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WO2001066746A2 - Nouvelles proteines et acides nucleiques codant pour ces proteines - Google Patents

Nouvelles proteines et acides nucleiques codant pour ces proteines Download PDF

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Publication number
WO2001066746A2
WO2001066746A2 PCT/US2001/007116 US0107116W WO0166746A2 WO 2001066746 A2 WO2001066746 A2 WO 2001066746A2 US 0107116 W US0107116 W US 0107116W WO 0166746 A2 WO0166746 A2 WO 0166746A2
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Prior art keywords
nucleic acid
amino acid
polypeptide
protein
seq
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WO2001066746A3 (fr
Inventor
Muralidhara Padigaru
Catherine E. Burgess
Kumud Majumder
Vishnu S. Mishra
Li Li
Jason C. Baumgartner
Kimberly A. Spytek
Velizar T. Tchernev
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CuraGen Corp
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CuraGen Corp
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Priority to EP01916415A priority Critical patent/EP1259612A2/fr
Priority to CA002401549A priority patent/CA2401549A1/fr
Priority to JP2001565900A priority patent/JP2003525633A/ja
Priority to AU4344401A priority patent/AU4344401A/xx
Publication of WO2001066746A2 publication Critical patent/WO2001066746A2/fr
Publication of WO2001066746A3 publication Critical patent/WO2001066746A3/fr
Anticipated expiration legal-status Critical
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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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the invention generally relates to nucleic acids and polypeptides. More particularly, the invention relates to nucleic acids encoding novel G-protein coupled receptor (GPCR) polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
  • GPCR G-protein coupled receptor
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as NOVX, or NOV1, NOV2, NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, and NOV10 nucleic acids and polypeptides.
  • NOVX nucleic acid or polypeptide sequences.
  • the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30.
  • the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30.
  • an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g., SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30) or a complement of said oligonucleotide.
  • a NOVX nucleic acid e.g., SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30
  • a complement of said oligonucleotide e.g., SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30
  • NOVX polypeptides SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
  • the invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a NOVX polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX. Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, or other disorders related to cell signal processing and metabolic pathway modulation.
  • the therapeutic can be, e.g., a NONX nucleic acid, a ⁇ ONX polypeptide, or a ⁇ ONX-specific antibody, or biologically-active derivatives or fragments thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability Disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, Retinal diseases including those involving photoreception, Cell growth rate disorders; cell shape disorders, feeding disorders; control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus ( ⁇ IDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeody
  • polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cD ⁇ A encoding ⁇ ONX may be useful in gene therapy, and ⁇ ONX may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HTV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention,- osteoporosis, Crohn's disease, multiple sclerosis, and treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to neoplasm; adenocarcinoma
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation.
  • the method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said
  • NOVX polypeptide Binding of the test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid.
  • NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly- expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome.
  • the expression of NONX polypeptide in both the test animal and the control animal is compared.
  • a change in the activity of ⁇ ONX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a ⁇ OVX polypeptide, a ⁇ OVX nucleic acid, or both, in a subject (e.g., a human subject).
  • the method includes measuring the amount of the ⁇ OVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the ⁇ OVX polypeptide present in a control sample.
  • An alteration in the level of the NONX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
  • the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a ⁇ OVX polypeptide, a ⁇ OVX nucleic acid, or a ⁇ ONX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
  • a subject e.g., a human subject
  • the disorder includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
  • the invention can be used in a method to identify the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
  • the invention is concerned with a method of screening a candidate substance interacting with an olfactory receptor polypeptide of SEQ ID ⁇ OS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, or 31, or fragments thereof.
  • the method involves providing an olfactory receptor polypeptide; obtaining a candidate substance; contacting the polypeptide and the candidate substance; and detecting the complexes formed, if any.
  • the invention is concerned with a method of screening ligand molecules interacting with an olfactory receptor polypeptide according to the invention.
  • the method involves providing a recombinant eukaryotic host cell containing a - nucleic acid molecule encoding the polypeptide; preparing membrane extracts of the recombinatnt eukaryotic host cell; contacting the membrane extracts with a selected ligand molecule; and detecting the production olevel of second messenger metabolites.
  • the method of screening ligand molecules comprises the steps of providing an adenovirus containing a nucleic acid encoding an olfactory receptor polypeptide; infecting an olfactory epithelium with the adenovirus; contacting the olactory epithelium with a selected ligand molecule; and detecting an increase, if any, of the response to the ligand molecule.
  • the invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides.
  • novel nucleic acids and their encoded polypeptides are referred to individually as NOV1, NOV2, NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, and NON 10.
  • the nucleic acids, and their encoded polypeptides are collectively designated herein as " ⁇ ONX”.
  • novel ⁇ ONX nucleic acids of the invention include the nucleic acids whose sequences are provided in Tables 1 A, 1C, IE, 2 A, 2C, 2E, 2G, 3 A, 4 A, 4C, 5 A, 6 A, 7 A, 8 A, 9 A, and 10 A, inclusive, or a fragment, derivative, analog or homolog thereof.
  • the novel novel ⁇ ONX nucleic acids of the invention include the nucleic acids whose sequences are provided in Tables 1 A, 1C, IE, 2 A, 2C, 2E, 2G, 3 A, 4 A, 4C, 5 A, 6 A, 7 A, 8 A, 9 A, and 10 A, inclusive, or a fragment, derivative, analog or homolog thereof.
