgagggcct 1920 ctgσtaσggc ctcttσgggg acctggcctt cgσσgσcaag gagagcctcg tggctgcσga 1980 gtcggggcgc aagccctaca ctggcgaccg gcgσcacccc atcttcttta agcgcaagct 2040 ggaccctgσc gagcccttσg caggcctσgσ σtccagtgcc ctcagtgtct ttgagacgga 2100 gccaggtggc ggcggcatgc gctggtttgt ggacacagct gagcgttatg cgctggctgg 2160 ccggccactg gccgagσtct gtgaccacaa cgσaaaggtg gσtσgagagσ ttggσcgcaa 2220 ccaggtggcg caaacgtgga ccatgctgcg gatcatctac tgcagccctg gcctagtgcc 2280 cactgcaaac ctcaacσaca gtgtgggcaa gggtggctσσ tgtggcctσσ cgσtσatgaa 2340 σagtttcaac ctgaaggata tggccccagg gttgggσagt gagacgcggσ tggaccgcag 2400 caaaggagat gcacggagcg acacagttσt gctσgaσtσc tσggccacac tcatcaccaa 2460 tgaggataac gaggaaaccg agggcagcga cgtacσtgσc gactaσctgc tgggtgacgt 2520 ggaaggtgag gaggacgagσ tgtacctgct ggatσσggaa cacgcgcacc ccgaggaccc 2580 tgagtgcgtg ctgccgcagg aggcctttcc gctgcgcσaσ gagatσgtgg aσaσgσσtcc 2640 cgggcccgag cacctgcagg aσaaggσσga σtσcccgcac gtgagcggca gcgaggcgga 2700

- tgtggcσtcc ctggcccccg tggaσtcctc cttctcgσtc σtgtσtgtσt σacacgcgct 2760 ctacgaσagσ cgcctgccgc ccgacttctt cggσgtgctg gtgcgcgaca tgσtgcactt 2820 ctacgctgag cagggcgacg tgcagatggc tgtgtctgtg σtσatcgtcc tgggtgaacg 2880 ggtgcgcaag gacatcgacg agcagaccca ggagcactgg tacacttcct acatcgacct 2940 gctgcagcgσ ttσσgσσtσt ggaaσgtgtσ caaσgaggtg gtσaagσtga gcacσagσσg 3000 σgccgtcagc tgcctcaacc aggcσtccac caccctgcac gtcaactgca gccactgcaa 3060 gcggcccatg agσagσσggg gσtgggtσtg σgaσaggtgσ σaσσgσtgcg ccagcatgtg 3120 tgccgtctgc caccacgtag tcaagggtct cttcgtgtgg tgccagggct gcagccacgg 3180 cggccacctg cagσaσatσa tgaagtggσt ggaaggcagc tcccactgtσ cσgcaggctg 3240 cggccacctc tgcgagtact cctgacgggg catctgctgg gcttgcccgg gcggccgcgt 3300 gcagaaσσcc gcctggtctt gtgcccgaga cgggcggagg ctggaacttg agaσσtσaat 3360 aaaggaagta gagccgctgt cggcactgcg tgtcacccca tagcgcggcg ggctcgccgt 3420 aggcσσgσgg ggcagctggc gggcgggggσ tttcggσggg gttgtσgσca tggtaaccgg 3480 cggcgcccat agtcgctctc ggggσggcct tcccagtgcc tcccgtagag gcctcttccg 3540 gggcgggcca aaggaggtca σttσσggggt σσgtcccggt tcccggcσgt t 3591

