CA2379801A1 - Receptors and associated proteins - Google Patents

Abstract

The invention provides human receptors and associated proteins (RECAP) and polynucleotides which identify and encode RECAP. 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 expression of RECAP.

Description

RECEPTORS AND ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of receptors and associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of neurological disorders; immunological disorders, including autoimmune/inflammatory disorders; and cell proliferative disorders, including cancer.
BACKGROUND OF THE INVENTION
The term receptor describes a protein that specifically recognizes other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell. Propagation of cellular signals, and transport and localization of proteins, all rely upon specific interactions between receptors and a variety of associated proteins. The term receptor may also be applied to proteins which bind to ligands with known or unknown chemical composition and which interact with other cellular components.
For example, the steroid hormone receptors bind to and regulate transcription of DNA.
Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines; peptide hormones; growth and differentiation factors; small peptide factors such as thyrotropin-releasing hormone; galanin, somatostatin, and tachykinins; and circulatory system-borne signaling molecules. Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell. This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transferrin, glucose- or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (Lodish, H. et al.
(1995) Molecular Cell Biolo~y, Scientific American Books, New York NY, p. 723; and Mikhailenko, I.
et al. (1997) J. Biol.
Chem. 272:6784-6791 ).
Signal transduction is the process of biochemical events by which cells are able to communicate with one another and respond to extracellular signals.
Extracellular signals are transduced through a biochemical cascade that begins with the binding of a signal molecule to a cell membrane receptor. The signal is propagated to effector molecules by intracellular signal transducing proteins and culminates with the activation of an intracellular target molecule. The process of signal transduction regulates a wide variety of cell functions including cell proliferation, differentiation, and SUBSTITUTE SHEET (RULE 26) gene transcription.
G~rotein Coupled Receptors (GPCRs) G-protein coupled receptors (GPCRs) are a class of molecules that participate in signal transduction in a variety of cell types. GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which span the plasma membrane and form a bundle of antiparallel alpha (a) helices. These proteins range in size from under 400 to over 1000 amino acids (Strosberg, A.D. (1991) Eur. J. Biochem. 196:1-10; Coughlin, S.R. (1994) Curr. Opin. Cell Biol.
6:191-197). The amino-terminus of the GPCR is extracellular, of variable length and often glycosylated;
the carboxy-terminus is cytoplasmic and generally phosphorylated.
Extracellular loops of the GPCR
alternate with intracellular loops and link the transmembrane domains. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account for structural and functional features of the receptor. In most cases, the bundle of a helices forms a binding pocket. In addition, the extracellular N-terminal segment or one or more of the three extracellular loops may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.
The activated receptor, in turn, interacts with an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or interactions with ion channel proteins.
(Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190; Watson, S. and S.
Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6. ) Hydrolysis of bound GTP by the G-protein completes the cycle, returning the G-protein to its inactive GDP-bound state.
GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, bombesin, bradykinin, endothelin, y-aminobutyric acid (GABA), hepatocyte growth factor, luteinizing hormone (LH), thrombin, thyroid stimulating hormone (TSH), melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, CSa anaphylatoxin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors. For example, the TPRA40 protein is a GPCR isolated liom mouse adipocytes and present in a number of mouse and human tissues, whose expression in adipose tissue is altered with aging and type 2 diabetes (Yang, H. (1999) Endocrinology 140:2859-2867).
SUBSTITUTE SHEET (RULE 26) GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Rhodopsin is the retinal photoreceptor which is located within the discs of the eye rod cell. Parma, J. et al. (1993, Nature 365:649-651) report that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggest that certain GPCRs susceptible to constitutive activation may behave as protooncogenes. Elevated levels of TSH receptor have been observed in brain tissue from Down syndrome and Alzheimer's disease patients, suggesting an apoptotic role for this receptor in neurodegenerative disorders (Labudova, O. et al. (1999) Life Sci. 64:1037-1044). Many clinically relevant drugs act on GPCRs, including a and ~3 Mockers which affect the activity of adrenergic receptors and are used in the treatment of hypertension and other cardiovascular disorders (Watson, supra, pp. 32-33).
Receptors Involved in the Immune System Examples of GPCRs implicated in inflammation and the immune response include the EGF
module-containing, mucin-like hormone receptor (Emrl) and CD97 receptor proteins. These GPCRs are members of the recently characterized EGF-TM7 receptors family. These seven transmembrane hormone receptors exist as heterodimers in vivo and contain between three and seven potential calcium-binding EGF-like motifs. CD97 is predominantly expressed in leukocytes, and is markedly upregulated on activated B and T cells. (McKnight, A. J. and Gordon, S. (1998) J. Leukoc. Biol.
63:271-280.) Irregularities in the GPCR signaling cascade may result in abnormal activation of leukocytes and lymphocytes, leading to the tissue damage and destruction seen in many inflammatory and autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis. Abnormal cell proliferation, including cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins (Meij, J.T.A. (1996) Mol. Cell. Biochem. 157:31-38; Aussel, C. et al. (1988) J. Immunol. 140:215-220).
T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate other immune cells. Although T cells collectively recognize a wide range of different antigens, a clonal line of T cells can only recognize a single antigen. Moreover, the antigen must be presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen-presenting cell. The TCR on most T cells consists of two polypeptide subunits, a and (3, which are immunoglobulin-like integral membrane glycoproteins of similar molecular weight. The TCRa and TCR(3 subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et SUBSTITUTE SHEET (RULE 26) al. (1984) Nature 309:757-762). The genes for the TCR subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A. (1991) Annu. Rev. Genet. 25: 487-510). Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg, A.C. et al. (1985) N.
Engl. J. Med. 313:529-533;
Olive, C. (1995) Immunol. Cell. Biol. 73:297-307; and Weiss, su ra).
Immunizations with peptides derived liom TCRs are effective treatment for some human T-cell-mediated autoimmune disease and in animal models of such illnesses, in particular, rheumatoid arthritis (Bridges, S.L. and Moreland, L.W.
(1998) Rheum. Dis. Clin. North Am. 24:641-650).
Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates immune regulation and inflammatory responses. The cellular responses triggered by TNF are initiated through its interaction with two distinct cell surface receptors, TNF-R1 and TNF-R2. (Tartaglia, L.A.
and Goeddel, D.V.
(1992) Immunol. Today 13:151-153). Both TNF receptors are part of the TNF
receptor (TNFR) superfamily, whose members include the Fas antigen, the p75 subunit of the NGF
receptor, the TRAIL
receptor, TRUNND, SalFl9R, CD27, CD30, and CD40. Members of the TNFR
superfamily share the TNFR/NGFR family cysteine-rich region signature, which consists of cysteine-rich pseudo-repeats in the extracellular domains. (ExPASy PROSITE document PDOC00561; Pan, G. et al.
(1998) FEBS Lett.
424:41-45; Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221; and Smith, C.A. et al. (1994) Cell 76:959-962). Polymorphisms in TNF-R2 are associated with systemic lupus erythematosus (Komata, T.
et al. (1999) Tissue Antigens 53:527-533). In addition, increased serum concentrations of soluble TNF-R1 have been observed in some patients with advanced gastric or colorectal cancer (Shibata, M. et al.
(1998) Surg. Today 28:884-888).
Another essential component of the immune response is the complement system, which responds to signals provided by antigen recognition by mobilizing effector activities including inflammation, phagocytosis, and cell lysis. Receptors on macrophages and neutrophils bind activated complement C3 on the surface of foreign particles such as bacteria, thus targeting the foreign particles for phagocytosis and destruction by lysosomal enzymes. Complement receptor 1 (CR1) has a wide cellular/tissue distribution, and mediates enhancement of phagocytosis, induction of IL-1 secretion and enhancement of B-cell differentiation. Defective expression of CR1 is associated with the autoimmune disease systemic lupus erythematosis. (Carroll, M.C. (1998) Annu. Rev. Immunol. 16:545-568.) Nuclear Receptors The nuclear receptors are another receptor family, and includes the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). RARs and RXRs can form heterodimers which are SUBSTITUTE SHEET (RULE 26) thought to have a signal transduction function. Retinoic acid (RA) is a biologically active metabolite of vitamin A (retinol), a fat-soluble vitamin found mainly in fish liver oils, liver, egg yolk, butter, and cream. Retinol cannot be synthesized in vivo and must be obtained from the diet. Retinol, RA, and other retinoids influence epithelial cell differentiation. A number of carrier proteins which bind retinol or other retinoids have been identified. These retinoid binding proteins (RBPs) appear to direct bound retinoid molecules to specific metabolic pathways. Specific receptors for RBPs mediate the cellular uptake of retinoids and the transfer of retinoids to intracellular RBPs (Sundaram, M. et al. (1999) J. Biol. Chem.
273:3336-3342).
Low Molecular Weight (LMW) G-proteins Low molecular weight (LMW) G-proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which are able to bind to and hydrolyze GTP, thus cycling between an inactive and an active state.
LMW G-proteins respond to extracellular signals from receptors and activating proteins by transducing mitogenic signals involved in various cell functions. The binding and hydrolysis of GTP regulates the response of LMW G-proteins and acts as an energy source during this process (Bokoch, G. M. and Der, C. J.
(1993) FASEB J.
7:750-759).
At least sixty members of the LMW G-protein superfamily have been identified and are currently grouped into the ras, rho, arf, sari, ran, and rab subfamilies. Activated ras genes were initially found in human cancers and subsequent studies confirmed that ras function is critical to receptor tyrosine kinase-mediated signal transduction pathways that determine whether cells continue to grow and divide, or whether they differentiate. Rho G-proteins control signal transduction pathways that link growth factor receptors to actin polymerization, which is necessary for normal cellular growth and division. The rab, arf, and sari families of proteins control the translocation of vesicles to and from membranes for protein localization, protein processing, and secretion. Ran G-proteins are located in the nucleus and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990) Science 249:635-640; Barbacid, M. (1987) Ann. Rev Biochem. 56:779-827; and Sasaki, T. and Takai, Y. (1998) Biochem. Biophys. Res. Commun. 245:641-645).
LMW G-proteins are GTPases which cycle between the active GTP-bound and inactive GDP-bound forms. At least three types of proteins regulate this process:
GTPase-activating proteins, (GAP), which stimulate GTP hydrolysis by the LMW G-protein; guanine nucleotide exchange factors (GEP), which facilitate the exchange of GDP bound to the LMW G-protein for GTP; and guanine nucleotide dissociation inhibitors (GDI), which inhibit this reaction (Ikeda, M. et al. (1998) J. Biol.
Chem. 273:814-821; Quilliam, L. A. (1995) Bioessays 17:395-404). The best characterized GEP is the mammalian homologue of the Drosophila Son-of-Sevenless protein. Both GEP and GAP activity may be SUBSTITUTE SHEET (RULE 26) affected by extracellular stimuli and modified by accessory proteins such as RalBP1 and POB1. Mutant Ras-family proteins, which bind but can not hydrolyze GTP, are permanently activated, and cause cell proliferation or cancer, as do GEP that. activate LMW G-proteins (Drivas, G.
T. et al. (1990) Mol. Cell.
Biol. 10:1793-1798; and Whitehead, I. P. et al. (1998) Mol Cell Biol. 18:4689-4697).
Olfactory GPCRs Another large subfamily of GPCRs are the olfactory receptors. These receptors share the seven hydrophobic transmembrane domains of other GPCRs and function by registering G
protein-mediated transduction of odorant signals. Numerous distinct olfactory receptors are required to distinguish different odors. Each olfactory sensory neuron expresses only one type of olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal pasages.
The discovery of new receptors and associated proteins 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, and neurological disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, receptors and associated proteins, referred to collectively as "RECAP" and individually as "RECAP-1," "RECAP-2," "RECAP-3,"
"RECAP-4,"
"RECAP-5," "RECAP-6," "RECAP-7," "RECAP-8," "RECAP-9," "RECAP-10," "RECAP-11,"
"RECAP-12," "RECAP-13," "RECAP-14," "RECAP-15," "RECAP-16," "RECAP-17," "RECAP-18,"
"RECAP-19," "RECAP-20," "RECAP-21," and "RECAP-22," In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-22. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO:1-22.
The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected tiom the group consisting of SEQ ID N0:1-22, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:1-22, and d) an immunogenic tiagment of an amino acid sequence selected tiom the group consisting of SEQ ID NO:1-22. In one alternative, the polynucleotide encodes a polypeptide selected from the SUBSTITUTE SHEET (RULE 26) group consisting of SEQ ID NO:1-22. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:23-44.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:I-22. 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:l-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ 1D NO:1-22. 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 comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID N0:1-22, and d) an immunogenic iiagment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22.
The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected Iiom the group consisting of SEQ ID N0:23-44, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous SUBSTITUTE SHEET (RULE 26) nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to 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 polynucleodde in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, b) a naturally occurring polynucleodde sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerise 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 pharmaceutical composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and a pharmaceutically acceptable excipient In one embodiment, the pharmaceutical composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-22. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional RECAP, comprising administering to a patient in need of such treatment the pharmaceutical composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino SUBSTITUTE SHEET (RULE 26) acid sequence selected from the group consisting of SEQ ID N0:1-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22. 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 pharmaceutical 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 RECAP, comprising administering to a patient in need of such treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected fiom the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22. 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 pharmaceutical 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 RECAP, comprising administering to a patient in need of such treatment the pharmaceutical composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, and d) an inununogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-22. 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 SUBSTITUTE SHEET (RULE 26) activity of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally occurnng amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22. 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 sequence selected from the group consisting of SEQ ID N0:23-44, 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 comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, ii) a naturally occurring polynucleotide sequence having at least 70%
sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to 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 comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:23-44, ii) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA
equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of the above polynucleotide sequence; 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 SUBSTITUTE SHEET (RULE 26) complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding RECAP.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of RECAP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding RECAP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the polynucleoddes 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.

SUBSTITUTE SHEET (RULE 26) DEFINITIONS
"RECAP" refers to the amino acid sequences of substantially purified RECAP
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 RECAP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of RECAP either by directly interacting with RECAP or by acting on components of the biological pathway in which RECAP
participates.
An "allelic variant" is an alternative form of the gene encoding RECAP.
Allelic variants may result liom 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 RECAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as RECAP or a polypeptide with at least one functional characteristic of RECAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding RECAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding RECAP. 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 RECAP. 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 RECAP 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 "anuno 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 SUBSTITUTE SHEET (RULE 26) 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 RECAP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of RECAP either by directly interacting with RECAP or by acting on components of the biological pathway in which RECAP
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 RECAP 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 "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 SUBSTITUTE SHEET (RULE 26) functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic RECAP, 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 polynucleodde 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 RECAP or fragments of RECAP 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., NaCI), 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 (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping eDNA, 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 ~a Gly, Ser ~'g His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val SUBSTITUTE SHEET (RULE 26) Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, 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 sequence 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.
A "fragment" is a unique portion of RECAP or the polynucleotide encoding RECAP
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 1D N0:23-44 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:23-44, for example, as distinct from any other sequence in the SUBSTITUTE SHEET (RULE 26) genome from which the fragment was obtained. A fragment of SEQ ID N0:23-44 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:23-44 from related polynucleotide sequences. The precise length of a fragment of SEQ ID
N0:23-44 and the region of SEQ ID N0:23-44 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 ID NO:1-22 is encoded by a fragment of SEQ 1D N0:23-44. A
fragment of SEQ ID NO:1-22 comprises a region of unique amino acid sequence that specifically identifies SEQ
ID NO:1-22. For example, a fragment of SEQ ID NO:1-22 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-22. The precise length of a fragment of SEQ ID NO:1-22 and the region of SEQ ID NO:1-22 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 polynucleodde 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 NCB1, Bethesda, MD, and on the Internet at SUBSTITUTE SHEET (RULE 26) http://www.ncbi.nlm.nih.govBLAST/. 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/gort%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 2Ø12 (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: S and Extension Gap: 2 penalties Gap x drop-off. 50 Expect: l0 Word Size: 11 Filter: nn Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a liagment 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 polypepdde sequences may be determined using the default parameters SUBSTITUTE SHEET (RULE 26) 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=1, 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 2Ø12 (Apr-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 ID 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 microchromosomes 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 alter the "washing" step(s). The washing steps) 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 SUBSTITUTE SHEET (RULE 26) 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 lrg/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 thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The T", 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 Tm 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"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleot:ides 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 Ng/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., Cot or Rot 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, Iilters, 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 SUBSTITUTE SHEET (RULE 26) systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of RECAP
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 RECAP
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 RECAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of RECAP.
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 andsense 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 RECAP 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 RECAP.
"Probe" refers to nucleic acid sequences encoding RECAP, 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 SUBSTITUTE SHEET (RULE 26) 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 polymerise 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"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current Protocols in Molecular Biolo~y, 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 InstitutelMIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays.
(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 oligonucleoddes 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 SUBSTITUTE SHEET (RULE 26) 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 artitlcial 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 nucleic acids encoding RECAP, or fragments thereof, or RECAP itself, 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 SUBSTITUTE SHEET (RULE 26) 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 liom 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), su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at SUBSTITUTE SHEET (RULE 26) 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 2Ø9 (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 95% or at least 98% 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 polynucleoddes due to alternative 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 generally will 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 2Ø9 (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 95%, or at least 98%
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 receptors and associated proteins (RECAP), the polynucleotides encoding RECAP, and the use of these compositions for the diagnosis, treatment, or prevention of neurological disorders; immunological disorders, including autoimmune/inflammatory disorders; and cell proliferative disorders, including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding RECAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each RECAP were identified, and column 4 shows the cDNA
libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries.
Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. In some cases, GenBank sequence identifiers are also shown in column 5. The Incyte clones and GenBank cDNA
sequences, where indicated, in column 5 were used to assemble the consensus nucleotide sequence of each RECAP and are useful as fragments in hybridization technologies.

SUBSTITUTE SHEET (RULE 26) The columns of Table 2 show various properties of each of the polypeptides of the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis along with relevant citations, all of which are expressly incorporated by reference herein in their entirety; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding RECAP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID N0:23-44 and to distinguish between SEQ ID N0:23-44 and related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue categories which express RECAP as a liaction of total tissues expressing RECAP. Column 4 lists diseases, disorders, or conditions associated with those tissues expressing RECAP as a fraction of total tissues expressing RECAP. Column 5 lists the vectors used to subclone each cDNA library. Of particular interest is the expression of SEQ ID N0:11 in hematopoietic/immune tissues and the expression of SEQ ID N0:14 in reproductive tissues.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding RECAP were isolated. Column 1 references the nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
The invention also encompasses RECAP variants. A preferred RECAP 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 RECAP amino acid sequence, and which contains at least one functional or structural characteristic of RECAP.
The invention also encompasses polynucleotides which encode RECAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected liom the group consisting of SEQ ID N0:23-44, which encodes RECAP. The polynucleotide sequences of SEQ ID N0:23-44, 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 RECAP. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least SUBSTITUTE SHEET (RULE 26) about 85 %, or even at least about 95 % polynucleotide sequence identity to the polynucleotide sequence encoding RECAP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:23-44 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
ID N0:23-44. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of RECAP.
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 RECAP, 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 RECAP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode RECAP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring RECAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding RECAP or its derivatives possessing a substantially different colon usage, e.g., inclusion of non-naturally occurring colons. Colons 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 colons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding RECAP 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 RECAP
and RECAP 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 RECAP 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 ID N0:23-44 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 SUBSTITUTE SHEET (RULE 26) embodiments of the invention. The methods may employ such enzymes as the Klenow fiagment of DNA
polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (PE
Biosystems, Foster City CA), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerises 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 (PE
Biosystems).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (PE
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 Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biolo~y and Biotechnolo~y, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding RECAP 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 SUBSTITUTE SHEET (RULE 26) 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, PE 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 RECAP may be cloned in recombinant DNA molecules that direct expression of RECAP, 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 RECAP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter RECAP-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 Number 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 RECAP, 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 tiagments. 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 SUBSTITUTE SHEET (RULE 26) 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 RECAP 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, RECAP 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
431A peptide synthesizer (PE Biosystems). Additionally, the amino acid sequence of RECAP, 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 RECAP, the nucleotide sequences encoding RECAP 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 RECAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequenceis~ encoding RECAP.
Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding RECAP 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.) SUBSTITUTE SHEET (RULE 26) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding RECAP 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, eh. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, eh. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding RECAP. 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; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; 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; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.
et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105;
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, LM. 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 RECAP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding RECAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORTl plasmid (Life Technologies). Ligation of sequences encoding RECAP into the vector's.multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identif canon 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 SUBSTITUTE SHEET (RULE 26) 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 RECAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of RECAP may be used. For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of RECAP. 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, su ra; Bitter, su ra; and Scorer, su ra.) Plant systems may also be used for expression of RECAP. Transcription of sequences encoding RECAP may be driven 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, su ra; Brogue, su ra; and Winter, su ra.) 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 Technolo~y (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 RECAP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses RECAP 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 RECAP in cell lines is preferred. For example, sequences encoding RECAP 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 SUBSTITUTE SHEET (RULE 26) 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 tk- 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),13 glucuronidase and its substrate I3-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, C.A. (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 RECAP is inserted within a marker gene sequence, transformed cells containing sequences encoding RECAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding RECAP 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 RECAP
and that express RECAP 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.
Inununological methods for detecting and measuring the expression of RECAP
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 RECAP is preferred, but a competitive binding assay may be SUBSTITUTE SHEET (RULE 26) employed. These and other assays are well known in the art. (See, e.g., Hampton, R, et al. (1990) Serological Methods, a Laborator~Manual, APS Press, St. Paul MN, Sect. IV;
Coligan, J.E. et al.
(1997) Current Protocols in lmmunolo~y, 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 RECAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding RECAP, 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 RECAP 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 RECAP may be designed to contain signal sequences which direct secretion of RECAP 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 RECAP 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 RECAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the SUBSTITUTE SHEET (RULE 26) screening of peptide libraries for inhibitors of RECAP 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-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-rrayc, 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-rrvyc, 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 RECAP encoding sequence and the heterologous protein sequence, so that RECAP
may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, eh. 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 RECAP 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.
RECAP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to RECAP. At least one and up to a plurality of test compounds may be screened for specific binding to RECAP. 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 RECAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, Coligan, J.E. et al. (1991) Current Protocols in lmmunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which RECAP
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 RECAP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing RECAP or cell membrane fractions which contain RECAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either RECAP
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 SUBSTITUTE SHEET (RULE 26) assay may comprise the steps of combining at least one test compound with RECAP, either in solution or affixed to a solid support, and detecting the binding of RECAP 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 compounds) may be free in solution or affixed to a solid support.
RECAP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of RECAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for RECAP
activity, wherein RECAP is combined with at least one test compound, and the activity of RECAP in the presence of a test compound is compared with the activity of RECAP in the absence of the test compound. A change in the activity of RECAP in the presence of the test compound is indicative of a compound that modulates the activity of RECAP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising RECAP under conditions suitable for RECAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of RECAP
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 RECAP 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 RECAP 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.
SUBSTITUTE SHEET (RULE 26) (1998) Science 282:1145-1147).
Polynucleotides encoding RECAP 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 RECAP 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 RECAP, e.g., by secreting RECAP 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 RECAP and receptors and associated proteins. 1n addition, the expression of RECAP is closely associated with cell proliferation, cancer, inflammation/trauma, and with neurological disorders.
Therefore, RECAP appears to play a role in neurological disorders;
immunological disorders, including autoimmune/inflammatory disorders; and cell proliferative disorders, including cancer. In the treatment of disorders associated with increased RECAP expression or activity, it is desirable to decrease the expression or activity of RECAP. In the treatment of disorders associated with decreased RECAP
expression or activity, it is desirable to increase the expression or activity of RECAP.
Therefore, in one embodiment, RECAP or a liagment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of RECAP. Examples of such disorders include, but are not limited to, 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, Down's syndrome, 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, 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;