  • ⁇ OVX proteins of the invention include the protein fragments whose sequences are provided in Tables IB, ID, IF, 2B, 2D, IF, 2H, 3B, 4B, 5B, 6B, 7B, 8B, 9B, and 10B, inclusive.
  • the individual ⁇ OVX nucleic acids and proteins are described below. Within the scope of this invention is a method of using these nucleic acids and peptides in the treatment or prevention of a disorder related to cell signaling or metabolic pathway modulation.
  • GPCRs G-Protein Coupled Receptor proteins
  • OMIM Online Mendelian Inheritance in Man
  • the olfactory receptor (OR) gene family constitutes one of the largest GPCR multigene families and is distributed among many chromosomal sites in the human genome. See Rouquier et al., Hum. Mol. Genet. 7(9):1337-45 (1998); Malnic et al, Cell 96:713-23 (1999). Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. See Vanderhaeghen et al., Genomics 39(3):239-46 (1997); Xie et al., Mamm. Genome 11(12):1070-78 (2000); Issel-Tarver et al., Proc. Natl. Acad. Sci.
  • chemoreceptors Other examples of seven membrane spanning proteins that are related to GPCRs are chemoreceptors. See Thomas et al., Gene 178(l-2):l-5 (1996). Chemoreceptors have been identified in taste, olfactory, and male reproductive tissues. See id; Walensky et al., J. Biol. Chem. 273(16):9378-87 (1998); Parmentier et al., Nature 355(6359):453-55 (1992); Asai et al., Biochem. Biophys. Res. Commun. 221(2):240-47 (1996).
  • NOV1 includes a family of three novel G-protein coupled receptor (“GPCR") proteins disclosed below.
  • GPCR G-protein coupled receptor
  • the disclosed proteins have been named NOV 1 a, NOV 1 b, and NOV 1 c and are each related to olfactory receptors.
  • a novel NOVla nucleic acid was identified on chromosome 6 by TblastN using CuraGen Corporation's sequence file for GPCR probes or homologs, and run against the
  • GenScanTM Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a disclosed NOVla nucleic acid of 993 (designated CuraGen Ace. No. dj408b20_B) nucleotides is shown in Table 1 A.
  • the disclosed NOVla open reading frame (“ORF") begins at the ATG initiation codon at nucleotides 15-17, shown in bold in Table 1 A.
  • the encoded polypeptide is referred to herein as NOVla polypeptide.
  • the disclosed NOVla ORF terminates at a TAA codon at nucleotides 954-956. As shown in Table 1 A, putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
  • Table IA NOVla nucleotide sequence (SEQ ID NO:l).
  • a disclosed encoded NOVla protein has 313 amino acid residues and is referred to as the NOVla protein.
  • the NOVla protein was analyzed for signal peptide prediction and cellular localization. SignalP results predict that NOVla is cleaved between position 38 and 39 of SEQ ID NO:2, i.e., at the slash in the amino acid sequence TVT-IF. Psort and Hydropathy profiles also predict that NOVla contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000), Golgi body (certainty of 0.400), endoplasmic reticulum (membrane) (certainty of 0.3000), and microbody (peroxisome) (certainity of 0.3000).
  • the disclosed NOVla polypeptide sequence is presented in Table IB- using the one-letter amino acid code. Table IB. Encoded NOVla protein sequence (SEQ ID NO:2).
  • NOVlb A target sequence identified previously, Accession Number dj408b20_B (NOV1 a), was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species.
  • the resulting amplicon was gel purified, cloned r and sequenced to high redundancy to provide the sequence reported below, which is designated NOVlb (Accession Number dj408b20B-l). There is one nucleotide change at position 365 of the sequence of dj408b20B-l.
  • a disclosed NOVlb nucleic acid of 943 nucleotides is shown in Table lC.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 2-4 and ending with a AAA codon at nucleotides 938-940.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table lC.
  • NOVlb includes a Kozak sequence.
  • Table 1C NOVlb Nucleic acid sequence (SEQ ID NO:3).
  • a disclosed encoded NOVlb protein has 313 amino acid residues (SEQ ID NO:4) and is referred to as the NOVlb protein.
  • the NOVlb protein was analyzed for signal peptide prediction and cellular localization. SignalP results predict that NOVlb is cleaved between position 38 and 39 of SEQ ID NO:4, i.e., at the slash in the amino acid sequence TNT-IF. Psort and Hydropathy profiles also predict that ⁇ ONlb contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000).
  • the disclosed ⁇ OVlb polypeptide sequence is presented in Table ID using the one-letter amino acid code.
  • the disclosed NOVlb protein differs from the disclosed NOVla protein at only one position. At positions 122, NOVla has P, while NOVlb has W.
  • NOVlc the target sequence identified previously, Accession Number dj408b20_B (NOVla), was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the resulting amplicons were gel purified, cloned and sequenced to high redundancy.
  • the resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp.
  • sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported below, which is designated NOVlc (Accession Number CG50369-01).
  • NOVlb This differs from the previously identified sequence of NOVlb (Accession Number dj408b20_B) in having 2 amino acid changes at the following positions: 270 (Gln->Arg) and 295 (Lys->Arg).
  • a disclosed NOVlc nucleic acid of 956 nucleotides is shown in Table IE.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 7-9 and ending with a TAA codon at nucleotides 946-948. Putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined in Table IE and the start and stop codons are in bold letters.
  • a disclosed NOVlc protein having 313 amino acid residues (SEQ ID NO:6) is presented using the one-letter code in Table IF.