<210> 38

<211> 3685

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 1773215CB1

<400> 38 gtcgccgcσt ggcσgggσgt agaσgσggtg gcagagcccg cgcggσgctg gaagσgagtg 60 gcggagcggc gggacctcgg cggaσtcgcc atggaggagg agggtgtgaa ggaagccggt 120 gagaagcctc ggggagcaσa gatggtggaσ aaggctggct ggatσaagaa gagcagtggg 180 ggcctcctgg gtttctggaa agaccgatat ctgctcctct gccaggccca gctgctggtc 240 tatgagaafcg aggatgatca gaagtgtgtg gagactgtgg agctgggσag σtatgagaag 300 tgccaggacc ttcgtgccct cctcaagcga aaacaccgct ttatcctgct gcgatcccca 360 gggaacaagg tcagcgacat caaattccag gcacσσaσσg gggaggagaa ggaatcctgg 420 atcaaagccc tcaatgaagg gattaaccga ggcaaaaaca aggctttcga tgaggtaaag 480 gtggacaaga gctgcgccct ggagcatgtg acacgggacc gggtgcgagg gggccagcga 540 cgccggccac caacgagagt ccacctgaag gaggtggcca gtgcagcttc tgacggtctt 600 ctgσgσσtgg atcttgatgt tccggacagt gggσcacσag tgtttgcccc cagcaatcat 660 gtcagtgaag cccaacctcg ggagacaccc cggcccctca tgcctcctac caagcctttc 720 ctagσaσσtg agaσσaσcag σcctggtgac agggtggaga cσσctgtggg ggagagagcc 780 ccaacccctg tctcagcaag ctctgaggtc tcccctgaga gccaagagga ctcagagacc 840 ccagcagagg aggacagtgg ctσtgagσag cctcccaaca gσgtσctgcc tgacaaaσtg 900 aaggtgagct gggagaaccc cagcccccag gaggcccctg ctgcagagag tgcagaaccg 960 tσσσaggσaσ σσtgttctga gacttctgag gctgccσσσa gggagggtgg gaagccccct 1020 acacccccac ccaagatctt atcagagaaa ctgaaagcct ccatgggtga gatgcaggct 1080 tctgggccac ctgctcσagg cacagtgcag gtctcagtga atggcatgga tgacagtσσt 1140 gagcctgcca agccctctca ggctgagggc accccaggaa ctcctccaaa ggatgcaaca 1200 aσatσσaσag σaσtgccccc σtgggaσσtg σσaσctcagt tσσatccσcg ctgctcctcc 1260 cttggggact tgcttgggga aggcccgcgg catcσcttgc agcccaggga acggctatat 1320 σgggcccagσ tggaggtgaa ggtggσσtσg gaaσagaσgg agaaactgtt gaacaaggtg 1380 ctgggcagtg agccggcccc tgttagtgcc gaaacattgc tcagccaggc tgtggagcag 1440 ctgaggcagg cσaσccaggt cctgσaggaa atgagagatt tgggagagct gagccaggaa 1500 gcacctgggc taagggagaa gcggaaggag ctggtgaccc tctacaggag aagtgcaccc 1560 tagggcσttσ tgggccagag gcaccatccc ttctggσσat σcatcaagtc catσaaggcσ 1620 cagccctgct gagaaatgtg cttctgcttc tacagcaatg gctgcaggag ggccattggg 1680 catgtσaggg tttggσσatg aσσσgaagag aσtσctggcg tccttcctac tctgctctgg 1740 ccagtggtgc caggtgccac ccagggctac tgcctggcta tctggσσtgg σctctgggct 1800 ggggctgggg