SUBSTITUTE SHEET (RULE 26) 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; an immunological disorder, including autoimmune/inflammatory disorders, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Breton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA
deficiency, severe combined immunodeficiency disease (SCID), immunodellciency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, and immunodeficiency associated with Cushing's disease, 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, hematopoietic cancers, including lymphoma, leukemia, and myeloma, and trauma; and 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.
In another embodiment, a vector capable of expressing RECAP 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 RECAP including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified RECAP 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 RECAP
including, but not limited SUBSTITUTE SHEET (RULE 26) to, those provided above.
In still another embodiment, an agonist which modulates the activity of RECAP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of RECAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of RECAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of RECAP.
Examples of such disorders include, but are not limited to, those neurological disorders;
immunological disorders, including autoimmune~inflammatory disorders; and cell proliferative disorders, including cancer, described above.
In one aspect, an antibody which specifically binds RECAP 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 RECAP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding RECAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of RECAP 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 RECAP may be produced using methods which are generally known in the art.
In particular, purified RECAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind RECAP. Antibodies to RECAP
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 RECAP 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.

SUBSTITUTE SHEET (RULE 26) It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to RECAP
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 RECAP 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 RECAP 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 RECAP-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 RECAP 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 RECAP and its specific SUBSTITUTE SHEET (RULE 26) antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering RECAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for RECAP. Affinity is expressed as an association constant, ICT, which is defined as the molar concentration of RECAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple RECAP epitopes, represents the average affinity, or avidity, of the antibodies for RECAP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular RECAP
epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the RECAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 10~ to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of RECAP, 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 ant:ibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of RECAP-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., su ra.) In another embodiment of the invention, the polynucleotides encoding RECAP, 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 modifed oligonucleotides) to the coding or regulatory regions of the gene encoding RECAP.
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 RECAP.
(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., SUBSTITUTE SHEET (RULE 26) Slater, J.E. et al. (1998) J. Allergy Clin. lmmunol. 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, s-u~ra; 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(1):217-225; Boado, R.J. et al.
(1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding RECAP 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 (SCID)-X1 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 VIII or Factor IX
deficiencies (Crystal, R.G.
(1995) Science 270:404-410; Verma, LM. and Somia, N. (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 falcinarum and Trypanosoma cruzi). In the case where a genetic deficiency in RECAP expression or regulation causes disease, the expression of RECAP 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 RECAP
are treated by constructing mammalian expression vectors encoding RECAP and introducing these vectors by mechanical means into RECAP-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;
Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).

SUBSTITUTE SHEET (RULE 26) Expression vectors that may be effective for the expression of RECAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX 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). RECAP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (i-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 H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding RECAP
from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION K1T, 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 RECAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding RECAP 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 ci.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 Number 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, SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26) 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 RECAP to cells which have one or more genetic abnormalities with respect to the expression of RECAP. 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 Number 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, LM. 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 RECAP to target cells which have one or more genetic abnormalities with respect to the expression of RECAP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing RECAP 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 Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
U.S. Patent Number 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 RECAP to target cells. The biology of the prototypic alphavirus, SUBSTITUTE SHEET (RULE 26) 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. Biotech. 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 RECAP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of RECAP-coding RNAs and the synthesis of high levels of RECAP 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 RECAP 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 liom 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 polymerises, 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, Futura 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 RECAP.
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 SUBSTITUTE SHEET (RULE 26) 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 RECAP. 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 RECAP. 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 RECAP expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding RECAP
may be therapeutically useful, and in the treament of disorders associated with decreased RECAP
expression or activity, a compound which specifically promotes expression of the polynucleotide encoding RECAP 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 SUBSTITUTE SHEET (RULE 26) chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding RECAP 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 RECAP 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 RECAP. 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 Schizosaccharomyces 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, M.L. 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 (Bruise, T.W. et al.
(1997) U.S. Patent No. 5,686,242; Bruise, 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 pharmaceutical 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 Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such pharmaceutical compositions may consist of RECAP, antibodies to RECAP, and mimetics, agonists, antagonists, or inhibitors of RECAP.
The pharmaceutical compositions utilized in this invention may be administered by any number SUBSTITUTE SHEET (RULE 26) of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Pharmaceutical 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.
Pharmaceutical 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 pharmaceutical compositions may be prepared for direct intracellular delivery of macromolecules comprising RECAP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, RECAP 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 RECAP
or fragments thereof, antibodies of RECAP, and agonists, antagonists or inhibitors of RECAP, 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 EDSo (the dose therapeutically effective in 50% of the population) or LDso (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 LDSO/EDSO ratio. Pharmaceutical 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 SUBSTITUTE SHEET (RULE 26) of circulating concentrations that includes the EDSO 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-acdng pharmaceutical 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 ~g to 100,000 /cg, 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 RECAP may be used for the diagnosis of disorders characterized by expression of RECAP, or in assays to monitor patients being treated with RECAP or agonists, antagonists, or inhibitors of RECAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for RECAP
include methods which utilize the antibody and a label to detect RECAP 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 RECAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of RECAP expression. Normal or standard values for RECAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to RECAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of RECAP
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 RECAP 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 SUBSTITUTE SHEET (RULE 26) quantify gene expression in biopsied tissues in which expression of RECAP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of RECAP, and to monitor regulation of RECAP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding RECAP or closely related molecules may be used to identify nucleic acid sequences which encode RECAP. 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 RECAP, 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 RECAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:23-44 or from genomic sequences including promoters, enhancers, and introns of the RECAP
gene.
Means for producing specific hybridization probes for DNAs encoding RECAP
include the cloning of polynucleotide sequences encoding RECAP or RECAP 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 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding RECAP may be used for the diagnosis of disorders associated with expression of RECAP. Examples of such disorders include, but are not limited to, 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, Down's syndrome, 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, 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 polymyosilis; inherited, metabolic, endocrine, and toxic myopathies; myasthenia SUBSTITUTE SHEET (RULE 26) 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; an immunological disorder, including autoimmune/inflammatory disorders, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeliciency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA
deficiency, severe combined immunodef ciency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, and immunodeficiency associated with Cushing's disease, 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, hematopoietic cancers, including lymphoma, leukemia, and myeloma, and trauma; and 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. The polynucleotide sequences encoding RECAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues Ii~om patients to detect altered RECAP
expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding RECAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide SUBSTITUTE SHEET (RULE 26) sequences encoding RECAP 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 signiFcantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding RECAP 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.
1n order to provide a basis for the diagnosis of a disorder associated with expression of RECAP, 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 RECAP, 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 RECAP
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 RECAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding RECAP, and will be employed under optimized conditions for identification of a spec;iflc 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 SUBSTITUTE SHEET (RULE 26) encoding RECAP 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 RECAP 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 RECAP 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. lmmunol. 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 in Seilhamer, J.J. et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No. 5,840,484, incorporated herein by reference. 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 SUBSTITUTE SHEET (RULE 26) display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, antibodies specific for RECAP, or RECAP or fragments thereof 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 Number 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 SUBSTITUTE SHEET (RULE 26) 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 isoelec;tric 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 RECAP
to quantify the levels of RECAP 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-SUBSTITUTE SHEET (RULE 26) 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;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-2155; and Heller, M.J. 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 RECAP
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 SUBSTITUTE SHEET (RULE 26) chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (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, e.g., 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, su ra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding RECAP 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 l 1q22-23, 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, RECAP, 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 RECAP 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 W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with RECAP, or fragments thereof, and washed. Bound RECAP is then defected by methods well known in the art.
Purified RECAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, SUBSTITUTE SHEET (RULE 26) 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 RECAP specifically compete with a test compound for binding RECAP.
In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with RECAP.
In additional embodiments, the nucleotide sequences which encode RECAP 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 preferred specific 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, in particular U.S. Ser. No. 60/145,232, U.S. Ser. No. 60/158,578, and U.S. Ser.
No. 60/165,192, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
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 CsCI
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, s-upra, units SUBSTITUTE SHEET (RULE 26) S.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 S 1000, 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), or pINCY plasmid (Incyte Genomics, Palo Alto CA). Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered liom 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 (PE 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 (PE 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 sequencing system (PE Biosystems) in conjunction with standard ABI protocols and base calling SUBSTITUTE SHEET (RULE 26) 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 V.
The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art.
Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 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, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments were generated using the 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.
The polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences 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 PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and 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 amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, 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, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID N0:23-44. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression SUBSTITUTE SHEET (RULE 26) 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, su ra, 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.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding RECAP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
V. Chromosomal Mapping of RECAP Encoding Polynucleotides The cDNA sequences which were used to assemble SEQ ID N0:23-44 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other SUBSTITUTE SHEET (RULE 26) implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ
ID N0:23-44 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (W1GR), and G~nethon 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 ID NO:, to that map location.
Genetic map locations are reported as 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 G6n~thon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. SEQ ID N0:24 maps to chromosome 1 within the interval from 12.8 to 22.9 centiMorgans. SEQ ID
N0:36 maps to chromosome 1 within the interval from 74.8 to 78.3 centiMorgans.
VI. Extension of RECAP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:23-44 were 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, 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 Mgz+, (NH4)2S04, and (3 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 l: 94°C, 3 nun; 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 nun; 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:

SUBSTITUTE SHEET (RULE 26) 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 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE and 0.5 p1 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 ~cl to 10 ~1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-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 Biotec;h).
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 polymerise (Stratagene) to till-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 polymerise (Amersham Pharmacia Biotech) and Pfu DNA polymerise (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 (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:23-44 are used to obtain 5' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes Hybridization probes derived liom SEQ ID N0:23-44 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 SUBSTITUTE SHEET (RULE 26) Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of [y-32P] adenosine triphosphate (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 10' 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 II, 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 dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
VIII. 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, su ra), 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; Shalom 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 SUBSTITUTE SHEET (RULE 26) 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/Nl oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/Ld RNase inhibitor, 500 ~M dATP, 500 ECM dGTP, 500 ~M dTTP, 40 ~M
dCTP, 40 NM 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 Nl 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 fig.
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 (V WR), 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 US
Patent No. 5,807,522, incorporated herein by reference. 1 lil of the array element DNA, at an average concentration of 100 ng/l.il, is loaded into the open capillary printing element by a high-speed robodc apparatus. The apparatus then deposits about 5 n1 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.

SUBSTITUTE SHEET (RULE 26) Hybridization Hybridization reactions contain 9 Lrl of sample mixture consisting of 0.2 irg each of Cy3 and Cy5 labeled cDNA synthesis products in SX 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 Erl of SX 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 (1X 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 CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Ine., 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 81477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two lluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the iluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per lluorophore 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 tluorophore, 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 tluorophores 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 SUBSTITUTE SHEET (RULE 26) 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 lluorophore'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).
IX. Complementary Polynucleotides Sequences complementary to the RECAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring RECAP. 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 RECAP. 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 RECAP-encoding transcript.
X. Expression of RECAP
Expression and purification of RECAP is achieved using bacterial or virus-based expression systems. For expression of RECAP 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 TS or 'f7 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 RECAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
Expression of RECAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto~ra~hica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding RECAP
by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infec;tivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera fru~iperda (SP9) 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, RECAP is synthesized as a fusion protein with, e.g., glutathione S-SUBSTITUTE SHEET (RULE 26) 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 japonicum, 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 RECAP 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, eh. 10 and 16).
Purified RECAP obtained by these methods can be used directly in the assays shown in Examples XI and XV.
XI. Demonstration of RECAP Activity Receptor activity of RECAP is determined in a ligand-binding assay using candidate ligand molecules in the presence of '2sI-labeled RECAP. RECAP is labeled with'ZSI
Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
Candidate ligand molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled RECAP, washed, and any wells with labeled RECAP complex are assayed. Data obtained using different concentrations of RECAP are used to calculate values for the number, affinity, and association of RECAP with the ligand molecules. The level of binding measured is proportional to the level of RECAP
activity.
In the alternative, activity of RECAP may be measured using an assay based upon the property of some GPCRs to support the in vitro proliferation of fibroblasts and tumor cells under serum-free conditions (Chiquet-Ehrismann, R, et al. (1986) Cell 47:131-139).
Wells in 96 well cluster plates (Falcon, Fisher Scientific, Santa Clara CA) are coated with RECAP by incubation with solutions at 50-100 pg/ml for 15 min at ambient temperature. The coating solution is aspirated, and the wells washed with Dulbecco's medium before cells are plated. Rat fibroblast cultures or rat mammary tumor cells are prepared as described and plated at a density of 104-lOs cells/ml in Dulbecco's medium supplemented with 10% fetal calf serum (FCS).
After three days the media are removed, and the cells washed three times with phosphate-buffered saline (PBS) before the addition of serum-free Dulbecco's medium containing 0.25 mg/ml bovine serum albunun (BSA, Fraction V, Sigma Chemical, St. Louis, MO). After 2 days the medium is aspirated, and 100 ~1 of [~H]thymidine (NEN) at 2 ~Ci/ml in fresh Dulbecco's medium containing 0.25 mg/ml BSA added. Parallel plates are fixed and stained to determine cell numbers. After 16 hr, the medium is aspirated, the cell layer washed with PBS, and the 10%
trichloroacetic acid-precipitable counts in the cell layer determined by liquid scintillation counting of radioisotope (normalized to relative cell numbers; Chiquet-Ehrismann, R. et al. (1986) supra). The rates of cell proliferation and SUBSTITUTE SHEET (RULE 26) [3H]thymidine uptake are proportional to the activity of RECAP in the sample.
In the alternative, the assay for RECAP activity is based upon the property of CD97/Emrl GPCR family proteins to modulate G protein-activated second messenger signal transduction pathways (e.g., cAMP; Gaudin, P., et al. (1998) J. Biol. Chem., 273:4990-4996). A
plasmid encoding full length RECAP is transfected into a mammalian cell line (e.g., COS-7 or Chinese hamster ovary (CHO-Kl) cell lines) using methods well-known in the art. Transfected cells are grown in 12-well trays in culture medium containing 2% FCS for 48 hours, the culture medium is discarded, then the attached cells are gently washed with PBS. The cells are then incubated in culture medium with 10% FCS or 2% FCS
for 30 minutes, then the medium is removed and cells lysed by treatment with 1 M perchloric acid.
The cAMP levels in the lysate are measured by radioimmunoassay using methods well-known in the art. Changes in the levels of cAMP in the lysate from 10% FCS-treated cells compared with those in 2% FCS-treated cells are proportional to the activity of RECAP present in the transfected cells.
In another alternative, an assay for RECAP activity is based on a prototypical assay for ligand/receptor-mediated modulation of cell proliferation. This assay measures the rate of DNA
synthesis in Swiss mouse 3T3 cells. A plasmid containing polynucleotides encoding RECAP is added to quiescent 3T3 cultured cells using transfection methods well known in the art.
The transiently transfected cells are then incubated in the presence of [3H]thymidine, a radioactive DNA precursor molecule. Varying amounts of RECAP ligand are then added to the cultured cells. Incorporation of [3H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a radioisotope counter, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold RECAP ligand concentration range is indicative of receptor activity. One unit of activity per milliliter is defined as the concentration of RECAP producing a 50% response level, where 100% represents maximal incorporation of [3H]thymidine into acid-precipitable DNA (McKay, I. and Leigh, L, eds.
(1993) Growth Factors: A
Practical Approach, Oxford University Press, New York, NY, p. 73.) In the alternative, the assay for RECAP activity is based upon the ability of GPCR family proteins to modulate G protein-activated second messenger signal transduction pathways (e.g., cAMP;
Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996). A plasmid encoding full length RECAP is transfected into a mammalian cell line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell lines) using methods well-known in the art. Transfected cells are grown in 12-well trays in culture medium for 48 hours, then the culture medium is discarded, and the attached cells are gently washed with PBS. The cells are then incubated in culture medium with or without ligand for 30 minutes, then the medium is removed and cells lysed by treatment with 1 M
perchloric acid. The cAMP levels in the lysate are measured by radioimmunoassay using methods well-known in the art.
Changes in the levels of cAMP in the lysate from cells exposed to ligand compared to those without SUBSTITUTE SHEET (RULE 26) ligand are proportional to the amount of RECAP present in the transfected cells.
XII. Functional Assays RECAP function is assessed by expressing the sequences encoding RECAP 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 ~cg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~g 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 RECAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding RECAP 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 inununoglobulin G (IgG). Transfected cells are efFciently 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 RECAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XIII. Production of RECAP Specific Antibodies RECAP 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 RECAP amino acid sequence is analyzed using LASERGENE
software SUBSTITUTE SHEET (RULE 26) (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 431A
peptide synthesizer (PE 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-RECAP
activity by, for example, binding the peptide or RECAP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIV. Purification of Naturally Occurring RECAP Using Specific Antibodies Naturally occurring or recombinant RECAP is substantially purified by immunoaffinity chromatography using antibodies specific for RECAP. An immunoaffinity column is constructed by covalently coupling anti-RECAP 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 RECAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of RECAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/RECAP 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 RECAP is collected.
XV. Identification of Molecules Which Interact with RECAP
RECAP, or biologically active fragments thereof, are labeled with'ZSI 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 mufti-well plate are incubated with the labeled RECAP, washed, and any wells with labeled RECAP complex are assayed. Data obtained using different concentrations of RECAP are used to calculate values for the number, affinity, and association of RECAP with the candidate molecules.
Alternatively, molecules interacting with RECAP 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).
RECAP 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 SUBSTITUTE SHEET (RULE 26) No. 6,057,101 ).
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.

