JP2003505083A - Receptors and related proteins - Google Patents

Abstract

  (57) [Summary] The present invention provides human receptors and related proteins (RECAP) and polynucleotides that identify and encode RECAP. The invention also provides expression vectors and host cells, antibodies, agonists, antagonists. Further, the present invention provides a method for diagnosing, treating, and preventing a disease associated with RECAP expression.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to nucleic acid sequences and amino acid sequences of receptors and related proteins, and also relates to immune disorders including neurological diseases, autoimmune diseases / inflammatory diseases, and cell proliferation abnormalities including cancer. It relates to the use of these sequences in diagnosis, treatment and prevention.

BACKGROUND OF THE INVENTION The term "receptor" refers to a protein that specifically recognizes other molecules. The category is broad and includes proteins with different functions. Most receptors are cell surface proteins that bind extracellular ligands and drive cellular responses in growth, differentiation, endocytosis, and immune response. Other receptors promote the selective transport of proteins from the endoplasmic reticulum, localizing the enzyme to specific sites on the cell. Signaling, protein trafficking and localization within cells all depend on the interaction of the receptor with various related proteins. The term "receptor"
Also refers to proteins that bind ligands with known or unknown chemical compounds and interact with other intracellular components. For example, the steroid hormone receptor is DN
It binds to A and regulates its DNA transcript.

Cell surface receptors are usually integral 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, tachykinin, and circulatory-mediated signaling molecules. Cell surface proteins on immune system cells are antigens, antibodies, and major histocompatibility complex (MHC)
Recognize bound peptides. Other cell surface receptors bind to the ligand and are taken up intracellularly. Such receptor-mediated endocytosis is associated with low density lipoprotein (LDL), transferrin, glucose terminal glycoprotein, mannose terminal glycoprotein, galactose terminal glycoprotein, immunoglobulin, phosphovitellogenin, fibrin, proteinase inhibitor. Occurs during uptake of complex, plasminogen activator, and thrombospondin (Lodish, H. et al. (1995) Molecular Cell Biology, Scientific Ame
rican Books, New York NY, p. 723; and Mikhailenko, I. et al. (1997) J. Biol.
Chem. 272: 6784-6791).

Signal transduction is one of the processes of biochemical phenomena whereby cells transduce each other and respond to extracellular signals. Extracellular signals are transmitted by a biochemical cascade that begins with the binding of signaling molecules to cell membrane receptors. This signal is transmitted to the effector molecule by the intracellular signal transduction protein, and finally the intracellular target molecule is activated. The process of signal transduction regulates a variety of cellular functions, including cell proliferation, differentiation, and gene transcription.

G Protein-Coupled Receptors (GPCRs) G protein-coupled receptors (GPCRs) are a class of molecules involved in signal transduction in various cell types. GPCRs are integral membrane proteins characterized by a hydrophobic seven-transmembrane domain that spans the cell membrane and forms a bundle of antiparallel α-helices. The sizes of these proteins range from less than 400 to 100
0 or more amino acids (Strosberg, AD (1991) Eur. J. Biochem. 196: 1-
10; Coughlin, SR (1994) Cuff. Opin. Cell Biol. 6: 191-197). The amino terminus of GPCRs is extracellular and their length is variable and often glycosylated. Its carboxyl terminus is intracellular and is usually phosphorylated. The extracellular loops of the GPCR alternate with the intracellular loops, connecting the transmembrane domains. GPCR
The most conserved domains of are the transmembrane domain and the first two cytoplasmic loops. The transmembrane domain governs the structural and functional properties of its receptor. In most cases, a bundle of α-helices forms the binding pocket. In addition, the extracellular N-terminal segment, or one or more of the three extracellular loops, may be involved in binding the ligand. Binding to the ligand alters the structure of the intracellular part of the receptor, which activates the receptor. The activated receptor then interacts with the intracellular heterotrimeric guanine nucleotide (G) protein complex. This protein complex mediates the production of second messengers, such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the intracellular signal transduction that interacts with ion channel proteins (Baldwin). , JM (1994) Curr. Opin. Cell Biol. 6: 180-190; W
atson, S. and S. Arkinstall (1994) The G-protein Linked Recentor Facts Book , Academic Press, San Diego CA, pp. 2-6). Hydrolysis of bound GTP by the G protein completes the cycle in which the G protein returns to its inactive, GDP-bound state.

[0006] GPCRs include a variety of receptors. Among them are sensory signal mediators (eg light and olfactory stimulators), adenosine, bombesin, bradykinin, endothelin, γ-aminobutyric acid (GABA), hepatocyte growth factor, luteinizing hormone (LH).
), Thrombin, thyroid stimulating hormone (TSH), melanocortin, neuropeptide Y
, Opioid peptides, opsin, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin, and biogenic amines (eg, dopamine, epinephrine, norepinephrine, histamine, glutamic acid (metabotropic), acetylcholine (muscarinic), and Serotonin), chemokines, lipid mediators of inflammation (eg prostaglandins and prostanoids, platelet activating factors, and leukotrienes), peptide hormones (eg calcitonin, C5a anaphylatoxin, follicle stimulating hormone (FSH),
It contains receptors for gonadotropin-releasing hormone (GnRH), neurokinin, thyroid-stimulating hormone-releasing hormone (TRH), and oxytocin). GPCRs that act as receptors for unidentified stimuli are called orphan receptors. For example, the TPRA40 protein is a GPCR isolated from mouse adipocytes and is present in multiple mouse tissues and human tissues. Expression of this protein in adipose tissue is altered by aging and type II diabetes (Yang, H. (1999) Endocrinol.
ogy 140: 2859-2867).

Mutations in GPCRs that can cause dysfunction and constitutive activation are associated with various human diseases (Coughlin, supra). For example, retinitis pigmentosa can be caused by mutations in the rhodopsin gene. Rhodopsin is a retinal photoreceptor located in the outer segment of retinal rod cells. Parma, J. et al. Have reported that somatic mutations in the thyroid-stimulating hormone receptor cause hyperactive thyroid adenomas and are prone to constitutive activation, and some GPCRs may act as proto-oncogenes. Reported (1993
, Nature 365: 649-651). Elevated levels of TSH receptors have been observed in Down's syndrome and Alzheimer's disease brain tissue, suggesting that this receptor plays a role in apoptosis in neurodegenerative diseases (Labudova, O. et al. (1999) Lif.
e Sci. 64: 1037-1044). Α and β affect the activity of adrenergic receptors
Clinically relevant agents, including blockers, act on GPCRs and are therefore used to treat hypertension and other cardiovascular diseases (Watson, pp. 32-33, supra).

Receptors Involved in the Immune System Examples of GPCRs involved in inflammation and immune responses include the mucin-like hormone receptor (Emr1) containing the EGF module and the CD97 receptor protein. These GPCRs are members of the EGF-TM7 receptor family whose characteristics have recently been elucidated. These 7-transmembrane hormone receptors exist in vivo as heterodimers and contain 3 to 7 potential calcium-binding EGF-like motifs. CD97 is mainly expressed on leukocytes and is significantly upregulated on activated B and T cells (
McKnight, AJ and Gordon, S. (1998) J. Leukoc. Biol. 20 63: 271-280).

Abnormalities in the GPCR signaling cascade can lead to abnormal activation of leukocytes and lymphocytes, leading to tissue damage and destruction in various inflammatory and autoimmune diseases. Examples of such diseases are rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis. Brain, thyroid, adrenal gland,
And aberrant cell proliferation, including cyclic AMP stimulation in gonadal tissues, is regulated by G proteins (Meij, JTA (1996) Mol. Cell. Biochem. 157:
31-38; Aussel, C. et al. (1988) J. Immunol. 140: 215-220).

T cells play two roles in the immune system as effectors and regulators. That is, it recognizes an antigen, induces cell death in infected cells, and transmits signals that stimulate other immune cells. T cells collectively recognize a variety of antigens, but clonal strains of T cells recognize only one type of antigen. In addition, the antigen must be presented to the T cell receptor as a complex of major histocompatibility complex (MHC) and peptide on the surface of antigen presenting cells. The TCR on most T cells consists of two polypeptide subunits, α and β. These subunits are immunoglobulin-like integral membrane glycoproteins with similar molecular weights. The TCRα and TCRβ subunits have an extracellular domain containing both variable and non-variable regions, a transmembrane domain that spans the membrane once, and a short intracellular domain (
Saito, H. et al. (1984) Nature 309: 757-762). The gene for the TCR subunit is formed by the rearrangement of different gene segments in somatic cells. The interaction of TCR with antigens in the proper location of MHC initiates a signaling cascade that induces cell proliferation, maturation, and cellular components of the immune system (Weiss, A. (1991).
Annu. Rev. Genet. 25: 487-510). Rearrangements in the TCR gene and altered TCR expression are found in lymphomas, leukemias, autoimmune diseases, and immunodeficiencies (Aisenber
g, AC et al. (1985) N. Engl. J. Med. 313: 529-533; Olive, C. (1995) Immunol
Cell. Biol. 73: 297-307; and Weiss, supra). Immunization with peptides derived from TCR is effective in treating certain human T-cell mediated autoimmune diseases and in animal models of such diseases, especially rheumatoid arthritis (Bridges, SL and
Moreland, LW (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 intracellular response initiated by TNF is distinctly different from TNF-R1
And TNF-R2, two cell surface receptors that interact with TNF (Tarta
glia, LA and Goeddel, DV (1992) Immunol. Today 13: 151-153). Both T
The NF receptor is a member of the TNF receptor (TNFR) superfamily. This TNF
Members of the R family also include Fas antigen, p75 subunit of NGF receptor, TRAIL receptor, TRUNND, SalF19R, CD27, CD30, and CD40. Members of the TNFR superfamily are TNFRs that consist of cysteine-rich pseudo-repeats of the extracellular domain.
/ NGFR family sharing high cysteine region signature (ExPASy PROSITE reference PDOC00561; Pan, G. et al. (1998) FEBS Lett. 424: 41-45; Bairoch, A. et al. (19
97) Nucleic Acids Res. 25: 217-221; and Smith, CA et al. (1994) Cell 76: 959.
-962). Polymorphisms in TNF-R2 are associated with systemic lupus erythematosus (Ko
mata, T. et al. (1999) Tissue Antigens 53: 527-533). In addition, elevated serum concentrations of soluble TNF-R1 were observed in patients with end-stage gastric or colorectal cancer (Shibata,
M. et al. (1998) Surg. Today 28: 884-888).

The complement system is another essential component of the immune response. The complement system responds to signals generated by antigen recognition by recruiting effector effects including inflammation, phagocytosis, and cytolysis. Receptors on macrophages and neutrophils bind to activated complement C3 on the surface of foreign particles such as bacteria, which causes them to be phagocytosed and degraded by lysosomal enzymes. Complement receptor 1 (CR1)
It is widely distributed in tissues and mediates promotion of phagocytosis, induction of IL-1 secretion, and promotion of B cell differentiation. Defective expression of CR-1 is associated with the autoimmune disease systemic lupus erythematosus (Carroll, MC (1998) Annu. Rev. Immunol. 16: 545-568).

Nuclear Receptors Nuclear receptors are another family of receptors, including the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). RAR and RXR form a heterodimer believed to have a signal transduction function. Retinoic acid (RA) is a vitamin A (
It is a biologically active metabolite of retinol) and a fat-soluble vitamin found in fish liver oil, liver, egg yolk, butter and cream. Retinol cannot be synthesized in vivo and must be taken from the diet. Retinol, RA,
And other retinoids affect the differentiation of epithelial cells. Several carrier proteins have been identified that bind retinol and other retinoids. These retinoid binding proteins (RBPs) are thought to directly bind retinoid molecules to specific metabolic pathways. RBP-specific receptors mediate intracellular uptake of retinoids and transport of retinoids into intracellular RBP (Sundaram, M.
(1999) J. Biol. Chem. 273: 3336-3342).

Low Molecular Weight (LMW) G Proteins Low molecular weight (LMW) G proteins control cell growth, cell cycle regulation, protein secretion, and intracellular vesicle interaction. The LMW G protein consists of a single polypeptide capable of binding to GTP and hydrolyzing it, thus cycling between inactive and active states. The LMW G protein responds to extracellular signals from the receptor and transduces mitogenic signals involved in various cellular functions to activate the protein. GTP binding and hydrolysis regulate the LMW G protein response and are an energy source in this process (Bokoch, GM and Der, CJ (1993
) FASEB J. 7: 750-759).

At least six members of the LMW G protein superfamily have been identified and are currently classified into the ras, rho, arf, sar1, ran, and rab subfamilies. The activated ras gene was first identified in human cancer tissue. Later studies found that the function of ras is crucial for the receptor tyrosine kinase-mediated signaling pathways that determine whether cells continue to grow, divide, or differentiate. Rho G protein is required for normal cell growth and division,
Controls the signaling pathways that connect growth factor receptors to actin polymerization. ra
The b, arf, and sar1 family of proteins translocate vesicles into and out of membranes for protein localization, protein processing, and secretion. Ran G protein is located in the nucleus, import of nuclear proteins, DNA
Plays an important role in the regulation of synthesis and cell cycle progression (Hall, A. (1990) Sc
ience 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).

The LMW G protein is a G that circulates between active and inactive GTP-bound forms.
It is TPase. At least three proteins control this process. The three proteins are GTP that promotes GTP hydrolysis by LMW G protein.
Ase-activating protein (GAP), a guanine nucleotide exchange factor that promotes the exchange of GDP bound to the LMW G protein for GTP, and a guanine nucleotide dissociation inhibitor (GDI) that suppresses this reaction (Ikeda, M. et al. 1998) J. Biol.
Chem. 273: 814-821; Quilliam, LA (1995) Bioessays 17: 395-404). The most characterized GEP is the mammalian homologue of the Drosophila Son-of-Sevenless protein. Both GEP and GAP activities are affected by extracellular stimuli,
It can be modified with accessory proteins such as RalBP1 and POB1. Mutated Ras family proteins that bind to GTP but cannot hydrolyze are constitutively activated and cause cell proliferation or cancer in the same way that GEP activates the LMW G protein (Drivas, GT et al. (1990 ) Mol. Cell. Biol. 1
0: 1793-1798; and Whitehead, IP et al. (1998) Mol Cell Biol. 18: 4689-4697.
).

Olfactory GPCRs Another large subfamily of GPCRs is the olfactory receptors. These receptors, like other GPCRs, have a hydrophobic seven-transmembrane domain and function when odorant signals are transduced by G proteins. Each olfactory neuron has only one
Expresses only one type of olfactory receptor. Distinct spatial areas of neurons expressing different receptors are found in the nasal cavity.

With the discovery of new receptors and related proteins and polynucleotides encoding them, novel novel useful for diagnosis / treatment / prevention of abnormal cell proliferation, autoimmune diseases / inflammatory diseases, and neurological diseases. By providing such a composition, the needs in the art are satisfied.

(Summary of Invention) The present invention is generically referred to as “RECAP”, and individually as “RECAP: 1” and “RECAP: 2”, respectively.
, "RECAP: 3", "RECAP: 4", "RECAP: 5", "RECAP: 6", "RECAP: 7", "REC
"AP: 8", "RECAP: 9", "RECAP: 10", "RECAP: 11", "RECAP: 12", "RECAP: 1"
3 "," RECAP: 14 "," RECAP: 15 "," RECAP: 16 "," RECAP: 17 "," RECAP: 18 "
, "RECAP: 19", "RECAP: 20", "RECAP: 21""RECAP:22" and "RECAP: 22"
Purified polypeptide that is a receptor and related proteins referred to as "."
In one embodiment of the invention: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 22 (SEQ ID NO: 1-22), and (b) consisting of SEQ ID NO: 1-22 A natural amino acid sequence having 90% or more sequence identity with an amino acid sequence selected from the group, and (c) a biologically active fragment of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-22. And (d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-22, and an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: .. Alternatively, an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-22 is provided.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. %
A natural amino acid sequence having the above sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID NO:
An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: an immunogenic fragment of the amino acid sequence selected from the group consisting of: 1-22. Alternatively, the polynucleotide is SEQ ID NO: 1.
It encodes a polypeptide selected from the group consisting of -22. Alternatively, the polynucleotide is selected from the group consisting of SEQ ID NOs: 23-44.

Further, the present invention provides (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. 90
A native amino acid sequence having a sequence identity of greater than or equal to%, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID
A promoter sequence operably linked to a polynucleotide encoding a polypeptide containing an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of NO: 1-22. Recombinant polynucleotides comprising are provided. Alternatively, the invention provides a cell transformed with this recombinant polynucleotide. In a further alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

The present invention also includes (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. 90
A native amino acid sequence having a sequence identity of greater than or equal to%, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID
Provided is a method for producing a polypeptide containing an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of NO: 1-22. This method comprises: (a) culturing cells transformed with a recombinant polynucleotide containing a promoter sequence operably linked to a polynucleotide encoding the polypeptide under conditions suitable for expression of the polypeptide. Steps to do, (b)
Recovering the polypeptide so expressed.

Furthermore, the present invention provides (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. 90
A native amino acid sequence having a sequence identity of greater than or equal to%, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID
An isolated antibody that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of NO: 1-22 .

Further, the present invention provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44 and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44. A natural polynucleotide sequence having 70% or more sequence identity with the sequence, (c) a polynucleotide sequence complementary to (a) above, (d) a polynucleotide sequence complementary to (b) above, (E) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of the RNA equivalents of (a) to (d) above is provided. Alternatively, the polynucleotide comprises at least 60 contiguous nucleotides.

The present invention further provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44, and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44. A natural polynucleotide sequence having a sequence identity of 70% or more with (c) a polynucleotide sequence complementary to (a) above, (d) a polynucleotide sequence complementary to (b) above, ( e) A method for detecting in a sample a target polynucleotide having a polynucleotide sequence containing a polynucleotide sequence selected from the group consisting of the RNA equivalents of (a) to (d) above. This method comprises the step of: (a) hybridizing the sample with a probe containing at least 20 consecutive nucleotides forming a sequence complementary to the target polynucleotide in the sample, wherein the probe and the target polynucleotide The step of specifically hybridizing the probe to the target polynucleotide under conditions in which a hybridization complex is formed with nucleotides or fragments thereof; and (b) the presence or absence of the hybridization complex. And optionally determining its yield if present. Alternatively, the probe comprises at least 60 contiguous nucleotides.

Further, the present invention provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44 and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44. A natural polynucleotide sequence having a sequence identity of 70% or more with (c) a polynucleotide sequence complementary to (a) above, (d) a polynucleotide sequence complementary to (b) above, ( e) A method for detecting in a sample a target polynucleotide having a polynucleotide sequence containing a polynucleotide sequence selected from the group consisting of the RNA equivalents of (a) to (d) above. The method comprises: (a) amplifying the target polynucleotide or fragment thereof using polymerase chain reaction amplification; and (b) detecting the presence of the amplified target polynucleotide or fragment thereof. Optionally, measuring the yield, if present.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. %
A natural amino acid sequence having the above sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID NO:
An effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of: 1-22 and a suitable pharmaceutical excipient. A pharmaceutical composition is provided. In one embodiment, there is provided a pharmaceutical composition comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. Furthermore, the present invention provides a method for treating a disease associated with decreased expression of functional RECAP or a symptom thereof, which comprises administering this pharmaceutical composition to a patient.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. %
A natural amino acid sequence having the above sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID NO:
The present invention provides a method for screening a compound effective as an agonist of a polypeptide containing an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of: 1-22. The method comprises the steps of (a) exposing a sample containing the polypeptide to a compound, and (b) detecting agonist activity of the sample. Alternatively, the invention provides a pharmaceutical composition comprising an agonist compound identified by this method and a suitable pharmaceutical excipient. In a further alternative, the invention comprises the administration of this pharmaceutical composition to a patient,
Disclosed is a method for treating a disease associated with reduced expression of functional RECAP and its symptoms.

Furthermore, the present invention provides (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. 90
A native amino acid sequence having a sequence identity of greater than or equal to%, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID
A method for screening a compound effective as an antagonist of a polypeptide containing an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of NO: 1-22 . The method comprises the steps of (a) exposing a sample containing the polypeptide to a compound, and (b) detecting antagonist activity of the sample. Alternatively, the invention provides
There is provided a pharmaceutical composition comprising an antagonist compound identified by this method and a suitable pharmaceutical excipient. In a further alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional RECAP, which comprises administering to a patient this pharmaceutical composition.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. %
A natural amino acid sequence having the above sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID NO:
A method for screening a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of: an immunogenic fragment of the amino acid sequence selected from the group consisting of: 1-22. The method comprises the steps of (a) binding the polypeptide to at least one compound under suitable conditions, and (b) detecting the binding of the polypeptide to the test compound to specifically bind the polypeptide. Identifying compounds that bind to.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22. %
A natural amino acid sequence having the above sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and (d) SEQ ID NO:
A method for screening a compound that regulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of: 1-22 and an immunogenic fragment of the amino acid sequence selected from the group consisting of This screening method comprises: (a) binding the polypeptide to at least one compound under conditions that permit its activity, and b) assessing the activity of the polypeptide in the presence of the test compound. The step of: (c) comparing the activity of this polypeptide in the presence of this test compound with the activity of this polypeptide in the absence of this test compound, in the presence of this test compound. The change in the activity of this polypeptide in γ is characteristic of suggesting the presence of a compound that regulates the activity of this polypeptide.