ctgggagcac acacgctggg acσtatgtgt ttgtgtggtc gttccaaact 1860 gccccagggc tttgggggcg gcacttgggg tttctgggaa tgacatcatc tctgttccσσ 1920 atσσσσagta gtttacattc ctgacttctg aatacagcac agctgagccc cctgcagσtc 1980 ccatctccag ctattcctag gcaaagagcc tcatggctaa ggcagcctca aagσcagccc 2040 σtcσtcccac ctattctgag tagσtgcaga ggσσttgggt cσaggσtcta ggttcatccσ 2100 tσagttgggg ggaacgtagg acccagctgg agcctcttga gggagatgag aggcctcttt 2160 gtgaggagga cattagσtgt gtggσσtσtc tσtσtttggσ σσtgtttσct tttttgcaaa 2220 acaaggacat tttctgcagc cccttcctct cagtgagcta tgattggagg gcttaggtct 2280 ggaggattca agagtggaag aggaatttaa ggggtcσcct agtctagtct ctgcccctgg 2340 atagtgtcca gccttgtata tttctgaaga ggtggatccc agagtggctc tgatgtccac 2400 attagaaaaa σttacttgta atgatcatgt cagσσttcag aagagaatcc cσacσaaσtt 2460 ctgtgcctcc tcagatgggg atttatctgg atctctgtgg ttccttctca gccgaaacag 2520 gtccagtatc ccagtσattt cttcaaatgσ tgataggggt atgttggaat ccgaagccac 2580 ttccccgcct tcaagcccca gatgggctgc tctcctgtaa ctttctagga gaagagacat 2640 tttcttcttt σcctttσctg gtccatcσct gcaσσctggt cctctcσcag cctctσσσσσ 2700 acattgtccc tgactctagg ggcacatcca gtctccatcg tgctgcagca gctggactga 2760 gggcagagcσ tgtaggtgca gaggccσtgg σtσccgaggt cσagσcaσtσ tσσσtggggc 2820 ctctggggtg agagcagctt ccgataggaσ ctgcccagat ttctgcatgt gcacttttgt 2880 ttactgaaag agagaaaggg gggggtcaca gcaacatgcc ctggcctttc tgctctgttc 2940 ccσaaccσca ctgaggcctg ctgcacaggt caatgccttc gttatcgtta ttgtactgtc 3000 actttgttct tgaggtagta gtcaaggatc aggaggggca gatgtσttct ctgggctgcg 3060 tggggccgga gcagaggtga gcagcaatgc actggttcgg gagcccccat cagcctcctt 3120 gtgσaaaσtg ggσcσσσatg σcacagtctg gσtttccctc catctgcccσ aggaσaagag 3180 caagaaggac atcagttgcc cagtcatgtg atcccctgcc atcttgcctt aggaacagcc 3240 ttcσcccacσ agcagccatg gctggctggg gctttagcca agccacctac tgcσaggaat 3300 tggagcctca gttccctcct gtgtcaagta gctaactgca gcagctggac tgagggcaga 3360 gtctgtgggt gcagagacσc tgcatgtagg tcaσaggttg aggcccagcc actctccσtg 3420 gggcctggtg ggtaggcaag tagctctggg gccacctcaa gtgaccaaat gctattaatt 3480 tcσatccttt agcaggσtgg gccctaggσa ggaagctggc ttσtgggaga ggagtgagaa 3540 cgtgcagggc ctgcctagct tgcgtgcttg aggaaggtgg cattccgtgc ttgcctcctt 3600 gaggagggtg gcattσtgtg tσttctgctt atgaagcgcσ tttcttaaag tttggcaata 3660 aatccatttt tatggaaaaa aaaaa 3685