  • the disclosed NOVlc sequence was derived by laboratory cloning of cDNA fragments covering the full length and/or part of the DNA sequence of the invention, and/or by in silico prediction of the full length and or part of the DNA sequence of the invention from public human sequence databases.
  • the laboratory cloning was performed using one or more of the methods summarized below:
  • SeqCallingTM Technology cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, cell lines, primary cells or tissue cultured primary cells and cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression for example, growth factors, chemokines, steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled with themselves and with public ESTs using bioinformatics programs to generate CuraGen's human SeqCalling database of SeqCalling assemblies.
  • Each assembly contains one or more overlapping cDNA sequences derived from one or more human samples. Fragments and ESTs were included as components for an assembly when the extent of identity with another component of the assembly was at least 95% over 50 bp. Each assembly can represent a gene and/or its variants such as splice forms and/or single nucleotide polymorphisms (SNPs) and their combinations.
  • SNPs single nucleotide polymorphisms
  • Exon Linldng The cDNA coding for the sequence was cloned by polymerase chain reaction (PCR) using the following primers: GAATTGGGTAAATGACAGCATC (SEQ ID NO:32) and TTAGCAAAGCTTATCATTTGC (SEQ ID NO:33) on the following pool of human cDNAs: Pool 1 - Adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • PCR polymerase chain reaction
  • Primers were designed based on in silico predictions for the full length or part (one or more exons) of the DNA protein sequence of the invention or by translated homology of the predicted exons to closely related human sequences or to sequences from other species. Usually multiple clones were sequenced to derive the sequence which was then assembled similar to the SeqCalling process. In addition, sequence traces were evaluated manually and edited for corrections if appropriate.
  • a variant sequence can include a single nucleotide polymo ⁇ hism (SNP).
  • SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
  • a SNP can arise in several ways.
  • a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion.
  • a SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele.
  • the polymo ⁇ hic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration of the amino acid encoded by the gene at the position of the SNP.
  • Intelomeres may also be silent, however, in the case that a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code.
  • SNPs occurring outside the region of a gene, or in an intron within a gene do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern for example, alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, stability of transcribed message.
  • NOVlc is expressed in at least the following tissues: Apical micro villi of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, co ⁇ us callosum, cardiac (atria and ventricle), caudate nucleus, central nervous system and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum,
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving- as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, potential obesity due to over-eating, potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NIDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HTV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, allergies, Parkinson's disease, acute heart failure, hypotension,
  • polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, asthma, allergies, bulimia, Parkinson's disease, acute heart failure, - hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease, multiple sclerosis; and Treatment of Albright hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, benign prostatic hypertrophy, and psychotic and neurological disorders (including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation) and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • the amino acids differences between the three NOV1 proteins are shown in Table 1G.
  • the differences between the three proteins appear to be localized to a few distinct regions. Thus, these proteins may have similar functions, such as serving as olfactory or chemokine receptors (see below).
  • NOVla lalM;IIW-.;WMBH SEQ ID NO:2
  • NOVlb lalHiWiiltUiallHi ⁇ SEQ ID NO: 4
  • NOVlc lalMJIUMsWiaflHin SEQ ID NO:6
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • All BLAST analyses described herein were performed using the NOVla amino acid sequence. As noted above, differences between the amino acid sequences of NOVla, NOVlb, and NOVlc were limited to discrete amino acids (e.g., positions 122, 270, and 295).
  • a BLASTX search was performed against public protein databases.
  • the full amino acid sequence of the protein of the invention was found to have 313 of 313 amino acid residues (100%) identical to, and 313 of 313 residues (100%) positive with, the 313 amino acid dJ408B20.2, a novel 7 transmembrane olfactory receptor protein from Homo sapien (emb
  • CAC14158.1 (AL133267), E e-177).
  • the disclosed NOVla protein (SEQ ID NO:2) also has good identity with a number of olfactory receptor proteins.
  • the disclosed NOVla has 254/310 (81%) amino acids identical with the 357 amino acid Homo sapien olfactory receptor (emb
  • CAC20513.1 (AJ302593) protein (Expect e-143), and 253/310 (81%)) amino acids identical, to the 310 amino acid Homo Sapien olfactory receptor (emb
  • CABl 1427.1 (Z98744)) (Expect e-142).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 1H.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • any reference to NOVl is assumed to encompass all variants. Residue differences between any NOVX variant sequences herein are written to show the residue in the "a" variant and the residue position with respect to the "a" variant.
  • NOV residues in all following sequence alignments that differ between the individual NOV variants are marked with the (o) symbol above the variant residue in all alignments herein.
  • the protein shown in line 1 of Table II depicts the sequence for NOVla, and the positions where NOVlb and NOVlc differs are marked with a (o) symbol (e.g., positions 122, 270, and 295).
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR_BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table IK. Table IK Amino Acid sequence for TM7 (SEQ ID NO:39).
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • NOVl RNA Expression information for NOVl RNA was derived using tissue sources including, but not limited to, proprietary database sources, public EST sources, literature sources, and/or RACE sources.
  • the nucleic acids and proteins of NOVl are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • OR olfactory receptor
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOVl protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • NOVX nucleic acids and proteins are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • a protein therapeutic e.g., a small molecule drug target
  • an antibody target therapeutic, diagnostic, drug targeting/cytotoxic antibody
  • nucleic acid useful in gene therapy gene delivery/gene ablation
  • composition promoting tissue regeneration in vitro and in vivo vi
  • biological defense weapon are further useful in the generation of antibodies that bind immuno- specifically to the novel NOVl substances for
  • NOV2 includes a family of four novel G-protein coupled receptor (“GPCR”) proteins disclosed below.
