EP1339748A2 - Recepteurs couples a la proteine g - Google Patents

Recepteurs couples a la proteine g

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Publication number
EP1339748A2
EP1339748A2 EP01999578A EP01999578A EP1339748A2 EP 1339748 A2 EP1339748 A2 EP 1339748A2 EP 01999578 A EP01999578 A EP 01999578A EP 01999578 A EP01999578 A EP 01999578A EP 1339748 A2 EP1339748 A2 EP 1339748A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
polypeptide
seq
gcrec
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01999578A
Other languages
German (de)
English (en)
Inventor
Deborah A. Kallick
Mariah R. Baughn
Dyung Aina M. Lu
Henry Yue
Richard C. Graul
Yan Lu
Li Ding
Catherine M. Tribouley
Y. Tom Tang
Ameena R. Gandhi
Michael Thornton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Genomics Inc
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Filing date
Publication date
Application filed by Incyte Genomics Inc filed Critical Incyte Genomics Inc
Publication of EP1339748A2 publication Critical patent/EP1339748A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to nucleic acid and amino acid sequences of G-protein coupled receptors and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of G-protein coupled receptors.
  • the present invention further relates to the use of specific G-protein coupled receptors to identify molecules that are involved in modulating taste or olfactory sensation.
  • Signal transduction is the general process by which cells respond to extracellular signals.
  • Signal transduction across the plasma membrane begins with the binding of a signal molecule, e.g., a hormone, neurotransmitter, or growth factor, to a cell membrane receptor.
  • the receptor thus activated, triggers an intracellular biochemical cascade that ends with the activation of an intracellular target molecule, such as a transcription factor.
  • This process of signal transduction regulates all types of cell functions including cell proliferation, differentiation, and gene transcription.
  • GPCRs G-protein coupled receptors
  • encoded by one of the largest families of genes yet identified play a central role in the transduction of extracellular signals across the plasma membrane. GPCRs have a proven history of being successful therapeutic targets.
  • GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha ( ⁇ ) helices. GPCRs range in size from under 400 to over 1000 amino acids (Strosberg, A.D. (1991) Eur. J. Biochem. 196:1-10; Coughlin, S.R. (1994) Curr. Opin. Cell Biol. 6:191-197).
  • the amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains.
  • Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists.
  • the most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops.
  • the transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of ⁇ helices forms a ligand-binding pocket.
  • the extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.
  • the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins.
  • G heterotrimeric guanine nucleotide binding
  • GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, ⁇ -aminobutyric acid (GAB A), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., bombesin, bradykinin, calcitonin, C5a anaphyl
  • the diversity of the GPCR family is further increased by alternative splicing.
  • Many GPCR genes contain introns, and there are currently over 30 such receptors for which splice variants have been identified. The largest number of variations are at the protein C-terminus. N-terminal and cytoplasmic loop variants are also frequent, while variants in the extracellular loops or transmembrane domains are less common. Some receptors have more than one site at which variance can occur.
  • the splice variants appear to be functionally distinct, based upon observed differences in distribution, signaling, coupling, regulation, and ligand binding profiles (Kilpatrick, GJ. et al. (1999) Trends Pharmacol. Sci. 20:294-301).
  • GPCRs can be divided into three major subfamilies: the rhodopsin-like, secretin-like, and metabotropic glutamate receptor subfamilies. Members of these GPCR subfamilies share similar functions and d e characteristic seven transmembrane structure, but have divergent amino acid sequences. The largest family consists of the rhodopsin-like GPCRs, which transmit diverse extracellular signals including hormones, neurotransmitters, and light. Rhodopsin is a photosensitive GPCR found in animal retinas. In vertebrates, rhodopsin molecules are embedded in membranous stacks found in photoreceptor (rod) cells.
  • Each rhodopsin molecule responds to a photon of light by triggering a decrease in cGMP levels which leads to the closure of plasma membrane sodium channels. In this manner, a visual signal is converted to a neural impulse.
  • Other rhodopsin-like GPCRs are directly involved in responding to neurotransmitters. These GPCRs include the receptors for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors), adenosine, galanin, and glutamate (N-methyl-D-aspartate/NMDA receptors).
  • adrenaline adrenergic receptors
  • acetylcholine muscarinic receptors
  • adenosine adenosine
  • galanin galanin
  • glutamate N-methyl-D-aspartate/NMDA receptors
  • the galanin receptors mediate the activity of the neuroendocrine peptide galanin, which inhibits secretion of insulin, acetylcholine, serotonin and noradrenaline, and stimulates prolactin and growth hormone release.
  • Galanin receptors are involved in feeding disorders, pain, depression, and Alzheimer's disease (Kask, K. et al. (1997) Life Sci. 60:1523-1533).
  • Other nervous system rhodopsin-like GPCRs include a growing family of receptors for lysophosphatidic acid and other lysophospholipids, which appear to have roles in development and neuropathology (Chun, J. et al. (1999) Cell Biochem. Biophys. 30:213-242).
  • the RAlc receptor which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium (Raming, K. et al. (1998) Receptors Channels 6:141-151).
  • olfactory-like receptors are not confined to olfactory tissues.
  • three rat genes encoding olfactory-like receptors having typical GPCR characteristics showed expression patterns not only in taste and olfactory tissue, but also in male reproductive tissue (Thomas, M.B. et al. (1996) Gene 178:1-5).
  • secretin-like GPCR subfamily have as their ligands peptide hormones such as secretin, calcitonin, glucagon, growth hormone-releasing hormone, parathyroid hormone, and vasoactive intestinal peptide.
  • the secretin receptor responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine
  • Secretin receptors are about 450 amino acids in length and are found in the plasma membrane of gastrointestinal cells. Binding of secretin to its receptor stimulates the production of cAMP.
  • Examples of secretin-like GPCRs implicated in inflammation and the immune response include the EGF module-containing, mucin-like hormone receptor (Emrl) and CD97 receptor proteins. These GPCRs are members of the recently characterized EGF-TM7 receptors subfamily. These seven transmembrane hormone receptors exist as heterodimers in vivo and contain between three and seven potential calcium-binding EGF-like motifs.
  • CD97 is predominantly expressed in leukocytes and is markedly upregulated on activated B and T cells (McKnight, A.J. and S. Gordon (1998) J. Leukoc. Biol. 63:271-280).
  • the third GPCR subfamily is the metabotropic glutamate receptor family.
  • Glutamate is the major excitatory neurotransmitter in the central nervous system.
  • the metabotropic glutamate receptors modulate the activity of intracellular effectors, and are involved in long-term potentiation (Watson, supra, p.130).
  • the Ca 2+ -sensing receptor which senses changes in the extracellular concentration of calcium ions, has a large extracellular domain including clusters of acidic amino acids which may be involved in calcium binding.
  • the metabotropic glutamate receptor family also includes pheromone receptors, the GABA B receptors, and the taste receptors.
  • GPCRs include two groups of chemoreceptor genes found in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. which are distantly related to the mammalian olfactory receptor genes.
  • GPCR mutations which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Furthermore, somatic activating mutations in the thyrotropin receptor have been reported to cause hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes (Parma, J. et al. (1993) Nature 365:649-651).
  • GPCR receptors for the following ligands also contain mutations associated with human disease: luteinizing hormone (precocious puberty); vasopressin V 2 (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); ⁇ 3 -adrenoceptor
  • GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure, and several cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mol. Med. 76:464-468).