The invention further provides a method of screening for compounds that are effective in altering the expression of a target polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 23-44, comprising: (a) Provided is a screening method comprising exposing a sample containing the target polynucleotide to a compound, and (b) detecting a change in the expression of the target polynucleotide.

The invention further provides a method of assessing the toxicity of a test compound. This method
(A) treating a biological sample containing nucleic acid with the test compound, and (b)
Hybridizing nucleic acid of the treated biological sample with a probe,
(C) measuring the yield of the hybridization complex, and (d) comparing the yield of the hybridization complex in the treated biological sample with the yield of the hybridization complex in the untreated biological sample. And the difference in yield of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. The probe in this method comprises (1) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44, and (2) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44. A native polynucleotide sequence having at least 70% sequence identity, (3
) A polynucleotide sequence complementary to (1) above, (4) a polynucleotide sequence complementary to (2) above, and (5) an RNA equivalent of (1) to (4) above. It comprises at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group. In addition, the hybridization is performed under the condition that a specific hybridization complex is formed between the probe and the target polynucleotide of the biological sample. The target polynucleotide is (1) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44, and (2) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 23-44. A natural polynucleotide sequence having a sequence identity of at least 70% with (3) a polynucleotide sequence complementary to (1) above, (4) above (
A polynucleotide sequence complementary to 2) and (5) the RNA equivalent of (1) to (5) above. Alternatively, the target polynucleotide is a fragment of the polynucleotide sequence.

(Description of the Present Invention) Before describing the protein and nucleic acid sequences and methods of the present invention, the present invention is not limited to the specific devices and materials and methods disclosed herein, and its embodiments are modified. Please understand what you can do. Also, the terminology used herein is for the purpose of describing particular embodiments only and is limited only by the scope of the following claims,
It should also be understood that it is not intended to limit the scope of the invention.

In the present specification and claims, “a”, “the (these, etc.)” represent the singular form.
Note that includes plural forms unless the context clearly indicates otherwise. Thus, for example, “a host cell” includes a plurality of host cells, “antibody” includes a plurality of antibodies, and equivalents well known to those skilled in the art.

All scientific and technical terms used herein, unless defined otherwise,
It has the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.
Although any apparatus and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, suitable apparatus and materials and methods are described herein.
All references cited herein are cited to describe and disclose the cell lines, protocols, reagents, vectors described in the references that may be used in connection with the present invention. The mere fact that the conventional invention is cited does not mean that the novelty of the present invention is impaired.

Definitions The term “RECAP” is obtained substantially from all species including natural, synthetic, semi-synthetic or recombinant (especially mammals including bovine, ovine, porcine, murine, equine and human). It refers to the amino acid sequence of purified RECAP.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of RECAP. This agonist either interacts directly with RECAP or acts with a component of the biological pathway in which RECAP is involved to regulate the activity of RECAP, including proteins, nucleic acids, carbohydrates, small molecules, any other compound or The composition may be included.

The term “allelic variant” refers to another form of the gene that encodes RECAP. An allelic variant sequence results from at least one mutation in a nucleic acid sequence,
Alternatively, it may be a mutated polypeptide, and its structure and function may or may not change. Some genes include those in which no naturally occurring allelic variant sequence is present, or one gene or many genes are present. Generally, mutations that give rise to allelic variants are
By natural deletion, addition, or substitution of nucleotides. Each of these mutations, either alone or concurrently with other mutations, occurs one or more times within a given sequence.

“Mutant” nucleic acid sequences encoding RECAP include various nucleotide deletions, insertions,
Alternatively, even if substitution occurs, it refers to the same polypeptide as RECAP, or a polypeptide having at least one functional property of RECAP. This definition includes inappropriate or unexpected hybridization of a RECAP-encoding polynucleotide sequence with an allelic variant at a position other than the normal chromosomal locus, as well as specific oligonucleotide probes of the RECAP-encoding polynucleotide. Includes polymorphisms that are easily or difficult to detect using. The encoded protein may also be mutated and may contain deletions, insertions, or substitutions of amino acid residues which result in silent changes and are functionally equivalent to RECAP. Intentional amino acid substitution is biologically or immunologically
To the extent that RECAP activity is retained, it can be based on similarities of residues for polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity. For example, negatively charged amino acids can include aspartic acid and glutamic acid, and positively charged amino acids can include lysine and arginine. Amino acids with similar hydrophilicity values and having polar uncharged side chains can include asparagine, glutamine, serine, threonine. Amino acids with similar hydrophilicity values and uncharged side chains include:
Leucine, isoleucine, valine, glycine, alanine, phenylalanine and tyrosine may be included.

The terms “amino acid” and “amino acid sequence” refer to oligopeptides, peptides, polypeptides, protein sequences, or any fragment thereof, including naturally occurring and synthetic molecules. When "amino acid sequence" refers to the sequence of a naturally occurring protein molecule,
"Amino acid sequence" and like terms are not intended to limit the amino acid sequence to the complete and intact amino acid sequence associated with the protein molecule described.

The term “amplification” refers to making copies of nucleic acid sequences. Amplification is generally performed by the polymerase chain reaction (PCR) technique well known in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of RECAP. Antagonists are antibodies, nucleic acids, carbohydrates, that interact directly with RECAP or interact with components of the biological pathways in which RECAP is involved to regulate RECAP activity.
It may include proteins such as small molecules, any other compound or composition.

The term “antibody” refers to intact molecules such as Fab and F (ab ′) ₂ and fragments and Fv fragments thereof that are capable of binding antigenic determinants. Antibodies that bind RECAP polypeptides can be produced using intact molecules, or fragments thereof, containing small peptides that immunize the antibody. The polypeptide or oligopeptide used to immunize an animal (eg, mouse, rat, or rabbit) can be synthesized from the translation of RNA or chemically and is optionally conjugated with a carrier protein. It is also possible. Commonly used carriers that are chemically linked to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemonean (KLH). The conjugated peptide is then used to immunize the animal.

The term “antigenic determinant” refers to the region (ie, epitope) of a molecule that makes contact with a particular antibody.
Refers to. When a protein or a fragment of a protein is used to immunize a host animal, various regions of this protein are antibodies that specifically bind to an antigenic determinant (a specific region on the protein or a three-dimensional structure). Can induce the production of The antigenic determinant may compete with the intact antigen (ie, the immunogen used to elicit an immune response) to bind the antibody.

As used herein, "antisense" refers to any composition capable of base pairing with the sense (coding) strand of a particular nucleic acid sequence. DNA in the antisense component
, RNA, peptide nucleic acid (PNA), and modified backbone such as phosphorothionate, methylphosphonate, and benzylphosphonate
e-oligonucleotide), an oligonucleotide having a modified sugar such as 2'-methoxyethyl sugar or 2'-methoxyethoxy sugar, and 5-methylcytosine or 2'-deoxyuracil, 7-deaza-2 '. -Oxynucleotides with modified bases such as deoxyguanosine may be included. Antisense molecules can be produced by any method, including chemical synthesis and transcription. Once introduced into a cell, the complementary antisense molecule base pairs with the natural nucleic acid sequence produced by the cell to form a duplex and inhibit transcription or translation. The expression "negative" or "minus" is the antisense strand, and the expression "positive" or "plus" is the sense strand.

The term “biologically active” refers to a protein having the structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, the term "immunologically active" or "immunogenic" means that natural or recombinant RECAP, synthetic RECAP, or any oligopeptide thereof, is responsible for the specific immune response of a suitable animal or cell. Refers to the ability to induce and bind to a particular antibody.

The term “complementary” refers to the relationship between two single-stranded nucleic acid sequences that anneal by base pairing. For example, the sequence "5'AGT3 '" is paired with the complementary sequence "3'TCA5'".

“Composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” refers broadly to any composition that comprises a given nucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution.
A composition comprising a polynucleotide sequence encoding a fragment of RECAP or RECAP,
It can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be bound to a stabilizer such as sugar.
In hybridization, the probe is developed in an aqueous solution containing salts (eg, NaCl) and detergents (eg, SDS: sodium dodecyl sulfate) and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). obtain.

The “consensus sequence” is obtained by repeatedly analyzing the DNA sequence in order to separate unnecessary bases, and 5 ′ and / or 5'and / or using the XL-PCR kit (PE Biosystems, Foster City CA).
Or nucleic acid sequence extended in the 3'direction and re-sequenced, or GEL
VIEW Fragment Construction System (GCG, Madison, WI) or Phrap (University of Wash
(ington, Seattle WA), etc., and refers to nucleic acid sequences constructed from one or more overlapping cDNAs or ESTs or genomic DNA fragments using a computer program for fragment construction. Some consensus sequences are constructed by both extension and overlap.

The term “conservative amino acid substitution” refers to a substitution that does not significantly alter the properties of the original protein. That is, the substitution does not significantly change the structure or function of the protein,
The structure of the protein, in particular its function, is preserved. The following shows conservative amino acid substitutions in which the original amino acid of one protein is replaced with another amino acid. Original residue Conservative substitution Ala Gly, Set Arg 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 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 In general, in the case of conservative amino acid substitutions, a) the backbone structure of the polypeptide in the substituted region, eg β sheet or α helix conformation, b) the charge of the molecule at the substituted site or Hydrophobic and / or c) most of the side chains are retained.

The term “deletion” refers to a change in an amino acid sequence that lacks one or more amino acid residues, or a change in a nucleic acid sequence that lacks one or more nucleotides.

The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of polynucleotide sequences include, for example, replacement of hydrogen with an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide has at least one biological or immunological function of a natural molecule (unmodified molecule).
Encodes a polypeptide that maintains one. A derivative polypeptide is a polypeptide modified by glycosylation, polyethylene glycolation, or any similar process that maintains at least one of the biological or immunological functions of the original polypeptide. That is.

“Detectable label” refers to a reporter molecule or enzyme that is capable of producing a measurable signal, either covalently or non-covalently attached to a polynucleotide or polypeptide.

The term “fragment” refers to a unique portion of RECAP or a polynucleotide encoding RECAP that is identical to its parent sequence but shorter than that sequence. The maximum length of a "fragment" is 1 nucleotide / from the parent sequence.
It is the length less amino acid residues. For example, a fragment contains 5 to 1000 consecutive nucleotides or amino acid residues. Probes, primers, antigens, therapeutic molecules, or fragments used for other purposes should be at least 5, 10, 15
, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250
Alternatively, the length is 500 consecutive nucleotides or amino acid residues. Fragments may also be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment may comprise a predetermined length of contiguous amino acids selected from the first 250 or 500 amino acids shown in the given sequence (or the first 25% or 50% of the polypeptide). . These lengths are examples, and any length described in the specification including the sequence listing and the tables and drawings can be included in the embodiments of the present invention.

A fragment of SEQ ID NO: 23-44 is, for example, a region of a unique polynucleotide sequence that uniquely identifies SEQ ID NO: 23-44, which differs from other sequences in the genome from which this fragment was obtained. including. Certain fragments of SEQ ID NO: 23-44 are useful, for example, in hybridization and amplification techniques, or similar methods of distinguishing SEQ ID NO: 23-44 from related polynucleotide sequences. The exact fragment length and region of SEQ ID NO: 23-44 that matches a given fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

A “full length” polynucleotide sequence is a sequence that has at least one translation initiation codon (eg, methionine), followed by an open reading frame and a translation stop codon. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence.

“Homology” is a sequence similarity between two or more polynucleotide sequences or between two or more polypeptide sequences. This sequence similarity can be translated into sequence identity.

Certain fragments of SEQ ID NO: 1-22 are encoded by certain fragments of SEQ ID NO: 23-44. Certain fragments of SEQ ID NO: 1-22 contain regions of unique amino acid sequence that specifically identify SEQ ID NO: 1-22. For example, certain fragments of SEQ ID NO: 1-22 are useful as immunogenic peptides for the production of antibodies that specifically recognize SEQ ID NO: 1-22. The exact fragment length or region of SEQ ID NO: 1-22 that matches a given fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. This includes partial similarity and complete similarity. The term “identity” can also be called “similarity”. Partially complementary sequences in which hybridization of the same sequence to the target nucleic acid is at least partially blocked are said to be "substantially similar." Inhibition of hybridization between the perfectly complementary sequence and the target sequence is tested using a hybridization assay (Southern or Northern blotting, solution hybridization, etc.) under mildly stringent conditions. Substantially similar sequences or hybridization probes competitively suppress binding of the perfectly similar (identical) sequence to the target sequence under mildly stringent conditions. This does not mean that non-specific binding is allowed under mildly stringent conditions, but under mild stringent conditions, the binding of two sequences to each other specifically (ie, selectively) with respect to each other. It has to work. A second target sequence, which is also less than partial complementarity (eg, less than 30% similarity or identity), can be used to test for the absence of non-specific binding. In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

The term “percent identity” or “% identity” with respect to polynucleotide sequences refers to matching residues between two or more polynucleotide sequences that are aligned using a standardized algorithm. It is a percentage. Such algorithms can insert gaps in the sequences to make a more meaningful comparison between the two sequences in a standardized and reproducible manner to optimize the alignment between the two sequences.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison W
I). This CLUSTAL V is for Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153, Higgins, DG et al. (1992) CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameter is Kt.
Set uple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. A "weighted" residue weight table was selected as the default. The percent identity is reported by CLUSTAL V as the "percentage similarity" between the aligned polynucleotide sequences.

Alternatively, a set of commonly used and freely available sequence comparison algorithms is
NCBI, Bethesda, MD, and Internet (http://WWW.ncbi.nlm.nih.gov/BLAS
National Center for Biotechnology Information (NCB)
I) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J
Mol. Biol. 215: 403-410). The suite of BLAST software includes various sequence analysis programs, including "blastn" used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool called "BLAST 2 Sequences" is available,
Used to directly compare pairs of two nucleotide sequences. "BLAST 2 Sequen
"ces" can be used interactively by accessing http://WWW.ncbi.nlm.nih.gov/gorf/b12.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). The BLAST program is commonly used with gaps and other parameters that set defaults. For example,
When comparing two nucleotide sequences, one may blast with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set to default parameters.
will use n. Such default parameters are, for example:

Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on For example, it may be measured for the entire length of a given sequence, determined by a particular sequence number, or for shorter lengths, for example, fragments obtained from a certain given sequence, for example, At least 20 or 30 consecutive
, 40, 50, 70, 100, 200 nucleotide fragments. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

Nucleic acid sequences that do not show high identity may still code for similar amino acid sequences due to the degeneracy of the genetic code. It is to be understood that degeneracy can be utilized to alter nucleic acid sequences to produce different nucleic acid sequences each encoding substantially the same protein.

The term “percent identity” or “% identity” as used in polypeptide sequences refers to matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. It is a percentage. Methods of polypeptide sequence alignment are well known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, described in detail above, generally conserve charge and hydrophobicity at the site of substitution, and result in polypeptide structure (and thus function) as well.
Is saved.

The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program (supra). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are Ktuple = 1, gap
Set penalty = 3, window = 5, and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. Similar to polynucleotide alignments, the percent identity of paired aligned polypeptide sequences is reported by CLUSTAL V as the "percentage of similarity."

Alternatively, the NCBI BLAST software suite is used. For example, when making a pairwise comparison of two polypeptide sequences, one would use blastp with the "BLAST 2 Sequences" tool Version 2.0.12 (Apr-21-2000) set with default parameters. . Such default parameters are, 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 is determined, for example, by a specific SEQ ID NO: It may be measured for the entire length of a given polypeptide sequence, or for shorter lengths, eg fragments obtained from one large given polypeptide sequence, eg at least 15, 20 consecutive or It may be measured against the length of a fragment of 30, 40, 50, 70, 150 residues. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

“Human Artificial Chromosome (HAC)” is a DNA of about 6 kb (kilobase) to 10 Mb in size.
A linear minichromosome containing all the elements necessary for the segregation and maintenance of a stable mitotic chromosome, which may include sequences.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been altered in order to resemble a more human antibody while retaining its original binding ability.

“Hybridization” refers to the following hybridization conditions.
A single-stranded polynucleotide is the process of annealing to form base pairs with a complementary single strand. Specific hybridization means that two nucleic acid sequences have high homology. Under conditions that permit annealing, specific hybridization complexes are formed and remain hybridized after the washing process. The washing process is particularly important in determining the stringency or stringency of the hybridization process, and under more stringent conditions, non-specific binding, ie binding of pairs of nucleic acid strands that are not perfectly matched. Is reduced. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those of skill in the art and are constant during hybridization, but the washing process involves changing the conditions during which to achieve the desired stringency. Is possible and hybridization specificity is obtained. Annealing conditions are, for example, at a temperature of 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and about 100 μg.
/ Ml of sheared and denatured salmon sperm DNA.

Generally, the stringency of hybridization also depends on the temperature at which the washing process is performed. The wash temperature is usually selected to be about 5-20 ° C below the thermal melting point (Tm) of the particular sequence at a given ionic strength and pH. This Tm is
It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The formula for calculating the Tm and the conditions for nucleic acid hybridization are well known, Sambrook, J. et al., 1989, Molecular Cloning : A Laboratory Manual , Volume 2 1-3, Cold Spring Harbor Press, Plainvi.
ew NY; Especially described in Chapter 2 of Volume 2.

High stringency hybridization between polynucleotides of the invention involves a wash step of about 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is carried out at temperatures of 65 ° C, 60 ° C, 55 ° C, 42 ° C. SSC concentrations range from about 0.1 to 2x SSC in the presence of about 0.1% SDS. Blocking reagents are usually used to prevent non-specific hybridization. Such blocking reagents include, for example, about 100 to 200 μg / ml of cleaved and denatured salmon sperm DNA. Organic solvents such as formamide at a concentration of about 35-50% v / v can be used in certain cases, such as, for example, RNA-DNA hybridizations. Useful modifications of these wash conditions are well known to those of skill in the art. Hybridization, particularly under highly stringent conditions, may suggest evolutionary similarities between the nucleotides. Such similarities strongly suggest that the nucleotides and encoded polypeptides play a similar role.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by hydrogen bonds between complementary bases. Hybridization complexes may be formed in solution (eg, C ₀ t or R ₀ t analysis), or one nucleic acid sequence in solution and a solid support (eg, paper, membrane, filter, chip, pin). ,
Or a slide glass, or any suitable substrate for immobilizing cells and their nucleic acids)
Can be formed with another nucleic acid sequence fixed to.

The term “insertion” or “addition” refers to a change in an amino acid sequence or nucleic acid sequence in which one or more amino acid residues or nucleotides are added, respectively.

“Immune response” refers to conditions associated with inflammatory diseases and trauma, immune disorders, infectious diseases, genetic diseases and the like. These conditions are characterized by the expression of various factors such as cytokines and chemokines, other signaling molecules that affect the cell and systemic defense systems.

The term “microarray” refers to the composition of multiple polynucleotides, polypeptides or other compounds on a substrate.

The term “element” or “array element” refers to a polynucleotide, polypeptide or other compound that has a uniquely designated position on a microarray.

The term “modulation” refers to a change in the activity of RECAP. For example, modulation results in a change in the protein activity or binding properties of RECAP, or other biological, functional or immunological properties.

The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides, or fragments thereof, single-or double-stranded, sense or antisense strand of genomic origin. Or DNA or RNA of synthetic origin
, Peptide nucleic acid (PNA), any DNA-like substance, and RNA-like substance.

“Operably linked” refers to the condition in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are very contiguous or contiguous if the regions encoding the two proteins must be joined in the same reading frame.

“Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent that comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. This terminal lysine renders the composition soluble. PNAs can bind preferentially to complementary single-stranded DNA or RNA to stop elongation of transcripts and polyethylene glycol to prolong life in cells.

“Post-translational modifications” of RECAP can include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modifications depend on the enzymatic environment of RECAP and may vary by cell type.

The “probe” is a nucleic acid sequence encoding RECAP, a complementary sequence thereof, or a fragment thereof, which is used for detecting the same or allelic nucleic acid sequence, or a related nucleic acid sequence. A probe is an oligonucleotide or polynucleotide that has been isolated with a detectable label or reporter molecule attached. Typical signs include
There are radioactive isotopes and ligands, chemiluminescent reagents, enzymes. "Primer"
Is a short nucleic acid, usually a DNA oligonucleotide, capable of forming complementary base pairs and annealing to a target polynucleotide. After the primer anneals to the polynucleotide, the target DNA is
It is extended along a single strand. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence in a PCR method.