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<110> INCYTE GENOMICS~, INC.
AU-YOUNG, Janice BANDMAN, Olga TANG, Y. Tom YUE, Henry AZIMZAI, Yalda BURFORD, Neil BAUGHN, Mariah R.
LU, Dyung Aina M.
HILLMAN, Jennifer L.
PATTERSON, Chandra LAL, Preeti <120> RECEPTORS AND ASSOCIATED PROTEINS
<130> PF-0726 PCT
<140> To Be Assigned <141> Herewith <150> 60/145,232; 60/158,578; 60/165,192 <151> 1999-07-21; 1999-10-07; 1999-11-12 <160> 44 <170> PERL Program <210> 1 <211> 411 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 209171CD1 <400> 1 Met Ala Gln Arg Gln Leu Leu Asn Lys Lys Gly Phe Gly Glu Pro Val Leu Pro Arg Pro Pro Ser Leu Ile Gln Asn Glu Cys Gly Gln Gly Glu Gln Ala Ser Glu Lys Asn Glu Cys Ile Ser Glu Asp Met Gly Asp Glu Asp Lys Glu Glu Arg Gln Glu Ser Arg Ala Ser Asp Trp His Ser Lys Thr Lys Asp Phe Gln Glu Ser Ser Ile Lys Ser Leu Lys Glu Lys Leu Leu Leu Glu Glu Glu Pro Thr Thr Ser His Gly Gln Ser Ser Gln Gly Ile Val Glu Glu Thr Ser Glu Glu Gly Asn Ser Val Pro Ala Ser Gln Ser Val Ala Ala Leu Thr Ser Lys Arg Ser Leu Val Leu Met Pro Glu Ser Ser Ala Glu Glu Ile Thr Val Cys Pro Glu Thr Gln Leu Ser Ser Ser Glu Thr Phe Asp Leu Glu Arg Glu Val Ser Pro Gly Ser Arg Asp Ile Leu Asp Gly Val Arg Ile Ile Met Ala Asp Lys Glu Val Gly Asn Lys Glu Asp Ala Glu Lys Glu Val Ala Ile Ser Thr Phe Ser Ser Ser Asn Gln vat Ser Cys Pro Leu Cys Asp Gln Cys Phe Pro Pro Thr Lys Ile Glu Arg His Ala Met Tyr Cys Asn Gly Leu Met Glu Glu Asp Thr Val Leu Thr Arg Arg Gln Lys Glu Ala Lys Thr Lys Ser Asp Ser Gly Thr Ala Ala Gln Thr Ser Leu Asp Ile Asp Lys Asn Glu Lys Cys Tyr Leu Cys Lys Ser Leu Val Pro Phe Arg Glu Tyr Gln Cys His Val Asp Ser Cys Leu Gln Leu Ala Lys Ala Asp Gln Gly Asp Gly Pro Glu Gly Ser Gly Arg Ala Cys Ser Thr Val Glu Gly Lys Trp Gln Gln Arg Leu Lys Asn Pro Lys Glu Lys Gly His Ser Glu Gly Arg Leu Leu Ser Phe Leu Glu Gln Ser Glu His Lys Thr Ser Asp Ala Asp Ile Lys Ser Ser Glu Thr Gly Ala Phe Arg Val Pro Ser Pro Gly Met Glu Glu Ala Gly Cys Ser Arg Glu Met Gln Ser Ser Phe Thr Arg Arg Asp Leu Asn Glu Ser Pro Val Lys Ser Phe Val Ser Ile Ser Glu Ala Thr Asp Cys Leu Val Asp Phe Lys Lys Gln Val Thr Val Gln Pro Gly Ser Arg Thr Arg Thr Lys Ala Gly Arg Gly Arg Arg Arg Lys Phe <210> 2 <211> 579 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 945430CD1 <400> 2 Met Phe Phe Arg Val Phe Leu His Phe Ile Arg Ser His Ser AIa Thr Ala Val Asp Phe Leu Pro Val Met Val His Arg Leu Pro Val Phe Lys Arg Tyr Met Gly Asn Thr Pro Gln Lys Lys Ala Val Phe Gly Gln Cys Arg Gly Leu Pro Cys Val Ala Pro Leu Leu Thr Thr Val Glu Glu Ala Pro Arg Gly Ile Ser Ala Arg Val Trp Gly His Phe Pro Lys Trp Leu Asn Gly Ser Leu Leu Arg Ile Gly Pro Gly Lys Phe Glu Phe Gly Lys Asp Lys Tyr Asn His Trp Phe Asp Gly Met Ala Leu Leu His Gln Phe Arg Met Ala Lys Gly Thr Val Thr Tyr Arg Ser Lys Phe Leu Gln Ser Asp Thr Tyr Lys Ala Asn Ser Ala Lys Asn Arg Ile Val Ile Ser Glu Phe Gly Thr Leu Ala Leu Pro Asp Pro Cys Lys Asn Val Phe Glu Arg Phe Met Ser Arg Phe Glu Leu Pro Gly Lys Ala Ala Ala Met Thr Asp Asn Thr Asn Val Asn Tyr Val Arg Tyr Lys Gly Asp Tyr Tyr Leu Cys Thr Glu Thr Asn Phe Met Asn Lys Val Asp Ile Glu Thr Leu Glu Lys Thr Glu Lys Val Asp Trp Ser Lys Phe Ile Ala Val Asn Gly Ala Thr Ala His Pro His Tyr Asp Pro Asp Gly Thr Ala Tyr Asn Met Gly Asn 230 235 ~ 240 Ser Phe Gly Pro Tyr Gly Phe Ser Tyr Lys Val Ile Arg Val Pro Pro Glu Lys Val Asp Leu Gly Glu Thr Ile His Gly Val Gln Val Ile Cys Ser Ile Ala Ser Thr Glu Lys Gly Lys Pro Ser Tyr Tyr His Ser Phe Gly Met Thr Arg Asn Tyr Ile Ile Phe Ile Glu Gln Pro Leu Lys Met Asn Leu Trp Lys Ile Ala Thr Ser Lys Ile Arg Gly Lys Ala Phe Ser Asp Gly Ile Ser Trp Glu Pro Gln Cys Asn Thr Arg Phe His Val Val Glu Lys Arg Thr Gly Gln Leu Leu Pro Gly Arg Tyr Tyr Ser Lys Pro Phe Val Thr Phe His Gln Ile Asn Ala Phe Glu Asp Gln Gly Cys Val Ile Ile Asp Leu Cys Ser Gln Asp Asn Gly Arg Thr Leu Glu Val Tyr Gln Leu Gln Asn Leu Arg Lys Ala Gly Glu Gly Leu Asp Gln Val His Asn Ser Ala Ala Lys Ser Phe Pro Arg Arg Phe Val Leu Pro Leu Asn Val Ser Leu Asn Ala Pro Glu Gly Asp Asn Leu Ser Pro Leu Ser Tyr Thr Ser Ala Ser Ala Val Lys Gln Ala Asp Gly Thr Ile Trp Cys Ser His Glu Asn Leu His Gln Glu Asp Leu Glu Lys Glu Gly Gly Ile Glu Phe Pro Gln Ile Tyr Tyr Asp Arg Phe Ser Gly Lys Lys Tyr His Phe Phe Tyr Gly Cys Gly Phe Arg His Leu Val Gly Asp Ser Leu Ile Lys Val Asp Val Val Asn Lys Thr Leu Lys Val Trp Arg Glu Asp Gly Phe Tyr Pro Ser Glu Pro Val Phe Val Pro Ala Pro Gly Thr Asn Glu Glu Asp Gly Gly Val Ile Leu Ser Val Val Ile Thr Pro Asn Gln Asn Glu Ser Asn Phe Leu Leu Val Leu Asp Ala Lys Asn Phe Glu Glu Leu Gly Arg Ala Glu Val Pro Val Gln Met Pro Tyr Gly Phe His Gly Thr Phe Ile Pro Ile <210> 3 <211> 370 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1305513CD1 <400> 3 Met Ala Asn Tyr Ser His Ala Ala Asp Asn Ile Leu Gln Asn Leu Ser Pro Leu Thr Ala Phe Leu Lys Leu Thr Ser Leu Gly Phe Ile Ile Gly Val Ser Val Val Gly Asn Leu Leu Ile Ser Ile Leu Leu Val Lys Asp Lys Thr Leu His Arg Ala Pro Tyr Tyr Phe Leu Leu Asp Leu Cys Cys Ser Asp Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe Val Phe Asn Ser Val Lys Asn Gly Ser Thr Trp Thr Tyr Gly Thr Leu Thr Cys Lys Val Ile Ala Phe Leu Gly Val Leu Ser Cys Phe His Thr Ala Phe Met Leu Phe Cys Ile Ser Val Thr Arg Tyr Leu Ala Ile Ala His His Arg Phe Tyr Thr Lys Arg Leu Thr Phe Trp Thr Cys Leu Ala Val Ile Cys Met Val Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val Gly Thr Tyr Ser Phe Ile Arg Glu Lys Asp Gln Cys Thr Phe Gln His Arg Ser Phe Arg Ala Asn Asp Ser Leu Gly Phe Met Leu Leu Leu Ala Leu Ile Leu Leu Ala Thr Gln Leu Val Tyr Leu Lys Leu Ile Phe Phe Val His Asp Arg Arg Lys Met Lys Pro Val Gln Phe Val Ala Ala Val Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Ser Gly Gln Ala Ala Ala Asn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu Leu Gly Ile Arg Gln Asn Ala Asn Thr Thr Gly Arg Arg Arg Leu Leu Val Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe Tyr Ile Met Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala Cys Tyr Trp Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe Leu Thr Ala Ala Val Trp Met Ser Phe Ala Gln Ala Gly Ile Asn Pro Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr Thr Leu Leu Tyr Cys Arg Lys Ser Arg Leu Pro Arg Glu Pro Tyr Cys Val Ile <210> 4 <211> 267 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1876283CD1 <400> 4 Met Ala Pro Trp Ala Leu Leu Ser Pro Gly Val Leu Val Arg Thr Gly His Thr Val Leu Thr Trp Gly Ile Thr Leu Val Leu Phe Leu His Asp Thr Glu Leu Arg Gln Trp Glu Glu Gln Gly Glu Leu Leu Leu Pro Leu Thr Phe Leu Leu Leu Val Leu Gly Ser Leu Leu Leu Tyr Leu Ala Val Ser Leu Met Asp Pro Gly Tyr Val Asn Val Gln Pro Gln Pro Gln Glu Glu Leu Lys Glu Glu Gln Thr Ala Met Val Pro Pro Ala Ile Pro Leu Arg Arg Cys Arg Tyr Cys Leu Val Leu Gln Pro Leu Arg Ala Arg His Cys Arg Glu Cys Arg Arg Cys Val Arg Arg Tyr Asp His His Cys Pro Trp Met Glu Asn Cys Val Gly Glu Arg Asn His Pro Leu Phe Val Val Tyr Leu Ala Leu Gln Leu Val Val Leu Leu Trp Gly Leu Tyr Leu Ala Trp Ser Gly Leu Arg Phe Phe Gln Pro Trp Gly Leu Trp Leu Arg Ser Ser Gly Leu Leu Phe Ala Thr Phe Leu Leu Leu Ser Leu Phe Ser Leu Val Ala Ser Leu Leu Leu Val Ser His Leu Tyr Leu Val Ala Ser Asn Thr Thr Thr Trp Glu Phe Ile Ser Ser His Arg Ile Ala Tyr Leu Arg Gln Arg Pro Ser Asn Pro Phe Asp Arg Gly Leu Thr Arg Asn Leu Ala His Phe Phe Cys Gly Trp Pro Ser Gly Ser Trp Glu Thr Leu Trp Ala Glu Glu Glu Glu Glu Gly Ser Ser Pro Ala Val <210> 5 <211> 951 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2470285CD1 <400> 5 Met Pro Gly Pro Leu Gly Leu Leu Cys Phe Leu Ala Leu Gly Leu Leu Gly Ser Ala Gly Pro Ser Gly Ala Ala Pro Pro Leu Cys Ala Ala Pro Cys Ser Cys Asp Gly Asp Arg Arg Val Asp Cys Ser Gly Lys Gly Leu Thr Ala Val Pro Glu Gly Leu Ser Ala Phe Thr Gln Ala Leu Asp Ile Ser Met Asn Asn Ile Thr Gln Leu Pro Glu Asp Ala Phe Lys Asn Phe Pro Phe Leu Glu Glu Leu Gln Leu Ala Gly Asn Asp Leu Ser Phe Ile His Pro Lys Ala Leu Ser Gly Leu Lys Glu Leu Lys Val Leu Thr Leu Gln Asn Asn Gln Leu Lys Thr Val Pro Ser Glu Ala Ile Arg Gly Leu Ser Ala Leu Gln Ser Leu Arg Leu Asp Ala Asn His Ile Thr Ser Val Pro Glu Asp Ser Phe Glu Gly Leu Val Gln Leu Arg His Leu Trp Leu Asp Asp Asn Ser Leu Thr Glu Val Pro Val His Pro Leu Ser Asn Leu Pro Thr Leu Gln Ala Leu Thr Leu Ala Leu Asn Lys Ile Ser Ser Ile Pro Asp Phe Ala Phe Thr Asn Leu Ser Ser Leu Val Val Leu His Leu His Asn Asn Lys Ile Arg Ser Leu Ser Gln His Cys Phe Asp Gly Leu Asp Asn Leu Glu Thr Leu Asp Leu Asn Tyr Asn Asn Leu Gly Glu Phe Pro Gln Ala Ile Lys Ala Leu Pro Ser Leu Lys Glu Leu Gly Phe His Ser Asn Ser Ile Ser Val Ile Pro Asp Gly Ala Phe Asp Gly Asn Pro Leu Leu Arg Thr Ile His Leu Tyr Asp Asn Pro Leu Ser Phe Val Gly Asn Ser Ala Phe His Asn Leu Ser Asp Leu His Ser Leu Val Ile Arg Gly Ala Ser Met Val Gln Gln Phe Pro Asn Leu Thr Gly Thr Val His Leu Glu Ser Leu Thr Leu Thr Gly Thr Lys Ile Ser Ser Ile Pro Asn Asn Leu Cys Gln Glu Gln Lys Met Leu Arg Thr Leu Asp Leu Ser Tyr Asn Asn Ile Arg Asp Leu Pro Ser Phe Asn Gly Cys His Ala Leu Glu Glu Ile Ser Leu Gln Arg Asn Gln Ile Tyr Gln Ile Lys Glu Gly Thr Phe Gln Gly Leu Ile Ser Leu Arg Ile Leu Asp Leu Ser Arg Asn Leu Ile His Glu Ile His Ser Arg Ala Phe Ala Thr Leu Gly Pro Ile Thr Asn Leu Asp Val Ser Phe Asn Glu Leu Thr Ser Phe Pro Thr Glu Gly Leu Asn Gly Leu Asn Gln Leu Lys Leu Val Gly Asn Phe Lys Leu Lys Glu Ala Leu Ala Ala Lys Asp Phe Val Asn Leu Arg Ser Leu Ser Val Pro Tyr Ala Tyr Gln Cys Cys Ala Phe Trp Gly Cys Asp Ser Tyr Ala Asn Leu Asn Thr Glu Asp Asn Ser Leu Gln Asp His Ser Val Ala Gln Glu Lys Gly Thr Ala Asp Ala Ala Asn Val Thr Ser Thr Leu Glu Asn Glu Glu His Ser Gln Ile Ile Ile His Cys Thr Pro Ser Thr Gly Ala Phe Lys Pro Cys Glu Tyr Leu Leu Gly Ser Trp Met Ile Arg Leu Thr Val Trp Phe Ile Phe Leu Val Ala Leu Phe Phe Asn Leu Leu Val Ile Leu Thr Thr Phe Ala Ser Cys Thr Ser Leu Pro Ser Ser Lys Leu Phe Ile Gly Leu Ile Ser Val Ser Asn Leu Phe Met Gly Ile Tyr Thr Gly Ile Leu Thr Phe Leu Asp Ala Val Ser Trp Gly Arg Phe Ala Glu Phe Gly Ile Trp Trp Glu Thr Gly Ser Gly Cys Lys Val Ala Gly Phe Leu Ala Val Phe Ser Ser Glu Ser Ala Ile Phe Leu Leu Met Leu Ala Thr Val Glu Arg Ser Leu Ser Ala Lys Asp Ile Met Lys Asn Gly Lys Ser Asn His Leu Lys Gln Phe Arg Val Ala Ala Leu Leu Ala Phe Leu Gly Ala Thr Val Ala Gly Cys Phe Pro Leu Phe His Arg Gly Glu Tyr Ser Ala Ser Pro Leu Cys Leu Pro Phe Pro Thr Gly Glu Thr Pro Ser Leu Gly Phe Thr Val Thr Leu Val Leu Leu Asn Ser Leu Ala Phe Leu Leu Met Ala Val Ile Tyr Thr Lys Leu Tyr Cys Asn Leu Glu Lys Glu Asp Leu Ser Glu Asn Ser Gln Ser Ser Met Ile Lys His Val Ala Trp Leu Ile Phe Thr Asn Cys Ile Phe Phe Cys Pro Val Ala Phe Phe Ser Phe Ala Pro Leu Ile Thr Ala Ile Ser Ile Ser Pro Glu Ile Met Lys Ser Val Thr Leu Ile Phe Phe Pro Leu Pro Ala Cys Leu Asn Pro Val Leu Tyr Val Phe Phe Asn Pro Lys Phe Lys Glu Asp Trp Lys Leu Leu Lys Arg Arg Val Thr Lys Lys Ser Gly Ser Val Ser Val Ser Ile Ser Ser Gln Gly Gly Cys Leu Glu Gln Asp Phe Tyr Tyr Asp Cys Gly Met Tyr Ser His Leu Gln Gly Asn Leu Thr Val Cys Asp Cys Cys Glu Ser Phe Leu Leu Thr Lys Pro Val Ser Cys Lys His Leu Ile Lys Ser His Ser Cys Pro Ala Leu Ala Val Ala Ser Cys Gln Arg Pro Glu Gly Tyr Trp Ser Asp Cys Gly Thr Gln Ser Ala His Ser Asp Tyr Ala Asp Glu Glu Asp Ser Phe Val Ser Asp Ser Ser Asp Gln Val Gln Ala Cys Gly Arg Ala Cys Phe Tyr Gln Ser Arg Gly Phe Pro Leu Val Arg Tyr Ala Tyr Asn Leu Pro Arg Val Lys Asp <210> 6 <211> 413 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2925789CD1 <400> 6 Met Gly Thr Phe Cys Ser Val Ile Lys Phe Glu Asn Leu Gln Glu Leu Lys Arg Leu Cys His Trp Gly Pro Ile Ile Ala Leu Gly Val Ile Ala Ile Cys Ser Thr Met Ala Met Ile Asp Ser Val Leu Trp Tyr Trp Pro Leu His Thr Thr Gly Gly Ser Val Asn Phe Ile Met Leu Ile Asn Trp Thr Val Met Ile Leu Tyr Asn Tyr Phe Asn Ala Met Phe Val Gly Pro Gly Phe Val Pro Leu Gly Trp Lys Pro Glu Ile Ser Gln Asp Thr Met Tyr Leu Gln Tyr Cys Lys Val Cys Gln g5 100 105 Ala Tyr Lys Ala Pro Arg Ser His His Cys Arg Lys Cys Asn Arg Cys Val Met Lys Met Asp His His Cys Pro Trp Ile Asn Asn Cys Cys Gly Tyr Gln Asn His Ala Ser Phe Thr Leu Phe Leu Leu Leu Ala Pro Leu Gly Cys Ile His Ala Ala Phe Ile Phe Val Met Thr Met Tyr Thr Gln Leu Tyr His Arg Leu Ser Phe Gly Trp Asn Thr Val Lys Ile Asp Met Ser Ala Ala Arg Arg Asp Pro Leu Pro Ile Val Pro Phe Gly Leu Ala Ala Phe Ala Thr Thr Leu Phe Ala Leu Gly Leu Ala Leu Gly Thr Thr Ile Ala Val Gly Met Leu Phe Phe Ile Gln Met Lys Ile Ile Leu Arg Asn Lys Thr Ser Ile Glu Ser Trp Ile Glu Glu Lys Ala Lys Asp Arg Ile Gln Tyr Tyr Gln Leu Asp Glu Val Phe Val Phe Pro Tyr Asp Met Gly Ser Arg Trp Arg Asn Phe Lys Gln Val Phe Thr Trp Ser Gly Val Pro Glu Gly Asp Gly Leu Glu Trp Pro Val Arg Glu Gly Cys His Gln Tyr Ser Leu Thr Ile Glu Gln Leu Lys Gln Lys Ala Asp Lys Arg Val Arg Ser Val Arg Tyr Lys Val Ile Glu Asp Tyr Ser Gly Ala Cys Cys Pro Leu Asn Lys Gly Ile Lys Thr Phe Phe Thr Ser Pro Cys Thr Glu Glu Pro Arg Ile Gln Leu Gln Lys Gly Glu Phe Ile Leu Ala Thr Arg Gly Leu Arg Tyr Trp Leu Tyr Gly Asp Lys Ile Leu Asp Asp Ser Phe Ile Glu Gly Val Ser Arg Ile Arg Gly Trp Phe Pro Arg Lys Cys Val Glu Lys Cys Pro Cys Asp Ala Glu Thr Asp Gln Ala Pro Glu Gly Glu Lys Lys Asn Arg <210> 7 <211> 144 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3099990CD1 <400> 7 Met Lys Gly Lys Ala Arg Lys Leu Phe Tyr Lys Ala Ile Val Arg Gly Glu Glu Thr Leu Arg Val Gly Asp Cys Ala Val Phe Leu Ser Ala Gly Arg Pro Asn Leu Pro Tyr Ile Gly Arg Ile Glu Ser Met Trp Glu Ser Trp Gly Ser Asn Met Val Val Lys Val Lys Trp Phe Tyr His Pro Glu Glu Thr Lys Leu Gly Lys Arg Gln Cys Asp Gly Lys Asn Ala Leu Tyr Gln Ser Cys His Glu Asp Glu Asn Asp Val gp 85 90 Gln Thr Ile Ser His Lys Cys Gln Val Val Ala Arg Glu Gln Tyr Glu Gln Met Ala Arg Ser Arg Lys Cys Gln Asp Arg Gln Asp Leu Tyr Tyr Leu Ala Gly Thr Tyr Asp Pro Thr Thr Gly Arg Leu Val Thr Ala Asp Gly Val Pro Ile Leu Cys <210> 8 <211> 174 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 103561CD1 <400> 8 Met Ala Pro Pro Val Arg Leu Glu Arg Pro Phe Pro Ser Arg Arg Phe Pro Gly Leu Leu Leu Ala Ala Leu Val Leu Leu Leu Ser Ser Phe Ser Asp Gln Cys Asn Val Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp Asp Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr Ser Ala Lys Asp Lys Cys Lys Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Ala His Val Ile Lys Asp Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Pro Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asn Thr Val Ile Trp Asp Asn Lys Thr Pro Val Cys Asp Ser Glu Leu Lys Tyr Ala Phe Leu Phe Leu Leu Pro Ile His Ser Asn Phe Ser Leu Glu <210> 9 <211> 449 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 288709CD1 <400> 9 Met Gln Leu Asp Trp Asn Gln Ala Gln Lys Ser Gly Asp Pro Gly Pro Ser Val Val Gly Leu Val Ser Ile Pro Gly Met Gly Lys Leu Leu Ala Glu Ala Pro Leu Val Leu Glu Pro Glu Lys Gln Met Leu Leu His Glu Thr His Gln Gly Leu Leu Gln Asp Gly Ser Pro Ile Leu Leu Ser Asp Val Ile Ser Ala Phe Leu Ser Asn Asn Asp Thr Gln Asn Leu Ser Ser Pro Val Thr Phe Thr Phe Ser His Arg Ser gp 85 90 Val Ile Pro Arg Gln Lys Val Leu Cys Val Phe Trp Glu His Gly Gln Asn Gly Cys Gly His Trp Ala Thr Thr Gly Cys Ser Thr Ile Gly Thr Arg Asp Thr Ser Thr Ile Cys Arg Cys Thr His Leu Ser Ser Phe Ala Val Leu Met Ala His Tyr Asp Val Gln Glu Glu Asp Pro Val Leu Thr Val Ile Thr Tyr Met Gly Leu Ser Val Ser Leu Leu Cys Leu Leu Leu Ala Ala Leu Thr Phe Leu Leu Cys Lys Ala Ile Gln Asn Thr Ser Thr Ser Leu His Leu Gln Leu Ser Leu Cys Leu Phe Leu Ala His Leu Leu Phe Leu Val Gly Ile Asp Arg Thr Glu Pro Lys Val Leu Cys Ser Ile Ile Ala Gly Ala Leu His Tyr Leu Tyr Leu Ala Ala Phe Thr Trp Met Leu Leu Glu Gly Val His Leu Phe Leu Thr Ala Arg Asn Leu Thr Val