  • the dopamine agonist L-dopa is used to treat Parkinson's disease
  • a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease.
  • Agonists and antagonists of adrenoceptors have been used for the treatment of asthma, high blood pressure, other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment of glaucoma and tachycardia; serotonin 5HT1D antagonists are used against migraine; and histamine HI antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn, supra).
  • the type 1 receptor for parathyroid hormone is a GPCR that mediates the PTH-dependent regulation of calcium homeostasis in the bloodstream. Study of PTH/receptor interactions may enable the development of novel PTH receptor ligands for the treatment of osteoporosis (Mannstadt, M. et al. (1999) Am. J. Physiol. 277:F665-F675).
  • chemokine receptor group of GPCRs have potential therapeutic utility in inflammation and infectious disease.
  • Chemokines are small polypeptides that act as intracellular signals in the regulation of leukocyte trafficking, hematopoiesis, and angiogenesis. Targeted disruption of various chemokine receptors in mice indicates that these receptors play roles in pathologic inflammation and in autoimmune disorders such as multiple sclerosis.
  • Chemokine receptors are also exploited by infectious agents, including herpesviruses and the human immunodeficiency virus (HTV-l) to facilitate infection.
  • HTV-l human immunodeficiency virus
  • a truncated version of chemokine receptor CCR5 which acts as a coreceptor for infection of T-cells by HTV-l, results in resistance to AIDS, suggesting that CCR5 antagonists could be useful in preventing the development of AIDS.
  • the invention features purified polypeptides, G-protein coupled receptors, referred to collectively as “GCREC” and individually as “GCREC-1 ,” “GCREC-2,” “GCREC-3,” “GCREC-4,” “GCREC-5,” “GCREC-6,” “GCREC-7,” “GCREC-8,” “GCREC-9,” “GCREC-10,” and “GCREC- 11.”
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1 , c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-ll, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:l-l 1.
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 11, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-11.
  • the polynucleotide is selected from the group consisting of SEQ ID NO
  • the invention additionally provides G-protein coupled receptors that are involved in olfactory and/or taste sensation.
  • the invention further provides polynucleotide sequences that encode said G- protein coupled receptors.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-l l, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -11 , and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
  • the invention provides a cell transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1 - 11 , c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -11 , and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1 - 11.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l -11 , b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -11 , c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-l l.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:12-22, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:12-22, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
  • the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1 - 11 , c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and a pharmaceutically acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1.
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-l l, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l -11 , c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l -11 , and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-l 1.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with overexpression of functional GCREC, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-ll, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-l l, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -1 1, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l -11.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides methods of using G-protein coupled receptors of the invention involved in olfactory and/or taste sensation, biologically active fragments thereof (including those having receptor activity), and amino acid sequences having at least 90% sequence identity therewith, to identify compounds that agonize or antagonize the foregoing receptor polypeptides. These compounds are useful for modulating, blocking and/or mimicking specific tastes and/or odors.
  • the present invention also relates to the use of olfactory and/or taste receptors of the invention, biologically active fragments thereof (including those having receptor activity), and polypeptides having at least 90% sequence identity therewith, in combination with one or more other olfactory and/or taste receptor polypeptides, to identify a compound or plurality of compounds that modulate, mimic, and/or block a specific olfactory and/or taste sensation.
  • the invention also relates to cells that express an olfactory or taste receptor polypeptide of the invention, a biologically active fragment thereof (including those having receptor activity), or a polypeptide having at least 90% sequence identity therewith, and the use of such cells in cell-based screens to identify molecules that modulate, mimic, and/or block specific olfactory or taste sensations.
  • the invention relates to a cell that co-expresses at least one olfactory or taste G- protein coupled receptor polypeptide of the invention, and a G-protein, and optionally one or more other olfactory and/or taste G-protein coupled receptor polypeptides, and the use of such a cell in screens to identify molecules that modulate, mimic, and/or block specific olfactory and/or taste sensations.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDN A library for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
  • GCREC refers to the amino acid sequences of substantially purified GCREC obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of GCREC.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GCREC either by directly interacting with GCREC or by acting on components of the biological pathway in which GCREC participates.
  • allelic variant is an alternative form of the gene encoding GCREC. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding GCREC include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as GCREC or a polypeptide with at least one functional characteristic of GCREC. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding GCREC, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding GCREC.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent GCREC.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of GCREC is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to ti e complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of GCREC.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of GCREC either by directly interacting with GCREC or by acting on components of the biological pathway in which GCREC participates.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind GCREC polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense”
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription.
  • the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic GCREC, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3 -TCA-5'.
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding GCREC or fragments of GCREC may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison Wl) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
  • a "fragment” is a unique portion of GCREC or the polynucleotide encoding GCREC which is identical in sequence to but shorter in length than the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ID NO:12-22 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 12-22, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO: 12-22 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO: 12-22 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO.12-22 and the region of SEQ ID NO:12-22 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:l-l 1 is encoded by a fragment of SEQ ID NO:12-22.
  • a fragment of SEQ ID NO:l -11 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:l-l l.
  • a fragment of SEQ ID NO: 1-11 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:l-l 1.
  • the precise length of a fragment of SEQ ID NO : 1 - 11 and the region of SEQ ID NO:l-ll to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
  • “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • BLAST Basic Local Alignment Search Tool
  • BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.n_lm.nih.gov/gorf/bl2.html.
  • the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and iydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s).
  • the washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T ⁇ for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or I ⁇ t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immunogenic response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • An "immunogenic fragment” is a polypeptide or oligopeptide fragment of GCREC which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of GCREC which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of GCREC. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of GCREC.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, ohgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • Post-translational modification of an GCREC may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of GCREC.
  • Probe refers to nucleic acid sequences encoding GCREC, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
  • this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing GCREC, nucleic acids encoding GCREC, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human G-protein coupled receptors (GCREC), the polynucleotides encoding GCREC, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabolic disorders, and viral infections.
  • GCREC G-protein coupled receptors
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank h ⁇ molog.
  • Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison Wl).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
  • SEQ ID NO:l is 32% identical to rat seven transmembrane receptor (GenBank ID g5525078) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1 Je-92, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ID NO:l also contains a seven transmembrane receptor (Secretin family) domain and a latrophilin CL-1-like GPS domain (an unusual family of ubiquitous G- protein-linked receptors) as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:2 is 48% identical to a mouse odorant receptor (GenBank ID gl 419016) as determined by BLAST, with a probability score of 5.5e-74.
  • SEQ ID NO:2 also contains a rhodopsin family 7 -transmembrane receptor domain as determined by searching for statistically significant matches in the HMM-based PFAM database.
  • SEQ ID NO:4 is 55% identical to a human olfactory receptor protein (GenBank ID g2370145) as determined by BLAST, with a probability score of 5.9e- 86.
  • SEQ ID NO:4 also contains a 7 -transmembrane receptor domain as determined by searching for statistically significant matches in the HMM-based PFAM database. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFTLESCAN analyses provide further corroborative evidence that SEQ ID NO:4 is a G-protein coupled receptor.
  • SEQ ID NO:8 is 77% identical to Mus musculus odorant receptor S25 (GenBank ID g4680264) as determined by BLAST, with a probability score of 8.6e-126.