The probes and primers used in the present invention include at least 15 of known sequences.
Of contiguous nucleotides. Longer probes and primers can be used to increase specificity. For example, at least 20 or 25, 30, 30, 40, 50, 60, 70, 80, 90, 100 contiguous of the disclosed nucleic acid sequences.
, 150 nucleotides. It should be understood that probes and primers that are considerably longer than the above examples can be used, and nucleotides of any length shown in the tables and drawings of the present specification and the sequence listing can also be used.

For the preparation and use of the probe and the primer, see, for example, Sambrook,
J. et al., 1989, Name "Molecular Cloning: A Laboratory Manual", 2nd
Editions 1-3 (Cold Spring Harbor Press, Plainview NY), or Ausubel, FM
In 1987, the name "Current Protocols in Molecular Biology" (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY), and Innis et al.,
In 1990, the name `` PCR Protocols, A Guide to Methods and Applications '' (Acade
mic Press, San Diego CA.). The set of primers for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research
, Cambridge MA), and the like, and can be derived from one known sequence using a computer program for such purpose.

Oligonucleotides used as primers are selected by computer programs well known in the art for primer selection. For example, OLIGO 4.06 software selects primer pairs for PCR, each up to 100 nucleotides,
And is useful for the analysis of large polynucleotides and oligonucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32,000 bases. Similar primer selection programs include additional features that expand capacity. For example, the PrimOU primer selection program (Genome Center at Uni
versity of Texas South West Medical Center, available from Dallas TX)
Since a specific primer can be selected from the megabase sequence, it is useful for designing a primer in a genome-wide scope. Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research,
(Available from Cambridge MA1) allows users to specify sequences that they would like to avoid as primer binding sites.
) ”Can be entered. In addition, Primer3 is especially useful for the selection of oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from each source and modified to meet the needs of users). Can also). PrimerGen Program (UK Human Genome Mapping Pro
(available from the ject Resource Center, Cambridge UK), which designs primers based on multiple sequence alignments, so that they will hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences. Can be selected. Therefore, this program is useful for identifying unique and conserved oligonucleotide or polynucleotide fragments. The oligonucleotide or polynucleotide fragment identified by any of the selection methods described above is, for example, a PCR method, a sequencing primer, a microarray element, or a specific identifying a polynucleotide that is completely or partially complementary to a sample nucleic acid. It is useful for the hybridization technique such as the probe.
The method for selecting the oligonucleotide is not limited to the above method.

As used herein, a “recombinant nucleic acid” is a sequence that is not a naturally occurring sequence, but is an artificial combination of two or more distant segments of a sequence. Often, this artificial combination is made by chemical synthesis, but it is more common to artificially manipulate distant segments of nucleic acid using genetic engineering techniques such as those described in Sambrook, supra. is there. The "recombinant nucleic acid" also includes a nucleic acid modified by simply adding or substituting a part of the nucleic acid. The recombinant nucleic acid may also include a nucleic acid sequence operably linked to a promoter sequence. Such recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such recombinant nucleic acid is a vaccinia virus-based virus that, when used to vaccinate a mammal expressing the recombinant nucleic acid, elicits a defensive immune response in the mammal. It can be part of the vector.

A “regulatory element” is a nucleic acid sequence usually derived from the untranslated region of a gene, including enhancers, promoters, introns and 5 ′ and 3 ′ untranslated regions (UTRs).
)including. Regulatory elements interact with host or viral proteins that regulate transcription, translation, or RNA stability.

A “reporter molecule” is a chemical or biochemical moiety used to label nucleic acids, amino acids or antibodies. Reporter molecules include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromophores, substrates, cofactors, inhibitors, magnetic particles and other components known in the art.

As used herein, the term “RNA equivalent” to a DNA sequence is composed of the same linear nucleic acid sequence as the reference DNA sequence, except that the nitrogenous base thymine is replaced with uracil to form a sugar chain. Its spine consists of ribose rather than deoxyribose.

The term “sample” is used in its broadest sense. Samples suspected of containing RECAP-encoding nucleic acid or fragments thereof, and RECAP itself include body fluids, extracts from cells, chromosomes or subcellular organelles isolated from cells, membranes, cells, and solutions. Genomic DNA, RNA, cDNA present in or immobilized on a substrate, tissues or tissue prints and the like may also be included.

The terms “specific binding” and “specifically bind” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. Point to. This interaction depends on the presence of a particular structure of the protein recognized by the binding molecule, eg, an antigenic determinant or epitope. For example, when the antibody is specific to the epitope "A", the reaction solution containing the unbound labeled "A" and the antibody is bound to the polypeptide containing the epitope A or the unbound unlabeled "A". Is present, the amount of labeled A bound to the antibody is reduced.

The term “substantially purified” refers to a nucleic acid sequence or amino acid sequence that has been removed from its natural environment and then isolated or separated, so that the composition to which it is naturally associated has at least about 60 amino acids. % Removed, preferably about 75% or more removed,
Most preferably, 90% or more is removed.

“Substitution” is the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microspheres. Includes particles and capillaries. The substrate has various surface morphologies such as walls or trenches, pins, channels, pores, to which polynucleotides and polypeptides bind.

“Transcriptional image” refers to the pattern of collective gene expression by a particular cell type or tissue under given conditions at a given time.

“Transformation” is the process by which exogenous DNA is introduced into recipient cells.
Transformation can occur under natural or artificial conditions by a variety of methods well known in the art and can be performed by any well known method of inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. You can The method of transformation is selected according to the type of host cell to be transformed. This method includes, but is not limited to, bacteriophage or virus infection, electroporation, lipofection, and particulate irradiation. "Transformed" cells include stably transformed cells in which the introduced DNA is capable of replicating autonomously, either as a plasmid or as part of a host chromosome. Further, a cell that transiently expresses the introduced DNA or the introduced RNA within a limited time is also included.

As used herein, the term “genetically modified organism” refers to any organism in which a human has introduced a heterologous nucleic acid into one or more cells of the organism using, for example, gene recombination techniques well known in the art. Yes, and includes, but is not limited to animals and plants. By careful genetic manipulation such as microinjection or infection with recombinant virus, the heterologous nucleic acid is introduced into the cell directly or indirectly into the precursor of the cell. “Genetic manipulation” refers to the introduction of recombinant DNA molecules, rather than the typical cross breeding or in vitro fertilization. Genetically modified organisms according to the invention include bacteria and cyanobacteria, fungi, plants, animals. The isolated DNA of the present invention can be introduced into a host by methods well known in the art, such as infection, transfection, transformation, transconjugation and the like. Techniques for introducing the DNA of the present invention into such organisms are well known and described in Sambrook et al. (1989) supra.

A “variant sequence” of a particular nucleic acid sequence refers to the default parameter setting “BLAST
2 Sequences "tool Version 2.0.9 (May-07-1999) shows that the specific nucleic acid sequence identity for a given length of a given nucleic acid sequence is at least 40%.
The nucleic acid sequence determined to be. Such nucleic acid pairs may, for example, be at least 50% or 60%, 70%, 80%, 85%, 90%, over a length.
It may exhibit 95%, 98%, or more identity. A variant sequence can be represented, for example, as an "allelic" variant sequence (described above) or a "splice" variant sequence, a "species" variant sequence, a "polymorphism" variant sequence. Splice variant sequences may have very high identities to the reference molecule, but alternative splicing of exons during mRNA processing may result in more or less polynucleotides. The corresponding polypeptide may have additional functional domains present in the reference molecule or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have a high degree of amino acid identity with each other. Polymorphic variant sequences differ in the polynucleotide sequence of a given gene from a given species and the species. The polymorphic variant sequence also includes one of the polynucleotide sequences
A "single nucleotide polymorphism" (SNP) that differs by two nucleotides may also be included. The presence of SNPs may suggest, for example, a population, a pathological condition, a propensity for a pathological condition.

A “variant” of a particular polypeptide sequence refers to the default parameter setting “
Blast 2 Sequences "tool version 2.0.9 (May-07-1999) by blastp, the identity of the particular polypeptide sequence to a certain length of a nucleic acid sequence is determined to be at least 40% polypeptide sequence That is. Such polypeptide pairs may, for example, be at least 50% or 60%, 7
It may exhibit 0%, 80%, 85%, 90%, 95%, 98%, or more identity.

Invention The present invention is based on the discovery of novel human receptors and related proteins (RECAPs) and polynucleotides encoding RECAPs, immune disorders including neurological diseases, autoimmune diseases / inflammatory diseases, And the use of these compositions in the diagnosis, treatment, and prevention of cell proliferative disorders, including cancer.

Table 1 shows the Incyte clones used to construct the full length nucleotide sequence encoding RECAP. Columns 1 and 2 show the SEQ ID NOs for the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone ID of the Incyte clone in which the nucleic acid encoding each RECAP was identified, and column 4 shows the cDNA library in which those clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. The Incyte clones for which the cDNA library is not shown are derived from the pooled cDNA library. The Incyte clones shown in column 5 were used to construct the consensus nucleotide sequence for each RECAP and are useful as fragments in hybridization techniques.

Each column of Table 2 shows various properties of each polypeptide of the invention. Column 1 is the SEQ ID NO, column 2 is the number of amino acid residues in each polypeptide, column 3 is the potential phosphorylation site, column 4 is the potential glycosylation site, and column 5 is the signature (sign).
ature) amino acid residues with sequence and motif, column 6 shows the homologous sequences identified by BLAST analysis, and the corresponding citations. In addition, reference is made to a part of this specification. Column 7 shows the method of analysis and possibly the searchable database to which the method of analysis can be applied. The analysis method in column 7 was used to characterize each polypeptide by sequence homology and protein motifs.

The columns of Table 3 show the tissue specificity and diseases, disorders and conditions associated with the nucleotide sequence encoding RECAP. Column 1 of Table 3 shows the nucleotide sequence numbers (SE
Q ID NO). Column 2 shows a fragment of the nucleotide sequence of column 1. These fragments are useful, for example, in hybridization or amplification techniques to identify SEQ ID NO: 23-44 and distinguish SEQ ID NO: 23-44 from related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 shows the name of the tissue expressing RECAP and its percentage in total tissues expressing RECAP. Column 4 shows diseases or disorders associated with tissues expressing RECAP, symptoms, and their proportion in total tissues expressing RECAP. Column 5 shows the vector used for subcloning each cDNA library. SEQ ID NO: 11 is expressed in hematopoietic / immune tissues, and SEQ ID NO:
Note also that 14 is expressed in reproductive tissues.

Each column of Table 4 is a description of the tissue used to create the cDNA library in which the clone of the cDNA encoding RECAP was isolated. Column 1 is the nucleotide SEQ
ID NO is shown, column 2 shows the cDNA library in which those clones were isolated, and column 3 shows the origin and details of the tissue corresponding to the cDNA library in column 2.

The present invention also includes variants of RECAP. Preferred RECAP variants have at least one of the functional or structural characteristics of RECAP, and have at least about 80% amino acid sequence identity to the RECAP amino acid sequence, or at least about 90%.
Amino acid sequence identity of, and also at least about 95% amino acid sequence identity.

The present invention also provides a polynucleotide encoding RECAP. In a particular embodiment, the invention provides a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 23-44 encoding RECAP. SEQ ID shown in the sequence listing
The NO: 23-44 polynucleotide sequence contains an equivalent RNA sequence in which the nitrogenous base thymine is replaced with uracil, and the backbone of the sugar chain is ribose rather than deoxyribose.

The invention also includes mutant sequences of the polynucleotide sequence encoding RECAP.
In particular, such variant sequences of the polynucleotide sequence have at least 70% polynucleotide sequence identity with the polynucleotide sequence encoding RECAP, or at least 85% polynucleotide sequence identity, and even at least 95%. Has polynucleotide sequence identity. A particular embodiment of the invention is SEQ ID N
SEQ: having at least 70% polynucleotide sequence identity, or at least 85% polynucleotide sequence identity, and even at least 95% polynucleotide sequence identity with a nucleic acid sequence selected from the group consisting of O: 23-44. ID NO: 23-
Provided is a variant sequence of a polynucleotide sequence comprising a sequence selected from the group consisting of 44. Each of the polynucleotide variants described above encodes an amino acid sequence having at least one functional or structural characteristic of RECAP.

It is noted that various polynucleotide sequences encoding RECAP that may be produced by the degeneracy of the genetic code include those that have minimal similarity to polynucleotide sequences of any known naturally-occurring gene. Those of ordinary skill in the art will understand. Thus, the present invention may include all of the possible polynucleotide sequence variations that may be created by the selection of combinations based on possible codon choices. These combinations are made on the basis of the standard triplet genetic code applied to the native RECAP polynucleotide sequence, and are considered to be the clear disclosure of all mutations.

The nucleotide sequence encoding RECAP and its variant sequences are generally hybridizable under suitable selected stringent conditions with the nucleotide sequence of native RECAP, but such as by including unnatural codons. It may be advantageous to make nucleotide sequences encoding RECAP or its derivatives with substantially different usage codons. Codons can be selected based on the frequency with which a particular codon is utilized by the host to increase the rate at which peptide expression occurs in a particular eukaryotic or prokaryotic host. RECAP without changing the encoded amino acid sequence
And another reason for substantially changing the nucleotide sequence encoding the derivative thereof is
Has desirable properties, such as a longer half-life, than transcripts made from native sequences
To make RNA transcripts.

The invention also includes the production of DNA sequences encoding RECAP and its derivatives, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can be inserted into any of the various available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry may be used to introduce mutations into the sequence encoding RECAP or any fragment thereof.

The present invention further provides a polynucleotide sequence capable of hybridizing to the claimed polynucleotide sequences, particularly SEQ ID NO: 23-44 and fragments thereof, under a variety of stringent conditions. Included (eg Wahl, GM and SL Berger
(1987) Methods Enzymol. 152: 399-407; and Kimmel. AR (1987) Methods En
zymol. 152: 507-511.). Hybridization conditions, including annealing and wash conditions, are described in the "Definitions".

Any of the embodiments of the present invention can be practiced using DNA sequencing methods well known in the art. This method includes, for example, the Klenow fragment of DNA polymerase I, SE
QUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems,
Foster City CA), thermostable T7 polymerase (Amersham, Pharmacia Biotech P
iscataway NJ) or ELONGASE amplification system (Life Technologies, Gaithersbu
Enzymes such as a combination of proofreading exonuclease and polymerase as found in rg MD) are used. Preferably, the MICROLAB 2200 liquid transfer system (Hamilt
on, Reno, NV), PTC200 Thermal Cycler200 (MJ Research, Watertown MA) and ABI CATALYST 800 (PE Biosystems). Next, ABI 373 or 377 DNA sequencing system (PE Biosystems)
, MEGABACE 1000 DNA Sequencing System (Molecular Dynamics. Sunnyval
e CA) or other methods well known in the art for sequencing. The resulting sequences are analyzed using various algorithms well known in the art (e.g., Ausubel
, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, N
See Meyers, RA (1995) Molecular Biology and Biotechn ology, Wiley VCH, New York NY, pp 856-853 a); ew York NY, unit 7.7 ...

A variety of PCR-based methods well known in the art can be used to extend the nucleic acid sequence encoding RECAP using a partial nucleotide sequence to extract upstream sequences such as promoters and regulatory elements. To detect. For example, one method utilizing restriction site PCR is to amplify an unknown sequence from genomic DNA in a cloning vector using common and nested primers (eg, S
arkar, G. (1993) PCR Methods Applic 2: 318-322). An alternative method using the inverse PCR method uses primers that extend in a wide range of directions and amplify unknown sequences from a circularized template. This template is derived from a restriction fragment containing a known genomic locus and sequences surrounding it (eg Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186.
See). The third method using the capture PCR method is human and yeast artificial chromosome D.
Includes PCR amplification of DNA fragments flanking a known sequence in NA (see, eg, Lagerstrom, M. et al. (1991) PCR Methods Applic 1: 111-119). This method allows digestion and ligation with multiple restriction enzymes to insert the recombinant double-stranded sequence into a region of unknown sequence prior to PCR. It is also possible to obtain the unknown sequence using other methods well known in the art. (For example, Parker, JD etc. (
1991) Nucleic Acids Res. 19: 3055-3060). In addition, using PCR, nested primers, and PROMOTER FINDER library (Clontech, Palo Alto CA)
It is possible to walk within the DNA. This method does not require screening the library and is useful for looking for intron / exon junctions. For all PCR-based methods, the primers are commercially available OLIGO 4.06 Primer Analysis softw
Using are (National Biosciences, Plymouth MN) or another suitable program, the length is 22 to 30 nucleotides, the GC content is 50% or more, about 68 ° C to 7
Designed to anneal to the mold at a temperature of 2 ° C.

When screening for full-length cDNAs, it is preferable to use libraries that are size-selected to contain large cDNAs. Furthermore, if the oligo d (T) library cannot produce a full-length cDNA, a randomly primed library, which often contains a sequence having the 5'region of the gene, is useful. Genomic libraries will be useful for extension of sequences into the 5'non-transcribed regulatory regions.

Sequencing or PCR using a commercially available capillary electrophoresis system
It is possible to analyze or confirm the size of the nucleotide sequence of the product. For more information,
Capillary sequencing uses a flowable polymer for electrophoretic separation, and a laser-activated fluorescent dye specific for four different nucleotides and a CCD camera used to detect emitted wavelengths. It is possible.
The output / light intensity is determined by the appropriate software (eg GENOTYPER and SEQUENCE NAVIGA
TOR, PE Biosystems) and converted into electrical signals, and the process from sample loading to computer analysis and electronic data display are computer controllable. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA, which may be present in small amounts in a particular sample.

In another embodiment of the invention, the polynucleotide sequence encoding RECAP or a fragment thereof is cloned into a recombinant DNA molecule to express RECAP, a fragment thereof or a functional equivalent in a suitable host cell. It is possible. Due to the inherent degeneracy of the genetic code, alternative DNA sequences can be made that encode substantially the same or a functionally equivalent amino acid sequence, which sequences are available for cloning and expression of RECAP.

The nucleotide sequences of the invention can be recombined using methods generally known in the art to alter the RECAP-encoding sequence for a variety of purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Nucleotide sequences can be recombined using DNA mixing with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon choices, create splice variants, and the like.

The nucleotides of the present invention were labeled with MOLECULAR BREEDING (Maxygen Inc., Santa Clara C
A; U.S. Pat.No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793.
-797; Christians, FC et al. (1999) Nat. Biotechnol. 17: 259-264; Crameri, A.
Other (1996) Nat. Biotechnol. 14: 315-319) and shuffling techniques such as RECAP's biological or enzymatic activity, or the ability to bind to other molecules or compounds. The biological properties of RECAP, such as, can be altered or improved. DNA shuffling is the process of creating a library of gene variants by recombination of gene fragments by the PCR method. The library is then selected or screened to identify genetic variants with the desired properties. These preferred variants are pooled and the DNA shuffling and selection / screening repeated. Therefore, a variety of genes are created by artificial breeding and rapid molecular evolution. For example, one gene fragment having a mutation at a random position can be subjected to recombination, screening, and shuffling until the desired property is optimized. Alternatively, a fragment of a given gene may be recombined with a fragment of a homologous gene of the same gene family of the same or different species, thereby regulating the genetic diversity of multiple natural genes in a regulatable manner in accordance with the protocol. Can be maximized.

According to another embodiment, the RECAP-encoding sequence can be wholly or partially synthesized using chemical methods well known in the art (eg, Carthers. MH et al. (198).
0) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Aci.
See ds Res. Symp. Ser. 225-232). Alternatively, chemical methods can be used to synthesize RECAP itself or fragments thereof. For example, peptide synthesis can be performed using various solid phase techniques (eg, Creighton, T. (1984) Proteins. Structures and Molecular Properties , WH Freeman, New York NY, pp. 55-60;
See Roberge, JY et al. (1995) Science 269: 202-204). Alternatively, automation of synthesis may be achieved using, for example, the ABI 431A Peptide Synthesizer (PE Biosystems). In addition, the amino acid sequence of RECAP, or any portion thereof, may be altered by direct synthetic changes, and / or by combination with sequences from other proteins or any portion thereof using chemical methods to produce the natural sequence. It is possible to produce a polypeptide having a polypeptide sequence or a variant polypeptide.

This peptide is used for preparative high performance liquid chromatography (eg, Chiez, RM).
And FZ Regnier (1990) Methods Enzymol. 182: 392-421)). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (see, for example, Creighton, supra, pp 28-53).