  • GPCR G-protein coupled receptor
  • the disclosed proteins have been named NOV2a, NOV2b, NOV2c, and NOV2d and are each related to olfactory receptors.
  • a disclosed NOV2a nucleic acid of 960 (designated CuraGen Ace. No. 6-L-19-D) nucleotides is shown in Table 2A.
  • the disclosed NOV2a open reading frame (“ORF") begins at the ATG initiation codon at nucleotides 14-16, shown in bold in Table 2 A.
  • the encoded polypeptide is referred to herein as NOV2a polypeptide.
  • the disclosed NOV2a ORF terminates at a TAG codon at nucleotides 955-957.
  • putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
  • NOV2a An open reading frame (ORF) for NOV2a was identified from nucleotides 14 to 955.
  • the disclosed NOV2a polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 is 314 amino acid residues and is presented using the one-letter code in Table 2B.
  • the NOV2a protein was analyzed for signal peptide prediction and cellular localization. SignalPep results predict that NOV2a is cleaved between position 43 and 44 of SEQ ID NO:8, i.e., at the slash in the amino acid sequence VVG-NL.
  • Psort and Hydropathy profiles also predict that NOV2a contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000) or possibly to Golgi body with a lower certainty.
  • Table 2B Encoded NOV2a protein sequence (SEQ ID NO:8).
  • a novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a target sequence identified previously as NOV2a was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries.
  • NOV2b Accession Number 6_L_19_D_dal
  • the resulting nucleotide sequence differs from that of NOV2a at nucleotides 344 and 900.
  • the encoded protein differs from that of NOV2a at residue 113.
  • a disclosed NOV2b nucleic acid of 954 nucleotides is shown in Table 2C.
  • the disclosed NOV2b open reading frame (“ORF") begins at the ATG initiation codon at nucleotides 7-9, shown in bold in Table 2C.
  • the encoded polypeptide is referred to herein as NOV2b polypeptide.
  • the disclosed NOV2b ORF terminates at a TGA codon at nucleotides 949-951. As shown in Table 2C, putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
  • NOV2b An open reading frame (ORF) for NOV2b was identified from nucleotides 7 to 949.
  • the disclosed NOV2b polypeptide (SEQ ID NO: 10) encoded by SEQ ID NO:9 is 314 amino acid residues and is presented using the one-letter code in Table 2D.
  • the NOV2b protein was analyzed for signal peptide prediction and cellular localization. SignalPep results predict that NOV2b is cleaved between position 43 and 44 of SEQ ID NO: 10, i.e., at the slash in the amino acid sequence WG-NL. Psort and Hydropathy profiles also predict that NOV2b contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000)
  • Table 2D Encoded NOV2b protein sequence (SEQ ID NO:10).
  • a target sequence identified previously as NOV2a was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • NOV2c and NOV2d differ from the previously identified sequence (NOV2a) at bp: 344, 386, 900 (NOV2d; 6_L_19_D_dal)) and 344, 386 (NOV2c; 6_L_19_D_da2).
  • a disclosed NOV2c nucleic acid of 954 nucleotides is shown in Table 2E.
  • the disclosed NOV2c open reading frame (“ORF") begins at the ATG initiation codon at nucleotides 7-9, shown in bold in Table 2E.
  • the encoded polypeptide is referred to herein as NOV2c polypeptide.
  • the disclosed NOV2c ORF terminates at a TGA codon at nucleotides 949-951.
  • putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
  • NOV2c An open reading frame (ORF) for NOV2c was identified from nucleotides 7 to 949.
  • the disclosed NOV2c polypeptide (SEQ ID NO: 12) encoded by SEQ ID NO:l 1 is 314 amino acid residues and is presented using the one-letter code in Table 2F.
  • the NOV2c protein was analyzed for signal peptide prediction and cellular localization. SignalPep results predict that NOV2c is cleaved between position 43 and 44 of SEQ ID NO:12, i.e., at the slash in the amino acid sequence WG-NL.
  • Psort and Hydropathy profiles also predict that NON2b contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000)
  • Table 2F Encoded ⁇ OV2c protein sequence (SEQ ID NO:12).
  • Possible SNPs for the disclosed NOV2c include:
  • a disclosed NOV2d nucleic acid of 954 nucleotides is shown in Table 2G.
  • the disclosed NOV2d open reading frame (“ORF") begins at the ATG initiation codon at nucleotides 7-9, shown in bold in Table 2G.
  • the encoded polypeptide is referred to herein as NOV2d polypeptide.
  • the disclosed NOV2d ORF terminates at a TGA codon at nucleotides 949-951.
  • putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
  • the nucleic acid sequence of NOV2d differs from NOV2b at position 386, which is C in NOV2b and T in NOV2d.
  • ORF open reading frame
  • the disclosed NOV2d polypeptide (SEQ ID NO: 14) encoded by SEQ ID NO: 13 is 311 amino acid residues and is presented using the one-letter code in Table 2H.
  • the NOV2d protein was analyzed for signal peptide prediction and cellular localization. SignalPep results predict that NOV2d is cleaved between position 43 and 44 of SEQ ID NO: 14, i.e., at the slash in the amino acid sequence VVG-NL. Psort and Hydropathy profiles also predict that NOV2b contains a signal peptide and is likely to be localized at the plasma membrane (certainty of 0.6000)
  • Possible SNPs for the disclosed NOV2d include: 62: A->G(2)
  • NOV2 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, deliriurii, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • infections such as bacterial, fungal, protoz
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer;
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • the amino acids differences between three of the NOV2 proteins are shown in Table 21.