  • SEQ ID NO:8 also contains a 7 -transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the HMM-based PFAM database. (See Table 3.) Data from BLIMPS, MOTIFS, and
  • SEQ ID NO:8 is a G-protein coupled receptor.
  • SEQ ID NO:3, SEQ ID NO:5-7, and SEQ ID NO:9-l 1 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO:l -11 are described in Table 7.
  • Table 4 the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:12-22 or that distinguish between SEQ ED NO:12-22 and related polynucleotide sequences.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST”).
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or "NT") or the NCBI RefSeq Protein Sequence Records (Le., those sequences including the designation "NP”).
  • polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as FL_XXXXX_N 1 _N 2 _YYYY_N S _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N 123 , if present, represent specific exons that may have been manually edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as F XXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
  • the invention also encompasses GCREC variants.
  • a preferred GCREC variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the GCREC amino acid sequence, and which contains at least one functional or structural characteristic of GCREC.
  • the invention also encompasses polynucleotides which encode GCREC.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:12-22, which encodes GCREC.
  • the polynucleotide sequences of SEQ ID NO: 12-22 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses a variant of a polynucleotide sequence encoding GCREC.
  • a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding GCREC.
  • a particular aspect of d e invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 12- 22 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12-22.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding GCREC.
  • a splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding GCREC, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
  • a splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding GCREC over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide sequence encoding GCREC. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of GCREC.
  • nucleotide sequences which encode GCREC and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring GCREC under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding GCREC or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode GCREC and GCREC derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding GCREC or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ED NO:12-22 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol.
  • Hybridization conditions including anneabng and wash conditions, are described in "Definitions.” Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Appbed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampbfication system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 bquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Appbed Biosystems), the MEGABACE 1000 DNA sequencing system
  • nucleic acid sequences encoding GCREC may be extended utibzing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampbfy unknown sequence from genomic DNA within a cloning vector.
  • Another method inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method involves PCR ampbfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR ampbfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and bgations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • hbraries When screening for full length cDNAs, it is preferable to use hbraries that have been size-selected to include larger cDNAs. In addition, random-primed hbraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an obgo d(T) bbrary does not yield a full-length cDNA. Genomic hbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ tlowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Appbed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in bmited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode GCREC may be cloned in recombinant DNA molecules that direct expression of GCREC, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express GCREC.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter GCREC-encoding sequences for a variety of purposes including, but not bmited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic obgonuclcotides may be used to engineer the nucleotide sequences.
  • obgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spbce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARB REEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of GCREC, such as its biological or enzymatic activity or its abibty to bind to other molecules or compounds.
  • MOLECULARB REEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians
  • DNA shuffling is a process by which a bbrary of gene variants is produced using PCR-mediated recombination of gene fragments. The bbrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • sequences encoding GCREC may be synthesized, in whole or in part, using chemical methods well known in the art.
  • chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • GCREC itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or sobd-phase techniques.
  • Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Appbed Biosystems). Additionally, the amino acid sequence of GCREC, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
  • the peptide may be substantially purified by preparative high performance bquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • nucleotide sequences encoding GCREC or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding GCREC.
  • regulatory sequences such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding GCREC.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding GCREC. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • a variety of expression vector/host systems may be utibzed to contain and express sequences encoding GCREC. These include, but are not bmited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., caubflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for debvery of nucleotide sequences to die targeted organ, tissue, or cell population.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding GCREC.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding GCREC can be achieved using a multifunctional E. cob vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies).
  • PBLUESCRIPT Stratagene, La Jolla CA
  • PSPORT1 plasmid Life Technologies
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of GCREC may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of GCREC.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of GCREC. Transcription of sequences encoding GCREC may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-31 1). Alternatively, plant promoters such as the small subunit of RUB ISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.
  • mammaban cells a number of viral-based expression systems may be utibzed.
  • sequences encoding GCREC may be bgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses GCREC in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaban host cells.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to debver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and debvered via conventional debvery methods (bposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • bposomes polycationic amino polymers, or vesicles
  • sequences encoding GCREC can be transformed into cell bnes using expression vectors which may contain viral origins of repbcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • expression vectors which may contain viral origins of repbcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems may be used to recover transformed cell bnes. These include, but are not bmited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11 :223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabobte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabobtes.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C.A . . (1995) Methods Mol. Biol.
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding GCREC is inserted within a marker gene sequence, transformed cells containing sequences encoding GCREC can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding GCREC under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding GCREC and that express GCREC may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not bmited to, DNA-DNA or DNA-RNA hybridizations, PCR ampbfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of GCREC using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-bnked immunosorbent assays
  • RTAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GCREC include obgolabebng, nick translation, end-labeling, or PCR ampbfication using a labeled nucleotide.
  • sequences encoding GCREC, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • a vector for the production of an mRNA probe Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison Wl), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionucbdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the bke.
  • Host cells transformed with nucleotide sequences encoding GCREC may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode GCREC may be designed to contain signal sequences which direct secretion of GCREC through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its abibty to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not bmited to, acetylation, carboxylation, glycosylation, phosphorylation, bpidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant nucleic acid sequences encoding GCREC may be bgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric GCREC protein containing a heterologous moiety that can be recognized by a commercially available antibody may facibtate the screening of peptide hbraries for inhibitors of GCREC activity.
  • Heterologous protein and peptide moieties may also facibtate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not bmited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodubn binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobibzed glutathione, maltose, phenylarsine oxide, calmodubn, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the GCREC encoding sequence and the heterologous protein sequence, so that GCREC may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facibtate expression and purification of fusion proteins.
  • synthesis of radiolabeled GCREC may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • GCREC of the present invention or fragments thereof may be used to screen for compounds that specifically bind to GCREC. At least one and up to a plurabty of test compounds may be screened for specific binding to GCREC. Examples of test compounds include antibodies, obgonucleotides, proteins (e.g., receptors), or small molecules.
  • the compound thus identified is closely related to the natural bgand of GCREC, e.g., a bgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • the compound can be closely related to the natural receptor to which GCREC binds, or to at least a fragment of the receptor, e.g., the bgand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express GCREC, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or K cob.
  • Cells expressing GCREC or cell membrane fractions which contain GCREC are then contacted with a test compound and binding, stimulation, or inhibition of activity of either GCREC or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with GCREC, either in solution or affixed to a sobd support, and detecting the binding of GCREC to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay may be carried out using cell-free preparations, chemical hbraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a sobd support.
  • GCREC of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of GCREC.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for GCREC activity, wherein GCREC is combined with at least one test compound, and the activity of GCREC in the presence of a test compound is compared with the activity of GCREC in the absence of the test compound. A change in the activity of GCREC in the presence of the test compound is indicative of a compound that modulates the activity of GCREC.
  • a test compound is combined with an in vitro or cell-free system comprising GCREC under conditions suitable for GCREC activity, and the assay is performed.
  • a test compound which modulates the activity of GCREC may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurabty of test compounds may be screened.
  • polynucleotides encoding GCREC or their mammaban homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
  • ES embryonic stem
  • mouse ES cells such as the mouse 129/SvJ cell bne
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Cbn.