In order to express biologically active RECAP, the nucleotide sequence encoding RECAP or a derivative thereof is inserted into a suitable expression vector. This expression vector
It contains the elements necessary to regulate the transcription and translation of the coding sequence inserted into a suitable host. These elements include enhancers in the polynucleotide sequence encoding the vector and RECAP, constitutive and expression-inducible promoters,
Regulatory sequences such as 5'and 3'untranslated regions are included. Such elements are
Its length and specificity vary. With a particular initiation signal, it is possible to achieve more efficient translation of the RECAP coding sequence. Such signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. RECA
If the P coding sequence and its initiation codon, upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal containing an in-frame ATG initiation codon must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of natural and synthetic sources. The efficiency of expression can be increased by the inclusion of enhancers suitable for the particular host cell system used.
(See, eg, Scharf, D. et al. (1994) Results Probl. Cell Differ. 201-18-162.).

Methods which are well known to those skilled in the art can be used to produce expression vectors containing sequences encoding RECAP and suitable transcriptional and translational regulatory elements. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination technology. (For example, Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, Chapters 4 and 8, and 16-17.
; And Ausubel, FM et al. (1995) Current Protocols in Molecular Biology ,
See John Wiley & Sons, New York NY, ch. 9 and 13-1-4).

A variety of expression vector / host systems may be utilized to retain and express the RECAP-encoding sequence. These include, but are not limited to, microorganisms such as recombinant bacteriophage, plasmids, or bacteria transformed with cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors. Insect cell lines infected with (eg, baculovirus) or plants transformed with viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg, Ti or pBR322 plasmids) Cell lines, animal cell lines, etc. are included (Sambrook, supra, Ausubel, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5.
503-5509, Bitter, GA et al. (1987) Methods Enzymol. 153: 516-544; Scorer, C.
A. et al. (1994) Bio / Technology 12:18 1-184; Engelhard, EK 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) EMBOJ. 6: 307-311, Coruzzi, G. et al. (1984)
) EMBOJ. 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) Mc
Graw Hill New York NY, pp.191-196, Logan, J. and T. Shenk (1984) Proc. N
atl. Acad. Sci. USA 81: 3655-3659, Harrington, JJ et al. (1997) Nat. Genet.
15: 345-355 etc.). Expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or expression vectors derived from various bacterial plasmids can be used to deliver nucleotide sequences to target organs, tissues or cell populations (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, B
uller, RM et al. (1985) Nature 317 (6040): 813-815; McGregor, DP et al. (1994) M
ol. Immunol. 31 (3): 219-226, Verma, IM and N. Somia (1997) Nature 389: 2.
39-242 etc.). The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for the RECAP-encoding polynucleotide sequence. For example, RECA
For routine cloning, subcloning and propagation of P-encoding polynucleotide sequences, multifunctional E. coli vectors such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (GIBCO BRL) can be used. Ligation of the RECAP-encoding sequence into multiple cloning sites of the vector resulted in lac
The Z gene is disrupted, allowing a colorimetric screening method for identification of transformed bacteria containing recombinant molecules. In addition, these vectors can be used to generate in vitro transcription of cloned sequences, dideoxin screening, single-stranded rescue by helper phage, and nested deletions (e.g., Van Heeke). , G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.
See). For example, if a large amount of RECAP is required for antibody production, then RECAP
Vectors that induce high levels of expression can be used. For example, a vector containing a strongly inducible T5 or T7 bacteriophage promoter can be used.

It is possible to use yeast expression systems to express RECAP. Various vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH promoter can be used for yeast Saccharomyces cerevisiae or Pichia pastoris . Furthermore, such vectors induce either the secretion of the expressed protein or its retention within the cell, integrating foreign sequences into the host genome for stable growth. (For example, Ausubel .; and Bitter, GA et al. (1987) Method above.
s Enzymol. 153: 51-794: Scorer. CA et al. (1994) Bio / Technology 121-181-18
Plant systems can also be used to express RECAP. Transcription of the RECAP-encoding sequence is, for example, by viral promoters such as the CaMV-derived 35S and 19S promoters alone or TMV (see, for example, Coruzzi, supra, Broglie, supra, Winter, supra).
Promoted in combination with the derived omega leader sequence. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, for example, The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill NY, pp. 191-196).

In mammalian cells, a variety of virus-based expression systems may be utilized. When adenovirus is used as an expression vector, the RECAP-encoding sequence can be linked to an adenovirus transcript / translation complex consisting of a late promoter and a triple leader sequence. By insertion into the nonessential E1 or E3 region of the viral genome,
It is possible to obtain live virus expressing RECAP in infected host cells (Logan
, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers, such as the Rous Sarcoma Virus (RSV) enhancer, can be used to increase expression in mammalian host cells. For high level expression of proteins, SV40 or EBV based vectors can be used.

Human artificial chromosomes (HACs) can be used to supply fragments of DNA that are larger than those expressed in the plasmid and contained therein. HACs of about 6kb-10Mb for treatment
Are prepared and delivered by conventional delivery methods (liposomes, polycationic amino polymers, or vesicles). (For example, Harrington.JJ et al. (1997) Nat Genet. 15: 3.
45-355.).

For long-term production of recombinant proteins in mammalian systems, stable expression of RECAP in cell lines is desirable. For example, an expression vector can be used to transform a cell line with a sequence encoding RECAP. Such expression vectors contain replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are grown in enriched medium for about 1-2 days before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selective mediators, and its presence allows growth and recovery of cells which reliably express the introduced sequences. Resistant clones of stably transformed cells can be propagated using tissue culture techniques suitable for that cell type.

Any number of selection systems can be used to recover transformed cell lines. The selection systems include, but are not limited to, include herpes simplex virus thymidine kinase gene and adenine phosphoribosyltransferase genes, respectively tk ^- or apr ^- used in the cell. (For example, Wigler, M. et al. (1
977) Cell 11: 223-232; and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, and als or pat confers resistance to chlorsulfuron (cRECAPsulfuron) and phosphinotricin acetyltransferase (eg, phosphinotricin acetyltransferase). , Wigl
er, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin,
F. et al. (1981) J. Mol. Biol. 150: 1-14). Further genes available for selection have been described in the literature, eg trpB and hisD which alter what the cell needs for metabolism (eg Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad.
. Sci. 85: 8047-51). Anitocyanin, green fluorescent protein (GFP; Clon
tech), β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin and other visible markers are used. Green fluorescent protein (GFP) (Clon
tech, Palo Alto, CA) can also be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression due to a particular vector system (eg, Rhodes, CA et al. (19
95) Methods Mol. Biol. 55: 121-131).

Even if the presence / absence of expression of the marker gene indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of the gene. For example, if a sequence encoding RECAP is inserted within a marker gene sequence, transformed cells containing the sequence encoding RECAP can be identified by the lack of marker gene function. Alternatively, the marker gene can be placed in tandem with the RECAP-encoding sequence under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

In general, a host cell that contains a nucleic acid sequence that encodes RECAP and that expresses RECAP can be identified using a variety of methods well known to those of skill in the art. These methods include DNA-DNA or DNA-RNA hybridizations, PCR methods, protein biological methods including membrane-based, solution-based, or chip-based techniques for the detection and / or quantification of nucleic acids or proteins. Includes, but is not limited to, test methods or immunological assays.

RE with either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring the expression of CAP are well known in the art. Such techniques include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence activated cell sorters (FACS) and the like. A two-site, monoclonal-based immunoassay using a monoclonal antibody that reacts with two non-interfering epitopes on RECAP.
) Is preferred, but competitive binding assays can also be used. These and other assays are well known in the art. (For example, Hampton.
R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Pa
ul. MN, Sect. IV; Coligan, JE et al Current Protocols in Immunology , Gree
ne Pub. Associates and Wiley-Interscience, New York. NY; and Pound, JD
(1990) Immunochemical Protocols , Humans Press, Totowa NJ).

Various labeling and conjugation techniques are well known to those of skill in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to RECAP-encoding polynucleotides include oligo-labeling, nick-translation, end-labeling, or labeled nucleotides. Includes the PCR amplification used.
Alternatively, the sequence encoding RECAP, or any fragment thereof, can be cloned into a vector to generate an mRNA probe. Such vectors, which are well known in the art and are commercially available, can be prepared using suitable R7 such as T7, T3, or SP6.
RNA in vitro by the addition of NA polymerase and labeled nucleotides
It can be used for probe synthesis. These methods are described, for example, in Amersham Pha.
rmacia Biotech and Promega (Madison WI), US Biochemical Corp (Clevelan
d OH) can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents and the like.

Host cells transformed with a nucleotide sequence encoding RECAP are cultured under conditions suitable for the expression and recovery of this protein in cell culture. Whether the protein produced by a transformed cell is secreted or remains intracellular depends on the sequence and / or the vector used. It will be appreciated by those skilled in the art that expression vectors containing a polynucleotide encoding RECAP can be designed to include a signal sequence that directs secretion of RECAP that penetrates prokaryotic and eukaryotic cell membranes.

In addition, a host cell strain may be chosen for its ability to regulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Modifications of such polypeptides include acetylation, carboxylation, glycosylation, phosphorylation, lipidation (
lipidation), and acylation, but is not limited thereto.
Post-translational processing which cleaves a "prepro" or "pro" form of the protein can be used to identify target proteins, folds and / or activities. A variety of host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular machinery and characteristic mechanisms for post-translational activity
It is available from the Collection (ATCC; Bethesda, MD) and is selected to ensure the correct modification and processing of foreign proteins.

In another embodiment of the invention, the natural or altered or recombinant nucleic acid sequence encoding RECAP is linked to a heterologous sequence which results in translation of the fusion protein of any of the host systems described above. For example, a chimera containing a heterologous moiety that can be recognized by a commercially available antibody
The RECAP protein may facilitate the screening of peptide libraries for inhibitors of RECAP activity. Also, the heterologous protein portion and the heterologous peptide portion can facilitate purification of the fusion protein using commercially available affinity substrates. Such moieties include glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6
-His, FLAG, c-mc, hemagglutinin (HA), but are not limited to these. GST and MBP, Trx, CBP, 6-His allow purification of cognate fusion proteins with immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelate resins, respectively.
FLAG, c-mc, and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Also, by engineering the fusion protein to include a proteolytic cleavage site between the RECAP encoding sequence and the heterologous protein sequence, RECAP can be cleaved from the heterologous moiety after purification. Methods for expressing and purifying fusion proteins are described in Ausubel. (1995, supra ch 10). A variety of commercially available kits can be used to facilitate expression and purification of the fusion protein.

In another embodiment of the invention, TNT rabbit reticulocyte lysate or wheat germ extraction system (Promega) can be used to synthesize radiolabeled RECAP in vitro . These systems connect transcription and translation of sequences encoding proteins that are operably linked to the T7 or T3, SP6 promoters. Translation occurs in the presence of a radiolabeled amino acid precursor, which is, for example, ³⁵ S methionine.

The RECAP of the present invention or a fragment thereof can be used to screen for compounds that specifically bind to RECAP. With at least one or more test compounds,
It is possible to screen for specific binding to RECAP. Examples of test compounds include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

In one example, the compound thus identified is closely linked to the natural ligand of RECAP, eg, the ligand or fragment thereof, or the natural substrate, structural or functional mimetic or natural binding partner. are related (Coligan, see Chapter 5, etc. of JE other (1991) Cu rrent Protocols in Immunology 1 (2)). Similarly, the compound is REC
It may be closely related to the natural receptor to which the AP binds, or at least some fragment of the receptor, eg, the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one example, screening for such compounds involves making suitable cells that express RECAP as either a secreted protein or a protein on the cell membrane. Suitable cells include cells from mammals, yeast, E. coli. Cells expressing RECAP or cell membrane fragments containing RECAP are contacted with a test compound and analyzed for binding, stimulation or inhibition of either RECAP or the compound.

Certain assays may simply experimentally bind the test compound to the polypeptide and detect binding by fluorophore, radioisotope, enzyme conjugate or other detectable label. For example, the assay can include the steps of binding at least one test compound to RECAP immobilized in solution or on a solid support, and detecting the binding of RECAP to the compound. Alternatively, detection and measurement of binding of the test compound in the presence of labeled competitor can be performed. In addition, this assay can be performed with cell-releasing agents, chemical libraries or natural product mixtures, where the test compound is released in solution or immobilized on a solid support.

The RECAP of the present invention or a fragment thereof can be used to screen for compounds that modulate the activity of RECAP. Such compounds include agonists, antagonists, partial or inverse agonists and the like. In one embodiment, RE
The assay is performed under conditions permitting the activity of RECAP where CAP binds at least one test compound and the activity of RECAP in the presence of the test compound is compared to the activity of RECAP in the absence of the test compound. The change in RECAP activity in the presence of the test compound is
It suggests the presence of compounds that modulate the activity of CAP. Alternatively, the test compound is bound to an in vitro or cell free system containing RECAP under conditions suitable for the activity of RECAP to perform the assay. In any of these assays, the test compound that modulates RECAP activity can bind indirectly and need not come into direct contact with the test compound. At least one and more than one test compound can be screened.

In another example, the polynucleotide encoding RECAP or a mammalian homologue thereof in an animal model system using homologous recombination in embryonic stem cells (ES cells) is used.
Knock out ”. Such techniques are well known in the art and are useful in the production of animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). For example, mouse ES cells such as 129 / SvJ cell line are derived from early mouse embryos,
It can be grown in medium. The ES cells are transformed with a vector containing the gene of interest disrupted with a marker gene such as the neomycin phosphotransferase gene (neo: Capecchi, MR (1989) Science 244: 1288-1292). This vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination is performed using the Cre-loxP system to knock out a gene of interest in a tissue-specific or developmental-stage-specific manner (Marth, JD (1996) Clin. Invest. 97: 1999-200).
2; Wagner, KU et al. (1997) Nucleic Acids Res. 25: 4323-43 30). The transformed ES cells are identified and microinjected into mouse cell blastocysts collected from, for example, the C57BL / 6 mouse line. Blastocysts are surgically introduced into pseudopregnant females, the inherited traits of the resulting chimeric progeny are determined and crossed to create a heterozygous or homozygous system. The transgenic animals thus produced can be tested for potential therapeutic or toxic agents.

Polynucleotides encoding RECAP can be engineered in vitro in human blastocyst-derived ES cells. Human ES cells have the potential to differentiate into at least eight distinct cell lineages, including endoderm, mesoderm and ectoderm cell types.
These cell lineages differentiate into, for example, neural cells, hematopoietic lineages and cardiomyocytes (Thom
son, JA et al. (1998) Science 282: 1145-1 147).

"Knock-in" humanized animals (pigs) or transgenic animals (mouse or rat) modeled for human disease using polynucleotides encoding RECAP
Can be produced. Using knock-in technology, a region of a polynucleotide encoding RECAP is injected into animal ES cells and the injected sequences are integrated into the animal cell genome. The transformed cells are injected into the blastula and the blastula is transplanted as described above. Study transgenic offspring or inbreds, treat with potential pharmaceuticals, and obtain information on treatment of human disease. Alternatively, mammalian inbred strains that overexpress RECAP, such as secreting RECAP into milk, can be a convenient source of protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4: 55- 74).

Treatment There are chemical and structural similarities between regions of RECAP and regions of receptors and related proteins, eg in the context of sequences and motifs. Furthermore, RECA
Expression of P is closely associated with cell proliferation, cancer, inflammation / trauma, and neurological disease.
Thus, RECAP is believed to play a role in neurological disorders, immune disorders including autoimmune / inflammatory disorders, and cell proliferation disorders including cancer. In treating diseases associated with increased RECAP expression or activity, it is desirable to reduce RECAP expression or activity. Further, in the treatment of a disease associated with a decrease in RECAP expression or activity, it is desirable to increase RECAP expression or activity.

Thus, in one embodiment, RECAP or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of disorders associated with decreased RECAP expression or activity. Examples of such disorders include, but are not limited to, neurological disorders, including epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasm, 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 neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis And other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system Disease and
Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-St
Prion diseases including raussler-Scheinker syndrome, fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma (cerebellor)
etinal hemangioblastomatosis), trigeminal neurovascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, peripheral nerve diseases, dermatomyositis and polymyositis, and hereditary , Metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic quadriplegia, mood and anxiety disorders, psychosis including schizophrenia, seasonal disorders (SAD), and quiz Disability, amnesia, catatonic disease, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease, progressive supranuclear palsy, cortical basal degeneration, It includes familial frontotemporal dementia and also immune disorders including autoimmune / inflammatory diseases, including acquired immune deficiency syndrome (AIDS), Breton-type agammaglobulinemia, Acquired immunity Brin hyperlipidemia (CVI), DiGeorge syndrome (
Thymic dysplasia), thymic dysplasia, IgA single deficiency, severe combined immunodeficiency (SCID), immunodeficiency with thrombocytopenia and eczema (Wiscott-Aldrich syndrome), Chediak-East disorder, chronic granulomatous disease , Hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic Anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candid ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymph Cytotoxic transient lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto thyroid Adenitis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatism Arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation,
Includes viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminthic infections, hematopoietic cancers including lymphoma and leukemia, myeloma, trauma, and abnormal cell proliferation. , Among them actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera , Psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, Ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid gland,
Includes uterine cancer.

In another embodiment, RECAP or a fragment or derivative thereof is expressed for the treatment or prevention of diseases associated with decreased expression or activity of RECAP, including but not limited to the diseases listed above. It is also possible to administer the resulting vector to a patient.

In yet another embodiment, RECAP, including but not limited to the diseases listed above.
For the treatment or prevention of the diseases associated with the decrease of the expression or activity of R., it is also possible to administer a pharmaceutical composition containing substantially purified RECAP to a patient together with a suitable pharmaceutical carrier.

In yet another embodiment, RECAP including but not limited to the diseases listed above.
It is also possible to administer to the patient an agonist that regulates the activity of RECAP, for the treatment or prevention of diseases associated with decreased expression or activity of.

In a further embodiment, an antagonist of RECAP can be administered to a patient for the treatment or prevention of disorders associated with increased RECAP expression or activity.
Examples of such disorders include, but are not limited to, the above-mentioned neurological disorders, immune disorders including autoimmune / inflammatory disorders, and cell proliferative disorders including cancer. In one embodiment, an antibody that specifically binds RECAP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism that delivers the drug to cells or tissues that express RECAP.

In another example, the complementary sequence of a polynucleotide encoding RECAP for the treatment or prevention of diseases associated with increased expression or activity of RECAP, including but not limited to the diseases listed above. It is also possible to administer a vector that expresses

In another example, any of the proteins, antagonists, antibodies, agonists, complementary sequences, vectors of the invention can be administered in combination with another suitable therapeutic agent. One of ordinary skill in the art can select suitable therapeutic agents for use in the combination therapy according to conventional pharmaceutical principles. The combination with therapeutic agents may bring about a synergistic effect in the treatment or prevention of the various diseases listed above. Using this method, it is possible to obtain a medicinal effect with a small amount of each drug, and it is possible to reduce the possibility of a wide range of side effects.

RECAP antagonists can be prepared using methods conventional in the art. Specifically, purified RECAP can be used to produce antibodies, or a library of therapeutic agents can be screened to identify those that specifically bind to RECAP. RECAP antibodies can also be produced using methods common in the art.
Such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies,
Included are single chains, Fab fragments, and fragments produced by Fab expression libraries. However, it is not limited to these. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly preferred for therapeutic use.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans and others are injected with RECAP or any fragment or oligopeptide thereof with immunogenic properties. Can be immunized with. Depending on the host species, various adjuvants may be used to enhance the immune response. Such adjuvants include Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and surfactants such as dinitrophenol. However, it is not limited thereto. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parv um it is particularly preferred.

Oligopeptides, peptides used to elicit antibodies to RECAP,
Alternatively, fragments preferably consist of at least about 5 amino acids, generally about 10 or more amino acids. These oligopeptides or peptides,
Alternatively, those fragments are preferably identical to a part of the amino acid sequence of the natural protein. A short stretch of RECAP amino acids can be fused with the sequence of another protein such as KLH to produce antibodies to the chimeric molecule.

Monoclonal antibodies to RECAP can be produced by any cell line in culture, using any technique which produces antibody molecules. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler.
, G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) .J. Immunol. M.
ethods 81-8-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-20.
30; Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques such as splicing the mouse antibody gene onto the human antibody gene developed to produce “chimeric antibodies” are used to obtain molecules with suitable antigen specificity and biological activity ( For example, Morrison, SL et al. (1984) Proc. N
atl. Acad. Sci. 81-4851-4855; Neuberger, MS et al. (1984) Nature 312: 604.
-608; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, applying the described techniques for the production of single chain antibodies using methods well known in the art,
Generates RECAP-specific single chain antibody. Antibodies with related specificities but of different idiotype composition can also be generated by chain mixing from random combinatorial immunoglobulin libraries (e.g., Burton DR (1991) Proc. Natl.
Acad. Sci. 88: 11120-3).