  • the differences between the three proteins appear to be localized to a few distinct regions. Thus, these proteins may have similar functions, such as serving as olfactory or chemokine receptors (see below).
  • the "E-value” or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • All BLAST analyses described herein were performed using the NOV2a amino acid sequence. As noted above, differences between the nucleic acid sequences of NOV2a, NOV2b, NOV2c, and NOV2d were limited to discrete nucleotides (e.g., positions 344, 386, and 900).
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 159 of 214 amino acid residues (74%) identical to, and 181 of 214 residues (84%) positive with, a 216 amino acid Homo sapien olfactory receptor protein (gi
  • (AF065860), E 2e-69).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 2J.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • any reference to NOV2 is assumed to encompass all variants. Residue differences between any NOVX variant sequences herein are written to show the residue in the "a" variant and the residue position with respect to the "a" variant.
  • NOV residues in all following sequence alignments that differ between the individual NOV variants are marked with the (o) symbol above the variant residue in all alignments herein.
  • the protein shown in line 1 of Table 2K depicts the sequence for NOV2a, and the positions where NOV2b, NOV2c, and NOV2d differ are marked with a (o) symbol (e.g., positions 130 and 147).
  • NOV2 The presence of identifiable domains in NOV2, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Inte ⁇ ro number by crossing the domain match (or numbers) using the Inte ⁇ ro website (http:www.ebi.ac.uk/ inte ⁇ ro).
  • DOMAIN results e.g., for NOV2a, were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR_BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 2M.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • the NOV2 homology to the olfactory receptors suggests that an endogenous small molecule ligand regulates this gene and hence drugs structurally similar to the endogenous ligand could serve
  • nucleic acids and proteins of NOV2 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • OR olfactory receptor
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV2 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • NOV3 An additional NOV-like protein of the invention, referred to herein as NOV3, is an Olfactory Receptor ("OR")-like protein.
  • OR Olfactory Receptor
  • a novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank. The nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full- length protein.
  • the novel nucleic acid of 957 nucleotides (6-L-19-E, SEQ ID NO:15) encoding a novel olfactory receptor-like protein is shown in Table 3 A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 13-15 and ending with a TAA codon at nucleotides 955-957.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 3 A, and the start and stop codons are in bold letters.
  • a disclosed NOV3 polypeptide (SEQ ID NO:16) encoded by SEQ ID NO:15 is 314 amino acid residues and is presented using the one-letter code in Table 3B.
  • the disclosed NOV3 protein was analyzed for signal peptide prediction and cellular localization. SignalP results predict that NOV3 is cleaved between position 43 and 44 of SEQ ID NO:16, i.e., at the slash in the amino acid sequence VLG-NL. Psort and Hydropathy profiles also predict that NOV3 contains a signal peptide and is likely to be localized to the endoplasmic reticulum (membrane) or the plasma membrane with comparable, high certainties.
  • Table 3B Encoded NOV3 protein sequence (SEQ ID NO:16).
  • the NOV3 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • infections such as bacterial, fungal, protozoal
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer;
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NOV3 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 153 of 216 amino acid residues (70%)) identical to, and 175 of 216 residues (80%) positive with, the 216 amino acid Homo sapien human olfactory receptor 5D4 (Olfactory Receptor 11-8C (OR11-8C) (gi
  • O5D4, E 7e-73).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 3C.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • NOV3 The presence of identifiable domains in NOV3, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Inte ⁇ ro number by crossing the domain match (or numbers) using the Inte ⁇ ro website (http:www.ebi.ac.uk/ inte ⁇ ro).
  • DOMAIN results e.g., for NOV3, were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 3F.
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • the nucleic acids and proteins of NON3 are useful in potential therapeutic applications implicated in various ⁇ OV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cD ⁇ A encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding ⁇ OV3 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification. Further, the protein similarity information, expression pattern, and map location for NOV3 suggests that NOV3 may have important structural and/or physiological functions characteristic of the NOV family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel NOV3 substances for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies” section below.
  • NOV4 includes a family of two nucleic acids disclosed below.
  • the disclosed nucleic acids encode a NOV-like protein.
  • a novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • NOV4a nucleic acid of 968 nucleotides (95-h-6-A, SEQ ID NO: 17) encoding a novel olfactory receptor-like protein is shown in Table 4A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 8-10 and ending- with a TAA codon at nucleotides 956-958. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters.
  • Table 4A NOV4a Nucleotide Sequence (SEQ ID NO:17).
  • a disclosed NOV4a polypeptide (SEQ ID NO: 18) encoded by SEQ ID NO: 17 is 316 amino acid residues and is presented using the one- letter code in Table 4B.
  • Table 4B Encoded NOV4 protein sequence (SEQ ID NO: 18).
  • NOV4b A target sequence identified previously as NOV4a (Accession Number 95-h-6-A) was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries.
  • the resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated NOV4b (Accession Number 95-h-6-Al). There is a single nucleotide difference between the two clones at base 274 that results in no change in amino acid sequence.
  • a disclosed NOV4b nucleic acid of 968 nucleotides (SEQ ID NO:19) encoding a novel olfactory receptor-like protein is shown in Table 4C.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 8-10 and ending with a TAA codon at nucleotides 956-958.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4C, and the start and stop codons are in bold letters.
  • the disclosed NOV4b polypeptide is identical to the disclosed NOV4a polypeptide (SEQ ID NO: 18). This polypeptide has 316 amino acid residues and is presented using the one-letter code in Table 4B.