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding GCREC may also be manipulated in vitro in ES cells derived from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate cell bneages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic bneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147). Polynucleotides encoding GCREC can also be used to create "knockin" humanized animals
  • a region of a polynucleotide encoding GCREC is injected into animal ES cells, and the injected sequence integrates into d e animal cell genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred bnes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress GCREC e.g., by secreting GCREC in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS
  • GCREC Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of GCREC and G-protein coupled receptors.
  • examples of tissues expressing GCREC can be found in Table 6. Therefore, GCREC appears to play a role in cell probferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabobc disorders, and viral infections.
  • GCREC appears to play a role in cell probferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabobc disorders, and viral infections.
  • GCREC In the treatment of disorders associated with decreased GCREC expression or activity, it is desirable to increase the expression or activity of GCREC.
  • GCREC or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC.
  • disorders include, but are not bmited to, a cell probferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gangba, gastrointestinal tract, heart, kidney,
  • a vector capable of expressing GCREC or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC including, but not bmited to, those described above.
  • compositions comprising a substantially purified GCREC in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC including, but not bmited to, those provided above.
  • an agonist which modulates the activity of GCREC may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of GCREC including, but not bmited to, those bsted above.
  • an antagonist of GCREC may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GCREC.
  • disorders include, but are not bmited to, those cell probferative, neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and metabobc disorders, and viral infections, described above.
  • an antibody which specifically binds GCREC may be used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to cells or tissues which express GCREC.
  • a vector expressing the complement of the polynucleotide encoding GCREC may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of GCREC including, but not bmited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of GCREC may be produced using methods which are generally known in the art.
  • purified GCREC may be used to produce antibodies or to screen hbraries of pharmaceutical agents to identify those which specifically bind GCREC.
  • Antibodies to GCREC may also be generated using methods that are well known in the art. Such antibodies may include, but are not bmited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression bbrary.
  • Neutrabzing antibodies i.e., those which inhibit dimer formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with GCREC or with any fragment or obgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not bmited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilb Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the obgopeptides, peptides, or fragments used to induce antibodies to GCREC have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these obgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of GCREC amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to GCREC may be prepared using any technique which provides for the production of antibody molecules by continuous cell bnes in culture. These include, but are not bmited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-cell hybridoma technique
  • EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobubn hbraries.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobubn hbraries or panels of highly specific binding reagents as disclosed in the bterature.
  • Antibody fragments which contain specific binding sites for GCREC may also be generated.
  • such fragments include, but are not bmited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression hbraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • K association constant
  • polyclonal antibody preparations may be further evaluated to determine the quabty and suitabibty of such preparations for certain downstream appbcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of GCREC-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidebnes for antibody quality and usage in various appbcations, are generally available. (See, e.g., Catty, supra, and Cobgan et al. supra.)
  • the polynucleotides encoding GCREC may be used for therapeutic pu ⁇ oses.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified obgonucleotides) to the coding or regulatory regions of the gene encoding GCREC.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified obgonucleotides
  • antisense obgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding GCREC.
  • Antisense sequences can be debvered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
  • polynucleotides encoding GCREC may be used for somatic or germbne gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- bnked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • diseases or disorders caused by deficiencies in GCREC are treated by constructing mammaban expression vectors encoding GCREC and introducing these vectors by mechanical means into GCREC-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) balbstic gold particle debvery, (iii) bposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J-L and H.
  • Expression vectors that may be effective for the expression of GCREC include, but are not bmited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • GCREC may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycbne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • Biotechnol. 9:451-456 commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding GCREC from a normal individual.
  • CommerciaUy available bposome transformation kits e.g., the PERFECT LIPID
  • TRANSFECTION KIT available from Invitrogen
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841 -845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammaban transfection protocols.
  • diseases or disorders caused by genetic defects with respect to GCREC expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding GCREC under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commercially available (Stratagene) and are based on pubbshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing cell bne (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61 :1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471 ; Zufferey, R.
  • VSVg vector producing cell bne
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell bnes and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
  • an adenovirus-based gene therapy debvery system is used to debver polynucleotides encoding GCREC to cells which have one or more genetic abnormabties with respect to the expression of GCREC.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art.
  • Repbcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268).
  • Potentially useful adenoviral vectors are described in U.S. Patent No.
  • Addenovirus vectors for gene therapy hereby inco ⁇ orated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, TM. and N. Somia (1997) Nature 18:389:239-242, both inco ⁇ orated by reference herein.
  • a he ⁇ es-based, gene therapy debvery system is used to debver polynucleotides encoding GCREC to target cells which have one or more genetic abnormabties with respect to the expression of GCREC.
  • the use of he ⁇ es simplex virus (HSV)-based vectors may be especially valuable for introducing GCREC to cells of the central nervous system, for which HS V has a tropism.
  • the construction and packaging of he ⁇ es-based vectors are well known to those with ordinary skill in the art.
  • a rephcation-competent he ⁇ es simplex virus (HSV) type 1 -based vector has been used to debver a reporter gene to the eyes of primates (Liu, X.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("He ⁇ es simplex virus strains for gene transfer"), which is hereby inco ⁇ orated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for pu ⁇ oses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby inco ⁇ orated by reference.
  • the manipulation of cloned he ⁇ esvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large he ⁇ esvirus genomes, the growth and propagation of he ⁇ esvirus, and the infection of cells with he ⁇ esvirus are techniques well known to those of ordinary skill in the art.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to debver polynucleotides encoding GCREC to target cells.
  • SFV Semhki Forest Virus
  • This subgenomic RNA repbcates to higher levels than the full length genomic RNA, resulting in the ove ⁇ roduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for GCREC into the alphavirus genome in place of the capsid-coding region results in the production of a large number of GCREC-coding RNAs and the synthesis of high levels of GCREC in vector transduced cells.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abibty to estabbsh a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repbcation of alphaviruses can be altered to suit the needs of the gene therapy appbcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses will allow the introduction of GCREC into a variety of cell types.
  • the specific transduction of a subset of ceUs in a population may require the sorting of cells prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
  • Obgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple hebx base-pairing methodology.
  • Triple hebx pairing is useful because it causes inhibition of the abibty of the double hebx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the bterature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Pubbshing, Mt. Kisco NY, pp. 163-177.)
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding GCREC.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the ohgonucleotide inoperable. The suitabibty of candidate targets may also be evaluated by testing accessibibty to hybridization with complementary obgonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing obgonucleotides such as sobd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding GCREC. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU bnes, cells, or tissues.
  • RNA molecules may be modified to increase intraceUular stabibty and half-bfe. Possible modifications include, but are not bmited to, the addition of flanking sequences at the 5' and/or 3 'ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase bnkages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding GCREC.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not bmited to, obgonucleotides, antisense obgonucleotides, triple hebx-forming obgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encoding GCREC may be therapeutically useful, and in the treatment of disorders associated with decreased GCREC expression or activity, a compound which specifically promotes expression of the polynucleotide encoding GCREC may be therapeutically useful.
  • At least one, and up to a plurabty, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary bbrary of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a bbrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding GCREC is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabihzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding GCREC are assayed by any method commonly known in the art.
  • 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 GCREC.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell bne such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
  • a particular embodiment of the present invention involves screening a combinatorial bbrary of obgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified obgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into ceUs or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
  • obgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified obgonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonaUy propagated for autologous transplant back into that same patient.
  • Debvery by transfection, by bposome injections, or by polycationic amino polymers may be achieved using methods which are web known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • compositions which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Pubbshing, Easton PA).