Antibodies can be induced by in vivo production in a population of lymphocytes, or by screening immunoglobulin libraries or a panel of highly specific binding reagents, as shown in the literature. (See, for example, Orlandoi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833.
-3837; Winter, G. et al. (1991) Nature 349: 293-299).

Antibodies containing specific binding sites for RECAP can also be produced. For example, such fragments include F (ab ') fragments produced by pepsin digestion of antibody molecules and Fa (ab') fragments produced by reducing the disulfide bridges of the fragments.
b fragments are included, but are not limited to. Alternatively, generating a Fab expression library allows for rapid and easy identification of monoclonal Fab fragments with the desired specificity (eg, Huse, WD et al. (1989) Science 254: 12.
See 75-1281).

Various immunoassays are used to screen to identify antibodies with the desired specificity. Numerous protocols for competitive binding, or immunoradioactivity, using either polyclonal or monoclonal antibodies with isolated specificity are well known in the art. Usually such immunoassays include RECA
Included is the measurement of complex regulation between P and its specific antibody. Two incoherent RECAP
Two-site monoclonal-based immunoassays are generally used with monoclonal antibodies reactive to the epitope, but competitive binding assays can also be used (Pound, supra).

Using various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques, R
Evaluate the affinity of the antibody for ECAP. The affinity is represented by a binding constant Ka, which is a value obtained by dividing the molar concentration of RECAP antibody complex by the molar concentration of free antibody and free antigen under equilibrium. The Ka of a polyclonal antibody drug having a heterogeneous affinity for many RECAP epitopes represents the average affinity or binding activity of the antibody for RECAP. The Ka of a monoclonal antibody drug monospecific to a particular RECAP epitope represents a true measure of affinity. A high affinity antibody drug having a Ka value of 10 ⁹ to 10 ¹² L / mol is preferably used in an immunoassay in which the RECAP antibody complex must withstand violent manipulation. The low affinity antibody drug having a Ka value of 10 ⁶ to 10 ⁷ L / mol is preferably used for immunopurification and similar treatments in which RECAP must be finally dissociated from the antibody in an activated state. (Catty, D. (19
88) Antibodies, Volume I: A Practical Approach .IRL Press, Washington, D
C; Liddell, JE and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies , John Wiley & Sons, New York NY).

To study the quality and suitability of such pharmaceuticals in certain downstream applications,
The antibody titer and binding activity of the polyclonal antibody drug are further evaluated. For example, a polyclonal antibody drug containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, is generally used in the treatment in which the RECAP antibody complex must be precipitated. Specificity of antibodies and antibody titer, binding activity, quality of antibodies and guidelines for usage in various applications are publicly available. (For example, C
See atty, supra, and Coligan et al., supra).

In another embodiment of the invention, the polynucleotide encoding RECAP, or any fragment or complementary sequence thereof, can be used for therapeutic purposes. In one embodiment, gene expression can be altered by designing sequences or antisense molecules (DNA and RNA, modified nucleotides) complementary to the coding and regulatory regions of the RECAP-encoding gene. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the sequence encoding RECAP, or from various positions along the coding region.

When used in therapy, any gene delivery system suitable for introducing antisense sequences into suitable target cells can be used. Antisense sequences can be delivered intracellularly in the form of expression plasmids that, upon transcription, express sequences complementary to at least a portion of the cellular sequences encoding the target protein (eg, Slater, JE et al.
(1998) J. Allergy Clin. Immunol. 102 (3): 469-475; and Scanlon, KJ et al. (1
995) 9 (13): 1288-1296.). The antisense sequence can also be introduced into cells using a viral vector such as a retrovirus or an adeno-associated viral vector (for example, Miller, AD (1990) Blood 76: 271; Ausubel,
Supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347). Other genetic delivery mechanisms include liposomal systems, artificial viral envelopes, and other systems known in the art (Rossi, JJ (1995) Br. Med.
Bull. 51 (1): 217-225; Boado, RJ et al. (1998) J. Pharm. Sci. 87 (11): 1308-1.
315; and Morris, MC et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736.).

In another embodiment of the invention, the polynucleotide encoding RECAP can be used for somatic or germ cell gene therapy. Gene therapy (i)
Gene deficiency (eg, X chromosome linked inheritance (Cavazzana-Calvo, M. et al. (2000) Sc
ience 288: 669-672), a severe combined immunodeficiency (SCID) -X1
), Hereditary adenosine-deaminase (ADA) deficiency (Blaese, RM et al. (1995) S
cience 270: 475-480; Bordignon, C. et al. (1995) Science 270: 470-475), severe combined immunodeficiency, cystic fibrosis (Zabner, J. et al. (1993) Cell 75: 207-). 2
16: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666; Crystal, RG
(1995) Hum. Gene Therapy 6: 667-703), thalassamia, familial hypercholesterolemia, hemophilia due to factor VIII or IX deficiency (Cr
ystal, 35 RG (1995) Science 270: 404-410; Verma, IM and Somia. N. (1
997) Nature 389: 239-242), (ii) expressing a conditionally lethal gene product (eg, in the case of cancer due to uncontrolled cell growth), and (iii) intracellular parasites. (For example, human immunodeficiency virus (HIV) (Baltimore, D. (1988) Na
ture 335: 395-396; Poescbla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:
11395-11399), hepatitis B or C virus (HBV, HCV), fungal parasites such as Candida albican s and Paracoccidioides brasiliensis , Plasmodium falciparum
And a protein having a protective function against protozoan parasites such as Trypanosoma cruzi ). If a deficiency of a gene required for expression or regulation of RECAP causes a disease, RECAP can be expressed from a suitable population of introduced cells to mitigate the symptoms caused by the gene deficiency.

In a further embodiment of the present invention, for diseases and abnormalities due to RECAP deficiency, mammalian expression vectors encoding RECAP are produced and these vectors are introduced into RECAP deficient cells by mechanical means. Treat by. Mechanical transfer techniques used for cells in vivo or in vitro include (i) microinjection of DNA into individual cells, (ii) implantation of gold particles, (iii) liposome-mediated transfection, (iv) ) Receptor-mediated gene transfer, and (v) DNA transposons (Morg
an, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217; Ivics,
Z. (1997) Cell 91: 501-510; Boulay, JL. And H. Recipon (1998) Curr. Op
in. Biotechnol. 9: 445-450).

Expression vectors that can influence RECAP expression include, but are not limited to, PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA.
), PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA), PTET-OF
F, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA) are included. In order to express RECAP, (i) a constitutively active promoter (for example, cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin gene), (ii) an inducible promoter (for example, a tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. contained in a commercially available T-REX plasmid (Invitrogen)).
Natl. Acad. Sci. USA 89: 5547-5551; Gossen, M. et al. (1995) Science 268: 1.
766-1769; Rossi, FMV and HM Blau (1998) Curr. Opin. Biotechnol. 9: 4
51-456)), ecdysone inducible promoter (commercially available plasmid PVGRXR
And PIND: Invitrogen), FK506 / rapamycin inducible promoter, or RU486 / mifepristone inducible promoter (Rossi, FMV a
nd HM Blau, supra), or (iii) it is possible to use the natural or tissue-specific promoter of the endogenous gene encoding RECAP from a normal individual.

Commercially available liposome transformation kits (eg PERFEC sold by Invitrogen)
T LIPID and TRANSFECTION KIT) allows one of skill in the art to introduce polynucleotides into target cells in culture without much reliance on experience. Alternatively, the calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52: 456-
467) or electroporation (Neumann, B. et al. (1982) EMBO J. 1: 841-845). These standard mammalian transfection protocols need to be modified to introduce DNA into primary cells.

In another embodiment of the present invention, the disease or disorder caused by the gene deficiency associated with RECAP expression comprises A Rev responsive element (RRE) with an encoding polynucleotide, (ii) a suitable RNA packaging signal, and (iii) additional retroviral cis-acting RNA sequences and coding sequences required for efficient vector propagation. ) And a retrovirus vector consisting of and can be treated. Retroviral vectors (eg PFB and PFBNEO)
Is available from Stratagene and published data (Riviere, I. et al. (1995) Pro
c. Natl. Acad. Sci. USA 92: 6733-6737). Reference to the above data is made a part of this specification. This vector is VSVg (Armentano,
D. et al. (1987) J. Virol. 61: 1647-1650; Bender, MA et al. (1987) J. Virol. 6
1: 1639-1646; Adam, MA and AD Miller (1988) J. Virol. 62: 3802-3806; D
ull, T. et al. (1998) J. Virol. 72: 8463-8471; Zufferey, R. et al. (1998) J. Viro
L. 72: 9873-9880) or a suitable vector-producing cell line (VPCL) expressing a promiscuous envelope protein or an envelope gene having an affinity for the receptor on the target cell. U.S. Pat.No. 5,910,434 issued to RIGG (`` Method for obtaining retrovirus packaging cell lines producing high
transducing efficiency retroviral cell "), a method for obtaining a retroviral packaging cell line is disclosed, which is incorporated herein by reference. Propagation of retroviral vectors, transduction of certain cell populations (eg, CD4 ⁺ T cells), and methods of returning the transduced cells to the patient include:
It is well known in the field of gene therapy and has been described in numerous publications (Ranga, U. et al.
(1997) J. Virol. 71: 7020-7029; Bauer, G. et al. (1997) Blood 89: 2259-2267;
Bonyhadi, ML (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
).

Alternatively, an adenovirus-based gene therapy delivery system is used to deliver a polynucleotide encoding RECAP to cells that have one or more genetic abnormalities associated with RECAP expression. The production and packaging of adenovirus-based vectors is well known in the art. Replication-defective adenovirus vectors have been shown to be variable for introducing genes encoding immunomodulatory proteins into intact pancreatic islets (Csete, ME et al. (1995) Transplantation 27: 263-268). A possible adenovirus vector is described in US Pat. No. 5,707,618 (“A
denovirus vectors for gene therapy "), which is incorporated herein by reference. Also for the adenovirus vector, Antinozz
i, PA et al. (1999) Annu. Rev. Nutr. 19: 511-544; and Verma, IM and N. So
See mia (1997) Nature 18: 389: 239-242, incorporated herein by reference.

Alternatively, a herpes gene therapy delivery system is used to deliver a polynucleotide encoding RECAP to a target cell that has one or more genetic abnormalities associated with RECAP expression. Herpes simplex virus (HSV) -based vectors are of particular importance in introducing RECAP into HSV-affected central neurons. The construction and packaging of herpes-based vectors is well known in the art. Replication competent herpes simplex virus (HSV
) Type I-based vectors have been used to deliver reporter genes to the primate eye (Liu, X. et al. (1999) Exp. Eye Res. 169: 385-395). Generation of the HSV-1 viral vector is described in US Pat. No. 5,804,413 to DeLuca (Herpes simplex v
irus swains for gene transfer), which is incorporated herein by reference. US Pat. No. 5,804,413 describes recombinant HSV d92 consisting of a genome containing at least one endogenous gene that is introduced into cells under the control of a suitable promoter for purposes including human gene therapy. There is. The patent also discloses methods of making and using recombinant HSV strains that are removed for ICP4, ICP27 and ICP22. Goins, WF for HSV vectors
Others (1999) J. Virol. 73: 519-532 and Xu, H. Others (1994) Dev. Biol. 163: 152-1
61, which is incorporated herein by reference. Manipulation of cloned herpesvirus sequences, passage of recombinant viruses after transfection of numerous plasmids containing different parts of the giant herpesvirus genome, growth and multiplication of herpesviruses, and into herpesvirus cells. Infection is a technique well known in the art.

Alternatively, an alphavirus (positive single stranded RNA virus) vector is used to deliver a polynucleotide encoding RECAP to target cells. Extensive biological research on the prototype α virus, Semliki Forest Virus (SFV), revealed that the gene transfer vector is based on the SFV genome (Garoff , H. and K.-J. Li (1998) Cun. Opin. Bi
otech. 9: 464-469). During replication of alphavirus RNA, subgenomic RNA that normally encodes the viral capsid protein is produced. Because this subgenomic RNA replicates at higher levels than full-length genomic RNA, capsid proteins are overproduced over viral proteins with enzymatic activity (eg, proteases and polymerases). Similarly, by introducing a RECAP-encoding sequence into the capsid-encoding region of the α virus genome, a large number of RECAP-encoding RNAs are produced in vector-transfected cells, and RECAP is synthesized at a high level. Α-virus infection is usually associated with cell lysis within 2-3 days, but Sindbis virus (
The ability of hamsters carrying SIN) to establish a persistent infection of normal kidney cells (BHK-21) may be favorably altered to allow alpha virus lytic replication to be applied in gene therapy. Suggesting that it is possible (Dryga, SA et al. (1997) V
irology 228: 74-83). Since the α virus can be introduced into various hosts, RECAP can be introduced into various types of cells. Specific transduction of a subset of cells in a population may require sorting of cells prior to transduction. Manipulation of alpha virus infectious cDNA clones, alpha virus cDNA and RNA transfection, and alpha virus infection methods are well known in the art.

[0177] For example, it is possible to inhibit the expression of a gene by using an oligonucleotide derived from the transcription start site from about -10 to about +10 from the start site. Similarly,
It can be inhibited using triple helix base pairing. The triple helix structure is useful because it prevents the double helix from expanding sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances with triplex DNA have been described in the literature (eg Gee, JE et al. (1994) In: Huber, BE and BI Car.
r, Molecular and ImmunologiRECAPproaches , Futura Publishing Co., Mt. Kis
See co, NY). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by blocking the binding of transcripts to ribosomes.

Ribozymes, enzymatic RNA molecules, can 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 nucleotide strand scission.
For example, recombinant hammerhead ribozyme molecules that specifically and effectively catalyze nucleotide breaks in the RECAP-encoding sequence are included.

A specific ribozyme cleavage site within any potential RNA target is followed by the sequence GUA.
, GUU, and GUC, are first identified by scanning the target molecule against ribozyme cleavage sites. Once identified, a short RNA sequence of 15 to 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is evaluated for secondary structural features that impair the function of the oligonucleotide. Is possible. The suitability of candidate targets can also be assessed by testing their ease of hybridization with complementary oligonucleotides using a ribonuclease protection assay.

Complementary ribonucleic acid molecules and ribozymes of the invention can be made for the synthesis of nucleic acid molecules using methods well known in the art. These methods include methods of chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, RNA molecules may be generated in vitro and in vivo by transcription of a DNA sequence encoding RECAP, such DNA sequences being prepared using various RNA polymerase promoters, such as T7 or SP6, in various vectors. It can be incorporated into. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.

Modification of RNA molecules can increase intracellular stability and prolong half-life. Possible modifications include the addition of flanking sequences at the 5'and / or 3'ends of the molecule, or the use of phosphorothionate or 2'O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. However, it is not limited thereto. Unique to PNA production, this concept is that adenine, cytidine, guanine, thymine, which are not readily recognized by endogenous endonucleases,
And the acetyl-, methyl-, thio-, and similar modified forms of uridine, as well as the inclusion of non-conventional bases such as inosine, queosine, and wybutosine, in whole of these molecules. Can be expanded to.

A further embodiment of the invention includes a method of screening for compounds that are effective in altering the expression of a polynucleotide encoding RECAP. Compounds effective in altering expression of a particular polynucleotide include, but are not limited to, non-polymeric chemicals, oligonucleotides, antisense oligonucleotides, triple helix-forming oligos capable of interacting with a particular polynucleotide sequence. Nucleotides, transcription factors and other polypeptide transcriptional regulators are included. Effective compounds may act as inhibitors or enhancers of polynucleotide expression and alter expression of the polynucleotide. Therefore, in the treatment of diseases associated with increased RECAP expression or activity, compounds that specifically inhibit the expression of the RECAP-encoding polynucleotide are therapeutically useful and are associated with decreased RECAP expression or activity. In treating disease, compounds that specifically enhance expression of a polynucleotide encoding RECAP may be therapeutically useful.

To test the efficacy of altered expression of a particular polynucleotide, at least 1
From one to a plurality of test compounds can be screened. The test compound is
It can be obtained by any method known in the art including chemical modification of effective compounds.
Such methods are based on the chemical and / or structural properties of the target polynucleotide when it alters the expression of the polynucleotide, when selected from commercially available or proprietary natural or unnatural compound libraries. It is useful for rationally designing compounds and for selecting from libraries of combinatorially or randomly generated compounds. A sample containing a polynucleotide encoding RECAP is obtained by exposure to at least one test compound. The sample can include, for example, intact cells, permeabilized cells, in vitro cell release systems or reconstituted biochemical systems. Changes in expression of a polynucleotide encoding RECAP are assayed by any method known in the art. Usually, 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 yield of hybridization is quantified and that value can serve as a basis for comparing the expression of polynucleotides exposed and unexposed to one or more test compounds. If an alteration in the expression of the polynucleotide exposed to the test compound is detected, it indicates that the test compound is effective in altering the expression of the polynucleotide. To examine compounds effective in altering the expression of a particular polynucleotide, for example, the Schizosaccharomyce spombe gene expression system (Atkins, D. et al. (1999) U.S. Pat.No. 5,932,435, Arndt,
GM et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells (
Clarke, ML et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13). Certain embodiments of the invention include screening combinatorial libraries of individual oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against specific polynucleotide sequences (Bruice , TW and others (1997)
U.S. Pat. No. 5,686,242, Bruice, TW et al. (2000) U.S. Pat. No. 6,022,691).

[0184] vectors available are a number of ways of introducing into a cell or tissue, in vivo, are equally suitable for use in in vitr o, and ex vivo. For ex vivo treatment, the vector is introduced into hepatocytes taken from a patient and cloned and propagated for autologous transplant back into the same patient. Transfection, ribosome injection or delivery by polycationic aminopolymers can be performed using methods well known in the art (eg Goldman, CK et al. (1997) Nature Biotechnology 15: 462.
-66 :).

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

Another embodiment of the present invention relates to the administration of a pharmaceutical or sterile composition in association with a pharmaceutically acceptable carrier for all of the above mentioned therapeutic effects. Such a pharmaceutical composition comprises RECAP, an antibody of RECAP, a mimetic, an agonist, an antagonist, an inhibitor of RECAP, and the like. The composition can be administered alone or with one or more additional agents such as stabilizers to a sterile biocompatible pharmaceutical carrier. Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water and the like. This composition can be administered alone or with another agent such as a drug or hormone.

The pharmaceutical composition used in the present invention can be administered by various routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, subarachnoid, intraventricular,
Includes, but is not limited to, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

Pharmaceutical compositions for pulmonary administration can be prepared in liquid or dry powder form. Such pharmaceutical compositions are typically aerosolized shortly before inhalation by the patient. Small molecule (
Aerosol delivery of fast-acting formulations is well known in the art (eg, for conventional low molecular weight organic drugs). In the case of macromolecules (for example, larger peptides and proteins), the technology of pulmonary transport via the alveolar region of the lung has improved in recent years, and it has become possible to actually transport drugs such as insulin into the blood ( Patton, JS et al., US Patent No. 5
, 997,848, etc.). Pulmonary transport is superior in that it can be administered without needle injection, obviating the need for potentially toxic penetration enhancers.

Suitable pharmaceutical compositions for use in the present invention include compositions which contain an effective amount of active ingredients to achieve the objectives. Those of ordinary skill in the art are well able to determine an effective dose with their own ability.

A particular form of pharmaceutical composition is prepared to deliver macromolecules, including RECAP or fragments thereof, directly into cells. For example, liposomal formulations containing cell-impermeable macromolecules can facilitate cell fusion and intracellular transport of macromolecules. Alternatively, RECAP or a fragment thereof can be attached to the cationic N-terminal part of the HIV Tat-1 protein. It has been confirmed that the fusion protein thus produced is transduced into cells of all tissues including the mouse model system brain (Schwarze, SR et al. (1999) Sci.
ence 285: 1569-1572).

For any composition, the therapeutically effective dose can be estimated initially either in tumor cell assays, eg, of tumor cells, or in animal models. Usually, mouse, rabbit, dog, monkey, pig or the like is used as the animal model. Animal models can also be used to determine the appropriate concentration range and route of administration. Based on such treatment, a beneficial dose and administration route for human can be determined.

A pharmaceutically effective dose is related to the amount of active ingredients, such as RECAP or a fragment thereof, an antibody of RECAP, an agonist or antagonist of RECAP, an inhibitor, etc. that ameliorates the symptoms or condition. Medicinal efficacy and toxicity are calculated by, for example, ED ₅₀ (dose that is medicinally effective in 50% of the population for taking) or LD ₅₀ (dose that is fatal in 50% of the population for taking) statistics. Can be determined by standard drug techniques in cell culture or animal studies. Dosage ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD _{50 /} ED _50. Pharmaceutical compositions that exhibit high therapeutic indices are desirable. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human application. Such compositions dosage included are toxic not including little or no, it is desirable that a range of circulating concentrations that include the ED _{5 0.} The dosage will vary within this range depending upon the dosage form employed, the patient sensitivity, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Dosage factors considered include disease severity, general health of the patient, age, weight and sex of the patient, time and frequency of administration, concomitant medications, response susceptibility, and treatment. Contains the response. A pharmaceutical composition having a long acting organ should be administered once every three or four days, once a week,
It can be administered once every two weeks depending on the half-life and clearance rate of the particular formulation.