Val Asn Tyr Ser Ser Ile Asn Arg Leu Met Lys Trp Ile Met Phe Pro Val Gly Tyr Gly Val Pro Ala Val Thr Val Ala Ile Ser Ala Ala Ser Trp Pro His Leu Tyr Gly Thr Ala Asp Arg Cys Trp Leu His Leu Asp Gln Gly Phe Met Trp Ser Phe Leu Gly Pro Val Cys Ala Ile Phe Ser Ala Asn Leu Val Leu Phe Ile Leu Val Phe Trp Ile Leu Lys Arg Lys Leu Ser Ser Leu Asn Ser Glu Val Ser Thr Ile Gln Asn Thr Arg Met Leu Ala Phe Lys Ala Thr Ala Gln Leu Phe Ile Leu Gly Cys Thr Trp Cys Leu Gly Leu Leu Gln Val Gly Pro Ala Ala Gln Val Met Ala Tyr Leu Phe Thr Ile Ile Asn Ser Leu Gln Gly Phe Phe Ile Phe Leu Val Tyr Cys Leu Leu Ser Gln Gln Val Gln Lys Gln Tyr Gln Lys Trp Phe Arg Glu Ile Val Lys Ser Lys Ser Glu Ser Glu Thr Tyr Thr Leu Ser Ser Lys Met Gly Pro Asp Ser Lys Pro Ser Glu Gly Asp Val Phe Pro Gly Gln Val Lys Arg Lys Tyr <210> 10 <211> 126 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 959893CD1 <400> 10 Met Glu Ser Phe Leu Gly Gly Val Leu Leu Ile Leu Trp Leu Gln Val Asp Trp Val Lys Ser Gln Lys Ile Glu Gln Asn Ser Glu Ala Leu Asn Ile Gln Glu Gly Lys Thr Ala Thr Leu Thr Cys Asn Tyr Thr Asn Tyr Ser Pro Ala Tyr Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Pro Val Phe Leu Leu Leu Ile Arg Glu Asn Glu Lys Glu Lys Arg Lys Glu Arg Leu Lys Val Thr Phe Asp Thr Thr Leu Lys Gln Ser Leu Phe His Ile Thr Ala Ser Gln Pro Ala Asp Ser Ala Asn Tyr Leu Cys Ala Leu Gly Gly Arg Gly Thr Asn Ser Pro Leu Gly Gln Ala Leu Ser <210> 11 <211> 273 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1414179CD1 <400> 11 Met Gly Arg Ser Arg Ser Arg Ser Ser Ser Arg Ser Lys His Thr Lys Ser Ser Lys His Asn Lys Lys Arg Ser Arg Ser Arg Ser Arg Ser Arg Asp Lys Glu Arg Val Arg Lys Arg Ser Lys Ser Arg Glu Ser Lys Arg Asn Arg Arg Arg Glu Ser Arg Ser Arg Ser Arg Ser Thr Asn Thr Ala Val Ser Arg Arg Glu Arg Asp Arg Glu Arg Ala Ser Ser Pro Pro Asp Arg Ile Asp Ile Phe Gly Arg Thr Val Ser Lys Arg Ser Ser Leu Asp Glu Lys Gln Lys Arg Glu Glu Glu Glu Lys Lys Ala Glu Phe Glu Arg Gln Arg Lys Ile Arg Gln Gln Glu Ile Glu Glu Lys Leu Ile Glu Glu Glu Thr Ala Arg Arg Val Glu Glu Leu Val Ala Lys Arg Val Glu Glu Glu Leu Glu Lys Arg Lys Asp Glu Ile Glu Arg Glu Val Leu Arg Arg Val Glu Glu Ala Lys Arg Ile Met Glu Lys Gln Leu Leu Glu Glu Leu Glu Arg Gln Arg Gln Ala Glu Leu Ala Ala Gln Lys Ala Arg Glu Glu Glu Glu Arg Ala Lys Arg Glu Glu Leu Glu Arg Ile Leu Glu Glu Asn Asn Arg Lys.Ile Ala Glu Ala Gln Ala Lys Leu Ala Glu Glu Gln Leu Arg Ile Val Glu Glu Gln Arg Lys Ile His Glu Glu Arg Met Lys Leu Glu Gln Glu Arg Gln Arg Gln Gln Lys Glu Glu Gln Lys Ile Ile Leu Gly Lys Gly Lys Ser Arg Pro Lys Leu Ser Phe Ser Leu Lys Thr Gln Asp <210> 12 <211> 140 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2197211CD1 <400> 12 Met Glu Lys Met Leu Glu Cys Ala Phe Ile Val Leu Trp Leu Gln Leu Gly Trp Leu Ser Gly Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu Arg Leu Gln Glu Gly Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val Ser Gly Leu Arg Gly Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly Pro Glu Phe Leu Phe Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys Glu Arg Leu Lys Ala Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile Thr Ala Pro Lys Pro Glu Asp Ser Ala Ser Tyr Leu Cys Ala Val Gln Gly Gly Ile Gly Asn Val Leu His Cys Gly Ser Gly Thr Gln Val Val Val Leu Pro His Ile Arg Asp Pro Gly Pro Ala Val <210> 13 <211> 479 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2263653CD1 <400> 13 Met Ala Val Leu Gly Val Gln Leu Val Val Thr Leu Leu Thr Ala Thr Leu Met His Arg Leu Ala Pro His Cys Ser Phe Ala Arg Trp Leu Leu Cys Asn Gly Ser Leu Phe Arg Tyr Lys His Pro Ser Glu Glu Glu Leu Arg Ala Leu Ala Gly Lys Pro Arg Pro Arg Gly Arg Lys Glu Arg Trp Ala Asn Gly Leu Ser Glu Glu Lys Pro Leu Ser Val Pro Arg Asp Ala Pro Phe Gln Leu Glu Thr Cys Pro Leu Thr Thr Val Asp Ala Leu Val Leu Arg Phe Phe Leu Glu Tyr Gln Trp Phe Val Asp Phe Ala Val Tyr Ser Gly Gly Val Tyr Leu Phe Thr Glu Ala Tyr Tyr Tyr Met Leu Gly Pro Ala Lys Glu Thr Asn Ile Ala Val Phe Trp Cys Leu Leu Thr Val Thr Phe Ser Ile Lys Met Phe Leu Thr Val Thr Arg Leu Tyr Phe Ser Ala Glu Glu Gly Gly Glu Arg Ser Val Cys Leu Thr Phe Ala Phe Leu Phe Leu Leu Leu Ala Met Leu Val Gln Val Val Arg Glu Glu Thr Leu Glu Leu Gly Leu Glu Pro Gly Leu Ala Ser Met Thr Gln Asn Leu Glu Pro Leu Leu Lys Lys Gln Gly Trp Asp Trp Ala Leu Pro Val Ala Lys Leu Ala Ile Arg Val Gly Leu Ala Val Val Gly Ser Val Leu Gly Ala Phe Leu Thr Phe Pro Gly Leu Arg Leu Ala Gln Thr His Arg Asp Ala Leu Thr Met Ser Glu Asp Arg Pro Met Leu Gln Phe Leu Leu His Thr Ser Phe Leu Ser Pro Leu Phe Ile Leu Trp Leu Trp Thr Lys Pro Ile Ala Arg Asp Phe Leu His Gln Pro Pro Phe Gly Glu Thr Arg Phe Ser Leu Leu Ser Asp Ser Ala Phe Asp Ser Gly Arg Leu Trp Leu Leu Val Val Leu Cys Leu Leu Arg Leu Ala Val Thr Arg Pro His Leu Gln Ala Tyr Leu Cys Leu Ala Lys Ala Arg Val Glu Gln Leu Arg Arg Glu Ala Gly Arg Ile Glu Ala Arg Glu Ile Gln Gln Arg Val Val Arg Val Tyr Cys Tyr Val Thr Val Val Ser Leu Gln Tyr Leu Thr Pro Leu Ile Leu Thr Leu Asn Cys Thr Leu Leu Leu Lys Thr Leu Gly Gly Tyr Ser Trp Gly Leu Gly Pro Ala Pro Leu Leu Ser Pro Asp Pro Ser Ser Ala Ser Ala Ala Pro Ile Gly Ser Gly Glu Asp Glu Val Gln Gln Thr Ala Ala Arg Ile Ala Gly Ala Leu Gly Gly Leu Leu Thr Pro Leu Phe Leu Arg Gly Val Leu Ala Tyr Leu Ile Trp Trp Thr Ala Ala Cys Gln Leu Leu Ala Ser Leu Phe Gly Leu Tyr Phe His Gln His Leu Ala Gly Ser <210> 14 <211> 99 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2504590CD1 <400> 14 Met Pro Arg Leu Lys Asp Pro Phe Phe Cys Tyr Gln Met Glu Ser His Cys Val Pro Arg Leu Glu Cys Ser Gly Ala Ile Ser Thr His Cys Lys Leu Cys Leu Pro Gly Ser Arg His Ser Pro Ala Ser Gly Ser Arg Val Ala Gly Thr Thr Gly Ala Arg His His Ser Trp Leu Ile Leu Phe Val Phe Ser Val Glu Thr Gly Tyr His His Val Ser Gln Asp Gly Leu Asp Leu Pro Asp Leu Val Ile Arg Pro Pro Gln Ser Pro Lys Val Leu Gly Leu Gln Ala <210> 15 <211> 349 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2529619CD1 <400> 15 Met Ser Ser Glu Met Val Lys Asn Gln Thr Met Val Thr Glu Phe Leu Leu Leu Gly Phe Leu Leu Gly Pro Arg Ile Gln Met Leu Leu Phe Gly Leu Phe Ser Leu Phe Tyr Val Phe Thr Leu Leu Gly Asn Gly Thr Ile Leu Gly Leu Ile Ser Leu Asp Ser Arg Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ala Val Val Asn Ile Ala Tyr Ala Cys Asn Thr Val Pro Gln Met Leu Val Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe Ala Gly Cys Met Thr Thr Thr Phe Leu Phe Leu Ser Phe Ala His Thr Glu Cys Leu Leu Leu Val Leu Met Ser Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr Phe Ile Ile Met Thr Trp Lys Val Cys Ile Thr Leu Ala Ile Thr Ser Trp Thr Cys Gly Ser Leu Leu Ala Met Val His Val Ser Leu Ile Leu Arg Leu Pro Phe Cys Gly Pro Arg Glu Ile Asn His Phe Phe Cys Glu Ile Leu Ser Val Leu Arg Leu Ala Cys Ala Asp Thr Trp Leu Asn Gln Val Val Ile Phe Ala Ala Cys Met Phe Ile Leu Val Gly Pro Leu Cys Leu Val Leu Val Ser Tyr Ser His Ile Leu Ala Ala Ile Leu Arg Ile Gln Ser Gly Glu Gly Arg Arg Lys Ala Phe Ser Thr Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Phe Gly Ser Ala Ile Val Met Tyr Met Ala Pro Lys Ser Arg His Pro Glu Glu Gln Gln Lys Val Leu Phe Leu Phe Tyr Ser Ser Phe Asn Pro Met Leu Asn Pro Leu Ile Tyr Asn Leu Arg Asn Val Glu Val Arg Cys Pro Glu Glu Ser Thr Val Gln Glu Lys Ser Phe Leu Arg Gly Val Thr Phe Glu Leu Pro Ala Ser Val Val Thr Trp Thr Leu Asp Ala Gln Leu Leu Pro Gln Ser Arg Lys Val Tyr Phe Ser Leu Ser Val Leu Tyr <210> 16 <211> 373 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 5467661CD1 <400> 16 Met Asp Thr Leu Glu Glu Val Thr Trp Ala Asn Gly Ser Thr Ala Leu Pro Pro Pro Leu Ala Pro Asn Ile Ser Val Pro His Arg Cys Leu Leu Leu Leu Tyr Glu Asp Ile Gly Thr Ser Arg Val Arg Tyr Trp Asp Leu Leu Leu Leu Ile Pro Asn Val Leu Phe Leu Ile Phe Leu Leu Trp Lys Leu Pro Ser Ala Arg Ala Lys Ile Arg Ile Thr Ser Ser Pro Ile Phe Ile Thr Phe Tyr Ile Leu Val Phe Val Val Ala Leu Val Gly Ile Ala Arg Ala Val Val Ser Met Thr Val Ser Thr Ser Asn Ala Ala Thr Val Ala Asp Lys Ile Leu Trp Glu Ile Thr Arg Phe Phe Leu Leu Ala Ile Glu Leu Ser Val Ile Ile Leu Gly Leu Ala Phe Gly His Leu Glu Ser Lys Ser Ser Ile Lys Arg Val Leu Ala Ile Thr Thr Val Leu Ser Leu Ala Tyr Ser Val Thr Gln Gly Thr Leu Glu Ile Leu Tyr Pro Asp Ala His Leu Ser Ala Glu Asp Phe Asn Ile Tyr Gly His Gly Gly Arg Gln Phe Trp Leu Val Ser Ser Cys Phe Phe Phe Leu Val Tyr Ser Leu Val Val Ile Leu Pro Lys Thr Pro Leu Lys Glu Arg Ile Ser Leu Pro Ser Arg Arg Ser Phe Tyr Val Tyr Ala Gly Ile Leu Ala Leu Leu Asn Leu Leu Gln Gly Leu Gly Ser Val Leu Leu Cys Phe Asp Ile Ile Glu Gly Leu Cys Cys Val Asp Ala Thr Thr Phe Leu Tyr Phe Ser Phe Phe Ala Pro Leu Ile Tyr Val Ala Phe Leu Arg Gly Phe Phe Gly Ser Glu Pro Lys Ile Leu Phe Ser Tyr Lys Cys Gln Val Asp Glu Thr Glu Glu Pro Asp Val His Leu Pro Gln Pro Tyr Ala Val Ala Arg Arg Glu Gly Leu Glu Ala Ala Gly Ala Ala Gly Ala Ser Ala Ala Ser Tyr Ser Ser Thr Gln Phe Asp Ser Ala Gly Gly Val Ala Tyr Leu Asp Asp Ile Ala Ser Met Pro Cys His Thr Gly Ser Ile Asn Ser Thr Asp Ser Glu Arg Trp Lys Ala Ile Asn Ala <210> 17 <211> 353 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 229740CD1 <400> 17 Met Leu Lys Met Met Glu Val Tyr Lys Glu Pro Arg Glu Gln Pro Ile Phe Thr Thr Arg Ala His Val Phe Gln Ile Asp Pro Asn Thr Lys Lys Asn Trp Met Pro Ala Ser Lys Gln Ala Val Thr Val Ser Tyr Phe Tyr Asp Val Thr Arg Asn Ser Tyr Arg Ile Ile Ser Val Asp Gly Ala Lys Val Ile Ile Asn Ser Thr Ile Thr Pro Asn Met Thr Phe Thr Asn Thr Ser Gln Thr Ser Gly Gln Trp Ala Asp Ser Arg Ala Asn Thr Val Phe Gly Leu Gly Phe Ser Ser Glu Gln Gln Leu Thr Lys Phe Ala Glu Lys Phe Gln Glu Val Lys Glu Ala Ala Lys Ile Ala Lys Asp Lys Thr Gln Glu Lys Ile Glu Thr Ser Ser Asn His Ser Gln Ala Ser Ser Val Asn Gly Thr Asp Asp Glu Lys Ala Ser His Ala Gly Pro Ala Asn Thr His Leu Lys Ser Glu Asn Asp Lys Leu Lys Ile Ala Leu Thr Gln Ser Ala Ala Asn Val Lys Lys Trp Glu Ile Glu Leu Gln Thr Leu Arg Glu Ser Asn Ala Arg Leu Thr Thr Ala Leu Gln Glu Ser Ala Ala Ser Val Glu Gln Trp Lys Arg Gln Phe Ser Ile Cys His Asp Glu Asn Asp Gln Leu Arg Asn Lys Ile Asp Glu Leu Glu Glu Gln Cys Ser Glu Ile Asn Arg Glu Lys Glu Lys Asn Thr Gln Leu Lys Arg Arg Ile Glu Glu Leu Glu Ala Glu Leu Arg Glu Lys Glu Thr Glu Leu Lys Asp Leu Arg Lys Gln Ser Glu Ile Ile Pro Gln Leu Met Ser Glu Cys Glu Tyr Val Ser Glu Lys Leu Glu Ala Ala Glu Arg Asp Asn Gln Asn Leu Glu Asp Lys Val Arg Ser Leu Lys Thr Asp Ile Glu Glu Ser Lys Tyr Arg Gln Arg His Leu Lys Val Glu Leu Lys Ser Phe Leu Glu Val Leu Asp Gly Lys Ile Asp Asp Leu His Asp Phe Arg Arg Gly Leu Ser Lys Leu Gly Thr Asp Asn <210> 18 <211> 441 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1317467CD1 <400> 18 Met Leu Leu Pro Gly Arg Ala Arg Gln Pro Pro Thr Pro Gln Pro Val Gln His Pro Gly Leu Arg Arg Gln Val Glu Pro Pro Gly Gln Leu Leu Arg Leu Phe Tyr Cys Thr Val Leu Val Cys Ser Lys Glu Ile Ser Ala Leu Thr Asp Phe Ser Gly Tyr Leu Thr Lys Leu Leu Gln Asn His Thr Thr Tyr Ala Cys Asp Gly Asp Tyr Leu Asn Leu Gln Cys Pro Arg His Ser Thr Ile Ser Val Gln Ser Ala Phe Tyr Gly Gln Asp Tyr Gln Met Cys Ser Ser Gln Lys Pro Ala Ser Gln Arg Glu Asp Ser Leu Thr Cys Val Ala Ala Thr Thr Phe Gln Lys Val Leu Asp Glu Cys Gln Asn Gln Arg Ala Cys His Leu Leu Val Asn Ser Arg Val Phe Gly Pro Asp Leu Cys Pro Gly Ser Ser Lys Tyr Leu Leu Val Ser Phe Lys Cys Gln Pro Asn Glu Leu Lys Asn Lys Thr Val Cys Glu Asp Gln Glu Leu Lys Leu His Cys His Glu Ser Lys Phe Leu Asn Ile Tyr Ser Ala Thr Tyr Gly Arg Arg Thr Gln Glu Arg Asp Ile Cys Ser Ser Lys Ala Glu Arg Leu Pro Pro Phe Asp Cys Leu Ser Tyr Ser Ala Leu Gln Val Leu Ser Arg Arg Cys Tyr Gly Lys Gln Arg Cys Lys Ile Ile Val Asn Asn His His Phe Gly Ser Pro Cys Leu Pro Gly Val Lys Lys Tyr Leu Thr Val Thr Tyr Ala Cys Val Pro Lys Asn Ile Leu Thr Ala Ile Asp Pro Ala Ile Ala Asn Leu Lys Pro Ser Leu Lys Gln Lys Asp Gly Glu Tyr Gly Ile Asn Phe Asp Pro Ser Gly Ser Lys Val Leu Arg Lys Asp Gly Ile Leu Val Ser Asn Ser Leu Ala Ala Phe Ala Tyr Ile Arg Ala His Pro Glu Arg Ala Ala Leu Leu Phe Val Ser Ser Val Cys Ile Gly Leu Ala Leu Thr Leu Cys Ala Leu Val Ile Arg Glu Ser Cys Ala Lys Asp Phe Arg Asp Leu Gln Leu Gly Arg Glu Gln Leu Val Pro Gly Ser Asp Lys Val Glu Glu Asp Ser Glu Asp Glu Glu Glu Glu Glu Asp Pro Ser Glu Ser Asp Phe Pro Gly Glu Leu Ser Gly Phe Cys Arg Thr Ser Tyr Pro Ile Tyr Ser Ser Ile Glu Ala Ala Glu Leu Ala Glu Arg Ile Glu Arg Arg Glu Gln Ile Ile Gln Glu Ile Trp Met Asn Ser Gly Leu Asp Thr Ser Leu Pro Arg Asn Met Gly Gln Phe Tyr <210> 19 <211> 310 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No. 2279267CD1 <400> 19 Met Gly Asp Asn Ile Thr Ser Ile Thr Glu Phe Leu Leu Leu Gly Phe Pro Val Gly Pro Arg Ile Gln Met Leu Leu Phe Gly Leu Phe Ser Leu Phe Tyr Val Phe Thr Leu Leu Gly Asn Gly Thr Ile Leu Gly Leu Ile Ser Leu Asp Ser Arg Leu His Ala Pro Met Tyr Phe Phe Leu Ser His Leu Ala Val Val Asp Ile Ala Tyr Ala Cys Asn Thr Val Pro Arg Met Leu Val Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe Ala Gly Arg Met Met Gln Thr Phe Leu Phe Ser Thr Phe Ala Val Thr Glu Cys Leu Leu Leu Val Val Met Ser Tyr Asp Leu Tyr Val Ala Ile Cys His Pro Leu Arg Tyr Leu Ala Ile Met Thr Trp Arg Val Cys Ile Thr Leu Ala Val Thr Ser Trp Thr Thr Gly Val Leu Leu Ser Leu Ile His Leu Val Leu Leu Leu Pro Leu Pro Phe Cys Arg Pro Gln Lys Ile Tyr His Phe Phe Cys Glu Ile Leu Ala Val Leu Lys Leu Ala Cys Ala Asp Thr His Ile Asn Glu Asn Met Val Leu Ala Gly Ala Ile Ser Gly Leu Val Gly Pro Leu Ser Thr Ile Val Val Ser Tyr Met Cys Ile Leu Cys Ala Ile Leu Gln Ile Gln Ser Arg Glu Val Gln Arg Lys Ala Phe Cys Thr Cys Phe Ser His Leu Cys Val Ile Gly Leu Phe Tyr Gly Thr Ala Ile Ile Met Tyr Val Gly Pro Arg Tyr Gly Asn Pro Lys Glu Gln Lys Lys Tyr Leu Leu Leu Phe His Ser Leu Phe Asn Pro Met Leu Asn Pro Leu Ile Cys Ser Leu Arg Asn Ser Glu Val Lys Asn Thr Leu Lys Arg Val Leu Gly Val Glu Arg Ala Leu <210> 20 <211> 438 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2436258CD1 <400> 20 Met Glu Val Gly Gly Asp Thr Ala Ala Pro Ala Pro Gly Gly Ala Glu Asp Leu Glu Asp Thr Gln Phe Pro Ser Glu Glu Ala Arg Glu Gly Gly Gly Val His Ala Val Pro Pro Asp Pro Glu Asp Glu Gly Leu Glu Glu Thr Glu Asp His Lys Leu Val Phe Leu Gln Gln Gly Pro Leu Leu Leu Val Ala Met Ser Arg Thr Ser Gln Ser Ala Ala Gln Leu Arg Gly Glu Leu Leu Ala Val His Ala Gln Ile Val Ser Thr Leu Thr Arg Ala Ser Val Ala Arg Ile Phe Ala His Lys Gln Asn Tyr Asp Leu Arg Arg Leu Leu Ala Gly Ser Glu Arg Thr Leu Asp Arg Leu Leu Asp Ser Met Glu Gln Asp Pro Gly Ala Leu Leu Leu Gly Ala Val Arg Cys Val Pro Leu Ala Arg Pro Leu Arg Asp Ala Leu Gly Ala Leu Leu Arg Arg Cys Thr Ala Pro Gly Leu Ala Leu Ser Val Leu Ala Val Gly Gly Arg Leu Ile Thr Ala Ala Gln Glu Arg Asn Val Leu Ala Glu Cys Arg Leu Asp Pro Ala Asp Leu Gln Leu Leu Leu Asp Trp Val Gly Ala Pro Ala Phe Ala Ala Gly Glu Ala Trp Ala Pro Val.Cys Leu Pro Arg Phe Asn Pro Asp Gly Phe Phe Tyr Ala Tyr Val Ala Arg Leu Asp Ala Met Pro Val Cys Leu Leu Leu Leu Gly Thr Gln Arg Glu Ala Phe His Ala Met Ala Ala Cys Arg Arg Leu Val Glu Asp Gly Met His Ala Leu Gly Ala Met Arg Ala Leu Gly Glu Ala Ala Ser Phe Ser Asn Ala Ser Ser Ala Ser Ala Pro Ala Tyr Ser Val Gln Ala Val Gly Ala Pro Gly Leu Arg His Phe Leu Tyr Lys Pro Leu Asp Ile Pro Asp His His Arg Gln Leu Pro Gln Phe Thr Ser Pro Glu Leu Glu Ala Pro Tyr Ser Arg Glu Glu Glu Arg Gln Arg Leu Ser Asp Leu Tyr His Arg Leu His Ala Arg Leu His Ser Thr Ser Arg Pro Leu Arg Leu Ile Tyr His Val Ala Glu Lys Glu Thr Leu Leu Ala Trp Val Thr Ser Lys Phe Glu Leu Tyr Thr Cys Leu Ser Pro Leu Val Thr Lys Ala Gly Ala Ile Leu Val Val Thr Lys Leu Leu Arg Trp Val Lys Lys Glu Glu Asp Arg Leu Phe Ile Arg Tyr Pro Pro Lys Tyr Ser Thr Pro Pro Ala Thr Ser Thr Asp Gln Ala Ala His Asn Gly Leu Phe Thr Gly Leu <210> 21 <211> 357 <212> PRT