<210> 39

<211> 3143

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No : 303698SCB1

<400> 39 ttcggctcga ggtaagactg cgacggtacc ggggcggcgg ggaaggaccg agaggcggga 60 ggagcagσgg ctcaggcgcc tgcaaaσtgg tggσσtgaac gaggtagaσσ atgaσtgtgg 120 tttcagtggc gtcactcgct gggσtgctct tcctgaggtt ttσctaagcc atcccctggc 180 ggaaccgccσ σcagtttttg tccagaagtg cttatagaaa agatggσtaa tattaaσσta 240 aaagaaataa ccttaatagt aggtgtggtt actgcctgct attggaacag cctcttttgt 300 ggttttgttt ttgatgatgt ttcagcaata ctggataaca aagacttgσa tccatctaca 360 σctttaaaaa ctttatttca aaatgacttc tggggaaccc ctatgtctga ggagagaagc 420 cacaagtctt accgtccctt aaσagtattg acatttσgσt taaattattt gttaagtgaa 480 σtaaaacσaa tgtcatatca tctcctgaafc atgattttfcc atgctgtggt tagtgtgata 540 tttctcaaag tatgcaaact ttttctggac aacaagagta gtgtgattgc ttctttactt 600 tttgcagtgc acccaataca cacagaagca gtaacaggag ttgttggaag agcagaactt 660 ttgtcatcta tcttttttct agσagctttt ttgtcatata ccagatcaaa aggaccagac 720 aattccataa tatggactcσ aattgccttg acagtgtttt tagtggctgt tgcaacatta 780 tgtaaagaac aaggaataac agttgtagga atttgσtgtg tgtatgaagt gtttattgcc 840 caggggtata ctttgccatt actatgtact actgctggaσ agtttctccg tggaaagggt 900 agcattccat tttctatgct gσagaσaσta gtaaaaσtca ttgtcttgat gttcagtaca 960 ttattacttg ttgtgattag agtccaggtt attcaatccc aacttσcagt attσaccagg 1020 tttgataacc cagctgσtgt aagσccaaσt cctacaaggc aactaacttt taactacσtσ 1080 σttcctgtga atgcttggtt gttattaaat ccttcagagc tctgctgtga ttggaccatg 1140 ggaaσaataσ σaσttataga gtcattacta gatattcgaa atctggccac atttactttc 1200 ttttgttttc tggggatgtt gggagtattc agtatcagat actctggtga ttcctσcaag 1260 actgttttaa tggcgctttg tttaatggca ttaccattta ttcctgcatc gaaccttttt 1320 tttcσagtfcg gatttgttgt tgcσgagcga gtattatatg ttcccagcat ggggttctgt 1380 attttggtag cccatggatg gcagaaaata tcaacaaaaa gtgtatttaa aaagctatcc 1440 tggatttgtc tgtctatggt gatactcact cattccttaa aaacattcca cagaaattgg 1500 gattgggagt ctgaatatac attgtttatg tcagccttga aggtaaataa aaataafcgcσ 1560 aaactttgga ataatgtggg tcatgctctg gaaaatgaaa agaactttga gagagctttg 1620 aaatacttct taσaggσtaσ σcatgttcag cσagatgata ttggtgccca tatgaatgta 1680 ggaagaactt ataaaaattt aaatagaacc aaagaagctg aagaatctta catgatggct 1740 aaatcaσtga tgcσtcaaat tattcctggt aaaaaatatg cagccagaat tgcccctaac 1800 cacctaaatg tttatatcaa tctggctaac ctgatccgag caaatgagtc ccgactggaa 1860 gaagcagatc agctgtaccg tcaagcaata agcatgaggc ccgacttcaa gσaggcttac 19-20 attagcagag gagaattgct tttaaaaatg aataaacctc ttaaagcaaa ggaagcatat 1980 cttaaagcac tagagctgga σagaaataat gcagatcttt ggtacaactt ggcaattgta 2040 catattgaac ttaaagaacc aaatgaagcc ctaaaaaact ttaatcgtgc tctggaacta 2100 aatσcaaagc ataaactagc attattcaac tctgctatag taatgσaaga atcaggtgag 2160 gttaaactca gacctgaagc tagaaaacga cttσtaagtt atataaatga agagccacta 2220 gatgctaatg ggtatttcaa tttgggaatg cttgσσatgg atgacaaaaa ggaσaatgaa 2280 gσagagattt ggatgaagaa agccataaag ttacaagccg acttccgaag tgctttgttt 2340 aatσtggσtσ tcctgtattc σσagactgca aaggaattaa aggσtttgcc aattttggag 2400 gagttactca gatactaccc tgatcatatc aagggcctca ttttaaaagg agacattctg 2460 atgaatcaaa agaaagatat actaggagca aaaaaatgtt ttgaaaggat tttggagatg 2520 gatccaagca atgtgcaagg aaaacacaat ctttgtgttg tttattttga agaaaaagac 2580 ttattaaaag ctgaaagatg cσttσttgaa acaσtggcat tagσaccaσa tgaagaatat 2640 attcagcgcc atttgaatat agtcagggat aagatttcct catctagttt tatagagcca 2700 atattcσσaa σσagtaagat ttcaagtgtg gaaggaaaga aaattccaac tgaaagtgta 2760 aaagaaatta gaggtgaatc cagacaaaca caaatagtaa aaacaagtga taataaaagt 2820 cagtctaaat cσaaσaaaca attaggaaaa aatggagacg aagagacacc cσaσaaaaσa 2880 acaaaagaca tcaaagaaat tgagaagaaa agagttgctg ctttaaaaag actagaagag 2940 attgaacgta ttttaaatgg tgaataacat taatatttat cgtgaσaatg gtatcaaaga 3000 acatcaatcc gtatcatgtg attgctttta ctgggagctt tgaaaaaaag ttcaagggtt 3060 cctaatggtc aatcatgagσ tgccttgaag taggatcaaa ataagatttt cattaaagac 3120 ctgtattatc ccaaaaaaaa aaa 3143