A typical dosage will vary depending on the route of administration, but is up to about 1 gram up to about 0.1-100,000 μg. Guidance on specific dosages and methods of delivery is provided in the literature and is generally available to practitioners in the art. Those skilled in the art will utilize formulations of nucleotides that are different than the protein or inhibitor. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Diagnosis In another embodiment, a patient who is diagnosed with a disease in which an antibody that specifically binds RECAP is characterized by RECAP expression, or is treated with a RECAP or a RECAP agonist or antagonist, inhibitor. Used in an assay to monitor Antibodies useful for diagnosis are formulated in the same manner as described in treatment. RECAP diagnostic assays include methods of detecting RECAP from human body fluids or from cells and tissues using antibodies and labels. These antibodies, used with or without modification, can be labeled by covalent or non-covalent attachment of reporter molecules. Various reporter molecules known in the art are used, some of which have been mentioned above.

Various protocols, including ELISA, RIA, and FACS for measuring RECAP are well known in the art and provide the basis for diagnosing altered or abnormal levels of RECAP expression. The value of normal or standard RECAP expression is determined by binding an antibody against RECAP to a body fluid or cell collected from a subject such as a normal mammal, for example, a human under conditions suitable for complex formation. To do. The amount of canonical complex formation can be quantified by various methods such as photometric. Amounts of RECAP expression, control and disease in the subject, samples from biopsy tissue are compared to baseline. The deviation between the reference value and the subject is an index for diagnosis.

According to another embodiment, the polynucleotide encoding RECAP can also be used for diagnosis. The polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. This polynucleotide is used to detect and quantify the expression of genes in biopsy tissues that express RECAP, which can be correlated with disease. This diagnostic assay is used to check for the presence of RECAP, as well as overexpression, and to monitor the regulation of RECAP levels during treatment.

In certain embodiments, it is possible to identify a nucleic acid sequence encoding RECAP by hybridization with a PCR probe capable of detecting a polynucleotide sequence comprising a gene sequence encoding RECAP or a closely related molecule. is there. For example, 5 '
The specificity of the probe, whether it is a highly specific region that is a regulatory region or a region with a slightly low specificity that is a conserved motif, and the stringency of hybridization or amplification, the probe determines RECAP. It will depend on whether to identify only the natural sequences that encode, or to identify only the natural sequences that encode the allele or related sequence.

The probe may also be used for detection of related sequences and may have at least 50% sequence identity with any sequence encoding RECAP. The desired hybridization probe of the present invention can be DNA or RNA and can be derived from the sequence of SEQ ID NO: 23-44, or the genomic sequence including the promoter, enhancer and intron of the RECAP gene.

As a method for producing a hybridization probe specific to RECAP-encoding DNA, there is a method of cloning a polynucleotide sequence encoding RECAP and a RECAP derivative into a vector for producing an mRNA probe. Such vectors are commercially available, well known to those skilled in the art, and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. The hybridization probe is, for example, ³² P.
Alternatively, it may be labeled with a radionuclide such as ³⁵ S, or a population of various reporters such as an enzyme label such as alkaline phosphatase linked to the probe by an avidin / biotin binding system.

Polynucleotide sequences encoding RECAP can be used to diagnose diseases associated with RECAP expression. Examples of such disorders include, but are not limited to, neurological disorders, including epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasm, 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 neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis And other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system Diseases and prion diseases including Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis Cerebelloretinal hemangioblastomatosis, trigeminal neurovascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, peripheral nerve diseases, dermatomyositis and multiple occurrences With myositis, hereditary, metabolic, endocrine,
And toxic myopathy, myasthenia gravis, periodic quadriplegia, mood and anxiety disorders, psychosis including schizophrenia, seasonal disorder (SAD), akathisia, amnesia, catatonia , Diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease, progressive supranuclear palsy, cortical basal degeneration, familial frontotemporal dementia and , And immune disorders including autoimmune / inflammatory diseases, including acquired immune deficiency syndrome (AIDS), Breton-type agammalobulinemia, and acquired immunoglobulinemia ( CVI), DiGeorge syndrome (thymic hypoplasia), thymic dysplasia, IgA single deficiency, severe combined immunodeficiency (SCID), immunodeficiency with thrombocytopenia and eczema (Wiscott-Aldrich syndrome), Chediat - East anomaly, chronic granulomatous disease, hereditary angioneurotic edema, immune deficiency associated with Cushing's disease, adrenal insufficiency, adult respiratory distress syndrome,
Allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candida ectodermal dystrophy (APECED), bronchi Inflammation, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphotoxin-induced temporary lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulus Nephritis, Goodpasture 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,
Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner's syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral and bacterial infections, fungi Infectious disease, parasitic infection,
Includes protozoal infections, helminthic infections, hematopoietic cancers including lymphomas and leukemias, myeloma, trauma, and abnormal cell proliferation, including actinic keratosis and atherosclerosis, gliding. Bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, Melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary , Cancer of the pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterus. The polynucleotide sequence encoding RECAP can be obtained by the Southern method, Northern method, dot blotting method, or other membrane-based technology, PCR method, dipstick, pin, ELISA assay,
And, it can be used for a microarray utilizing body fluid or tissue collected from a patient to detect the expression of mutant RECAP. Such qualitative or quantitative methods are well known in the art.

In one embodiment, the nucleotide sequence encoding RECAP is associated with a disease
It will be particularly useful in assays to detect the above mentioned diseases. The nucleotide sequence encoding RECAP may be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the samples are washed and the signal is quantified and compared to the reference value. If the amount of signal in the patient sample is significantly altered compared to the control sample, the level of mutation of the RECAP-encoding nucleotide sequence in the sample reveals the presence of the associated disease. Such assays can be used to estimate the effectiveness of a particular treatment in animal studies, clinical trials, or monitoring individual patient treatment.

A normal or canonical expression profile is established to provide a basis for diagnosis of diseases associated with expression of RECAP. This can be achieved by combining a body fluid or cell extracted from a normal subject, either animal or human, with a sequence or a fragment thereof encoding RECAP under conditions suitable for hybridization or amplification. . Standard hybridizations can be quantitated by comparing the values obtained from normal subjects to those from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared with those obtained from subjects who exhibit symptoms of the disease. The deviation between the reference value and the subject's value is used to determine whether or not the patient is affected.

Once the presence of the disease is established and the treatment protocol is initiated, the hybridization assay is repeated on a regular basis to estimate whether the level of expression in the subject has begun to approach the values shown in normal patients. Is possible. The results of repeated assays can be used to see the effect of treatment over a period of days to months.

In cancer, abnormal amounts of transcripts in living tissue from an individual predispose to the development of the disease and may provide a method of detecting the disease before actual clinical symptoms develop. is there. This type of clearer diagnosis allows medical professionals to use preventative or aggressive therapies early to prevent the development or progression of cancer.

Further diagnostic uses of oligonucleotides designed from sequences encoding RECAP can include the use of PCR. Such oligomers can be chemically synthesized, enzymatically produced, or produced in vitro . The oligomer preferably contains a fragment of a polynucleotide encoding RECAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding RECAP, and is used to identify a specific gene or condition under the optimum conditions. It Further, the oligomer can be used for detecting and / or quantifying a closely related DNA or RNA sequence under slightly mild stringent conditions.

In certain embodiments, oligonucleotide primers derived from the polynucleotide sequence encoding RECAP can be used to detect single nucleotide polymorphisms (SNPs). SNP is
Nucleotide substitutions that often cause congenital or acquired genetic disease in humans,
Insertions and deletions. Methods of detection of SNPs include, but are not limited to, single-stranded conformational polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, DNA is amplified by polymerase chain reaction (PCR) using an oligonucleotide primer derived from a polynucleotide sequence encoding RECAP. This DNA can be derived, for example, from lesions or normal tissue, biopsy samples, body fluids and the like. The SNPs in this DNA make a difference in the secondary and tertiary structure of the PCR product in single-stranded form. This difference can be detected using gel electrophoresis in a non-denaturing gel. In fSCCP, by fluorescently labeling an oligonucleotide primer, it becomes possible to detect an amplimer with a high throughput device such as a DNA sequencing device. In addition, a sequence database analysis method called in silico SNP (isSNP) can identify polymorphisms by comparing the sequences of individual overlapping DNA fragments used to construct a common consensus sequence. . These computer-based methods eliminate sequence variability due to automated analysis of DNA sequence chromatograms and sequencing errors using statistical models and DNA adjustments in the laboratory. Alternatively, for example, the high throughput MASSARRAY system (Sequenom, I
nc., San Diego CA) to detect and characterize SNPs by mass spectrometry.

Methods that can be used to quantify RECAP expression include radiolabeling or biotin labeling of nucleotides, coamplification of regulatory nucleic acids, and standard curves plus results. (For example, Melby, PC et al. (1993) J. I.
mmunol. Methods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The quantitation rate of a large number of samples was accelerated by using a high-throughput assay in which the oligomer or polynucleotide of interest was included in various dilutions and the quantitation was rapid by spectrophotometry or non-color response.

In yet another example, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein can be used as targets in microarrays. Microarrays can be used in transcription imaging techniques to simultaneously monitor the relative expression levels of multiple genes. In this regard, U.S. Pat. No. 5,840,484 issued to Seilhamer, JJ et al.
Native Gene Transcript Analysis ”), which is incorporated herein by reference. Microarrays can also be used to identify genetic mutations, mutations and polymorphisms. This information can be used to determine gene function, elucidate the genetic basis of the disease, diagnose the disease, monitor the progression / regression of diseases associated with gene expression, and develop and activate therapeutic agents in the treatment of disease. Can be monitored. In particular, this information can be used to develop a pharmacogenomic profile of a patient in order to select the optimal and effective treatment regimen for the patient. For example, based on the patient's pharmacogenomic profile, therapeutic agents can be selected that are highly effective but have few side effects on the patient.

In another example, an antibody specific for RECAP, RECAP or a fragment thereof can be used as an element on the microarray. Microarrays can be used to monitor and measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.

Certain examples relate to the use of the polynucleotides of the invention to produce a transcriptional image of a tissue or cell type. Transcriptional images represent global patterns of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number and relative abundance of genes expressed at a given time under a given condition (Seilliamer et al., US Patent No. 5,840,484, "Comparative Gene Transc").
ript analysis ", which is hereby incorporated by reference for this patent), and thus the entire transcript or reverse transcript of a particular tissue or cell type is A hybrid image can be generated by hybridizing its complementary sequences, hi one embodiment, the polynucleotides of the invention or their complementary sequences hybridize in a high-throughput fashion to form a subset of multiple elements on a microarray. The resulting transcriptional image can be a profile of gene activity.

Transcript images can be generated using transcripts isolated from tissues, cell lines, biopsy samples, or other biological samples. Thus, the transcriptional image reflects gene expression in vivo for tissue or biopsy samples, or in vitro for cell lines.

Transcriptional images showing the expression profile of the polynucleotides of the invention can also be used in connection with in vitro model systems and preclinical evaluation of drugs, as well as toxicity studies of synthetic or natural compounds. All compounds cause characteristic gene expression patterns, often referred to as molecular fingerprints or toxicity signatures, that suggest mechanisms of action and toxicity (Nuwaysir, EF et al. (1999) Mol.
Carcinog. 24:15 3-159, Steiner, S. and NL Anderson (2000) Toxicol. Let
t. 112-113: 467-471, and also incorporated herein by reference). A test compound is likely to share toxic properties if it has the same signature as the signature of a known compound with toxicity. The more useful and accurate the fingerprint or signature contains expression information from more genes and gene families. Ideally, the expression should be measured across the genome and provide the highest quality signature. Similarly important are genes whose expression is not altered by any test compound. It is because the expression levels of these genes can be used to normalize the rest of the expression data. Normalized treatment is useful for comparing expression data after treatment with different compounds. Although assigning gene function to elements of a toxicity signature helps to elucidate the mechanism of toxicity, statistical knowledge of signatures leading to toxicity prediction requires no knowledge of gene function (eg, February 29, 2000). National Institute of Environmenta
See Press Release 00-02 published by Health Sciences. This is available at http://www.niehs.nih.gov/oc/news/toxchip.htm).
Therefore, it is important and desirable to include all expressed gene sequences in toxicity screens using the toxicity signature.

In certain embodiments, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acid with the test compound. Nucleic acids expressed in the treated biological sample can be hybridized with one or more probes specific for the polynucleotide of the invention, thereby quantifying the transcription level corresponding to the polynucleotide of the invention. The transcription level in the treated biological sample is compared to the level in the untreated biological sample. The difference in the transcription level of both samples is indicative of the toxic reaction that the test compound causes in the treated samples.

Another example relates to analyzing the proteome of a tissue or cell type using the polypeptide sequences of the invention. The term "proteome" refers to the global pattern of protein expression in a particular tissue or cell type. Each protein that makes up the proteome can be further analyzed individually. The proteome expression pattern or profile is analyzed by quantifying the number of proteins expressed under a given condition at a given time and their relative abundance. Therefore,
A proteomic profile of a cell can be generated by isolating and analyzing polypeptides of a particular tissue or cell type. In some embodiments, such separation is performed by two-dimensional gel electrophoresis. In this two-dimensional gel electrophoresis method, first,
Proteins are separated from the sample by one-dimensional isoelectric focusing and then by molecular weight by two-dimensional sodium dodecyl sulfate slab gel electrophoresis (
Steiner and Anderson). These proteins are visualized in the gel as spots in discrete discrete locations, usually by staining the gel with stains such as Coomassie blue or silver or fluorescent stains. The optical density of each protein spot is usually proportional to the protein level in the sample. The optical densities of co-located protein spots obtained from different samples, eg, biological samples, either treated or untreated with a test compound or therapeutic agent, are compared to determine changes in protein spot density associated with treatment. The proteins in the spot are
For example, chemical or enzymatic cleavage followed by partial sequencing using standard methods of mass spectrometry. The identity of a protein within a spot can be determined by comparing its partial sequence, which is preferably at least 5 consecutive amino acid residues, to a polypeptide sequence of the invention. In some cases, additional sequences for definitive protein identification are obtained.

Proteome profiles can also be generated by quantifying RECAP expression levels using RECAP-specific antibodies. In one example, protein expression levels are quantified by using antibodies as elements on the microarray and exposing the microarray to a sample to detect the level of protein binding to each array element (Lueking, A. et al. (1999). ) Anal. Biochern. 270: 103-111, Mend
Oze, LG et al. (1999) Biotechniques 27: 778-788). Detection can be accomplished by a variety of methods known in the art, for example, thiol-reactive or amino-reactive fluorescent compounds can be used to react proteins in a sample to detect the amount of fluorescent binding at each array element. .

The toxicity signature at the proteome level is also useful for toxicological screening and should be analyzed in parallel with the toxicity signature at the transcription level. For some proteins in some tissues, the abundance of transcripts and protein abundance may be low (Anderson, NL and J. Seilhamer (1997) Electrophores
18: 533-537), the proteome toxicity signature may be useful in the analysis of compounds that do not significantly affect the transcriptional image but alter the proteome profile. Furthermore, analysis of transcription in body fluids is difficult due to the rapid degradation of mRNA. Therefore, proteome profiling in such cases is more reliable and informative.

In another example, the toxicity of a test compound is assessed by treating a biological sample containing the protein with the test compound. The proteins 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 corresponding amount of protein in the untreated biological sample. Differences in the amount of protein in both samples indicate a toxic response to the test compound in the treated samples. Individual proteins sequence their amino acid residues,
These partial sequences are identified by comparison with the polypeptide of the invention.

In another example, the toxicity of a test compound is assessed by treating a biological sample containing the protein with the test compound. Proteins obtained from biological samples are incubated with antibodies specific for the polypeptides of the invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of protein in the untreated biological sample. The difference in the amount of protein in both samples is indicative of a toxic response to the test compound in the treated sample.

Microarrays are prepared, used, and analyzed by methods well known in the art. (For example, Brennan, TM et al. (1995) US Pat. No. 5,474,796; Schena, M. et al. (1996) Pro.
c. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) PCT application number W
O95 / 251116; Shalon, D. et al. (1995) PCT application number WO95 / 35505; Heller, RA et al. (1
997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Heller, MJ et al. (1997) U.S. Pat. No. 5,605,662). Various types of microarrays are well known and for details see DNA Microarrays: A Practical Approach , M. Schena, ed. (199
9) Described in Oxford University Press, London. In addition, this document is incorporated by reference.

In another embodiment of the invention, nucleic acid sequences encoding RECAP can also be used to make hybridization probes useful for mapping the native genomic sequence. Either coding or non-coding sequences can be used, although in some cases non-coding sequences are preferred over coding sequences. For example, conserved coding sequences between members of a multigene family can result in unwanted cross-hybridization during chromosome mapping. This sequence can be a specific chromosome, a specific region of a chromosome or an artificial chromosome, such as a human artificial chromosome (HAC), a yeast artificial chromosome (YAC),
Mapped to bacterial artificial chromosomes (BACs), bacterial P1 products, or single-chromosomal cDNA libraries (Harrington, JJ et al. (1997) Nat Genet. 15: 345-355, Pr.
ice, CM (1993) Blood Rev. 7: 127-134, Trask, BJ (1991) Trends Genet.
7: 149-154 etc.). Once mapped, the nucleic acid sequences of the present invention can be used to create a gene linkage map that correlates, for example, the inheritance of a disease state with a particular chromosomal region or restriction fragment length polymorphism (RFLP) inheritance (Lander). , ES and D. Botste
in (1986) Proc. Natl. Acad. Sci. USA 83: 7353-7357).

In sit fluorescence hybridization (FISH) can be correlated with other physical and genetic map data (eg Heinz-Ulrich, et al. (1995) in Meyers, supra,
pp. 965-968). Examples of genetic map data are available in various scientific journals or Online
It can be found on the World Wide Web site of Mendelian Inheritance in Man (OMIM). Correlation of the position of the gene encoding RECAP on the physical chromosomal map with a particular disease, or a predisposition to a particular disease, helps determine the DNA region associated with such a disease, so additional locations are needed. The cloning to determine is performed.

Genetic maps can also be extended using physical mapping techniques such as in sit hybridization of chromosomal preparations and binding analysis using established chromosomal markers. Placing a gene on the chromosome of another mammal, such as a mouse, often reveals related markers, even if the precise human chromosome location is not known. This information is valuable to researchers investigating genetic disorders using positional cloning or other gene discovery techniques. The location of one or more genes involved in a disease or syndrome is
When roughly determined by gene binding to a specific gene region such as 11q22-23,
Any sequence mapping to that region represents a relevant or regulatory gene for further investigation (eg Gatti, RA et al. (1988) Nature 336: 57.
See 7-580). Further, the target nucleotide sequence of the present invention may be used to detect a difference in chromosomal position between a normal subject and a carrier, that is, an infected subject due to translocation, inversion, or the like.

In another embodiment of the invention, RECAP, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen a library of compounds in any of a variety of drug screening techniques. Fragments used in such screening may be free in solution, immobilized on a solid support, retained on the surface of cells, or present intracellularly. The formation of complexes due to the binding of RECAP to the agent being tested may be measured.

Another method used for drug screening is used to increase the screening throughput of compounds with suitable binding affinities for the protein of interest (eg, Geysen, et al. (1984) PCT Application No. See WO84 / 03564). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is reacted with RECAP, or a fragment thereof, and then washed. Bound RECAP is then detected by methods well known in the art.
Purified RECAP can also be coated directly on plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another example, competitive drug screening assays can be used in which neutralizing antibodies capable of binding RECAP specifically compete with the test compound for binding RECAP. In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with RECAP.

In another example, nucleotide sequences encoding RECAP are used in developing molecular biology techniques, including, but not limited to, currently known nucleotides such as the triplet code and specific base pair interactions. New technologies can be provided that depend on the characteristics of the sequence.

Those skilled in the art will be able to make full use of the present invention without further explanation given the preceding description. Therefore, the specific preferred embodiments described below are for purposes of illustration and not limitation of the present invention.

All patent applications, patents, publications mentioned above and below, especially US patent application Ser. Nos. 60 / 145,232, 60 / 158,578, 60 / 165,192, are incorporated herein by reference.

(Example) 1 Preparation of cDNA library RNA was purchased from Clontech or isolated from the tissues listed in Table 4. First, while homogenizing a part of this tissue and dissolving it in a guanidinium isothiocyanate solution, homogenizing another part of this tissue and dissolving it in phenol, or TRIZOL (Life Technologies), guanidinium Dissolved in a mixture of suitable modifiers such as a single phase solution of thiocyanate and phenol. The lysate was extracted by centrifugation over cesium chloride or with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.