  • the NOV4 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • infections such as bacterial, fungal, protozoal
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HTV-l or HTV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma;
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • the amino acid differences between the two NOV4 proteins are shown in Table 4D. Thus, these proteins may have similar functions, such as serving as olfactory or chemokine receptors (see below).
  • NOV4a *1QNQSFVTEFVLLGLSQNPNVQEIVFVVFLFVYIATVGGNMLIVVTILSSPALLVSPMYI
  • NOV4b •10NOSFVTEFVLLGLSONPNVOEIVFVVFLFVYTATVGGNMLIVVTILSSPALLVSPMYI
  • nucleotide differences between the two NOV4 nucleic acids are shown in Table 4E.
  • the differences between the nucleic acids appear to be localized to a few distinct regions.
  • NOV4a ATGATCGTTATGTGGCCATTTGCAAGCCCTTGCATTACTCTTCTATCATGAACAGGA
  • NOV4a EH ⁇ ESJSSHSEammM ⁇ MimuM ⁇ i
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • All BLAST analyses described herein were performed using the NOV4a amino acid sequence. As noted above, there are no differences between the amino acid sequences of NOV4a and NOV4b.
  • the amino acid sequence of the NOV4 protein of the invention was found to have 187 of 301 amino acid residues (62%) identical to, 228 of 301 residues (70%) positive, and 1 gap out of 301 residues, with, the 308 amino acid odorant receptor 16, an olfactory receptor protein from Mus musculus (gi
  • ref]NP_067343.1, E le-93).
  • the disclosed NOV4 protein (SEQ ID NO: 18) also has good identity with a number of olfactory receptor proteins.
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 4F.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • any reference to NOV4 is assumed to encompass all variants.
  • NON4 The presence of identifiable domains in NON4, as well as all other ⁇ ONX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Inte ⁇ ro number by crossing the domain match (or numbers) using the Inte ⁇ ro website (http:www.ebi.ac.uk/ inte ⁇ ro).
  • DOMAIN results e.g., for NON4a, were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR_BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 41.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • nucleic acids and proteins of NOV4 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV4 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a- small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel NOV4 substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • a novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a disclosed nucleic acid of 946 nucleotides (95-h-6-B, SEQ ID NO:20) encoding a novel NOV5 olfactory receptor-like protein is shown in Table 5A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 7-9 and ending with a TAA codon at nucleotides 937-939. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 5A, and the start and stop codons are in bold letters.
  • a disclosed NOV5 polypeptide (SEQ ID NO:21) encoded by SEQ ID NO:20 is 308 amino acid residues and is presented using the one-letter code in Table 5B.
  • Table 5B Encoded NOV5 protein sequence (SEQ ID NO:21).
  • the NOV5 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIN-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • infections such as bacterial, fungal, protozo
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NOV5 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 157 of 302 amino acid residues (51%) identical to, and 215 of 302 residues (70%) positive with, the 308 amino acid odorant receptor 16 from Mus musculus (gi
  • , E 8e-77).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 5C.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • NOV5 The presence of identifiable domains in NOV5, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Inte ⁇ ro number by crossing the domain match (or numbers) using the Inte ⁇ ro website (http:www.ebi.ac.uk/ inte ⁇ ro).
  • DOMAIN results e.g., for NOV3, were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amin ⁇ acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 5F.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 173O143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • nucleic acids and proteins of NOV5 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV5 protein, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • a novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually corrected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a disclosed NOV6 nucleic acid of 1000 nucleotides (95-h-6-C, SEQ ID NO:22) encoding a novel NOV6 olfactory receptor-like protein is shown in Table 6A.
  • ORF open reading frame
  • a disclosed NOV6 polypeptide (SEQ ID NO:23) encoded by SEQ ID NO:22 is 326 amino acid residues and is presented using the one-letter code in Table 6B.
  • the NOV6 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • infections such as bacterial, fungal, protozoal
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer;
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NOV6 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 163 of 300 amino acid residues (54%) identical to, and 216 of 300 residues (71 %) positive with, the 308 amino acid odorant receptor 16 from Mus musculus (gi
  • , E le-77).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 6C.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • CDD conserveed Domain Database
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR_BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 6F.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, - 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • nucleic acids and proteins of NOV6 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV6 protein, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • a novel NOV7 nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually co ⁇ ected for apparent inconsistencies, thereby obtaining the sequences encoding the protein fragment.
  • a disclosed nucleic acid of 868 nucleotides (95-h-6-D, SEQ ID NO:24) encoding a novel NOV7 olfactory receptor-like protein is shown in Table 7A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 11-13 extending through nucleotide 868. Putative untranslated regions upstream from the initiation codon are underlined in Table 7A, and the start codon is in bold letters.
  • the disclosed NOV7 protein was analyzed for signal peptide prediction and cellular localization.
  • SignalP results predict that NOV7 is cleaved between position 50 and 51 of SEQ ID NO:25, i.e., at the slash in the amino acid sequence CLS-NL.
  • Table 7B Encoded NOV7 protein sequence (SEQ ID NO:25).