<213> Homo Sapiens <220>

<221> misc_feature <223> Incyte No:2681738CD1 ID

<400> 21 Met Ala Thr ValProAsp GlyCysArg AsnGly LeuLys Ser Thr Lys Tyr Tyr LeuCysAsp LysAlaGlu AlaTrp GlyIle Val Arg Leu Glu Thr AlaThrAla GlyValVal ThrSer ValAla Phe Val Met Leu Thr ProIleLeu ValCysLys ValGln AspSer Asn Leu Arg Arg Lys Met Leu Pro Thr Gln Phe Leu Phe Leu Leu Gly Val Leu Gly Ile Phe Gly Leu Thr Phe Ala Phe Ile Ile Gly Leu Asp Gly Ser Thr Gly Pro Thr Arg Phe Phe Leu Phe Gly Ile Leu Phe Ser Ile Cys Phe Ser Cys Leu Leu Ala His Ala Val Ser Leu Thr Lys Leu Val Arg Gly Arg Lys Pro Leu Ser Leu Leu Val Ile Leu Gly Leu Ala Val Gly Phe Ser Leu Val Gln Asp Val Ile Ala Ile Glu Tyr Ile Val Leu Thr Met Asn Arg Thr Asn Val Asn Val Phe Ser Glu Leu Ser Ala Pro Arg Arg Asn Glu Asp Phe Val Leu Leu Leu Thr Tyr Val Leu Phe Leu Met Ala Leu Thr Phe Leu Met Ser Ser Phe Thr Phe Cys Gly Ser Phe Thr Gly Trp Lys Arg His Gly Ala His Ile Tyr Leu Thr Met Leu Leu Ser Ile Ala Ile Trp Val Ala Trp Ile Thr Leu Leu Met Leu Pro Asp Phe Asp Arg Arg Trp Asp Asp Thr Ile Leu Ser Ser Ala Leu Ala Ala Asn Gly Trp Val Phe Leu Leu Ala Tyr Val Ser Pro Glu Phe Trp Leu Leu Thr Lys Gln Arg Asn Pro Met Asp Tyr Pro Val Glu Asp Ala Phe Cys Lys Pro Gln Leu Val Lys Lys Ser Tyr Gly Val Glu Asn Arg Ala Tyr Ser Gln Glu Glu Ile Thr Gln Gly Phe Glu Glu Thr Gly Asp Thr Leu Tyr Ala Pro Tyr Ser Thr His Phe Gln Leu Gln Asn Gln Pro Pro Gln Lys Glu Phe Ser Ile Pro Arg Ala His Ala Trp Pro Ser Pro Tyr Lys Asp Tyr Glu Val Lys Lys Glu Gly Ser <210> 22 <211> 1069 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2859482CD1 <400> 22 Met Asp Asp Lys Ala Ser Val Gly Lys Ile Ser Val Ser Ser Asp Ser Val Ser Thr Leu Asn Ser Glu Asp Phe Val Leu Val Ser Arg Gln Gly Asp Glu Thr Pro Ser Thr Asn Asn Gly Ser Asp Asp Glu Lys Thr Gly Leu Lys Ile Val Gly Asn Gly Ser Glu Gln Gln Leu Gln Lys Glu Leu Ala Asp Val Leu Met Asp Pro Pro Met Asp Asp Gln Pro Gly Glu Lys Glu Leu Val Lys Arg Ser Gln Leu Asp Gly Glu Gly Asp Gly Pro Leu Ser Asn Gln Leu Ser Ala Ser Ser Thr Ile Asn Pro Val Pro Leu Val Gly Leu Gln Lys Pro Glu Met Ser Leu Pro Val Lys Pro Gly Gln Gly Asp Ser Glu Ala Ser Ser Pro Phe Thr Pro Val Ala Asp Glu Asp Ser Val Val Phe Ser Lys Leu Thr Tyr Leu Gly Cys Ala Ser Val Asn Ala Pro Arg Ser Glu Val Glu Ala Leu Arg Met Met Ser Ile Leu Arg Ser Gln Cys Gln Ile Ser Leu Asp Val Thr Leu Ser Val Pro Asn Val Ser Glu Gly Ile Val Arg Leu Leu Asp Pro Gln Thr Asn Thr Glu Ile Ala Asn Tyr Pro Ile Tyr Lys Ile Leu Phe Cys Val Arg Gly His Asp Gly Thr Pro Glu Ser Asp Cys Phe Ala Phe Thr Glu Ser His Tyr Asn Ala Glu Leu Phe Arg Ile His Val Phe Arg Cys Glu Ile Gln Glu Ala Val Ser Arg Ile Leu Tyr Ser Phe Ala Thr Ala Phe Arg Arg Ser Ala Lys Gln Thr Pro Leu Ser Ala Thr Ala Ala Pro Gln Thr Pro Asp Ser Asp Ile Phe Thr Phe Ser Val Ser Leu Glu Ile Lys Glu Asp Asp Gly Lys Gly Tyr Phe Ser Ala Val Pro Lys Asp Lys Asp Arg Gln Cys Phe Lys Leu Arg Gln Gly Ile Asp Lys Lys Ile Val Ile Tyr Val Gln Gln Thr Thr Asn Lys Glu Leu Ala Ile Glu Arg Cys Phe Gly Leu Leu Leu Ser Pro Gly Lys Asp Val Arg Asn Ser Asp Met His Leu Leu Asp Leu Glu Ser Met Gly Lys Ser Ser Asp Gly Lys Ser Tyr Val Ile Thr Gly Ser Trp Asn Pro Lys Ser Pro His Phe Gln Val Val Asn Glu Glu Thr Pro Lys Asp Lys Val Leu Phe Met Thr Thr Ala Val Asp Leu Val Ile Thr Glu Val Gln Glu Pro Val Arg Phe Leu Leu Glu Thr Lys Val Arg Val Cys Ser Pro Asn Glu Arg Leu Phe Trp Pro Phe Ser Lys Arg Ser Thr Thr Glu Asn Phe Phe Leu Lys Leu Lys Gln Ile Lys Gln Arg Glu Arg Lys Asn Asn Thr Asp Thr Leu Tyr Glu Val Val Cys Leu Glu Ser Glu Ser Glu Arg Glu Arg Arg Lys Thr Thr Ala Ser Pro Ser Val Arg Leu Pro Gln Ser Gly Ser Gln Ser Ser Val Ile Pro Ser Pro Pro Glu Asp Asp Glu Glu Glu Asp Asn Asp Glu Pro Leu Leu Ser Gly Ser Gly Asp Val Ser Lys Glu Cys Ala Glu Lys Ile Leu Glu Thr Trp Gly Glu Leu Leu Ser Lys Trp His Leu Asn Leu Asn Val Arg Pro Lys Gln Leu Ser Ser Leu Val Arg Asn Gly Val Pro Glu Ala Leu Arg Gly Glu Val Trp Gln Leu Leu Ala Gly Cys His Asn Asn Asp His Leu Val Glu Lys Tyr Arg Ile Leu Ile Thr Lys Glu Ser Pro Gln Asp Ser Ala Ile Thr Arg Asp Ile Asn Arg Thr Phe Pro Ala His Asp Tyr Phe Lys Asp Thr Gly Gly Asp Gly Gln Asp Ser Leu Tyr Lys Ile Cys Lys Ala Tyr Ser Val Tyr Asp Glu Glu Ile Gly Tyr Cys Gln Gly Gln Ser Phe Leu Ala Ala Val Leu Leu Leu His Met Pro Glu Glu Gln Ala Phe Ser Val Leu Val Lys Ile Met Phe Asp Tyr Gly Leu Arg Glu Leu Phe Lys Gln Asn Phe Glu Asp Leu His Cys Lys Phe Tyr Gln Leu Glu Arg Leu Met Gln Glu Tyr Ile Pro Asp Leu Tyr Asn His Phe Leu Asp Ile Ser Leu Glu Ala His Met Tyr Ala Ser Gln Trp Phe Leu Thr Leu Phe Thr Ala Lys Phe Pro Leu Tyr Met Val Phe His Ile Ile Asp Leu Leu Leu Cys Glu Gly Ile Ser Val Ile Phe Asn Val Ala Leu Gly Leu Leu Lys Thr Ser Lys Asp Asp Leu Leu Leu Thr Asp Phe Glu Gly Ala Leu 770 775 . 780 Lys Phe Phe Arg Val Gln Leu Pro Lys Arg Tyr Arg Ser Glu Glu Asn Ala Lys Lys Leu Met Glu Leu Ala Cys Asn Met Lys Ile Ser Gln Lys Lys Leu Lys Lys Tyr Glu Lys Glu Tyr His Thr Met Arg Glu Gln Gln Ala Gln Gln Glu Asp Pro Ile Glu Arg Phe Glu Arg Glu Asn Arg Arg Leu Gln Glu Ala Asn Met Arg Leu Glu Gln Glu Asn Asp Asp Leu Ala His Glu Leu Val Thr Ser Lys Ile Ala Leu Arg Lys Asp Leu Asp Asn Ala Glu Glu Lys Ala Asp Ala Leu Asn Lys Glu Leu Leu Met Thr Lys Gln Lys Leu Ile Asp Ala Glu Glu Glu Lys Arg Arg Leu Glu Glu Glu Ser Ala Gln Leu Lys Glu Met Cys Arg Arg Glu Leu Asp Lys Ala Glu Ser Glu Ile Lys Lys Asn Ser Ser Ile Ile Gly Asp Tyr Lys Gln Ile Cys Ser Gln Leu Ser Glu Arg Leu Glu Lys Gln Gln Thr Ala Asn Lys Val Glu Ile Glu Lys Ile Arg Gln Lys Val Asp Asp Cys Glu Arg Cys Arg Glu Phe Phe Asn Lys Glu Gly Arg Val Lys Gly Ile Ser Ser Thr Lys Glu Val Leu Asp Glu Asp Thr Asp Glu Glu Lys Glu Thr Leu Lys Asn Gln Leu Arg Glu Met Glu Leu Glu Leu Ala Gln Thr Lys Leu Gln Leu Val Glu Ala Glu Cys Lys Ile Gln Asp Leu Glu His His Leu Gly Leu Ala Leu Asn Glu Val Gln Ala Ala Lys Lys Thr Trp Phe Asn Arg Thr Leu Ser Ser Ile Lys Thr Ala Thr Gly Val Gln Gly Lys Glu Thr Cys <210> 23 <211> 1995 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 209171CB1 <400> 23 ggaccgtctt cccagtccca tcaagagaaa accacagact ctgggctcac tgaaggcata 60 tggcagctgg tacctccatc actgtttaaa ggctcacata tcagtcaggg aaacgaggct 120 gaggaaagag aggagccttg ggaccacact gaaaaaactg aagaggagcc ggtctctggc 180 agctcaggaa gctgggacca gtcaagccag ccagtgtttg agaatgtgaa cgttaaatct 240 tttgacagat gtactggcca ctcggctgag cacacacagt gtgggaagcc acaggaaagt 300 actgggaggg gttctgcttt tctcaaagct gtccagggta gcggggacac atctaggcac 360 tgtctaccta ccctagcaga tgccaaaggt ctccaggaca ctgggggcac tgtgaactat 420 ttctggggta ttccattctg ccctgatgga gtagacccta accagtatac caaggtcatt 480 ctctgccagt tggaggttta tcaaaagagc ctgaaaatgg ctcagaggca gctccttaat 540 aaaaaaggtt ttggggaacc agtgttacct agacctcctt ctctgatcca gaatgaatgt 600 ggccaaggag agcaggctag tgagaaaaat gaatgcatct cagaagatat gggagatgaa 660 gacaaagagg agaggcagga gtctagggca tctgactggc actcaaaaac caaggatttc 720 caggaaagct caattaaaag cttgaaagag aaacttttgt tggaggaaga accaacaacc 780 agtcatggtc agtcttccca agggattgtt gaagaaactt ctgaagaggg aaactctgta 840 cctgcttcac aaagtgttgc tgctttgacc agtaagagaa gcttagtcct tatgccagag 900 agttctgcag aagaaatcac tgtttgtcct gagacccagc taagttcctc tgaaactttt 960 gaccttgaaa gagaagtctc tccaggtagc agagatatct tggatggagt cagaataata 1020 atggcagata aggaggttgg taacaaggaa gatgctgaga aggaagtagc tatttctacc 1080 ttctcatcca gtaaccaggt atcctgcccg ctatgtgacc aatgctttcc acccacaaag 1140 attgaacgac atgccatgta ctgcaatggt ctgatggagg aagatacagt attgactcgg 1200 agacaaaaag aggccaagac caagagtgac agtgggacag ctgcccagac ttctctagac 1260 attgacaaga atgagaagtg ttacctctgt aaatccctgg tcccatttag agagtatcag 1320 tgtcatgtgg actcctgtct ccagcttgca aaggctgacc aaggagatgg acctgaaggg 1380 agtggaagag catgttcaac tgtggagggg aagtggcagc agaggctgaa gaacccaaag 1440 gaaaaaggcc acagtgaagg ccgactcctt agtttcttgg aacagtctga gcacaagact 1500 tcagatgcag acatcaagtc ttcagaaaca ggagccttca gggtgccttc accagggatg 1560 gaagaggcag gctgcagcag agagatgcag agttctttca cacgtcgtga cttaaatgaa 1620 tctcccgtca agtcttttgt ttccatttca gaagccacag attgcttagt ggactttaaa 1680 aagcaagtta ctgtccagcc aggtagtcgg acacggacca aagctggcag aggaagaagg 1740 agaaaattct gaatttctag ggtccaaaag ttgacaaaac cattagtagg aggggtgggc 1800 catgttcatt aagccatagt ggtccctagt tcattgttga gcaagtttta gccctgcagt 1860 tttcaccacc agcacctacc cagcattctg gtttttatgt tttttatgat ctatgcagac 1920 aactgtgtat tctgttttat aacagtttgt ttgaatttac ttacagttaa aaaatttaaa 1980 tataaaaaaa aaaaa 1995 <210> 24 <211> 2051 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 945430CB1 <400> 24 tcgaccacgc gtccgtgcgt gttcactgtt tagtagtact caaaactgcc agtgtgagag 60 gatttggaaa tcactggatc tgctcaatac aaaaatgttt tttcgagtct ttctccattt 120 tatcaggagt cattctgcca ctgcagtgga tttccttcct gtgatggtgc accggctccc 180 agttttcaaa aggtacatgg gaaatactcc tcagaaaaaa gccgtctttg ggcagtgtcg 240 gggtctgcca tgtgttgcac cgctgctgac cacagtggaa gaggctccac ggggcatctc 300 tgctcgagtc tggggacatt ttcctaagtg gctcaatggc tctctacttc gaattggacc 360 tgggaaattc gagtttggga aggataagta caatcattgg tttgatggga tggcgctgct 420 tcaccagttc agaatggcaa agggcacagt gacatacagg agcaagtttc tacagagtga 480 tacatataag gccaacagtg ctaaaaaccg aa~agtgatc tcagaatttg gcacactggc 540 tctcccggat ccatgcaaga atgtttttga acgtttcatg tccaggtttg agctgcctgg 600 taaagctgca gccatgactg acaatactaa tgtcaactat gtgcggtaca agggtgatta 660 ctacctctgc actgagacca actttatgaa taaagtggac attgaaactc tggaaaaaac 720 agaaaaggta gattggagca aatttattgc tgtgaatgga gcaactgcac atcctcatta 780 tgacccggat ggaacagcat acaatatggg gaactccttt gggccatatg gtttctccta 840 taaggttatt cgggttcctc cagagaaggt ggaccttggg gagacaatcc atggagtcca 900 ggtgatatgt tctattgctt ctacagagaa agggaaacct tcttactacc atagctttgg 960 aatgacaagg aactatataa ttttcattga acaacctcta aagatgaacc tgtggaaaat 1020 tgccacttct aaaattcggg gaaaggcctt ttcagatggg ataagctggg aaccccagtg 1080 taatacgcgg tttcatgtgg tggaaaaacg cactggacag ctccttccag ggagatacta 1140 cagcaaacct tttgttacat ttcatcaaat caatgccttt gaggaccagg gctgtgttat 1200 aattgatttg tgctctcaag ataatggaag aaccctagaa gtttaccagt tacagaatct 1260 caggaaggct ggggaagggc ttgatcaggt ccataattca gcagccaaat ctttccctcg 1320 aaggtttgtt ttgcctttaa atgtcagttt gaatgcccct gagggagaca acctgagtcc 1380 attgtcctat acttcagcca gtgctgtgaa acaggctgat ggaacgatct ggtgctctca 1440 tgaaaatcta catcaggagg acctagaaaa ggaaggaggc attgaatttc ctcagatcta 1500 ctatgatcga ttcagtggca aaaagtatca tttcttttat ggctgtggct ttcggcattt 1560 agtgggggat tctctgatca aggttgatgt ggtgaataag acactgaagg tttggagaga 1620 agatggcttt tatccctcag aacctgtttt tgttccagca ccaggaacca atgaagaaga 1680 tggtggggtt attctttctg tggtgatcac tcccaaccag aatgaaagca attttctcct 1740 agttttggat gccaagaact ttgaagagct gggccgagca gaggtacctg tgcagatgcc 1800 ttatgggttc catggtacct tcatacccat ctgatgggac aaccacaagg tctggaaact 1860 aggtttaaaa taagtgtgca cttggacata aagactggag aaataaacac tgaggactcc 1920 aaaagggggg caaggaggaa gaggggcagg ggttaaaaag ctacctattg aatactatgt 1980 tccctatttg ggtgatgggt tcattagaag tccaaacctc agcagcacac aatatactca 2040 tgtaacaagg g <210> 25 <211> 2067 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1305513CB1 <400> 25 ggcaaaaagc atgcagaaaa agaagcagac gttttacatt gggaattaat gaaagcgtgt 60 ctgctagttt tgggtaggag aactgggaag ttgttgctta aaattttata tcacctccac 120 aaacaaaact cttcggaaat ggtaaaataa gaaaatgcat gattctagag gcattcctaa 180 gcacccacgt gtcgggcttt gtggtgtctg tggtatcatc cgaccgtttg gactggttag 240 ggcttactga gagctccatt tctggaaagc cttacaagac tgaggaatat cagactgcga 300 atcaccggga acggttcctt tgcagcacag aagcaatctc tctccccatc ttcgcatatt 360 ctgatggcaa aacaagtgga agaaaagagg aagcatgact gcagatcaga tcagttctct 420 ttgtggatta tattttcagt aaaatgtatg gatctatctt ttccttgttc ttatatctag 480 atcatgagac ttgactgagg ctgtatcctt atcctccatc catctatggc gaactatagc 540 catgcagctg acaacatttt gcaaaatctc tcgcctctaa cagcctttct gaaactgact 600 tccttgggtt tcataatagg agtcagcgtg gtgggcaacc tcctgatctc cattttgcta 660 gtgaaagata agaccttgca tagagcacct tactacttcc tgttggatct ttgctgttca 720 gatatcctca gatctgcaat ttgtttccca tttgtgttca actctgtcaa aaatggctct 780 acctggactt atgggactct gacttgcaaa gtgattgcct ttctgggggt tttgtcctgt 840 ttccacactg ccttcatgct cttctgcatc agtgtcacca gatacttagc tatcgcccat 900 caccgcttct atacaaagag gctgaccttt tggacgtgtc tggctgtgat etgtatggtg 960 tggactctgt ctgtggccat ggcatttccc ccggttttag acgtgggcac ttactcattc 1020 attagggaga aagatcaatg caccttccaa caccgctcct tcagggctaa tgattcctta 1080 ggatttatgc tgcttcttgc tctcatcctc ctagccacac agcttgtcta cctcaagctg 1140 atatttttcg tccacgatcg aagaaaaatg aagccagtcc agtttgtagc agcagtcagc 1200 cagaactgga cttttcatgg tcctggagcc agtggccagg cagctgccaa ttggctagca 1260 ggatttggaa ggggtcccac accacccacc ttgctgggca tcaggcaaaa tgcaaacacc 1320 acaggcagaa gaaggctatt ggtcttagac gagttcaaaa tggagaaaag aatcagcaga 1380 atgttctata taatgacttt tctgtttcta accttgtggg gcccctacct ggtggcctgt 1440 tattggagag tttttgcaag agggcctgta gtaccagggg gatttctaac agctgctgtc 1500 tggatgagtt ttgcccaagc aggaatcaat ccttttgtct gcattttctc aaacagggag 1560 ctgaggcgct gtttcagcac aacccttc~t tactgcagaa aatccaggtt accaagggaa 1620 ccttactgtg ttatatgagg gagcatctgt aaatctttag ccttgtgaaa actaaccttc 1680 tctgctgagc aattgtggcc catagccata ttttgagaag aaattcaaga atggaatcag 1740 cagttttaag gatttgggca acattctgca gtctttgcaa tagttcacct ataatcctat 1800 tttaaatctc agagtgatcc tgctgactgc cagcaaaggt ttgtaattaa gaagggactg 1860 aaccactgcc ctaagtttct ttatgtggtc aaaaactaga taatgaaagt agcaggtgct 1920 aagtatcagt gctaaatgct ctgtatgtca ctacatatga aaaaacatca aaaaacaatt 1980 agcattggac atcttaataa attaagttga catgaggtaa atgtgttgat aaaaactaat 2040 tttagaagtt tgaagacttt aaaacag 2067 <210> 26 <211> 1165 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1876283CB1 <400> 26 cttcttccgg gtggggcccc gggccgaggc gatggcgccc tgggcgctcc tcagccctgg 60 ggtcctggtg cggaccgggc acaccgtgct gacctgggga atcacgctgg tgctcttcct 120 gcacgatacc gagctgcggc aatgggagga gcagggggag ctgctcctgc ccctcacctt 180 cctgctcctg gtgctgggct ccctgctgct ctacctcgct gtgtcactca tggaccctgg 240 ctacgtgaat gtgcagcccc agcctcagga ggagctcaaa gaggagcaga cagccatggt 300 tcctccagcc atccctcttc ggcgctgcag atactgcctg gtgctgcagc ccctgagggc 360 tcggcactgc cgtgagtgcc gccgttgcgt ccgccgctac gaccaccact gcccctggat 420 ggagaactgt gtgggagagc gcaaccaccc actctttgtg gtctacctgg cgctgcagct 480 ggtggtgctt ctgtggggcc tgtacctggc atggtcaggc ctccggttct tccagccctg 540 gggtctgtgg ttgcggtcca gcgggctcct gttcgccacc ttcctgctgc tgtccctctt 600 ctcgttggtg gccagcctgc tcctcgtctc gcacctctac ctggtggcca gcaacaccac 660 cacctgggaa ttcatctcct cacaccgcat cgcctatctc cgccagcgcc ccagcaaccc 720 cttcgaccga ggcctgaccc gcaacctggc ccacttcttc tgtggatggc cctcagggtc 780 ctgggagacc ctctgggctg aggaggagga agagggcagc agcccagctg tttagggttg 840 ctggaggccg ggctaccgtc ttgtgcctga aaaccacggg gcctgtcccc agctggggtg 900 agcgctcaga gggcctgggg ccctcactcc tgcccacgcc tcccagaccc cagaacggag 960 cttcaagtca gacagatccc tgccttggtg ggcagttctg ccttccaagg aagaagggga 1020 agaaaaggac ctgtgggtgg ctcaggccca agcagacccc gggctccacc ccagccccgc 1080 ccaggctgct gccagtgcac acttttacaa atttaatata aagcaagtcc agtcttaaaa 1140 agacaaacca taaaaataaa aaaaa 1165 <210> 27 <211> 3523 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2470285CB1 <220>