  • Such compositions may consist of GCREC, antibodies to GCREC, and mimetics, agonists, antagonists, or inhibitors of GCREC.
  • compositions utibzed in this invention may be administered by any number of routes including, but not bmited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subbngual, or rectal means.
  • compositions for pulmonary administration may be prepared in bquid or dry powder form. These compositions are generaUy aerosobzed immediately prior to inhalation by the patient.
  • smaU molecules e.g. traditional low molecular weight organic drugs
  • aerosol debvery of fast- acting formulations is well-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary debvery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
  • the determination of an effective dose is well within the capabibty of those skilled in the art.
  • Speciabzed forms of compositions may be prepared for direct intracellular debvery of macromolecules comprising GCREC or fragments thereof.
  • macromolecules comprising GCREC or fragments thereof.
  • bposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular debvery of the macromolecule.
  • GCREC or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initiaUy either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example GCREC or fragments thereof, antibodies of GCREC, and agonists, antagonists or inhibitors of GCREC, which ameborates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with bttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • I mg-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-bfe and clearance rate of the particular formulation. Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • antibodies which specifically bind GCREC may be used for the diagnosis of disorders characterized by expression of GCREC, or in assays to monitor patients being treated with GCREC or agonists, antagonists, or inhibitors of GCREC.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for GCREC include methods which utibze the antibody and a label to detect GCREC in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • GCREC a variety of protocols for measuring GCREC, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of GCREC expression.
  • Normal or standard values for GCREC expression are estabbshed by combining body fluids or cell extracts taken from normal mammaban subjects, for example, human subjects, with antibodies to GCREC under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of GCREC expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabbshes the parameters for diagnosing disease.
  • the polynucleotides encoding GCREC may be used for diagnostic pu ⁇ oses.
  • the polynucleotides which may be used include ohgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of GCREC may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of GCREC, and to monitor regulation of GCREC levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding GCREC or closely related molecules may be used to identify nucleic acid sequences which encode GCREC.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampbfication wiU determine whether the probe identifies only naturally occurring sequences encoding GCREC, allebc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the GCREC encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 12-22 or from genomic sequences including promoters, enhancers, and introns of the GCREC gene.
  • Means for producing specific hybridization probes for DNAs encoding GCREC include the cloning of polynucleotide sequences encoding GCREC or GCREC derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucbdes such as 32 P or 35 S, or by enzymatic labels, such as alkabne phosphatase coupled to the probe via avidin/biotin coupbng systems, and the bke.
  • radionucbdes such as 32 P or 35 S
  • enzymatic labels such as alkabne phosphatase coupled to the probe via avidin/biotin coupbng systems, and the bke.
  • Polynucleotide sequences encoding GCREC may be used for the diagnosis of disorders associated with expression of GCREC.
  • disorders include, but are not bmited to, a cell probferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangba, gastrointestinal tract, heart, kidney, bver, lung, muscle,
  • the polynucleotide sequences encoding GCREC may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-bke assays; and in microarrays utibzing fluids or tissues from patients to detect altered GCREC expression. Such quahtative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding GCREC may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding GCREC may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
  • the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding GCREC in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in cbnical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of
  • GCREC a normal or standard profile for expression is estabbshed. This may be accompbshed by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding GCREC, under conditions suitable for hybridization or ampbfication. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabbsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual cbnical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • obgonucleotides designed from the sequences encoding GCREC may involve the use of PCR. These obgomers may be chemically synthesized, generated enzymatically, or produced in vitro. Obgomers will preferably contain a fragment of a polynucleotide encoding GCREC, or a fragment of a polynucleotide complementary to the polynucleotide encoding GCREC, and wiU be employed under optimized conditions for identification of a specific gene or condition. Obgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • ohgonucleotide primers derived from the polynucleotide sequences encoding GCREC may be used to detect single nucleotide polymo ⁇ hisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not bmited to, single-stranded conformation polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymo ⁇ hism
  • fSSCP fluorescent SSCP
  • ohgonucleotide primers derived from the polynucleotide sequences encoding GCREC are used to amphfy DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the bke.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the ohgonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • sequence database analysis methods termed in sibco SNP (isSNP) are capable of identifying polymo ⁇ hisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • Methods which may also be used to quantify the expression of GCREC include radiolabebng or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the obgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • obgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymo ⁇ hisms.
  • This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • GCREC fragments of GCREC, or antibodies specific for GCREC may be used as elements on a microarra .
  • the microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al, "Comparative Gene Transcript Analysis," U.S. Patent No.
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totabty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurabty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, cell bnes, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell bne.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and precbnical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturaUy-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular finge ⁇ rints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al.
  • test compound has a signature similar to that of a compound with known toxicity, it is bkely to share those toxic properties.
  • These finge ⁇ rints or signatures are most useful and refined when they contain expression information from a large number of genes and gene famibes. Ideally, a genome- wide measurement of expression provides the highest quabty signature. Even genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normabze the rest of the expression data.
  • the normabzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in inte ⁇ retation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
  • the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or cell type.
  • proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
  • a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuahzed in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generally proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for GCREC to quantify the levels of GCREC expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lucking, A. et al. (1999) Anal. Biochem. 270:103-111 ; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, NX. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profibng may be more rehab le and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appbcation W095/251116; Shalon, D. et al. (1995) PCT appbcation WO95/35505; Heller, R.A. et al (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al.
  • nucleic acid sequences encoding GCREC may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA hbraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA hbraries.
  • the nucleic acid sequences of the invention may be used to develop genetic bnkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymo ⁇ hism (RFLP).
  • RFLP restriction fragment length polymo ⁇ hism
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendeban Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding GCREC on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • OMIM Online Mendeban Inheritance in Man
  • In situ hybridization of chromosomal preparations and physical mapping techniques, such as bnkage analysis using estabbshed chromosomal markers, may be used for extending genetic maps.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • GCREC its catalytic or immunogenic fragments, or obgopeptides thereof can be used for screening hbraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a sobd support, borne on a cell surface, or located intracellularly. The formation of binding complexes between GCREC and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different small test compounds are synthesized on a sobd substrate. The test compounds are reacted with GCREC, or fragments thereof,. and washed. Bound GCREC is then detected by methods well known in the art. Purified GCREC can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutrabzing antibodies can be used to capture the peptide and immobibze it on a sobd support.
  • nucleotide sequences which encode GCREC may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not bmited to, such properties as the triplet genetic code and specific base pair interactions. Without further elaboration, it is bebeved that one skilled in the art can, using the preceding description, utibze the present invention to its fullest extent.
  • Incyte cDNAs were derived from cDNA hbraries described in the LEF ⁇ SEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A)+ RNA was isolated using obgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constructed the corresponding cDNA hbraries. Otherwise, cDNA was synthesized and cDNA hbraries were constructed with the
  • UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1 -6.6.) Reverse transcription was initiated using obgo d(T) or random primers. Synthetic ohgonucleotide adapters were bgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300- 1000 bp) using SEPHACRYL SI 000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were bgated into compatible restriction enzyme sites of the polybnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
  • Recombinant plasmids were transformed into competent E. cob cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies.
  • Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
  • a Magic or WIZARD Minipreps DNA purification system Promega
  • an AGTC Miniprep purification kit Edge Biosystems, Gaithersburg MD
  • Plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophihzation, at 4°C Alternatively, plasmid DNA was ampbfied from host cell lysates using direct bnk PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycbng steps were carried out in a single reaction mixture.
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Appbed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or suppbed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Appbed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Appbed Biosystems) in conjunction with standard ABI protocols and base calbng software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 1.1). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VTTI.
  • the polynucleotide sequences derived from Incyte cDNAs were vabdated by removing vector, bnker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubbc databases such as the GenBank primate, rodent, mammaban, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae. Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • pubbc databases such as the GenBank primate, rodent, mammaban, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM
  • PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus
  • HMM is a probabibstic approach which analyzes consensus primary structures of gene famihes. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to full length.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where appbcable, the scores, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty value, the greater the identity between two sequences).
  • Genscan is a general-pu ⁇ ose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karbn (1997) J. Mol. Biol 268:78-94, and Burge, C. and S. Karbn (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
  • the encoded polypeptides were analyzed by querying against PFAM models for G-protein coupled receptors. Potential G-protein coupled receptors were also identified by homology to Incyte cDNA sequences mat had been annotated as G-protein coupled receptors.
  • Genscan-predicted sequences were then compared by BLAST analysisHo the genpept and gbpri pubbc databases.
  • Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or pubbc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
  • Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • Full length polynucleotide sequences were obtained by assembbng Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubbc cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible spbce variants that were subsequently confirmed, edited, or extended to create a fuh length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
  • Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubbc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of GCREC Encoding Polynucleotides
  • sequences which were used to assemble SEQ ID NO: 12-22 were compared with sequences from the Incyte LTFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ID NO: 12-22 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubbc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WTGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • pubbc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WTGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normabzed value between 0 and 100, and is calculated as follows: the BLAST score is multipbed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quabty in a BLAST abgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding GCREC are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA sequence is derived from a cDNA bbrary constructed from a human tissue.
  • Each human tissue is classified into one of the following organ tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaba, female; genitaba, male; germ cells; hemic and immune system; bver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of hbraries in each categoiy is counted and divided by the total number of hbraries across all categories.
  • each human tissue is classified into one of die following disease/condition categories: cancer, cell line, developmental, inflammation, neurological trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding GCREC.
  • cDNA sequences and cDNA bbrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
  • Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using ohgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 rain; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ were as follows: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C
  • the plate was scanned in a Fluoroskan ⁇ (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l ahquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WT), and sonicated or sheared prior to rebgation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WT
  • sonicated or sheared prior to rebgation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were rebgated using T4 bgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. cob cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37° C in 384- well plates in LB/2x carb bquid media. The cells were lysed, and DNA was ampbfied by PCR using Taq DNA polymerase
  • Step 1 94°C, 3 min
  • Step 2 94°C, 15 sec
  • Step 3 60°C, 1 min
  • Step 4 72°C, 2 min
  • Step 5 steps 2, 3, and 4 repeated 29 times
  • Step 6 72°C, 5 min
  • Step 7 storage at 4°C.
  • DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampbfied using the same conditions as described above.
  • Hybridization probes derived from SEQ ID NO:12-22 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labehng of obgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentially the same procedure is used with larger nucleotide fragments. Obgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each obgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled obgonucleotides arc substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl ⁇ , Eco RI, Pst I, Xba I, or Pvu fl (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x sabne sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuabzed using autoradiography or an alternative imaging means and compared.
  • the bnkage or synthesis of array elements upon a microarray can be achieved utibzing photobthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and sobd with a non-porous surface (Schena (1999), supra). Suggested substrates include sibcon, sibca, glass sbdes, glass chips, and sibcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and bnk elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines well known to those of ordinary skiU in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or obgomers thereof may comprise the elements of the microarray. Fragments or obgomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the obgo-(dT) cellulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l obgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMB RIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labehng) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampbfied from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR ampbfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampbfied in thirty cycles of PCR from an initial quantity of 1 -2 ng to a final quantity greater than 5 ⁇ g.
  • Ampbfied array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified array elements are immobibzed on polymer-coated glass sbdes. Glass microscope sbdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass sbdes are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated sbdes are cured in a 110°C oven. Array elements are appbed to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 inco ⁇ orated herein by reference.
  • 1 ⁇ l of the array element DNA is loaded into the open capillary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of array element sample per sbde.
  • Microarrays are UV-crossbnkcd using a STRATALINKER UV-crossbnker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered sabne (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distilled water as before.
  • PBS phosphate buffered sabne
  • Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral bnes at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser bght is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the shde containing the array is placed on a computer-controUed X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentially. Emitted hght is spbt, based on wavelength, into two photomultipher tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipher tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typically cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the cabbration is done by labehng samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipher tube is digitized using a 12-bit RTI-835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a Unear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
  • Sequences complementary to the GCREC-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring GCREC.
  • obgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate obgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of GCREC.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary ohgonucleotide is designed to prevent ribosomal binding to the GCREC-encoding transcript.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not bmited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express GCREC upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • GCREC GCREC in eukaryotic cells
  • infecting insect or mammaban cell bnes with recombinant Autographica cabfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus AcMNPV
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding GCREC by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
  • GCREC is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • FLAG an 8-amino acid peptide
  • 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified GCREC obtained by these methods can be used directly in the assays shown in Examples XVI, XVII, and XVi ⁇ , where appbcable. XIII. Functional Assays
  • GCREC function is assessed by expressing the sequences encoding GCREC at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammaban expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell hne, for example, an endothebal or hematopoietic cell line, using either bposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a rebable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward hght scatter and 90 degree side bght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry. Oxford, New York NY.
  • GCREC The influence of GCREC on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding GCREC and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobubn G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding GCREC and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • the GCREC amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding obgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • obgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Appbed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • ABI 431 A peptide synthesizer Appbed Biosystems
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the obgopeptide- KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-GCREC activity by, for example, binding the peptide or GCREC to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant GCREC is substantially purified by immunoaffinity chromatography using antibodies specific for GCREC.
  • An immunoaffinity column is constructed by covalently coupling anti-GCREC antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupbng, the resin is blocked and washed according to the manufacturer's instructions.
  • GCREC Media containing GCREC are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of GCREC (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/GCREC binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCREC is collected.
  • a chaotrope such as urea or thiocyanate ion
  • GCREC or a fragment thereof, is labeled with 1 5 I Bolton-Hunter reagent.
  • a fragment of GCREC includes, for example, a fragment comprising one or more of the three extracellular loops, the extracellular N-terminal region, or the third intracellular loop.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled GCREC, washed, and any weUs with labeled GCREC complex are assayed. Data obtained using different concentrations of GCREC are used to calculate values for the number, affinity, and association of GCREC with the candidate bgand molecules.
  • GCREC may also be used in the PATHCALLENG process (CuraGen Co ⁇ ., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • GCREC agonists or antagonists may be tested for activation or inhibition of GCREC receptor activity using the assays described in sections XVEE and XVEEE.
  • Candidate molecules may be selected from known GPCR agonists or antagonists, peptide hbraries, or combinatorial chemical hbraries.
  • Methods for detecting interactions of GCREC with intracellular signal transduction molecules such as G proteins are based on the premise that internal segments or cytoplasmic domains from an O ⁇ han G protein-coupled seven transmembrane receptor may be exchanged with the analogous domains of a known G protein-coupled seven transmembrane receptor and used to identify the G- proteins and downstream signabng pathways activated by the o ⁇ han receptor domains (Kobilka, B.K. et al. (1988) Science 240:1310-1316).