  • the NOV7 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • infections such as bacterial, fungal, protozoal
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients " suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NOV7 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 159 of 286 amino acid residues (55%) identical to, and 192 of 286 residues (66%) positive with, the 308 amino acid odorant receptor 16 taMi ⁇ musculus (gi
  • , E 2e-78).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 7C.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 7F.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, ⁇ opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, ⁇ opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • nucleic acids and proteins of NOV7 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV7 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid usefiil in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • NOV8 A novel NOV8 nucleic acid was identified on chromosome 11 by TblastN using
  • CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually co ⁇ ected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a disclosed nucleic acid of 948 nucleotides (95-h-6-E, SEQ ID NO:26) encoding a novel NOV8 olfactory receptor-like protein is shown in Table 8 A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TAA codon at nucleotides 943-945. Putative untranslated regions upstream from the initiation codon and downstream from the stop codon are underlined in Table 8A, and the start and stop codons are in bold letters.
  • the disclosed NOV8 polypeptide (SEQ ID NO:27) encoded by SEQ ID NO:26 is 311 amino acid residues and is presented using the one-letter code in Table 8B.
  • Table 8B Encoded NOV8 protein sequence (SEQ ID NO:27).
  • NOV8 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NOV8 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 189 of 299 amino acid residues (63%>) identical to, and 234 of 299 residues (74%) positive with, the 302 amino acid gene for odorant receptor A16 from Mus musculus (gi
  • reflNP_065261.11, E 2e-96).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 8C.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • NOV8 The presence of identifiable domains in NOV8, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Inte ⁇ ro number by crossing the domain match (or numbers) using the Inte ⁇ ro website (http:www.ebi.ac.uk/ interpro).
  • DOMAIN results e.g., for NOV8, were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections.
  • the "strong” group of r conserved amino acid residues may be any one of the following groups of amino acids: STA,
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR_BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 8F.
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • nucleic acids and proteins of NOV 8 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV8 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification. Further, the protein similarity information, expression pattern, and map location for NOV8 suggests that NOV8 may have important structural and/or physiological functions characteristic of the NOV family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • a novel NOV9 nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually co ⁇ ected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a disclosed NOV9 nucleic acid of 944 nucleotides (95-h-6-F, SEQ ID NO:28) encoding a novel NOV9 olfactory receptor-like protein is shown in Table 9A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 7-9 and ending with a TAG codon at nucleotides 934-936. Putative untranslated regions upstream from the initiation codon and downstream from the stop codon are underlined in Table 9A, and the start and stop codons are in bold letters.
  • the disclosed NOV9 polypeptide (SEQ ID NO:29) encoded by SEQ ID NO:28 is 309 amino acid residues and is presented using the one-letter code in Table 9B.
  • NOV9 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies
  • infections such as bacterial, fungal, protozoal and viral infections (
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer;
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NOV9 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 168 of 308 amino acid residues (54%) identical to, 227 of 308 residues (73%) positive, and gaps at 2 of 308 residues, with the 308 amino acid odorant receptor A16 from Mus musculus (gi
  • , E 4e-80).
  • the disclosed protein is also similar to the olfactory proteins disclosed in Table 9C.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • NOV9 The presence of identifiable domains in NOV9, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Inte ⁇ ro number by crossing the domain match (or numbers) using the Inte ⁇ ro website (http:www.ebi.ac.uk/ inte ⁇ ro).
  • DOMAIN results e.g., for NOV9, were collected from the conserveed Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR BOVIN, accession P20288", gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 9F.
  • the 7 transmembrane receptor family includes a number of different.proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454,
  • nucleic acids and proteins of NOV9 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV9 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • the NOVX nucleic acids and proteins are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic pu ⁇ ura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • a protein therapeutic e.g., a small molecule drug target
  • an antibody target therapeutic, diagnostic, drug targeting/cytotoxic antibody
  • nucleic acid useful in gene therapy gene delivery/gene ablation
  • composition promoting tissue regeneration in vitro and in vivo vi
  • biological defense weapon are further useful in the generation of antibodies that bind immunospecifically to the novel NON9 substances for
  • a novel ⁇ OV10 nucleic acid was identified on chromosome 11 by TblastN using CuraGen Co ⁇ oration's sequence file for GPCR probe or homolog, and run against the Genomic Daily Files made available by GenBank.
  • the nucleic acid was further predicted by the program GenScanTM, including selection of exons. These were further modified by means of similarities using BLAST searches. The sequences were then manually co ⁇ ected for apparent inconsistencies, thereby obtaining the sequences encoding the full-length protein.
  • a disclosed NOV10 nucleic acid of 1003 nucleotides (AC023078_A, SEQ ID NO:30) encoding a novel NOV 10 olfactory receptor-like protein is shown in Table 10 A.
  • An open reading frame (ORF) was identified beginning with an ATG initiation codon at nucleotides 5-7 and ending with a TGA codon at nucleotides 995-997. Putative untranslated regions upstream from the initiation codon and downstream from the stop codon are underlined in Table 10 A, and the start and stop codons are in bold letters.
  • the disclosed NOV10 polypeptide (SEQ ID NO:31) encoded by SEQ ID NO:30 is 330 amino acid residues and is presented using the one-letter code in Table 10B.
  • Table 10B Encoded ⁇ OV10 protein sequence (SEQ ID ⁇ O:31).
  • the NOV10 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in used in the treatment of infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological-- .
  • infections such as bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anor
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIN-1 or HIN-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • E-value or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. All BLAST analyses described herein were performed using the NON 10 amino acid sequence.
  • a BLASTX search was performed against public protein databases.
  • the amino acid sequence of the protein of the invention was found to have 89 of 265 amino acid residues (33%o) identical to, 146 of 265 residues (54%) positive, and gaps at.10 of 265 residues, with the 324 amino acid putative protein from Mus musculus (gi
  • (AK015036)), E 6e-26).