<221> unsure <222> 3393, 3419, 3435, 3443 <223> a, t, c, g, or other <400> 27 gcggccgcag caatgccggg cccgctaggg ctgctctgct tcctcgccct ggggctgctc 60 ggctcggccg ggcccagcgg cgcggcgccg cctctctgcg cggcgccctg cagctgcgac 120 ggcgaccgtc gggtggactg ctccgggaag gggctgacgg ccgtgcccga ggggctcagc 180 gccttcaccc aagcgctgga tatcagtatg aacaacatta ctcagttgcc agaagatgca 240 tttaagaact ttccttttct agaagagcta caattggcgg gcaacgacct ttcttttatc 300 cacccaaagg ccttgtctgg gttgaaagaa ctcaaagttc taacgctcca gaataatcag 360 ttgaaaacag tacccagtga agccattcga gggctgagtg ctttgcagtc tttgcgttta 420 gatgccaacc atattacctc agtccccgag gacagttttg aaggacttgt tcagttacgg 480 catctgtggc tggatgacaa cagcttgacg gaggtgcctg tgcaccccct cagcaatctg 540 cccaccctac aggcgctgac cctggctctc aacaagatct caagtatccc tgactttgca 600 tttaccaacc tttcaagcct ggtagttctg catcttcata acaataaaat tagaagcctg 660 agtcaacact gttttgatgg actagataac ctggagacct tagacttgaa ttataataac 720 ttgggggaat ttcctcaggc tattaaagcc cttcctagcc ttaaagagct aggatttcat 780 agtaattcta tttctgttat ccctgatgga gcatttgatg gtaatccact cttaagaact 840 atacatttgt atgataatcc tctgtctttt gtggggaact cagcatttca caatttatct 900 gatcttcatt ccctagtcat tcgtggtgca agcatggtgc agcagttccc caatcttaca 960 ggaactgtcc acctggaaag tctgactttg acaggtacaa agataagcag catacctaat 1020 aatttgtgcc aagaacaaaa gatgcttagg actttggact tgtcttacaa taatataaga 1080 gaccttccaa gttttaatgg ttgccatgct ctggaagaaa tttctttaca gcgtaatcaa 1140 atctaccaaa taaaggaagg cacctttcaa ggcctgatat ctctaaggat tctagatctg 1200 agtagaaacc tgatacatga aattcacagt agagcttttg ccacacttgg gccaataact 1260 aacctagatg taagtttcaa tgaattaact tcctttccta cggaaggcct gaatgggcta 1320 aatcaactga aacttgtggg caacttcaag ctgaaagaag ccttagcagc aaaagacttt 1380 gttaacctca ggtctttatc agtaccatat gcttatcagt gctgtgcatt ttggggttgt 1440 gactcttatg caaatttaaa cacagaagat aacagcctcc aggaccacag tgtggcacag 1500 gagaaaggta ctgctgatgc agcaaatgtc acaagcactc ttgaaaatga agaacatagt 1560 caaataatta tccattgtac accttcaaca ggtgctttta agccctgtga atatttactg 1620 ggaagctgga tgattcgtct tactgtgtgg ttcattttct tggttgcatt atttttcaac 1680 ctgcttgtta ttttaacaac atttgcatct tgtacatcac tgccttcgtc caaattgttt 1740 ataggcttga tttctgtgtc taacttattc atgggaatct atactggcat cctaactttt 1800 cttgatgctg tgtcctgggg cagattcgct gaatttggca tttggtggga aactggcagt 1860 ggctgcaaag tagctgggtt tcttgcagtt ttctcctcag aaagtgccat atttttatta 1920 atgctagcaa ctgtcgaaag aagcttatct gcaaaagata taatgaaaaa tgggaagagc 1980 aatcatctca aacagttccg ggttgctgcc cttttggctt tcctaggtgc tacagtagca 2040 ggctgttttc cccttttcca tagaggggaa tattctgcat cacccctttg tttgccattt 2100 cctacaggtg aaacgccatc attaggattc actgtaacgt tagtgctatt aaactcacta 2160 gcatttttat taatggccgt tatctacact aagctatact gcaacttgga aaaagaggac 2220 ctctcagaaa actcacaatc tagcatgatt aagcatgtcg cttggctaat cttcaccaat 2280 tgcatctttt tctgccctgt ggcgtttttt tcatttgcac cattgatcac tgcaatctct 2340 atcagccccg aaataatgaa gtctgttact ctgatatttt ttccattgcc tgcttgcctg 2400 aatccagtcc tgtatgtttt cttcaaccca aagtttaaag aagactggaa gttactgaag 2460 cgacgtgtta ccaagaaaag tggatcagtt tcagtttcca tcagtagcca aggtggttgt 2520 ctggaacagg atttctacta cgactgtggc atgtactcac atttgcaggg caacctgact 2580 gtttgcgact gctgcgaatc gtttctttta acaaagccag tatcatgcaa acacttgata 2640 aaatcacaca gctgtcctgc attggcagtg gcttcttgcc aaagacctga gggctactgg 2700 tccgactgtg gcacacagtc ggcccactct gattatgcag atgaagaaga ttcctttgtc 2760 tcagacagtt ctgaccaggt gcaggcctgt ggacgagcct gcttctacca gagtagagga 2820 ttccctttgg tgcgctatgc ttacaatcta ccaagagtta aagactgaac tactgtgtgt 2880 gtaaccgttt cccccgtcaa ccaaaatcag tgtttataga gtgaacccta ttctcatctt 2940 tcatctggga agcacttctg taatcactgc ctggtgtcac ttagaagaag gagaggtggc 3000 agtttatttc tcaaaccagt cattttcaaa gaacaggtgc ctaaattata aattggtgaa 3060 aaatgcaatg tccaagcaat gtatgatctg tttgaaacaa atatatgact tgaaaaggat 3120 cttaggtgta gtagagcaat ataatgttag ttttttctga tccataagaa gcaaatttat 3180 acctatttgt gtattaagca caagataaag aacagctgtt aatatttttt aaaaatctat 3240 tttaaaatgt gattttctat aactgaagaa aatatcttgc taattttacc taatgtttca 3300 tccttaatct caggacaact tactgcaggg ccaaaaaagg gactgtccca gctagaactg 3360 tgagagtata cataggcatt actttattat gtnttcactt gccatccttg acataagang 3420 actataaatt ttgtntaagc canttataaa tctaaacctg gtagatgttt taaaccatat 3480 tacagctgtt aggttaaaaa atagctggac attggtttca gtt 3523 <210> 28 <211> 2179 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: P2925789CB1 <400> 28 gtcctggcga gggcgctggc cgagaggtgc tcggcttgta gcaggtcccg cactccagcc 60 tctcgctgcc agggtttgct ctctgcttgt cctgggctga ggtgtccatg acggagtcat 120 ccaaggagga aaaaatctgt tccgggtgag cccaggccgc cccggatatg cgatggctga 180 ggagcagaca ccagggacca cactgaggtt gggtttcaga ccaagacact ggattctcct 240 agttaagata aagagctttg ggtgcctgac agtgaaaatg gtgtaatctg cgttaacagt 300 tcacagcttg aaggcatgac aattaaagaa cacacatgga cttgtggcac atggaaatgt 360 gcgcacagaa aaaggaaatc tataattctt ttaaagtagg aaggcattct tccttgccaa 420 aatgggtaca ttctgttcgg ttatcaagtt tgaaaatcta caagaattaa agagactgtg 480 tcactggggt cccatcatag cccttggtgt tatagcaata tgttctacca tggccatgat 540 tgactctgtg ttgtggtatt ggcccttaca tacaactgga ggaagtgtga atttcatcat 600 gttgataaat tggactgtca tgattcttta taattacttc aatgccatgt ttgtcggtcc 660 gggctttgtc cctctggggt ggaaaccgga aatttctcag gataccatgt atctccagta 720 ttgtaaagtc tgccaagcat acaaggcacc acgttcacat cactgcagaa agtgtaacag 780 atgtgtgatg aagatggacc atcactgtcc ttggatcaac aactgttgtg gttaccaaaa 840 tcatgcttcg ttcacactgt ttctcctttt agcaccactg ggttgtatcc atgctgcttt 900 catttttgtg atgactatgt acacacagct ttatcatcgg ctctcctttg ggtggaacac 960 agtgaagatc gacatgagtg cagcccggag agatcctctt ccaattgttc catttggatt 1020 agctgcattt gctaccacct tgtttgcctt gggattagct ttaggaacaa ccatagctgt 1080 tgggatgttg ttttttatcc agatgaaaat aattctcaga aacaaaactt ctattgagtc 1140 atggattgaa gagaaggcta aagatcgaat tcagtattat caactagatg aagtctttgt 1200 ttttccatat gatatgggaa gtagatggag gaactttaaa caggtattta cgtggtcagg 1260 ggtccctgaa ggagatggac ttgagtggcc agtaagagaa ggctgtcacc aatacagctt 1320 aacaatagaa cagttgaaac aaaaagcaga taagagagtc agaagtgttc gctataaagt 1380 aatagaagat tatagtggtg cctgctgccc tctgaataaa ggaatcaaaa ccttcttcac 1440 aagtccctgc accgaagagc ctcgaataca gctgcaaaaa ggggaattca ttttagccac 1500 aagaggttta cgatactggt tatatggaga caaaattctt gatgattcct ttatagaagg 1560 tgtttcaaga ataaggggtt ggttccctag aaaatgtgtg gaaaagtgtc cctgtgatgc 1620 tgaaacagat caagccccag agggggagaa gaaaaataga tagctgctgt taaaacaaaa 1680 ttatccttta agtctgctta attacttgaa aattgtacat attactaaag aattatgcaa 1740 tgagcctact ctggttaaga tgttcttttc ctcaaaggtg ccctagtgcc atgatttaaa 1800 tatttttatt accattttga aatggagaag ccattctgca tatgcctttg aattcctgcc 1860 cctctttacc acctcttcct ccccctcaaa ggaaaaacat ttcatccaag taagttaacg 1920 gcattttctg taggattttc ttatgcactg cacactctgg acctcacctg cagatacagt 1980 tccccccttg ccaggagcat ctgcatgtgg tacttctctt ttccctcagt tgatatttct 2040 tatatgatat tctagatact atagaactca atttgtcaga ttcagtataa cctcagattt 2100 tgttacctgt cttttaaaaa tgcagatttt gtcaaatcaa ataaagatca atggatgttg 2160 ggtataaaaa aaaaaaaaa 2179 <210> 29 <211> 645 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3099990CB1 <400> 29 cggccaaaga tctcagcctt cctgcccgcc cggcagctct ggaagtggtc ggggaatccc 60 acacagcggc gtggcatgaa ggggaaggcc cggaagctgt tctacaaggc catcgtgcgg 120 ggcgaggaga ccctgcgtgt cggggactgt gccgtcttcc tgtcagctgg gcggcccaac 180 ctcccctaca tcggccgcat cgagagcatg tgggagtcgt ggggcagcaa catggtggtc 240 aaggtcaagt ggttctacca ccctgaggag accaagctgg gcaagaggca gtgcgacggc 300 aagaatgcgc tgtaccagtc ctgccacgag gatgagaacg acgtgcagac catctcccac 360 aagtgccagg tcgtggcgcg cgagcagtat gagcagatgg cccggagccg caagtgccag 420 gaccggcagg acctctacta cctggcgggc acctacgacc ccaccaccgg gcgcctggtg 480 acggctgatg gcgtgcccat cctatgctga gccgcccacc gcagatgcct cccacgtgcg 540 ccagggaccc tgtgtgcgga cctggcgtcg gccaagccac cgggcaggag gcagccccgg 600 cctcccaagg gcgcatctga gcaaatatgc aaaagcccac agggc 645 <210> 30 <211> 627 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 103561CB1 <400> 30 cggactcaga agggacttcc ctgctcggct ggctttcggt ttctctgctc acctccggat 60 aaatcacggg gtctcccgcg ccgctcatgg cgcctcccgt ccgtctcgag cgtccctttc 120 cttcccggcg ctttcctggg ttgcttctgg cggccctggt gttgctgctg tcctccttct 180 ccgatcaatg caatgtcccg gaatggcttc catttgccag gcctaccaac ctaactgatg 240 actttgagtt tcccattggg acatatctga actatgaatg ccgccctggt tattccggaa 300 gaccgttttc tatcatctgc ctaaaaaact cagtctggac aagtgctaag gacaagtgca 360 aacgtaaatc atgtcgtaat cctccagatc ctgtgaatgg catggcacat gtgatcaaag 420 acatccagtt cggatcccaa attaaatatt cttgtcctaa aggataccga ctcattggtt 480 cctcgtctgc cacatgcatc atctcaggca acactgtcat ttgggataat aaaacacctg 540 tttgtgacag tgagttgaaa tatgcattcc tatttctttt accgatacat tctaattttt 600 ctctggaata ataaaaatct taaccga 627 <210> 31 <211> 1858 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 288709CB1 <400> 31 ctggatgagc tgctggaggc cctggggacc tggagaccct gccccgctta cagcagcact 60 gtgtggccag tcacctgctg gatggcctag aggatgtcct cagaggcctg agcaagaacc 120 tttccaatgg gctgttgaac ttcagttatc ctgcaggcac agaattgtcc ctggaggtgc 180 agaagcaagt agacaggagt gtcaccttga gacagaatca ggcagtgatg cagctcgact 240 ggaatcaggc acagaaatct ggtgacccag gcccttctgt ggtgggcctt gtctccattc 300 cagggatggg caagttgctg gctgaggccc ctctggtcct ggaacctgag aagcagatgc 360 ttctgcatga gacacaccag ggcttgctgc aggacggctc ccccatcctg ctctcagatg 420 tgatctctgc ctttctgagc aacaacgaca cccaaaacct cagctcccca gttaccttca 480 ccttctccca ccgttcagtg atcccgagac agaaggtgct ctgtgtcttc tgggagcatg 540 gccagaatgg atgtggtcac tgggccacca caggctgcag cacaataggc accagagaca 600 ccagcaccat ctgccgttgc acccacctga gcagctttgc cgtcctcatg gcccactacg 660 atgtgcagga ggaggatccc gtgctgactg tcatcaccta catggggctg agcgtctctc 720 tgctgtgcct cctcctggcg gccctcactt ttctcctgtg taaagccatc cagaacacca 780 gcacctcact gcatctgcag ctctcgctct gcctcttcct ggcccacctc ctcttcctcg 840 tggggattga tcgaactgaa cccaaggtgc tgtgctccat catcgccggt gctttgcact 900 atctctacct ggccgccttc acctggatgc tgctggaggg tgtgcacctc ttcctcactg 960 cacggaacct gacagtggtc aactactcaa gcatcaatag actcatgaag tggatcatgt 1020 tcccagtcgg ctatggcgtt cccgctgtga ctgtggccat ttctgcagcc tcctggcctc 1080 acctttatgg aactgctgat cgatgctggc tccacctgga ccagggattc atgtggagtt 1140 tccttggccc agtctgtgcc attttctctg cgaatttagt attgtttatc ttggtctttt 1200 ggattttgaa aagaaaactt tcctccctca atagtgaagt gtcaaccatc cagaacacaa 1260 ggatgctggc tttcaaagca acagctcagc tcttcatcct gggctgcaca tggtgtctgg 1320 gcttgctaca ggtgggtcca gctgcccagg tcatggccta cctcttcacc atcatcaaca 1380 gcctccaagg cttcttcatc ttcttggtct actgcctcct cagccagcag gtccagaaac 1440 aatatcaaaa gtggtttaga gagatcgtaa aatcaaaatc tgagtctgag acatacacac 1500 tttccagcaa gatgggtcct gactcaaaac ccagtgaggg ggatgttttt ccaggacaag 1560 tgaagagaaa atattaaaac tagaatattc aactccatat ggaaaatcat atccatggat 1620 ctctttggca ttatgaagaa tgaagctaag gaaaagggaa ttcattaaac atatcatcct 1680 tggagaggaa gtaatcaacc tttacttccc aagctgtttg ttctccacaa taggctctca 1740 acaaatgtgt ggtaaattgc atttctcttc actatggtgt attcagtcaa tgcttgtccc 1800 tggaaaccca aagcatgacc actgcaaata tttccttgac tttttgtaaa aaaaaaaa 1858 <210> 32 <211> 539 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 959893CB1 <400> 32 tttttggctg agaaggctgg gtctacattt caggccacat ttggggagac gaatggagtc 60 attcctggga ggtgttttgc tgattttgtg gcttcaagtg gactgggtga agagccaaaa 120 gatagaacag aattccgagg ccctgaacat tcaggagggt aaaacggcca ccctgacctg 180 caactataca aactattccc cagcatactt acagtggtac cgacaagatc caggaagagg 240 ccctgttttc ttgctactca tacgtgaaaa tgagaaagaa aaaaggaaag aaagactgaa 300 ggtcaccttt gataccaccc ttaaacagag tttgtttcat atcacagcct cccagcctgc 360 agactcagct aactacctct gtgctctagg gggaagagga acaaactcac ctttgggaca 420 ggcactcagc tagaagtggg actcaatatc cagaaccctg accctgccgt gtacaagctg 480 agagactcta aatccagtga caagtctgtc tgcctattaa cggttttgat tctcaaaaa 539 <210> 33 <211> 1126 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1414179CB1 <400> 33 tgccatctta ggggcgcctg gcgctacggg tttctcgttg gaggcggcct tcgtggcagc 60 tgtagacgcc gggaaaaggc ataaagtccg ttggccgaca cctttctttc ctccggcctc 120 ggtagaaccg ccagcccgcg tccgaaggcg gaggcgaggg gaactggccg cgtgaggggc 180 ctgaggcgag cggttagagc gtctcccgga aggatgggcc ggtctcggag ccggagctcg 240 tcccgctcca agcacaccaa gagcagcaag cacaacaaga agcgcagccg gtcccggtcg 300 cgatcccggg acaaggagcg cgtgcggaag cgttccaaat ctcgggaaag taaacggaac 