  • domains of the o ⁇ han receptor may be cloned as a portion of a fusion protein and used in binding assays to demonstrate interactions with specific G proteins.
  • the third intraceUular loop of G protein-coupled seven transmembrane receptors is important for G protein interaction and signal transduction (Conkbn, B.R. et al (1993) Cell 73:631-641).
  • the DNA fragment corresponding to the third intraceUular loop of GCREC may be ampbfied by the polymerase chain reaction (PCR) and subcloned into a fusion vector such as pGEX (Pharmacia Biotech).
  • PCR polymerase chain reaction
  • pGEX Pharmacia Biotech
  • the construct is transformed into an appropriate bacterial host, induced, and the fusion protein is purified from the ceU lysate by glutathione-Sepharose 4B (Pharmacia Biotech) affinity chromatography.
  • cell extracts containing G proteins are prepared by extraction with 50 mM Tris, pH 7.8, 1 mM EGTA, 5 mM MgCl 2 , 20 mM CHAPS, 20% glycerol, 10 ⁇ g of both aprotinin and leupeptin, and 20 ⁇ l of 50 mM phenylmethylsulfonyl fluoride.
  • the lysate is incubated on ice for 45 min with constant stirring, centrifuged at 23,000 g for 15 min at 4°C, and the supernatant is collected.
  • GST glutathione S-transferase
  • the [ 32 P]ADP-labeled proteins are separated on 10% SDS-PAGE gels, and autoradiographed.
  • the separated proteins in these gels are transferred to nitrocellulose paper, blocked with blotto (5% nonfat dried milk, 50 mM Tris-HCl (pH 8.0), 2 mM CaCl 2 , 80 mM NaCl, 0.02% NaN 3 , and 0.2% Nonidet P-40) for 1 hour at room temperature, followed by incubation for 1.5 hours with G ⁇ subtype selective antibodies (1:500; Calbiochem-Novabiochem). After three washes, blots are incubated with horseradish peroxidase (HRP) -conjugated goat anti-rabbit immunoglobubn (1 :2000, Cappel,
  • HRP horseradish peroxidase
  • An assay for GCREC activity measures the expression of GCREC on the cell surface.
  • cDNA encoding GCREC is transfected into an appropriate mammaban cell bne.
  • Cell surface proteins are labeled with biotin as described (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed using GCREC-specific antibodies, and immunoprecipitated samples are analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of GCREC expressed on the cell surface.
  • an assay for GCREC activity is based on a prototypical assay for bgand/receptor-mediated modulation of ceU probferation. This assay measures the rate of DNA synthesis in Swiss mouse 3T3 cells.
  • a plasmid containing polynucleotides encoding GCREC is added to quiescent 3T3 cultured cells using transfection methods well known in the art.
  • the transiently transfected ceUs are then incubated in the presence of [ 3 H]fhymidine, a radioactive DNA precursor molecule.
  • Varying amounts of GCREC bgand are then added to the cultured cells.
  • Inco ⁇ oration of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a radioisotope counter, and the amount inco ⁇ orated is directly proportional to the amount of newly synthesized DNA.
  • a hnear dose-response curve over at least a hundred-fold GCREC bgand concentration range is indicative of receptor activity.
  • One unit of activity per milbbter is defined as the concentration of GCREC producing a 50% response level, where 100% represents maximal inco ⁇ oration of [ 3 H]thymidine into acid-precipitable DNA (McKay, I. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach. Oxford University Press, New York NY, p. 73.)
  • the assay for GCREC activity is based upon the abibty of GPCR family proteins to modulate G protein-activated second messenger signal transduction pathways (e.g., cAMP; Gaudin, P.
  • a plasmid encoding full length GCREC is transfected into a mammaban ceU bne (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell bnes) using methods well-known in the art.
  • a mammaban ceU bne e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell bnes
  • Transfected cells are grown in 12-weU trays in culture medium for 48 hours, then the culture medium is discarded, and the attached cells are gently washed with PBS. The cells are then incubated in culture medium with or without bgand for 30 minutes, then the medium is removed and cells lysed by treatment with 1 M perchloric acid.
  • cAMP levels in the lysate are measured by radioimmunoassay using methods weU-known in the art. Changes in the levels of cAMP in the lysate from cells exposed to ligand compared to those without bgand are proportional to the amount of GCREC present in the transfected cells.
  • the cells are grown in 24-well plates containing lxlO 5 cells/well and incubated with inositol-free media and [ 3 H]myoinositol, 2 ⁇ Ci/well, for 48 hr.
  • the culture medium is removed, and the cells washed with buffer containing 10 mM LiCl followed by addition of bgand.
  • the reaction is stopped by addition of perchloric acid.
  • Inositol phosphates are extracted and separated on Dowex AG1-X8 (Bio-Rad) anion exchange resin, and the total labeled inositol phosphates counted by bquid scintillation.
  • GCREC is expressed in a eukaryotic cell bne such as CHO (Chinese Hamster Ovary) or HEK (Human Embryonic Kidney) 293 which have a good history of GPCR expression and which contain a wide range of G-proteins allowing for functional coupbng of the expressed GCREC to downstream effectors.
  • the transformed cells are assayed for activation of the expressed receptors in the presence of candidate hgands.
  • Activity is measured by changes in intracellular second messengers, such as cycbc AMP or Ca 2+ . These may be measured directly using standard methods well known in the art, or by the use of reporter gene assays in which a luminescent protein (e.g.
  • firefly luciferase or green fluorescent protein is under the transcriptional control of a promoter responsive to the stimulation of protein kinase C by the activated receptor (Milbgan, G. et al. (1996) Trends Pharmacol. Sci. 17:235-237).
  • Assay technologies are available for both of these second messenger systems to allow high throughput readout in multi-well plate format, such as the adenylyl cyclase activation FlashPlate Assay (NEN Life Sciences Products), or fluorescent Ca 2+ indicators such as Fluo-4 AM (Molecular Probes) in combination with the FLTPR fluorimetric plate reading system (Molecular Devices).
  • GCREC may be coexpressed with the G-proteins G ⁇ l5/16 which have been demonstrated to couple to a wide range of G-proteins (Offermanns, S. and M.I. Simon (1995) J. Biol. Chem. 270:15175-15180), in order to funnel the signal transduction of the GCREC through a pathway involving phosphohpase C and Ca 2+ mobilization.
  • G-proteins Offermanns, S. and M.I. Simon (1995) J. Biol. Chem. 270:15175-15180
  • GCREC may be expressed in engineered yeast systems which lack endogenous GPCRs, thus providing the advantage of a null background for GCREC activation screening. These yeast systems substitute a human GPCR and G ⁇ protein for the corresponding components of the endogenous yeast pheromone receptor pathway.
  • Downstream signabng pathways are also modified so that the normal yeast response to the signal is converted to positive growth on selective media or to reporter gene expression (Broach, J.R. and J. Thorner (1996) Nature 384 (supp.):14-16).
  • the receptors are screened against putative ligands including known GPCR Ugands and other naturally occurring bioactive molecules.
  • Biological extracts from tissues, biological fluids and cell supernatants are also screened.