RNA extraction with phenol and precipitation were repeated as many times as necessary to increase the purity of the RNA. In some cases, RNA is treated with a DNA degrading enzyme. Most libraries have oligo d (T) -linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN).
Valencia CA), OLIGOTEX mRNA purification kit (QIAGEN) was used to isolate poly (A +) RNA. Alternatively, another RNA isolation kit such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX) was used to isolate directly from tissue lysates.

[0232] In some cases, RNA was provided to Stratagene and a cDNA library corresponding to Stratagene was created. Otherwise, UNIZAP vector system (Stratagene)
Alternatively, a cDNA library was prepared by using the SUPERSCRIPT plasmid system (Life Technologies) to synthesize cDNA by a recommended method known in the art or a similar method.
(See, eg, Ausubel, 1997, supra, Units 5.1-6.6). Reverse transcription is oligo d (T)
Or started with random primers. A synthetic oligonucleotide adapter was attached to the double-stranded cDNA and the cDNA was digested with a suitable restriction enzyme or restriction enzymes. For most libraries, SEPHACRYL S 1000 or SEPHAROSE
CL2B, SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech
), And the size of the cDNA (300-1000 bp) was selected by agarose gel electrophoresis. PBLUESCRIPT plasmid (Stratagene) or pSPORT1 plasmid (Life Technolo
gies), pcDNA2.1 plasmid (Invitrogen Carlsbad CA), plNCY plasmid (Incyt
The cDNA was attached to the compatible restriction enzyme sites of the polylinker of a suitable plasmid such as ePharmaceuticals, Palo Alto CA). Use this recombinant plasmid with XL1-Blue, XL1-BIueMRF, SOLR from Stratagene, or DH5α or DH from Life Technologies.
10B, ELECTROMAX DH 10B was introduced into competent E. coli cells and incorporated.

2 Isolation of cDNA clones The plasmids obtained as described in Example 1 above were UNIZAP vector system (S
tratagene) or by in vivo excision using cell lysis. Magic or WIZARD Minipreps DNA Purification System (Promega), and AGTC Minip
rep purification kit (Edge Biosystems, Gaithersburg MD), QIAGEN's QIAWELL 8 Pla
smid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid Purification System, REAL
Plasmids were purified using at least one of the Prep 96 plasmid kits. After precipitation, it was resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without freeze-drying.

Alternatively, plasmid DNA was amplified from host cell lysates by a high throughput direct ligation PCR method. (Rao, VB (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and transferred to 384-well plates for storage and the concentration of amplified plasmid DNA is quantified using PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). Measured.

3 Sequencing and Analysis The sequencing method of Incyte cDNA recovered in plasmids as described in Example 2 is as follows. The cDNA sequencing reaction can be performed by standard methods, or with ABI CATALYST 800 (PE Biosystems) thermal cycler or PTC-200 thermal cycler (MJ Research) and HYDRA microdispenser (Robb
ins Scientific) or MICROLAB 2200 (Hamilton) liquid transfer system in combination with a high throughput instrument. To prepare the cDNA sequencing reaction, use Amersham Pharmacia Biotech's reagent or ABI PRISM BIGDYE Termi.
The reagents included in the ABI sequencing kit such as the nator cycle sequencing ready reaction kit (PE Biosystems) were used. MEGABACE 1000 DNA for electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides.
A Sequencing System (Molecular Dynamics), ABI PRISM 373 or 377 Sequencing Systems (PE Biosystems) using standard ABI protocol and base pair calling software, and other sequence analysis systems known in the art were used. cDN
The open reading frame for the A sequence was determined using standard methods (Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected and extended by the method described in 5 of this Example.

The construction and analysis of the polynucleotide sequences obtained from the sequencing of the cDNA was carried out in combination with software utilizing algorithms well known to those skilled in the art.
Table 5 outlines the tools, software, algorithms utilized, their description, references, and threshold parameters used. Column 1 of Table 5 is the tools and programs used, algorithms, column 2 is a brief description thereof, column 3 is a reference cited as part of this specification by reference, column 4 is a sequence of two sequences. The score, probability value, and other parameters used in the evaluation of the degree of coincidence are shown (the higher the probability value, the higher the homology between sequences). MACDNASIS PRO software (Hitachi Softwa
re Engineering, S. San Francisco CA) and LASER GENE software (DNASTAR)
Was used.

Confirmation of the polynucleotide sequence is performed by using BLAST and dynamic programming, programs and algorithms based on dinucleotide nearest neighbor analysis to remove vectors and linkers, poly A sequences and mask ambiguous base pairs. I went there. next,
Using databases based on BLAST, FASTA and BLIMPS, public databases such as GenBank primates and rodents, mammals, vertebrates, eukaryotic databases and BLOCKS, PRINTS, DOMO, PRODOM and PFAM databases. Annotations were obtained by querying these sequences against sequences selected from. These sequences were constructed into full-length polynucleotide sequences using programs based on Phred and Phrap, Consed and screened for open reading frames with programs based on BLAST and FASTA, BLIMPS. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, and the GenBank database (above) and databases such as SwissProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite,
Alternatively, these full-length sequences were analyzed by querying a protein family database based on Hidden Markov Models (HMM) such as PFAM. HMMs use probabilities to analyze consensus primary structure of gene families (eg, Ed
dy, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365).

The programs described above for the construction and analysis of full length polynucleotide and amino acid sequences can also be used to identify fragments of the polynucleotide sequence from SEQ ID NOs: 23-44. Fragments of up to about 20-4000 nucleotides are useful for hybridization and amplification and have been described in the invention above.

4 Analysis of Polynucleotide Expression Northern analysis is an experimental technique used to detect the presence of transcripts of a gene, labeling the membrane bound RNA from a particular cell type or tissue. (See, eg, Sambrook, supra,
Chapter 7; and Ausubel. FM et al., Supra, Chapters 4 and 16).

Using similar computer technology for BLAST, GenBank or LIFESEQ (Inc
Search for identical or related molecules in cDNA databases such as yte Pharmaceuticals). This assay is much faster than many membrane-based hybridizations. Further, the sensitivity of computer searches can be modified to determine whether any particular match is classified as an exact match or a homologous match. Search criteria are

[0241]

[Equation 1]

Is the product score defined as The product score is a standardized value of 0-100 and is calculated as follows. Multiply the BLAST score by the percent identity of the nucleotide sequences,
The product is divided by 5 times the shorter length of the two sequences. The BLAST score is calculated by assigning a score of +5 to each base that matches in the high scoring segment pair (HSP) and a score of -4 to each mismatch. The two sequences may share more than one HSP (separated by a gap). If there are two or more HSPs, the base score with the highest BLAST score is used to calculate the product score. The product score represents the balance between fragmental overlap and quality of BLAST alignments. For example, the product score 100 is
Only obtained if there is 100% match over the shorter length of the two sequences compared. The product score 70 is either 100% coincidence and 70% overlap at one end, or 88% coincidence and 100% overlap at the other end. The product score 50 is either 100% coincidence and 50% overlap at one end, or 79% coincidence and 100% overlap at the other end.

The results of the Northern analysis are reported as the percent distribution of the library in which transcripts encoding RECAP occurred. The analysis also includes classification of the cDNA library by organ / tissue and disease. Organ / tissue categories include cardiovascular, skin, development, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive, urological. Disease categories include cancer, inflammation, trauma, cell proliferation, nerves, pooled. By category, the number of libraries expressing the sequence of interest was counted and divided by the number of libraries in the entire range. Table 3 shows the ratio (percentage) of specific expression in each tissue and the ratio of expression in each disease.

5 Chromosomal Mapping of a Polynucleotide Encoding RECAP The cDNA sequence used to construct SEQ ID NO: 23-44 was prepared using BLAST and Smith-Water.
The man algorithm was used to compare the sequences with the Insight LIFESEQ database and the public domain database. Sequences from these databases that match SEQ ID NO: 23-44 were assembled into a cluster of contiguous and overlapping sequences using a construction algorithm such as Phrap (Table 5). Stanford Human Genonse Center (SHG
C), Whitehead Institute for Genome Research (WIGR) and radiation hybrid and gene mapping data available from public sources such as Genethon will be used to determine if the clustered sequences have already been mapped. If the cluster contained mapped sequences, all sequences of that cluster (including the particular SEQ ID NO) were assigned to that mapping position.

The location of the genetic map is described as a section or range of the human chromosome. The range of mapping positions shown in centimorgan is measured from the end of the short arm (p) of the chromosome (centimorgan (cM) is a unit indicating the distance based on the crossover rate between genes on the same chromosome. On average, 1 cM is approximately equal to 1 megabase of human chromosomes, but can vary greatly due to the high recombination rate and the low recombination rate.) distance
cM is based on genetic markers mapped by Genethon that can detect the boundaries of radiation hybrid markers whose sequences are contained in their respective clusters. SEQ ID NO: 24 maps to chromosome 1 in the range 12.8 to 22.9 centimorgans, and SEQ ID NO: 36 maps to chromosome 1 in the range 74.8 to 78.3 centimorgans.

6 Extension of a Polynucleotide Encoding a RECAP The full length nucleic acid sequence of SEQ ID NO: 23-44 was prepared using oligonucleotide primers designed from suitable fragments of the full length molecule to The fragment was extended and produced. One primer was synthesized to initiate the 5'extension of the known fragment and the other primer was synthesized to initiate the 3'extension of the known fragment. The starting primer was approximately 22 to 30 nucleotides in length and approximately 5 nucleotides in length using OLIGO 4.06 software (National Biosciences) or other suitable program.
It had a GC content of 0% or higher and was designed to anneal to the target sequence at a temperature of about 68-72 ° C. Nucleotides were extended so that hairpin structures and primer-primer dimers did not occur.

This sequence was extended with selected human cDNA libraries. If more than one extension is required or desired, additional or nested primer sets are designed.

Amplification was performed with high fidelity by the PCR method using a method known in the art. PCR is PTC-200
It was performed in a 96-well block plate using a thermal cycler (MJ Research, Inc.). The reaction mixture consisted of template DNA, 200 nmol of each primer, Mg ²⁺ and (NH ₄ ) ₂ SO ₄ and β.
− Buffer containing mercaptoethanol, Taq DNA polymerase (Amersham Pha
rmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (St
ratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 60 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Storage at 4 ° C Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 57 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Store at 4 ° C.

The DNA concentration in each well was dissolved in 1X TE and 0.5 μl of undiluted PCR product.
100 μl PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugen
e OR) is distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagent and be measured. Fluoroskan this plate
Scan with II (Labsystems Oy, Helsinki, Finland) and measure the fluorescence of the sample to quantify the concentration of DNA. A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reaction was successful in extending the sequence.

The extended nucleotides are desalted and concentrated before being transferred to a 384 well plate and Cvi
JI Cholera virus endonuclease (Molecular Biology Research, Madison W
Digested with I) and sonicated or sheared before religation to pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel to cut the fragments and the agar was digested with Agar ACE (Promega). T4 Ligase (New England B
The clones extended with iolabs, Beverly MA) were cloned into pUC 18 vector (Amersham Pha
rmacia Biotech) and treated with Pfu DNA polymerase (Stratagene) extension of restriction sites to transfect competent E. coli cells. The transfected cells were selected and transferred to a medium containing antibiotics, each colony was cut off, and cultured in a 384-well plate of LB / 2X carbenicillin culture solution at 37 ° C. overnight.

The cells were lysed and the DNA was PCR amplified using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) as follows. Step 1 3 minutes at 94 ° C Step 2 15 seconds at 94 ° C Step 3 1 minute at 60 ° C Step 4 2 minutes at 72 ° C Step 5 Repeat steps 2, 3 and 4 29 times Step 6 5 minutes at 72 ° C Step 7 Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with poor DNA recovery were amplified again under the above conditions. Samples were diluted with 20% dimethysulphoxide (1: 2, v / v) and used in the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cycle se.
Sequencing was performed using the quencing ready reaction kit (PE Biosystems).

Similarly, following the procedure described above, utilizing the nucleotide sequence of SEQ ID NO: 23-44, 5'with the oligonucleotide designed for this extension and a suitable genomic library.
The regulatory sequence was obtained.

7 Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO: 23-44 are used to screen cDNA, mRNA, or genomic DNA. Special mention is made of labeling of oligonucleotides of about 20 base pairs, but essentially the same procedure is used for larger cDNA fragments. Oligonucleotides were designed using state-of-the-art software, such as OLIGO 4.06 software (National Bioscience), with 50 pmol of each oligomer and 250 μCi of [γ- ³² P] adenosine triphosphate (A
mersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN, Bost)
on MA) and used in combination for labeling. The labeled oligonucleotide was loaded onto a SEPHADEX G-25 ultrafine exclusion dextran bead column (Amersham Phar
Substantially purified using macia Biotech). An aliquot containing the labeled probe at 10 ⁷ counts per minute was added to the following endonucleases: Ase I, Bgl II, Eco R.
Human genome cleaved with one of I, Pst I, Xba1 or Pvu II (DuPont NEN)
Used in typical membrane-based hybridization analysis of DNA.

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are sequentially washed at room temperature, eg, under conditions of max 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. The hybridization patterns are visualized and compared by autoradiography or another imaging means.

8 Microarrays Linking or synthesizing array elements on a microarray is accomplished using photolithography, piezo printing (inkjet printers, see Baldeschweiler et al., Supra), mechanical microspotting techniques and the like. It is possible. In each of the above techniques, the substrate should be a solid, non-porous solid (Schena (1999). Supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, arrays similar to dot or slot blots can be utilized to place and bond the elements to the surface of the substrate by thermal, ultraviolet, or chemical or mechanical bonding means. Regular arrays can be made using available methods and machinery and can contain any suitable number of elements (Schena, M. et al. (1995) S
cience 270: 467-470, Shalon. D. et al. (1996) Genome Res. 6: 639-645, Marshall.
, A. and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31.).

A full-length cDNA, an expressed gene sequence fragment (EST), or a fragment or oligomer thereof can be an element of a microarray. Fragments and oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array element is hybridized with the polynucleotide in the biological sample. The polynucleotide in the biological sample is attached to a fluorescent label or other molecular tag to facilitate detection. After hybridization, unhybridized nucleotides are removed from the biological sample and the fluorescence scanner detects the hybridization at each array element. Alternatively, laser desorption and mass spectrometry may also be used to detect hybridization. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to the elements on the microarray can be calculated. The preparation and use of the microarray in one embodiment is detailed below.

Preparation of Tissue or Cell Samples 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 contained MMLV reverse transcriptase, 0.05 pg / μl oligo (dT) primer (21mer), 1x
First strand buffer, 0.03 units / μl RNase inhibitor, 500 μM dATP, 5
Reverse transcribing with 00 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). This reverse transcription reaction was performed using the GEMBRIGHT kit (Incyte) in 25 ml containing 200 ng of poly (A) ⁺ RNA.
Do with capacity. A specific control poly (A) ⁺ RNA is synthesized from non-coding yeast genomic DNA by in vitro transcription. After incubation at 370 ° C for 2 hours, each reaction sample (one labeled with Cy3 and the other labeled with Cy5) was treated with 2.5 ml of 0.5M sodium hydroxide and incubated at 850 ° C for 20 minutes to stop the reaction. To denature the RNA. Samples are purified using two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA). After conjugation, the two reaction samples were treated with 1 ml of glycogen (1 mg / ml), 6 ml.
Ethanol precipitate with 0 ml sodium acetate and 300 ml 100% ethanol. Samples of SpeedVAC (Savant Instruments Inc., Holbrook NY
) To finish and resuspend in 14 μl 5 × SSC / 0.2% SDS.

Microarray Preparation Arrays of the invention are used to make array elements. Each array element is amplified from bacterial cells containing the vector containing the cloned cDNA insert.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified by PCR for 30 cycles from an initial amount of 1-2 ng to a final amount of more than 5 μg. The amplified array element is SEPHACRYL-40
Purify using 0 (Amersham Pharmacia Biotech).

The purified array elements are immobilized on polymer-coated glass slides. Microscope glass slides (Corning) are washed with a large amount of distilled water during and after the treatment and ultrasonically washed in 0.1% SDS and acetone. 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), W
est Chester PA), washed extensively in distilled water,
Coat with 0.05% aminopropylsilane (Sigma) in% ethanol. The coated glass slide is cured by heating at 110 ° C.

Array elements are added to a coated glass substrate using the method described in US Pat. No. 5,807,522. References to this patent are incorporated herein by reference. 1 μl of the array element DNA with an average concentration of 100 ng / μl was opened using an open capillary printing element (open capillary) using a high-speed mechanical device.
Fill the printing element). The instrument then dispenses approximately 5 nl of array element sample per slide.

For microarrays, STRATALINKER UV crosslinker (Stratagene) is used for UV crosslinking. The microarray is washed once with 0.2% SDS at room temperature and three times with distilled water. The non-specific binding site is phosphate buffered saline (PBS) (Trop
Microarrays are incubated in 0.2% casein (IX, Inc., Bedford MA) for 30 minutes at 60 ° C., then blocked by washing with 0.2% SDS and distilled water as described above.

Hybridization The hybridization reaction solution contained 9 μl of the sample mixture containing 0.2 μg of each of the Cy3 and Cy5 labeled cDNA synthesis products in 5 × SSC, 0.2% SDS hybridization buffer. . The sample mixture is heated at 65 ° C. for 5 minutes, aliquoted onto the microarray surface and covered with a 1.8 cm ² cover glass. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. Add 140 μl of 5 × SSC to the corner of the chamber and adjust the humidity in the chamber to 10
Hold at 0%. The chamber containing this array is incubated at 60 ° C. for approximately 6.5 hours. Arrays were prepared in the first wash buffer (1 × SSC, 0.1% SDS) 45
Wash 3 times for 10 minutes each in the second wash buffer (0.1 x SSC) at 45 ° C for 10 minutes, then dry.

Detection Reporter Labeled Hybridization Complex Can Generate Spectral Lines at 488 nm for Exciting Cy3 and 632 nm for Exciting Cy3 Inn
Detection with a microscope equipped with an ova 70 gas 10 W laser (Coherent, Inc., Santa Clara CA). A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the excitation laser light on the array. The slide containing this array is placed on the computer-controlled XY stage of the microscope and raster-scanned through the objective lens. The 1.8 cm × 1.8 cm array used in this example is scanned at a resolution of 20 μm.

In two different scans, the mixed gas multi-line laser excites two fluorophores sequentially. The emitted light corresponds to two fluorophores based on wavelength, two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Brid
gewater NJ). The signal is filtered using a suitable filter placed between the array and the photomultiplier tube. The maximum emission of the phosphor used is Cy3
Is 565 nm, and Cy5 is 650 nm. The instrument can record spectra from both phosphors at the same time, but with a suitable filter for the laser source, one scan per phosphor and usually two scans of each array.

Scan sensitivity is usually calibrated using the signal intensity generated by a cDNA control species added to a sample mixture of known concentration. A specific position on the array contains a complementary DNA sequence such that the intensity of the signal at that position correlates to a weight ratio of the hybridizing species of 1: 100,000. Two samples from different samples (eg representing test cells and control cells) are each labeled with a different fluorophore and hybridized to a single array to identify genes that are differentially expressed. The calibration is carried out by labeling a sample of calibrating cDNA with two fluorophores and adding equal volumes to the hybridization mixture.

The output of the photomultiplier tube is a 12-bit RTI-835H analog-digital (AID) conversion board (Analog Device) installed in an IBM compatible PC computer.
s, Inc., Norwood MA). The digitized data has a signal intensity of blue (low signal) to red (using a linear 20-color conversion).
It is displayed as an image that is mapped into the pseudo color range up to (high signal). The data is also analyzed quantitatively. If two different fluorophores are excited at the same time for measurement, then the emission spectrum of each fluorophore is used to first correct the data for optical crosstalk between the fluorophores (due to the overlapping emission spectra). .

The grid is superimposed on the fluorescent signal image so that the signal from each spot is centered on each element of the grid. The fluorescent signals within each element are integrated and a number corresponding to the average intensity of the signal is obtained. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).

9 A sequence complementary to the sequence encoding the complementary polynucleotide RECAP, or any portion thereof, is used to reduce or inhibit the expression of native RECAP. About 15 to about 3
Although the use of oligonucleotides containing 0 base pairs is described, essentially the same method can be used for smaller or larger sequence fragments. Ol
Appropriate oligonucleotides are designed using igo4.06 software (National Biosciences) and RECAP coding sequences. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5'sequence and used to prevent the promoter from binding to the coding sequence. To prevent translation,
A complementary oligonucleotide is designed to prevent the ribosome from binding to the RECAP-encoding transcript.