360 cggcggcggg agtcgcggtc ccgttcgcgc tccaccaaca cggccgtgtc ccggcgcgag 420 cgggaccggg agcgcgcctc gtccccgccc gaccgcatcg acatcttcgg gcgcacggtg 480 agcaagcgca gcagcctgga cgagaagcag aagcgagagg aggaggagaa gaaagcggag 540 ttcgagcggc agcgaaaaat tcgacagcaa gaaatagaag aaaaactcat cgaggaagaa 600 acagcacgaa gagtagaaga attggtagca aaaagggtgg aggaagaact ggagaaaagg 660 aaggatgaaa ttgaacgaga agttctccga agggtggagg aagccaaacg catcatggaa 720 aagcagttgc tcgaagaact cgagcgacag agacaagctg agcttgccgc acaaaaagct 780 agagaggagg aagaacgtgc aaaacgtgag gagctagagc gaatactgga agagaataac 840 cgaaaaattg cagaagcaca agccaaactg gccgaagaac agttgagaat tgttgaagaa 900 caaagaaaga ttcatgagga aaggatgaaa ctagaacaag aacgacaacg tcaacaaaaa 960 gaagaacaaa aaattatcct gggcaagggg aagtccaggc caaaactgtc cttctcatta 1020 aaaacccagg attaaattgc aaactctgaa ctttttacaa agaaaaatgg aaaaactttg 1080 tatggtagct tcatgttgaa gtggtttttt gtttttggtt ttgttt 1126 <210> 34 <211> 490 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2197211CB1 <400> 34 gcctctgaga aaagaaggtt ggaattatcg taatttgttt ctaggctgag ataccagcat 60 ggagaaaatg ttggagtgtg cattcatagt cttgtggctt cagcttggct ggttgagtgg 120 agaagaccag gtgacgcaga gtcccgaggc cctgagactc caggagggag agagtagcag 180 tctcaactgc agttacacag tcagcggttt aagagggctg ttctggtata ggcaagatcc 240 tgggaaaggc cctgaattcc tcttcaccct gtattcagct ggggaagaaa aggagaaaga 300 aaggctaaaa gccacattga caaagaagga aagctttctg cacatcacag cccctaaacc 360 tgaagactca gccagttatc tctgtgctgt gcaggggggc attgggaatg tgctgcattg 420 cgggtccggc actcaagtgg ttgttttacc acatatccgg gaccctggac ctgccgtgta 480 acagctgaga 490 <210> 35 <211> 1799 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2263653CB1 <400> 35 ctaggccggt gggtgagtgc accgcgttct cgcacgcgtc atggcggtcc tcggagtaca 60 gctggtggtg accctgctca ctgccaccct catgcacagg ctggcgccac actgctcctt 120 cgcgcgctgg ctgctctgta acggcagttt gttccgatac aagcacccgt ctgaggagga 180 gcttcgggcc ctggcgggga agccgaggcc cagaggcagg aaagagcggt gggccaatgg 240 ccttagtgag gagaagccac tgtctgtgcc ccgagatgcc ccgttccagc tggagacctg 300 ccccctcacg accgtggatg ccctggtcct gcgcttcttc ctggagtacc agtggtttgt 360 ggactttgct gtgtactcgg gcggcgtgta cctcttcaca gaggcctact actacatgct 420 gggaccagcc aaggagacta acattgctgt gttctggtgc ctgctcacgg tgaccttctc 480 catcaagatg ttcctgacag tgacacggct gtacttcagc gccgaggagg ggggtgagcg 540 ctctgtctgc ctcacctttg ccttcctctt cctgctgctg gccatgctgg tgcaagtggt 600 gcgggaggag accctcgagc tgggcctgga gcctggtctg gccagcatga cccagaactt 660 agagccactt ctgaagaagc agggctggga ctgggcgctt cctgtggcca agctggctat 720 ccgcgtggga ctggcagtgg tgggctctgt gctgggtgcc ttcctcacct tcccaggcct 780 gcggctggcc cagacccacc gggacgcact gaccatgtcg gaggacagac ccatgctgca 840 gttcctcctg cacaccagct tcctgtctcc cctgttcatc ctgtggctct ggacaaagcc 900 cattgcacgg gacttcctgc accagccgcc gtttggggag acgcgtttct ccctgctgtc 960 cgattctgcc ttcgactctg ggcgcctctg gttgctggtg gtgctgtgcc tgctgcggct 1020 ggcggtgacc cggccccacc tgcaggccta cctgtgcctg gccaaggccc gggtggagca 1080 gctgcgaagg gaggctggcc gcatcgaagc ccgtgaaatc cagcagaggg tggtccgagt 1140 ctactgctat gtgaccgtgg tgagcttgca gtacctgacg ccgctcatcc tcaccctcaa 1200 ctgcacactt ctgctcaaga cgctgggagg ctattcctgg ggcctgggcc cagctcctct 1260 actatccccc gacccatcct cagccagcgc tgcccccatc ggctctgggg aggacgaagt 1320 ccagcagact gcagcgcgga ttgccggggc tctgggtggc ctgcttactc ccctcttcct 1380 ccgtggcgtc ctggcctacc tcatctggtg gacggctgcc tgccagctgc tcgccagcct 1440 tttcggcctc tacttccacc agcacttggc aggctcctag ctgcctgcag accctcctgg 1500 ggccctgagg tctgttcctg gggcagcggg acactagcct gccccctctg tttgcgcccc 1560 cgtgtcccca gctgcaaggt ggggccggac tccccggcgt tcccttcacc acagtgcctg 1620 acccgcggcc ccccttggac gccgagtttc tgcctcagaa ctgtctctcc tgggcccagc 1680 agcatgaggg tcccgaggcc attgtctccg aagcgtatgt gccaggtttg agtggcgagg 1740 gtgatgctgg ctgctcttct gaacaaataa aggagcatgc cgatttttaa aaaaaaaaa 1799 <210> 36 <211> 1435 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2504590CB1 <400> 36 catagcgcga ggttcaccat gttggccagg ctggtcttga actcctgtac cttgtgaaat 60 gcctgcctca gcctcccaaa gtgctgggat tacaggcatg agccactatg cccaggctca 120 aagatccttt tttttgttat cagatggagt ctcactgtgt ccccaggctg gagtgcagtg 180 gcgcgatctc aactcactgc aagctctgcc tcccgggttc acgccattct cctgcctcag 240 gctcccgagt agctgggact acaggcgccc gccaccactc ctggctaatt ttgtttgtat 300 tttcagtaga gacgggatat caccatgtta gccaggatgg tctcgatctg cctgacctcg 360 tgatccgccc tcctcagtct cccaaagtat taggattaca ggcgtgagcc accgcccccg 420 gccctcaaag attgttatga gtcctcacag atgtcagagg acgatgctgt gacctatatt 480 tgcaaacgcc cggaagactt aaagctattg ttttgcaaat gcacagataa caataataac 540 atgatgctgt ggatgagcat gaaagttttt ctcttactcc taattataat gttttccctc 600 ttactgcttt gccctgctga tggaattcag taaaggaatc tctaccagca gacacttggt 660 ttttaccctt cctaggacct ttaatagata tctttttatt actaatcttt gctctttgct 720 tgtttaacct ccttgtaaag tttgtgtctt ccagattaca ataattccat gtaaagatga 780 tgctggcaca aggctttcaa cccatcccct cttctgaccc agaagataaa gacatcctac 840 ctttgagcct tttagaacag gtatccaggg attttacctc tccagtgcta ggcagggtct 900 atgcccataa catcagcagg aagcagttac agaagatgaa cctccgccct ~ctgcaagcc 960 ccttaagatt aaggaggagt atataatctc tgatggggaa atgaggtagg agaccagaag 1020 gacttatttt ccattcccaa ccccattgaa cagagcagga tctggtcaaa acagggtgca 1080 gtggagaagc ctgctgaaac cagcagatga tgatgaaagt gacctctagt tgccctcact 1140 gcttatgagc ataaagacac taccactggg accatggcca gtttacaaat gtcatggcaa 1200 cacaccttgg caatggcctg gaagttactt tatatggttc tggaaactcc ctgccccttt 1260 cccagaaagt tctgaataac ctacctctta attggcatgc aattaaaagt ggctctaaat 1320 acaactagct agtagcccac aggcaccaac tctgggcaca ctgcctatag gttagccctg 1380 ctctgcaaga agtagcacca gttcaataaa agttgctttc tctcaaaaaa aaaaa 1435 <210> 37 <211> 1792 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2529619CB1 <400> 37 acacgcgtcc ggagctaacc aggcacatct tcacaatgaa atcttcacaa gctcagaacc 60 atcaacttgc tacactgtgt tcctccagtg cccagttcac aggaagcact caagagtagg 120 atcaattgtt atcaacctct agcagattat tgaatagacg gtcgttacat gaaactgaca 180 ctcagttctt catggtccac gatagttctt atatccagaa aacgtactta cgttatcaac 240 tctgactcca taagacaact ttgcaaagtc ttggctcata actataaatc aaggctgaat 300 caaacaggac tactgttctg aaagaatact cagactatga gtagtgaaat ggtgaaaaat 360 cagacaatgg tcacagagtt cctcctactg ggatttctcc tgggcccaag gattcagatg 420 ctcctctttg ggctcttctc cctgttctat gtcttcaccc tgctggggaa tgggaccatc 480 ctggggctca tctcactgga ctccagactc cacaccccca tgtacttctt cctctcacac 540 ctggccgtcg tcaacatcgc ctatgcctgc aacacagtgc cccagatgct ggtgaacctc 600 ctgcatccag ccaagcccat ctcctttgct ggctgcatga caactacctt tctctttttg 660 agttttgcac atactgaatg cctcctgttg gtgctgatgt cctacgatcg gtacgtggcc 720 atctgccacc ctctccgata tttcatcatc atgacctgga aagtctgcat cactctggcc 780 atcacttcct ggacatgtgg ctccctcctg gctatggtcc atgtgagcct catcctaaga 840 ctgccctttt gtgggcctcg tgaaatcaac cacttcttct gtgaaatcct gtctgtcctc 900 aggctggcct gtgctgatac ctggctcaac caggtggtca tctttgcagc ctgcatgttc 960 atcctggtgg gaccactctg cctggtgctg gtctcctact cacacatcct ggcggccatc 1020 ctgaggatcc agtctgggga gggccgcaga aaggccttct ccacctgctc ctcccacctc 1080 tgcgtagtgg gactcttctt tggcagcgcc atcgtcatgt acatggcccc taagtcccgc 1140 catcctgagg agcagcagaa ggtccttttt ctattttaca gttctttcaa cccgatgcta 1200 aaccccctga tttacaacct gaggaatgta gaggtcaggt gccctgagga gagcactgtg 1260 caagaaaagt cattcctaag aggtgtgaca tttgaactgc cagcctcagt tgtcacgtgg 1320 actcttgatg cccaattatt gcctcaatcc agaaaagttt acttctcttt atctgtgctt 1380 tactgacaga agggcaagtc ttctctcgtt ttttgcagat aaaattttag atgtgttgca 1440 ttcattgggt ttctatgaga tgtggtttta tcagacaatt ttttctttta tttcacaatt 1500 actttaatat ctttgaccag caaatatctc caccctccag ggagaggtag tagcttctag 1560 ggaaaccatc ttggagaggg tcctgtcttc ccctgaggtg ggctctgaat ccagcactct 1620 tcccctttct tggagggtca cttggaacca gctaactttt cagggtcctt tcttcctagt 1680 tctgctcata catctgtcat gtaacacttt agtgttctat atttgcatag ctgtatcctt 1740 ccattagttt gtatagagct gacttagtat ttttcggacg cagttggtcg at 1792 <210> 38 <211> 1817 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5467661CB1 <400> 38 gacggtccgg ccgcggccca ggccgggagg tggagcgcgg ctgccgccag ctggtccact 60 gacaactgct gccagctgga aggcgcgtag ggcgagaggt ggcgcgtcct cagctttcca 120 gtcttcgatt ccgcccgctt tccgagtgag aaacgcaggc ctctctcttc cgtgaagcag 180 cgtcttgtct ctgcacctcc tctgtgctgt ctctttaaga gggctggctg ctggcgggat 240 ggacaccctg gaggaggtga cttgggccaa tgggagcaca gcgctacccc cacccctggc 300 accaaacatc agtgtgcctc atcgctgcct gctgctgctc tacgaagaca ttggcacctc 360 cagggtccgg tactgggacc tcttgctgct catccccaat gtgctcttcc tcatcttcct 420 gctctggaag cttccatctg ctcgggcgaa gatccgcatc acctccagcc ccatttttat 480 caccttctac atcctggtgt ttgtggtggc gctggtgggc attgcccggg ccgtggtatc 540 catgacggtg agcacctcga acgctgcaac tgttgctgat aagatcctgt gggagatcac 600 ccgcttcttc ctgctggcca tcgagctgag tgtgatcatc ctgggcctgg cctttggcca 660 cctggagagt aagtccagca tcaagcgggt gctggccatc accacagtgc tgtccctggc 720 ctactctgtc acccagggga ccctggagat cctgtaccct gatgcccatc tctcagctga 780 ggactttaat atctatggcc atgggggccg ccagttctgg ctggtcagct cctgcttctt 840 cttcctggtc tactctctgg tggtcatcct tcccaagacc ccgctgaagg agcgcatctc 900 cctgccttct cggaggagct tctacgtgta tgcgggcatc ctggcactgc tcaacctact 960 gcaggggctg gggagtgtgc tgctgtgctt cgacatcatc gaggggctct gctgtgtaga 1020 tgccacaacc ttcctgtact tcagcttctt cgctccgctc atctacgtgg ctttcctccg 1080 gggcttcttc ggctcggagc ccaagatcct cttctcctac aaatgccaag tggacgagac 1140 agaggagcca gatgtacacc taccccagcc ctacgctgtg gcccgacggg agggcctgga 1200 ggctgcaggg gctgctgggg cctcagctgc cagctactcg agcacgcagt tcgactctgc 1260 cggcggggtg gcctacctgg atgacatcgc ttccatgccc tgccacactg gcagcatcaa 1320 cagcacagac agcgagcgct ggaaggccat caatgcctga gggcagctgc cagggcctgt 1380 ggaggacagg ccagagagga ggccagcagg cccagagtcc ccaggggagg aggaccaggt 1440 caagggacgt tctgtgggca gtagccctgt gtggccctgt tcccaccatg agtctggagg 1500 ccccacctcc ctggggctcc caatcccctt tgccatctct gctctcactg gggaccctcc 1560 tccccttccc acctgctctc atactgctca gtgacatggc ccaggctttc cttccagggc 1620 catgcttggc aaggttgget gagggcaccc tccttctctg cacccttggc acgagggcag 1680 ggctggctct cccaatgcct ccatcccatc cccatggtgc tttggcctcc tcaaagcatc 1740 caccatggtg gatggactga agtgtgtata ttttcttgat ctatttttta ataaaaagga 1800 aaaggagcaa aaaaaaa 1817 <210> 39 <211> 1820 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 229740CB1 <400> 39 ctttgagatg ctgaagatga tggaagtcta taaggaacct agagaacagc ccatcttcac 60 cacccgagcg catgtcttcc agattgaccc caacaccaag aagaactgga tgcctgcgag 120 caagcaggcg gtcaccgttt cctacttcta tgatgtcaca aggaacagct atcggatcat 180 cagtgtggac ggagccaagg tgatcataaa cagcacaatc acaccgaata tgaccttcac 240 caacacgtca cagacgtctg ggcagtgggc cgacagcaga gccaacacag tgtttggttt 300 ggggttttcc tctgagcagc agctgacaaa gtttgcagag aaattccagg aggtgaaaga 360 agctgccaag atagccaaag acaagacgca ggagaaaatc gagacctcaa gtaatcattc 420 ccaagcatcc agtgtcaacg ggacggacga tgaaaaggcc tctcacgccg gtccagccaa 480 cacacacctg aagtctgaga atgacaagct gaagattgcc ttgacgcaga gcgcagccaa 540 cgtgaagaag tgggagatcg agctgcagac ccttcgggag agcaatgcac ggctgaccac 600 agcactgcag gagtcggcag ccagtgtgga gcagtggaag aggcagttct ccatctgcca 660 tgatgagaat gaccagctcc gcaacaagat tgatgagctg gaagaacaat gcagtgagat 720 caacagagag aaggagaaga acacgcagct gaagaggagg atcgaggagc tggaggcaga 780 gctccgagaa aaggagacag agctgaaaga tctccgaaaa caaagtgaaa tcatacctca 840 gctcatgtca gagtgcgaat atgtctctga gaagctagag gcggcagaga gagacaatca 900 aaacctggaa gacaaagtgc gttccttaaa gacagacatt gaggagagca aataccgaca 960 gcgccacctg aaggtggagt tgaagagctt cctggaggtg ctggacggga agattgacga 1020 cctgcatgac ttccgccgag ggctctccaa gctgggcacc gataactagg gctggccgag 1080 gcccaggccc cgcccgtgag tcccaagcgt gtgtgcgaga ccagatagct ctaggacgtt 1140 cttctgtgtg cattgcttct gtaaatgcag gcgcagtttg tcgtgtttcc aaaccagttg 1200 tgccgtccac tcactccttt tcagaataga aatctcctct cgcttctctg gccttgtgag 1260 gttgtggaca actggaagat tctgactcag gaatccagaa ctaggtctac cttcaacatt 1320 tatgcagtca gggcagggat gtttatatct ttcataaggg ctgttgcaac catatgaact 1380 gaaaaaacac gcattttgta atccaaatat tgatattctt tacaccaagc catcaggctc 1440 cttttatcaa atagcattca gagtatttga atgtccacca gacaccagcc ccggggggca 1500 cagagagaac aacattcctc tctgtcaaca tcgagaggct ttaaaacaac tgtttagtgg 1560 aaactttctg agagatggaa aacaagcttc tggtgggtgc attttctggc ccggagttgc 1620 ctgcatccac gctactgccc cctgcccccc gcccccccag tttgtacggt tgcaacagtg 1680 ttccttttct tggttttaat ttctgagcag atgattgtgc tgtgggaaca gcacacagtg 1740 agggtgccta gcacaatgtc tggcacaaag taggtgctta ataaatattt gttcaattaa 1800 atgtgaaaaa aaaaaaaaaa 1820 <210> 40 <211> 1620 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1317467CB1 <400> 40 tccccgccat gtgacgccgt ccttagccct gcgaccccca gcgcgtcccg ggcctgcgcc 60 tccgccccgc cgcgcagcac gatgcttctg ccgggacgcg cacgccaacc gccgacgccc 120 cagcccgtgc agcatcccgg cctccgccgg caggtagagc cgccggggca gctcctgcgc 180 ctcttctact gcactgtcct ggtctgctcc aaagagatct cagcgctcac cgacttctct 240 ggttacctaa ccaaactcct gcaaaaccac accacctatg cctgtgatgg ggactatttg 300 aatctacagt gccctcggca ttctacgata agtgtccaat cggcatttta tgggcaagat 360 taccaaatgt gtagttccca gaagcctgcc tcccagaggg aagacagctt aacctgtgtg 420 gcagccacca ccttccagaa ggtgctggac gaatgccaga accagcgggc ctgccacctc 480 ctggtcaata gccgtgtttt tggacctgac ctttgtccag gaagcagtaa atacctccta 540 gtctccttta aatgccaacc taatgaatta aaaaacaaaa ccgtgtgtga agaccaggag 600 ctgaaactgc actgccatga atccaagttc ctcaacatct actctgcgac ctacggcagg 660 aggacccagg aaagggacat ctgctcctcc aaggcagagc ggctcccccc tttcgattgc 720 ttgtcttact cagctttgca agtcctatcc cgaaggtgct atgggaagca gagatgcaaa 780 atcatcgtca acaatcacca ttttggaagc ccctgtttgc caggcgtgaa aaaatacctc 840 actgtgacct acgcatgtgt tcccaagaac atactcacag cgattgatcc agccattgct 900 aatctaaaac cttctttgaa gcagaaagat ggtgaatatg gtataaactt cgacccaagc 960 ggatcgaagg ttctgaggaa agatggaatt cttgttagca actctctggc