  • N-terminus is non-cytosolic
  • G-protein coupled receptor BL00649 BLIMPS- T376-K403, F802-T847, C787-L812, BLOCKS Y873-L902, L914-G935, T956-F981
  • EGF-like domain signature 2 MOTIFS C134-C147
  • M25-I45, T57-I77, Q101-F124, M137-F165, A199-L227, E270-K296 N-terminus is non-cytosolic
  • G-protein coupled receptors ProfileScan signature F103-V147
  • Rhodopsin-like GPCR superfamily BLIMPS- signature PR00237 PRINTS
  • Olfactory receptor signature PR00245 BLIMPS- M59-L80, F178-D192, Y239-G254, PRINTS V275-L286, S292-L306
  • Olfactory G-protein coupled receptor BLAS - PD000921 L167-L247 PRODOM
  • Olfactory G-protein coupled receptor BLAST- PD149621 V248-F309 PRODOM
  • G-protein coupled receptors BLAST-DOMO
  • MOTIFS signature A111-I127
  • Rhodopsin-like GPCR superfamily BLIMPS- signature PR00237 T27-R51, M60-K81, PRINTS
  • Olfactory receptor signature PR00245 BLIMPS- M60-K81, F178-S192, F239-G254, PRINTS A274-L285, S291-M305
  • MOTIFS signature S111-V127
  • TMAP T8-L28, L35-T55, G62-L82, TMAP Q101-Y124, I137-C165, F201-F221, G234-C254
  • G-protein coupled receptor BL00237 BLIMPS- K91-P130, I208-Y219, K235-R261, BLOCKS T282-K298
  • G-protein coupled receptors signature PROFILESCAN g_protein_receptor .prf : F103-G153 Rhodopsin-like GPCR superfamily BLIMPS- signature PR00237: L27-R51, M60-Q81, PRINTS F105-I127, L200-V223, Q272-K298
  • OLFACTORY RECEPTOR BLAST- PD149621: I247- 308
  • PRODOM PD000921 L166-L246
  • MOTIFS signature S110-I126
  • G-protein coupled recept BL00237 BLIMPS- K90-P129, T280-K296 BLOCKS
  • Olfactory receptor signature PR00245 BLIMPS- M59-K80, Y177-D191, F238-G253, PRINTS F272-L283, S289-L303
  • Vasopressin receptor signature BLIMPS- PR00896B L55-L66 PRINTS
  • OLFACTORY RECEPTOR BLAST- PD000921: F168-L245
  • PRODOM PD149621 T246-L303
  • MOTIFS signature T110-I126
  • MOTIFS signature S110-I126
  • T135 T175 family G40-Y291 T205
  • TMAP C31-Y59, S92-R120, I139-H165, TMAP
  • Olfactory receptor signature PR00245 BLIMPS- M58-K79, T175-D189, L236-V251, PRINTS L275-L286
  • RECEPTOR OLFACTORY PROTEIN PD000921 BLAST- L164-I243 PRODOM
  • GPCRDPV02 PCR2- Library was constructed using pooled cDNA from different donors.
  • cDNA was generated TOPOTA using mRNA isolated from the following: aorta, cerebellum, lymph nodes, muscle, tonsil (lymphoid hyperplasia) , bladder tumor (invasive grade 3 transitional cell carcinoma.), breast (proliferative fibrocystic changes without atypia characterized by epithilial ductal hyperplasia, testicle tumor (embryonal carcinoma) , spleen, ovary, parathyroid, ileum, breast skin, sigmoid colon, penis tumor (fungating invasive grade 4 squamous cell carcinoma) , fetal lung, , breast, fetal small intestine, fetal liver, fetal pancreas, fetal lung, fetal skin, fetal penis, fetal bone, fetal ribs, frontal brain tumor (grade 4 gemistocytic astrocytoma)
  • stromal hyperthecosis bladder, bladder tumor (invasive grade 3 transitional cell carcinoma) , stomach, lymph node tumor (metastatic basaloid squamous cell carcinoma) , tonsil (reactive lymphoid hyperplasia) , periosteum from the tibia, fetal brain, fetal spleen, uterus tumor, endometrial (grade 3 adenosquamous carcinoma) , seminal vesicle, liver, aorta, adrenal gland, lymph node (metastatic grade 3 squamous cell carcinoma) , glossal muscle, esophagus, esophagus tumor (invasive grade 3 adenocarcinoma), ileum, pancreas, soft tissue tumor from the skull (grade 3 ependymoma) , transverse colon,
  • rectum tumor grade 3 colonic adenocarcinoma
  • rib tumor grade 3 osteosarcoma
  • osteosarcoma metal-static grade 3 osteosarcoma
  • lung heart, placenta, thymus, stomach, spleen (splenomegaly with congestion) , uterus, cervix (mild chronic cervicitis with focal squamous metaplasia)
  • spleen tumor malignant lymphoma, diffuse large cell type, B-cell phenotype with abundant reactive T-cells and marked granulomatous response
  • umbilical cord blood mononuclear cells upper lobe lung tumor, (grade 3 squamous cell carcinoma)
  • endometrium secretory phase
  • kidney transformed embryonal cell line (293-EBNA) (treated with 5Aza- 2deoxycytidine for 72 hours)
  • HMEC cells mammary epithelial cells
  • peripheral blood monocytes treated with IL-10 at time 0, lOng/ml, LPS was added at 1 hour at
  • GPCRDPV02 5ng/ml Incubation 24 hrs) , peripheral blood monocytes (treated with anti-IL-10 at (cont . ) time 0, lOng/ml, LPS was added at 1 hour at 5ng/ml .
  • ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
  • fastx score 100 or greater
  • HMM Probability valuer hidden Markov model
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
  • TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
  • HMM hidden Markov model

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Abstract

L'invention concerne des récepteurs humains couplés à la protéine G (GCREC) et des polynucléotides qui identifient et codent pour CGREC. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention concerne en outre des méthodes de diagnostic, de traitement ou de prévention de troubles associés à une expression aberrante de GCREC.
EP01999578A 2000-12-08 2001-12-05 Recepteurs couples a la proteine g Withdrawn EP1339748A2 (fr)

Applications Claiming Priority (9)

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US25432300P 2000-12-08 2000-12-08
US254323P 2000-12-08
US25556400P 2000-12-13 2000-12-13
US255564P 2000-12-13
US25771600P 2000-12-21 2000-12-21
US257716P 2000-12-21
US26284801P 2001-01-19 2001-01-19
US262848P 2001-01-19
PCT/US2001/046659 WO2002046230A2 (fr) 2000-12-08 2001-12-05 Récepteurs couplés à la protéine g

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US7803982B2 (en) 2001-04-20 2010-09-28 The Mount Sinai School Of Medicine Of New York University T1R3 transgenic animals, cells and related methods
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CA2342833A1 (fr) * 1998-09-17 2000-03-23 Incyte Pharmaceuticals, Inc. Proteines gpcr humaines
WO2000020590A2 (fr) * 1998-10-06 2000-04-13 Incyte Pharmaceuticals, Inc. Proteines de recepteurs couples a des proteines g
AU2001264579B2 (en) * 2000-05-12 2006-04-06 Lexicon Pharmaceuticals, Inc. Seven transmembrane proteins and polynucleotides encoding the same

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See references of WO0246230A2 *

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AU2002220231A1 (en) 2002-06-18

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