<210> 40

<211> 1759

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<223> Incyte ID No: 2041080CB1

<400> 40 ggggctctgc gcgagcgagg gacgacggaa gggacgggca ggtgtgggcg cggggccacg 60 cagccσgaσg gcgggagtcg caggtgctgg gtgcatgggσ σagtgaggac gcacagagat 120 ccctcgccgc gcggaggagg agcagcgcgg gagccaggcg ctgccccaag accctgcctg 180 cgtσσgagσg agσggaaσσt σgcgσttcgc ccggggacaa tσσgaagtσσ gσgctatgga 240 agaggagaaa tatttgcctg agctgatggc agagaaagat agcctggatc catcttttgt 300 gcatgcgtcg cgccttttgg cagaagaaat tgaaaagttt σaaggttσtg atggaaaaaa 360 ggaagacgaa gaaaagaagt atcttgatgt catcagcaac aaaaacataa agctctcaga 420 aagagtactg attσσtgtca agcagtatcc aaagttcaat tttgtgggga aattgcttgg 480 accaagagga aactccttga agaggctaca ggaagaaaca ggtgctaaaa tgtctatcct 540 gggcaaagga tcaatgagag ataaagctaa ggaagaagaa σtaaggaaga gtggggaagσ 600 caaatatgcc cacttgagtg atgagcttca tgtattaatt gaagtgtttg ctccacctgg 660 ggaagcttat- tσaσgtatga gtcatgσatt ggaagagatt aaaaaattcc tggttcctga 720 ctacaatgat gaaattcgtc aggaacaact acgtgaatta tcttacttaa atggctcaga 780 ggaσtctggt σgtggcagag gtattagagg cagagggatσ agaatagσtσ σcacagσtcc 840 ttcaaggggc cgtgggggtg ccattcctcc tcccccacca cctggacgag gtgttctcac 900 cσσtσgggga agσaσtgtaa cccgtggagc gcttσσagtg ccacctgtag caagaggtgt 960 ccctacccct cgagcccggg gggcaccaac agtgccagga tacagggcac ctcctcctcc 1020 agcccatgaa gcttatgaag aatatggtta tgatgatggσ tacgggggtg aatatgatga 1080 ccagacctat gagacttatg ataacagcta tgcgacccaa acacaaagtg tgcctgaata 1140 ctatgactaσ ggtcatggag taagtgagga tgcctatgac agσtacgcac cagaagaatg 1200 ggccaσaacc cgctctagct tgaaggcacc accgcagagg tcagccagag ggggatacag 1260 ggaacaσccc tatggtagat attgaaggtc σttσccaσct gtgacctσaσ σtcaaagaσa 1320 attcatagcc tgtggtctcc acataaacag caacaagaca agtacccaca aacaaagaac 1380 acataattga ataaccgatg tgatttgcta agaaggatgg aggtgaagat gtatgataga 1440 aaaacgaaaa actggaataa acccaagact ggctgtttgg aggtttaatg tttttaacaa 1500 tggaggccaa aaaagaagat aatttggatt tgaaccaata ttttcttσat σtcaactσaσ 1560 ctcaccacta cagatccaag cctgacacag agcctaacag aaatactttc ctccaaatac 1620 aatσctgttc accatggaag acattaaaaσ cttcagtggt tgctgσtggg ggcσttσacc 1680 aaaatgcagc cattcacctt gctgctgggg gccttcacca aaatgcagcc atfccaccttc 1740 tttgcttttg ctctaccta 1759