agcctttgct 1020 tacattagag cccacccgga gagagctgcc ctgctgttcg tgtccagtgt ctgcatcggc 1080 ctggccctca cactgtgcgc cctggtcatc agagagtcct gtgccaagga cttccgcgac 1140 ttgcagctgg ggagggagca gctggtgcca ggaagtgaca aggtcgagga ggacagcgag 1200 gatgaagaag aggaggagga cccctctgag tctgatttcc caggggaact gtcggggttc 1260 tgtaggactt catatcctat atacagttcc atagaagctg cagagctcgc agaaaggatt 1320 gagcgcaggg agcaaatcat tcaggaaata tggatgaaca gtggtttgga cacctcgctc 1380 ccaagaaaca tgggccagtt ctactgaaaa ccacatgcat cttgatgcga tcgcactttc 1440 tgaagaagga aggatcccaa atgcccctcc agttctggtt cacctgtacc ttctatgaag 1500 gagaattcgt catgtcattc aacactcgtg aggccaggaa gctattaaag ggatgtttca 1560 agctgtttct agcacattcc aaaataaatg aggagggaag agtctttgtt ttctgtaaaa 1620 <210> 41 <211> 974 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2279267CB1 <400> 41 atgggggaca atataacatc catcacagag ttcctcctac tgggatttcc cgttggccca 60 aggattcaga tgctcctctt tgggctcttc tccctgttct acgtcttcac cctgctgggg 120 aacgggacca tactggggct catctcactg gactccagac tgcacgcccc catgtacttc 180 ttcctctcac acctggcggt cgtcgacatc gcctacgcct gcaacacggt gccccggatg 240 ctggtgaacc tcctgcatcc agccaagccc atctcctttg cgggccgcat gatgcagacc 300 tttctgtttt ccacttttgc tgtcacagaa tgtctcctcc tggtggtgat gtcctatgat 360 ctgtacgtgg ccatctgcca ccccctccga tatttggcca tcatgacctg gagagtctgc 420 atcaccctcg cggtgacttc ctggaccact ggagtccttt tatccttgat tcatcttgtg 480 ttacttctac ctttaccctt ctgtaggccc cagaaaattt atcacttttt ttgtgaaatc 540 ttggctgttc tcaaacttgc ctgtgcagat acccacatca atgagaacat ggtcttggcc 600 ggagcaattt ctgggctggt gggacccttg tccacaattg tagtttcata tatgtgcatc 660 ctctgtgcta tccttcagat ccaatcaagg gaagttcaga ggaaagcctt ctgcacctgc 720 ttctcccacc tctgtgtgat tggactcttt tatggcacag ccattatcat gtatgttgga 780 cccagatatg ggaaccccaa ggagcagaag aaatatctcc tgctgtttca cagcctcttt 840 aatcccatgc tcaatcccct tatctgtagt cttaggaact cagaagtgaa gaatactttg 900 aagagagtgc tgggagtaga aagggcttta tgaaaaggat tatggcattg tgactgacag 960 tgacctagga agtt 974 <210> 42 <211> 1561 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2436258CB1 <400> 42 gttaaaacag ggatgtgcag atggaggtcg gaggagacac tgctgccccg gcccccgggg 60 gcgcggagga cttggaggac acgcagttcc ccagtgagga agctagagaa ggtggagggg 120 ttcacgcggt cccgccggat cccgaagacg agggcctgga ggaaacagag gaccacaagc 180 tggtgttcct acaacagggc ccactgttgc tcgtggccat gtcacggact tctcagtcag 240 cagcccagct gcggggggag ctgctagctg tgcacgcaca gatcgtgagc acacttacac 300 gtgcaagtgt cgcccgcatc ttcgcacaca agcagaacta tgacctccgc cgcctgctgg 360 ctggttcaga gcgcacactg gaccgacttc tggacagtat ggagcaggac ccaggagccc 420 tgctcctggg tgccgtgcgc tgtgtgcccc ttgcccgccc gctgcgagac gcactaggtg 480 cgctcctccg acgttgcaca gcgcctggcc tggcgctgtc agtgctggca gtaggcggtc 540 gacttataac agcagcccag gagcggaatg tgctggccga gtgccggctg gacccagctg 600 acctgcagtt gctgctcgac tgggtgggtg caccagcctt tgcggcgggt gaggcttggg 660 cacctgtgtg cctgccccgc ttcaaccctg atggtttttt ctacgcctac gtggcccgcc 720 tggatgctat gcctgtctgc ctgctgctgc ttggcaccca acgtgaagcc ttccatgcca 780 tggccgcctg ccggcgcctg gttgaagatg ggatgcatgc ccttggtgcc atgcgtgccc 840 ttggggaggc tgccagcttc tctaatgcct catcagccag tgctcctgcc tacagcgtgc 900 aggctgtcgg ggcgccgggc ctccggcact tcctgtataa gccgctggac atccctgacc 960 accaccgcca actgccccag tttaccagcc ctgagctaga ggccccctac agcagagagg 1020 aggagcggca gcggctgtcg gacctgtacc accgcctgca tgctcgtctc cacagcacct 1080 cccgacccct gcgcctcatt taccacgtgg ctgagaagga gacactactg gcctgggtga 1140 cctccaaatt cgagctctat acctgcctca gccctctggt gaccaaggca ggtgcaatct 1200 tggtagtgac caaactcctg cgctgggtga agaaagagga ggaccggctc ttcattcgtt 1260 acccacccaa gtactccaca ccaccagcca cctctacgga ccaagctgcc cataatggct 1320 tgttcactgg actctgatag ttggagctcc cagaccaggc agtgctggga gcaaccacct 1380 ttgtttttta ccttctgtct accctggaaa tgtgtgtggg ggtgtgtctg tggccagtca 1440 ttgtctccct aagcaatggg gcaaggtctg agggcccacc gatgagagag atggtggcag 1500 ccgacagggg agcaggctgc tttccctgcc cagtcatgca ccttcccctc tggggaaatt 1560 c 1561 <210> 43 <211> 1619 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2681738CB1 <400> 43 tgcgagggcg ggatagctgt ccaaggtctc ccccagcact gaggagctcg cctgctgccc 60 tcttgcgcgc gggaagcagc accaagttca cggccaacgc cttggcacta gggtccagaa 120 tggctacaac agtccctgat ggttgccgca atggcctgaa atccaagtac tacagacttt 180 gtgataaggc tgaagcttgg ggcatcgtcc tagaaacggt ggccacagcc ggggttgtga 240 cctcggtggc cttcatgctc actctcccga tcctcgtctg caaggtgcag gactccaaca 300 ggcgaaaaat gctgcctact cagtttctct tcctcctggg tgtgttgggc atctttggcc 360 tcaccttcgc cttcatcatc ggactggacg ggagcacagg gcccacacgc ttcttcctct 420 ttgggatcct cttttccatc tgcttctcct gcctgctggc tcatgctgtc agtctgacca 480 agctcgtccg ggggaggaag cccctttccc tgttggtgat tctgggtctg gccgtgggct 540 tcagcctagt ccaggatgtt atcgctattg aatatattgt cctgaccatg aataggacca 600 acgtcaatgt cttttctgag ctttccgctc ctcgtcgcaa tgaagacttt gtcctcctgc 660 tcacctacgt cctcttcttg atggcgctga ccttcctcat gtcctccttc accttctgtg 720 gttccttcac gggctggaag agacatgggg cccacatcta cctcacgatg ctcctctcca 780 ttgccatctg ggtggcctgg atcaccctgc tcatgcttcc tgactttgac cgcaggtggg 840 atgacaccat cctcagctcc gccttggctg ccaatggctg ggtgttcctg ttggcttatg 900 ttagtcccga gttttggctg ctcacaaagc aacgaaaccc catggattat cctgttgagg 960 atgctttctg taaacctcaa ctcgtgaaga agagctatgg tgtggagaac agagcctact 1020 ctcaagagga aatcactcaa ggttttgaag agacagggga cacgctctat gccccctatt 1080 ccacacattt tcagctgcag aaccagcctc cccaaaagga attctccatc ccacgggccc 1140 acgcttggcc gagcccttac aaagactatg aagtaaagaa agagggcagc taactctgtc 1200 ctgaagagtg ggacaaatgc agccgggcgg cagatctagc gggagctcaa agggatgtgg 1260 gcgaaatctt gagtcttctg agaaaactgt acaagacact acgggaacag tttgcctccc 1320 tcccagcctc aaccacaatt cttccatgct ggggctgatg tgggctagta agactccagt 1380 tcttagaggc gctgtagtat tttttttttt ttttggctca tccttaggat acttctttta 1440 agtgggagtc tcaggcaact caagtttaga cccttactct ttttgtttgt tttttgaaac 1500 aggatcttgc tctgtcaccc aggcttgagt gcagtggtgc gatcacagcc cagtgcagcc 1560 tcgaccacct gtgctcaagc aatcctccca tctccatctc ccaaagtgct gggatgaca 1619 <210> 44 <211> 3691 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2859482CB1 <400> 44 ggcgagcggc gggcggcgga ggaggagacg gcaggtcgga ctgaccaaat atggaagtgt 60 gcaagtcttg aacacattag tttatacaat gtggcctgaa agtttccctc ctctttccaa 120 atcaaccata atattctgag gcattaaaaa atatttaatc attcatgtgt tgagactcat 180 tcttgagtta tggatgacaa ggcttctgtt ggaaaaatca gtgtctcttc agactcagta 240 tctactctta atagtgaaga ttttgtcttg gtttccaggc aaggagatga gacaccatct 300 acaaataatg gaagtgatga tgagaaaaca ggactcaaga ttgtagggaa tggaagtgaa 360 cagcagctgc aaaaagagct agcagatgta ctgatggatc ctccaatgga cgaccagcca 420 ggggaaaagg agcttgtgaa aaggtcacaa ctggatggtg aaggagatgg gcctctttct 480 aatcagctct ccgcttcatc caccattaac cctgtgccat tagtagggct ccaaaaacca 540 gagatgagcc taccagtgaa acctggacaa ggagattctg aagcttcaag tcctttcaca 600 ccagtggccg atgaggacag cgtagttttc agtaaactga cttacttagg ctgtgcctcg 660 gtaaatgctc ccaggagtga agtggaagcc ttaaggatga tgtccatctt aagaagccag 720 tgtcagattt cactagatgt taccctttca gtgccgaatg tgtctgaagg aattgtgaga 780 ctcttagatc ctcagacaaa cactgaaata gcaaactacc ctatctacaa aatcctcttc 840 tgtgtcagag ggcatgatgg aactcctgag agtgactgtt ttgctttcac tgaaagtcat 900 tacaatgcag agctcttcag aatacacgtc ttccggtgtg aaatacaaga agctgtaagc 960 cggatacttt acagttttgc cactgccttc cgccgttctg ccaagcagac cccactttca 1020 gccactgctg caccccagac tcctgacagt gacatcttta ccttctctgt gtctttagaa 1080 ataaaagaag atgatggtaa aggttatttt agtgcagttc ccaaagataa ggacagacag 1140 tgctttaaac tacgccaagg aattgataag aagattgtca tctatgtgca gcaaacaact 1200 aataaagaac ttgccattga aaggtgtttt ggtcttctcc ttagtccagg aaaagatgta 1260 cgaaatagtg acatgcactt attagatttg gaatctatgg gcaaaagttc agatggaaag 1320 tcgtatgtta ttacggggag ctggaatcca aaatccccac attttcaagt tgtaaatgaa 1380 gaaactccta aagataaagt cctgtttatg accacagctg tagatttggt aataacagaa 1440 gtacaggagc ctgttcgatt tctcctggag acaaaagtcc gcgtttgctc acctaatgaa 1500 agattattct ggcccttcag caaacgtagt actactgaaa atttcttttt gaaactaaaa 1560 cagataaagc aaagggagag aaagaataat actgacactt tatatgaagt tgtatgcttg 1620 gaaagtgaat cagaaagaga gaggaggaaa actacagcca gtccttcagt tcgcctgcca 1680 cagtctggat cgcaaagttc agtgatacct tctcctccag aagatgatga agaggaagat 1740 aatgatgaac ctctcctgag tggatctggt gatgtatcca aagaatgtgc agaaaaaatt 1800 cttgaaacat ggggagaact gttgtcaaaa tggcatctca acttgaatgt gagaccgaag 1860 cagttgtcat ccttagtaag aaacggtgtc cctgaagctc ttcgaggaga agtctggcag 1920 ctgctagcag gctgtcataa caatgaccac ctggtagaga aataccgcat tcttatcaca 1980 aaggagtctc cccaggacag tgctatcacc cgggatatta accgaacatt cccagcccat 2040 gactacttta aggacacagg aggagatgga caagattcct tatataaaat atgcaaggct 2100 tattctgtgt atgatgaaga gattggttat tgccagggcc agtcatttct tgctgctgtg 2160 ctccttctcc atatgcctga agaacaggca ttcagtgttc tggtcaagat catgtttgac 2220 tatgggctca gggaactttt caagcaaaac ttcgaagatt tgcattgcaa attttaccag 2280 ttggagcgcc tcatgcagga atacattcct gacctgtaca accacttcct ggatataagc 2340 cttgaagcac acatgtatgc ctcccagtgg tttcttactc ttttcactgc aaaattccct 2400 ctctacatgg tcttccatat catcgacctg cttttatgtg agggaataag tgttattttt 2460 aatgtcgccc ttggattatt aaagacttcg aaagatgacc tgctgttgac agactttgaa 2520 ggtgccttga agttctttag ggttcagctt cctaagagat accgctcaga agaaaatgca 2580 aaaaaactaa tggaattagc ctgcaacatg aagattagtc agaagaagtt gaaaaaatac 2640 gagaaagaat atcacaccat gagggaacag caggcccagc aagaagaccc catcgagcga 2700 tttgagcggg agaataggcg tctacaagaa gctaacatga ggttggaaca ggaaaacgat 2760 gacttagccc atgagctggt gaccagcaag attgcactac ggaaggacct ggataacgct 2820 gaggaaaagg cagatgctct gaataaggag ctgctgatga ccaaacagaa gttgattgat 2880 gcagaagaag agaaaagacg gctggaagaa gagtctgctc agttaaaaga aatgtgccgt 2940 cgggaactcg acaaggcaga atctgagatt aaaaaaaaca gttctatcat tggtgactat 3000 aagcagattt gttctcagtt gagtgaaaga ttggagaagc agcagacagc caataaggtg 3060 gaaattgaga aaattcggca aaaagtggat gactgtgagc ggtgccggga atttttcaac 3120 aaagaagggc gtgtaaaagg cataagctca accaaggagg ttttagatga ggacacggat 3180 gaagagaaag agacgctcaa gaaccagctg agagaaatgg agctagaact ggcacagacc 3240 aaactccagc tggtggaggc cgagtgtaag atacaggact tggaacacca tttagggctt 3300 gccctcaatg aggtgcaggc agccaagaag acgtggttta accgaacact gagctccata 3360 aagacagcaa ccggggttca agggaaagag acttgctgag agcagctgcc gcctcccgac 3420 accttcagaa aacacgacac cttttgttgc cttctttggc cagatgtgtg attctgtgac 3480 ttgtcccagg accagaatgt acctaagtca gatccataga cgcatgttgg taggtcactg 3540 gaccagagct tgtgaagcag gcaacctctg gggtaagact actgatacta acaggcctgc 3600 tagctcagcc gacgctctgg acactctaga aatcactcct cagtgtgacc tcccaggcct 3660 cttccccgtg tacgtcaaca cctcacccag c 3691

Claims (28)

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

a) an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID
NO:22, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:22, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ 1D NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, and SEQ ID NO:22, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, and SEQ ID NO:22.

2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID
NO:22.

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 selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, and SEQ ID NO:44.

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

11. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide sequence selected from the group consisting of SEQ ID
NO:23, SEQ ID
NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID
NO:44, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ
ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID
NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID
NO:44, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).

12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 11.

13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, 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.

14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.

15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, 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.

16. A pharmaceutical composition comprising an effective amount of a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

17. A pharmaceutical composition of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ
ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ 1D
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:22.

18. A method for treating a disease or condition associated with decreased expression of functional RECAP, comprising administering to a patient in need of such treatment the pharmaceutical composition of claim 16.

19. A method for 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.

20. A pharmaceutical composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.

21. A method for treating a disease or condition associated with decreased expression of functional RECAP, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 20.

22. A method for 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.

23. A pharmaceutical composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.

24. A method for treating a disease or condition associated with overexpression of functional RECAP, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 23.

25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of:

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.

26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said 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.

27. A method for 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, and b) detecting altered expression of the target polynucleotide.

28. 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 of claim 11 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 11 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.