10 Expression of RECAP Expression and purification of RECAP can be performed using a bacterial or virus-based expression system. For expression of RECAP in bacteria, the cDNA is subcloned into a suitable vector containing an inducible promoter that enhances antibiotic resistance and the transcription level of the cDNA. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host such as BL21 (DE3). Bacteria with antibiotic resistance are induced by isopropyl β-D thiogalactopyranoside (IPTG)
Express. Expression of RECAP in eukaryotic cells is carried out by infecting insect or mammalian cell lines with the Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with the cDNA encoding RECAP by either homologous recombination or bacterial mediated gene transfer with transfer plasmid mediated. Viral infectivity is maintained and the strong polyhedrin promoter results in high levels of cDNA transcription. Recombinant baculovirus is often Spodoptera frugiperda
(Sf9) Used to infect insect cells, but also used to infect human hepatocytes. In the case of the latter infection, further genetic modification of the baculovirus is required. (For example, Engelhard.EK 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 used, for example, glutathione S transferase (GST).
, Or a fusion protein synthesized with a peptide epitope tag such as FLAG or 6-His, the affinity-based purification of the recombinant fusion protein from crude cell lysate can be performed rapidly in one step. The 26-kilodalton enzyme GST from Schistosoma japonicum allows purification of fusion proteins with immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharma
cia Biotech). After purification, the GST moiety can be proteolytically cleaved from RECAP at specific manipulation sites. FLAG, a peptide of 8 amino acids, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). A stretch of 6 consecutive histidine residues, 6-His, allows purification on a metal chelate resin (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995, supra, ch 10, 16). RECAP purified by these methods can be used directly to perform the assays of Examples 11 and 15 below.

Demonstration of 11 RECAP activity RECAP receptor activity is measured in a ligand binding assay using candidate ligand molecules in the presence of ¹²⁵ I-labeled RECAP. Labeling RECAP with ¹²⁵ I Bolton Hunter Reagent (Bolton, AE and WM Hunter (1973) Biochem. 1. 133: 529-539)
. Labeled candidate ligand molecules pre-placed in the wells of a multi-well plate
Incubate with RECAP, then wash and assay all wells in which labeled RECAP complex is present. Data from various concentrations of RECAP are used to calculate values for the number and affinity, association of RECAP bound to the candidate molecule. The level of binding measured is proportional to the level of RECAP activity.

Alternatively, RECAP activity is tested in serum-free conditions using an assay based on the in vitro fibroblast and cancer cell-supporting properties of certain GPCRs (Ch
iquet-Ehrismann, R, et al. (1986) Cell 47: 131-139). 96-well cluster plate (Falcon, Fisher Scientitic, Santa Clara CA), 50-100 μg / well
Incubate at ambient temperature and coat with RECAP using ml solution.
Prior to transferring cells, aspirate coating solution and wash with Dulbecco's medium. Rat fibroblast culture cells or rat breast cancer cells are prepared as described above and transferred to Dulbecco's medium supplemented with 10% fetal calf serum (FCS) at a concentration of 10 ⁴ -10 ⁵ .

After 3 days the medium was removed and the cultured cells were incubated with phosphate buffered saline (PBS) before adding serum-free Dulbecco's medium (containing 0.25 mg / ml bovine serum albumin).
) Three times (BSA, Fraction V, Sigma Chemical, St. Louis, MO). After 2 days, the medium was aspirated and 2 μCi / ml [ ³ H] was added to a new Dulbecco's medium containing 0.25 mg / ml PBS.
Add 100 μl of thymidine (NEN). Parallel plates are fixed and stained for cell number. After 16 hours, the medium was aspirated, the cell layer was washed with PBS, and the 10% trichloroacetic acid insoluble count in the cell layer was measured by radioisotope (Chiquet-Ehrisrnann, R. et al. (1986) normalized to the cell number). Determined by liquid scintillation counter method. The rate of cell proliferation and [ ³ H] thymidine incorporation is proportional to the activity of RECAP in the sample.

Alternative assays for RECAP activity are based on the property of CD97 / Emr1 GPCR family proteins to regulate the G protein-activated second messenger signaling pathway (eg, cAMP; Gaudin, P., et al. 1998) 1. Biol. Chem., 273
: 4990-4996). Plasmids encoding full length RECAP are transfected into mammalian cell lines (eg, COS-7 or Chinese Hamster Ovary (CHO-K1) cell lines) using methods well known in the art. The transfected cells are grown in a 12-well tray of medium containing 2% FCS for 48 hours, then the medium is eliminated and then the attached cells are gently washed with PBS. Next, the cells are mixed with 10% FCS.
Alternatively, after incubating for 30 minutes in a medium containing 2% FCS, the medium is removed, and 1
Lyse cells by treatment with M perchloric acid. The level of cyclic AMP in the lysate is measured by radioimmunoassay by methods well known in the art. 2% FCS
The difference in the levels of cyclic AMP in the lysates between treatment with and with 10% FCS is proportional to the activity of RECAP present in the transfected cells.

An alternative assay for RECAP activity is based on a prototype assay for ligand / receptor-mediated alterations in cell proliferation. In this assay, Sw
The percentage of DNA synthesis in iss mouse 3T3 cells is measured. A plasmid containing a polynucleotide encoding RECAP is added to quiescent 3T3 cultured cells by methods well known in the art. The transiently transfected cells are then incubated in the presence of the radioactive DNA precursor molecule, [ ³ H] thymidine. Various amounts of RECAP ligand are added to transfected cells. [ ³ H] thymidine incorporated into acid-insoluble DNA at appropriate time intervals is measured by a radioisotope counter method. The amount of [ ³ H] thymidine incorporated is proportional to the amount of newly synthesized DNA. At least 10
A linear dose-effect curve for 0-fold RECAP ligand concentration shows the activity of the receptor. One unit of activity per ml is defined as the concentration of RECAP that produces a response level of 50%. Here, the 100% response level refers to the maximum amount of [ ³ H] thymidine incorporated into acid-insoluble DNA (McKay, I. and Leigh, I., eds. (1993) Growth Factors: A Practical Approach. , Oxford University Press, New York, NY, p. 73
).

An alternative assay for RECAP activity is based on the property of GPCR family proteins to regulate the G protein-activated second messenger signaling pathway (eg, cAMP; Gaudin, P., et al. (1998) 1 Biol. Chem., 273: 4990-4996). The full-length RECAP-encoding plasmid is transduced into a mammalian cell line (eg, Chinese Hamster Ovary (CHO-K1) cell line or human embryonic kidney (HEK-293) cell line) using methods well known in the art. To be perfect. The transfected cells are allowed to grow for 48 hours in a 12-well tray in medium before the medium is eliminated and then the attached cells are gently washed with PBS. The cells are then incubated for 30 minutes in medium with or without ligand, after which the medium is removed and treated with 1M perchloric acid to lyse the cells. The level of cyclic AMP in the lysate is measured by radioimmunoassay by methods well known in the art. The difference in cyclic AMP levels in the lysates with and without ligand exposure is proportional to the activity of RECAP present in transfected cells.

12 Functional Assays RECAP function is assessed by expression of sequences encoding RECAP at physiologically elevated levels in mammalian cell culture systems. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Such vectors include pCMV SPORT ^™ (Life Technologies.) And pCR 3.1.
(Invitrogen, Carlsbad, CA), both containing the cytomegalovirus promoter. For example, 5 to 10 μg of the recombinant vector is transiently transfected into a human cell line derived from endothelium or hematopoiesis by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows to distinguish between transfected and non-transfected cells. In addition, by the expression of the labeled protein,
The expression of the cDNA from the recombinant vector can be accurately predicted. Such labeled proteins include green fluorescent protein (GFP; Clontech), and CD64 or CD64-GFP fusion protein. Identify the transfected cells expressing GFP or CD64-GFP using automated flow cytometry (FCM) utilizing a technique based on laser optics,
Assess the cell's apoptotic state and other cellular characteristics. In addition, FCM detects and weighs uptake of fluorescent molecules that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena included changes in nuclear DNA content as measured by staining DNA with propidium iodide, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, and specific antibody Changes in cell surface and intracellular protein expression, as measured by the reactivity of A., and changes in plasma membrane composition measured by the binding of fluorescent complex annexin V protein to the cell surface.
Flow cytometry by Ormerod, MG (1994) Flow Cytometry Oxford, New
It is listed in York, NY.

The effect of RECAP on gene expression depends on the sequence encoding RECAP and CD64 or CD64.
-A highly purified cell population transfected with either GFP can be evaluated. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or antibodies against CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods well known in the art. Expression of mRNAs encoding RECAP and other genes of interest can be analyzed by Northern analysis or microarray technology.

13 Production of Antibodies Specific for RECAP Polyacrylamide gel electrophoresis (PAGE; eg, Harrington, MG (1990)
Methods Enzymol. 1816-3088-495) or other purification techniques and substantially purified RECAP is used to immunize rabbits using standard protocols to generate antibodies.

Alternatively, the RECAP amino acid sequence may be analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions and the corresponding oligopeptides synthesized and used to determine those methods well known to those of skill in the art. To produce antibodies. Selection of suitable epitopes, such as those near the C-terminus or within adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11).

Oligopeptides, typically about 15 residues in length, were applied to ABI 431A from Applied Biosystems.
Synthesized by chemistry of fmoc method using a peptide synthesizer (PE Biosystems), and N-maleimidobenzoyl-N-hydroxysuccinimide ester (
MBS) to bind to KLH (Sigma-Aldrich, St. Louis MO),
Enhances immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant.
To test the anti-peptide activity and anti-RECAP activity of the obtained antiserum, the peptide or RECAP was bound to the substrate, blocked with 1% BSA, reacted with rabbit antiserum and washed, and then radioactive React with goat anti-rabbit IgG labeled with iodine.

Purification of native RECAP with 14 specific antibody Native RECAP or recombinant RECAP is substantially purified by immunoaffinity chromatography using an antibody specific for RECAP. The immunoaffinity column is formed by covalently binding an anti-RECAP antibody with a resin for activation chromatography such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.

The culture medium containing RECAP is passed through an immunoaffinity column, and the column is washed under conditions capable of preferentially adsorbing RECAP (for example, with a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and RECAP (for example, with a buffer of pH 2-3 or with a high concentration of chaotropic ion such as urea or thiocyanate ion), and RECAP is recovered.

15 Identification of Molecules that Interact with RECAP RECAP or biologically active fragments thereof were labeled with ¹²⁵ I Bolton-Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). Label with. Candidate molecules pre-arranged in multiwell plates were labeled with labeled RE.
Incubate with CAP, wash and assay all wells with labeled RECAP complex. The data obtained at various RECAP concentrations are used to calculate values for the number and affinity, association of RECAP bound to the candidate molecule.

Alternatively, a molecule that interacts with RECAP may be prepared by the method described in Fields, S. and O. Song (1989, Natur.
e 340: 245-246) (yeast two-hybrid sys).
tem) and the MATCHMAKER system (Clontech) and other commercially available kits based on 2-hybrid systems.

RECAP also uses the high-throughput yeast two-hybrid system for P
The ATHCALLING process (CuraGen Corp., New Haven CT) can be used to determine all interactions between proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) US Patent No. No. 6,057,101).

Those skilled in the art will be able to make various modifications of the described methods and systems of the present invention without departing from the scope and spirit of the invention. Although the present invention has been described with reference to particular preferred embodiments, it should be understood that the scope of the invention should not be unduly limited to such particular 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 within the scope of the following claims.

BRIEF DESCRIPTION OF THE TABLES Table 1 lists the polypeptide and nucleotide sequences used to generate the full-length sequences encoding RECAP (SEQ ID NO), clone identification number, cDN
A library and a cDNA fragment are shown.

Table 2 lists the characteristics of each polypeptide sequence, including latent motifs and homologous sequences, and
The method, algorithm, and searchable database used for RECAP analysis are shown.

Table 3 lists selected fragments of each nucleic acid sequence, tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, diseases, disorders and conditions associated with these tissues, and each DNA. Shows the cloned vector.

Table 4 shows the tissues used to create the cDNA library in which the cDNA clone encoding RECAP was isolated.

Table 5 lists the tools used to analyze the polynucleotides and polypeptides of the invention,
The programs and algorithms, their description, references, and threshold parameters are shown.

[0293]

[Table 1]

[0294]

[Table 2]

[0295]

[Table 3]

[0296]

[Table 4]

[0297]

[Table 5]

[0298]

[Table 6]

[0299]

[Table 7]

[0300]

[Table 8]

[0301]

[Table 9]

[0302]

[Table 10]

[0303]

[Table 11]

[0304]

[Table 12]

[0305]

[Table 13]

[0306]

[Table 14]

[0307]

[Table 15]

[0308]

[Sequence list]

[Procedure amendment]

[Submission date] May 1, 2002 (2002.5.1)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (reference) A61P 1/16 A61P 3/00 4C084 1/18 3/10 4H045 3/00 5/00 3/10 7 / 06 5/00 9/10 7/06 11/06 9/10 11/16 11/06 13/12 11/16 17/00 13/12 19/02 17/00 19/10 19/02 25/00 19 / 10 101 25/00 25/02 101 25/04 25/02 25/06 25/04 25/14 25/06 25/16 25/14 25/18 25/16 25/20 25/18 25/28 25 / 20 31/00 25/28 31/10 31/00 31/18 31/10 33/00 31/18 35/00 33/00 37/00 35/00 37/02 37/00 37/06 37/02 37/08 37/06 43/00 111 37/08 C07K 14/705 43/00 111 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21 / 02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 C1 2N 15/00 ZNAA 5/00 A 33/566 A61K 37/02 (31) Priority claim number 60/165, 192 (32) Priority date November 12, 1999 (November 11, 1999) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB , BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, G M, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Tung, Wai Tom, California 95118 San Jose Runwick Court 4230 (72) Inventor Yue, Henry USA California 94087, Sunnyvale Lewis Avenue 826 (72) Inventor Azimzai , Yarda California, USA 94552 · Castro Valley Boulder Canyon Live 5518 (72) Inventor Burford, Neil Califo USA Lunia 94122 San Francisco Force Avenue 1308 (72) Inventor Bogun, Mariah Earl USA California 94577 San Leandro Santiago Road 14244 (72) Inventor Ryu, Dung Aina Em USA California 95136 San Jose Park Belmont Place 55 (72) Inventor Hillman, Jennifer El, United States 94040, Mountain View # 17, Monrode Live 230 (72) Inventor Patterson, Chandra, United States 94025, Menlo Park # 1, Sherwood Way 490 ( 72) Inventor Lal, Pretty, California, USA 95054 Santa Clara Russ Drive 2382 F term (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA63 CA01 GA11 HA12 4B063 QA01 QA1 8 QQ42 QR32. ZA55 ZA59 ZA60 ZA66 ZA81 ZA89 ZA94 ZA96 ZA97 ZB05 ZB07 ZB08 ZB13 ZB15 ZB26 ZB35 ZB37 ZC02 ZC03 ZC31 ZC35 4H045 AA10 AA11 AA20 BA10 DA50 EA20 EA50 FA74

Claims (28)

[Claims] 1. An isolated polypeptide comprising: (a) 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
An amino acid sequence selected from the group consisting of NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 22; and (b) 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
A natural amino acid having at least 90% sequence identity with an amino acid sequence selected from the group consisting of NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 22. And (c) 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
A biologically active fragment of an amino acid sequence selected from the group consisting of NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 22; d) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, S
EQ 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 and an immunogenic fragment of an amino acid sequence selected from the group consisting of: An isolated polypeptide comprising a different amino acid sequence. 2. 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 I
The isolated polypeptide of claim 1 selected from the group consisting of D NO: 22. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. An isolated polynucleotide encoding the polypeptide of claim 2. 5. 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,
The isolated claim 4 selected from the group consisting of 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. Polynucleotides. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 7. A cell transformed with the recombinant polynucleotide of claim 6. 8. A genetically modified organism containing the recombinant polynucleotide according to claim 6. 9. A method for producing the polypeptide of claim 1, which is (a) operably linked to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. The polypeptide of claim 1, comprising the steps of culturing cells transformed with a recombinant polynucleotide containing the expressed promoter sequence, and (b) recovering the polypeptide so expressed. Production method. 10. An isolated antibody that specifically binds to the polypeptide of claim 1. 11. An isolated polynucleotide comprising: (a) SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID N.
O: 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 N
A polynucleotide sequence selected from the group consisting of O: 44, and (b) SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID N
O: 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 N
A natural polynucleotide sequence having a sequence identity of at least 70% with a polynucleotide sequence selected from the group consisting of O: 44; (c) a polynucleotide sequence complementary to (a) above; An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence complementary to (b) and (e) an RNA equivalent of (a) to (d) above. 12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 11. 13. A method for detecting a target polynucleotide having the polynucleotide sequence according to claim 11 in a sample, comprising: (a) at least 20 comprising a sequence complementary to the target polynucleotide in the sample. Hybridizing the sample with a probe comprising a number of contiguous nucleotides, wherein the probe binds to the target polynucleotide under conditions where a hybridization complex of the probe with the target polynucleotide or fragment thereof is formed. Specifically hybridizing to a nucleotide, and (b) detecting the presence or absence of the hybridization complex and optionally measuring the yield if present. A method for detecting a target polynucleotide. 14. The method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides. 15. A method of detecting a target polynucleotide having the polynucleotide sequence of claim 11 in a sample, the method comprising: (a) amplifying the target polynucleotide or a fragment thereof using polymerase chain reaction amplification. And (b) detecting whether or not the amplified target polynucleotide or a fragment thereof is present, and if present, optionally measuring the yield thereof, the target polynucleotide. Detection method. 16. A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 17. The polypeptide is 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, S
EQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ
The pharmaceutical composition according to claim 16, which is a polypeptide comprising an amino acid sequence selected from the group consisting of ID NO: 20 and SEQ ID NO: 22. 18. A method for treating a disease associated with decreased expression of functional RECAP (receptor and related protein) or a symptom thereof, which comprises providing the pharmaceutical composition according to claim 16 to a patient in need of such treatment. A method of treatment comprising administering. 19. A method of screening a compound effective as an agonist of the polypeptide of claim 1, comprising the step of: (a) exposing a sample containing the polypeptide of claim 1 to the compound; Detecting the agonist activity of the sample. 20. A pharmaceutical composition comprising an agonist compound identified by the screening method of claim 19 and a pharmaceutically acceptable excipient. 21. A method of treating a disease associated with reduced expression of functional RECAP or a symptom thereof, comprising administering the pharmaceutical composition of claim 20 to a patient in need of such treatment. Characterized treatment method. 22. A method of screening a compound effective as an antagonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound; Detecting antagonist activity in the sample. 23. A pharmaceutical composition comprising an antagonist compound identified by the screening method of claim 22 and a pharmaceutically acceptable excipient. 24. A method for treating a disease associated with overexpression of functional RECAP or a symptom thereof, which comprises administering the pharmaceutical composition according to claim 23 to a patient in need of such treatment. Characterized treatment method. 25. A method for screening a compound that specifically binds to the polypeptide of claim 1, comprising the step of: (a) binding the polypeptide of claim 1 to at least one test compound under suitable conditions. And (b) the step of detecting the binding between the polypeptide of claim 1 and the test compound to identify the compound that specifically binds to the polypeptide of claim 1. . 26. A method of screening a compound that modulates the activity of the polypeptide of claim 1, comprising: (a) at least one test compound of the polypeptide of claim 1 under conditions permitting its activity. And (b) evaluating the activity of the polypeptide of claim 1 in the presence of the test compound, and (c) the activity of the polypeptide of claim 1 in the presence of the test compound. And comparing the activity of the polypeptide of claim 1 in the absence of the test compound with the change in activity of the polypeptide of claim 1 in the presence of the test compound. A method for screening, which is characterized by suggesting the presence of a compound that regulates the activity of the polypeptide of. 27. A method of screening a compound effective for altering the expression of a target polynucleotide comprising the sequence of claim 5, comprising: (a) exposing a sample containing the target polynucleotide to the compound. And (b) a step of detecting a change in expression of the target polynucleotide. 28. A method for assessing the toxicity of a test compound, the method comprising: (a) treating a biological sample containing a nucleic acid with the test compound; and (b) treating the nucleic acid of the biological sample. Hybridizing under a condition that a specific hybridization complex is formed between the probe containing at least 20 contiguous nucleotides of said polynucleotide and the target polynucleotide of said biological sample. And wherein the target polynucleotide comprises the polynucleotide sequence of the polynucleotide of claim 11 or a fragment thereof, (c) measuring the yield of hybridization complexes, and (d) in the treated biological sample. The yield of hybridization complex was determined by Comparing the yield of the hybridization complex in the pull, and the difference in the yield of the hybridization complex in the treated biological sample is indicative of the toxicity of the test compound. .