  • the disclosed protein is also similar to the proteins disclosed in Table IOC.
  • the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be mutated to a much broader extent without altering protein structure or function.
  • the "strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • 118205 VNPKIAKIFEMQSMPNGKTRTSLKTMSRRKLSQQKEKKATQMLAI VIlGVFIICfflpBFIT NOVIO DNWCQfl -MDFLTAvB
  • the representative member of the 7 transmembrane receptor family is the D2 dopamine receptor from Bos taurus (SWISSPROT: locus D2DR_BOVIN, accession P20288; gene index 118205).
  • the D2 receptor is an integral membrane protein and belongs to Family 1 of G-protein coupled receptors. The activity of the D2 receptor is mediated by G proteins, which inhibit adenylyl cyclase. Chio et al, Nature 343:255-269 (1990). Residues 51-427 of this 444 amino acid protein are considered to be the representative TM7 domain, shown in Table 10F.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • nucleic acids and proteins of NOV10 are useful in potential therapeutic applications implicated in various NOV- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding NOV10 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hype ⁇ lasia, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopemc puxpura, immunodeficiencies, graft versus host disease, bronchial asthma, and other diseases, disorders and conditions of the like.
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy. Additional NOV-related diseases and - disorders are mentioned throughout the Specification.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or fenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
  • an NOVX nucleic acid can encode a mature NOVX polypeptide.
  • a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the co ⁇ esponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal of the N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved would have the residues from residue M+1 to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step o5f post-translational modification other than a proteolytic cleavage event.
  • additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides co ⁇ esponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1
  • NOS:l 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide).
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains.
  • Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a prefe ⁇ ed identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, as well as a polypeptide possessing NOVX biological activity. Various biological activities ofthe NOVX proteins are described below.
  • An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX nucleic acid.
  • ORF co ⁇ esponds to a nucleotide sequence that could potentially be translated into a polypeptide.
  • a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
  • An ORF that represents the coding sequence for a full protein begins with an ATG "start” codon and terminates with one ofthe three “stop” codons, namely, TAA ⁇ TAG, or TGA.
  • an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.
  • a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequences determined from the cloning ofthe human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15,.
  • Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
  • a polypeptide having a biologically-active portion of an NOVX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically- active portion of NOVX” can be prepared by isolating a portion SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 that encodes a polypeptide having an NOVX biological activity (the biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
  • nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequences ofthe NOVX polypeptides may exist within a population (e.g., the human population).
  • Such genetic polymo ⁇ hism in the NOVX genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX genes. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe NOVX polypeptides, are intended to be within the scope ofthe invention. Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules co ⁇ esponding to natural allelic variants and homologues of the NOVX cDNAs ofthe invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
  • an isolated nucleic acid molecule ofthe invention hybridizes to the coding region.
  • the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding NOVX proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PNP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm D ⁇ A at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences of SEQ ID ⁇ OS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 co ⁇ esponds to a naturally-occurring nucleic acid molecule.
  • a . "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 or fragments, analogs or derivatives thereof, under conditions of moderate stringency.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • a non- limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02%) Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences ofthe NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • amino acid residues that are conserved among the NOVX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
  • nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity.
  • NOVX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • the protein encoded by the nucleic acid molecule is at least about 60%) homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31; more preferably at least about 70% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31; and most preferably at least about 95% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS.2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity.
  • SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • the relatedness of amino acid families may also be determined based on side chain _ interactions.
  • Substituted amino acids may be fully conserved "strong” residues or fully conserved “weak” residues.
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • the "weak” group of conserved residues may be any one ofthe following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within each group represent the single letter amino acid code.
  • a mutant NOVX protein can be assayed for (i) the ability to form protei protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (HI) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
  • a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
  • Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an NOVX protein.
  • coding region refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence - encoding the NOVX protein.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also refe ⁇ ed to as 5' and 3' untranslated regions).
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA.
  • the antisense oligonucleotide can be complementary to the region su ⁇ ounding the translation start site of NOVX mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression ofthe protein (e.g., by inhibiting transcription and/or translation).
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are prefe ⁇ ed.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other. See, e.g., Gaultier, et al, 1987. Nucl. Acids Res. 15: 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a r chimeric RNA-DNA analogue (see, e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330. .
  • Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in
  • Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA.
  • a ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30).
  • a derivative of a Tetrahymena L- 19 INS R ⁇ A can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an ⁇ OVX-encoding mR ⁇ A.
  • ⁇ OVX mR A can also be used to select a catalytic R ⁇ A having a specific ribonuclease activity from a pool of R ⁇ A molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
  • ⁇ OVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe ⁇ OVX nucleic acid (e.g., the ⁇ OVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe ⁇ OVX gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe ⁇ OVX nucleic acid e.g., the ⁇ OVX promoter and/or enhancers
  • the ⁇ OVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23.
  • peptide nucleic acids or "P ⁇ As” refer to nucleic acid mimics (e.g., D ⁇ A mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of P ⁇ As has been shown to allow for specific hybridization to D ⁇ A and R ⁇ A under conditions of low ionic strength.
  • the synthesis of P ⁇ A ohgomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
  • P ⁇ As of ⁇ OVX can be used in therapeutic and diagnostic applications.
  • P ⁇ As can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation a ⁇ est or inhibiting replication.
  • P ⁇ As of ⁇ OVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., P ⁇ A directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., nucleases (see, Hyrup, et al, 1996. supra); or as probes or primers for D ⁇ A sequence and hybridization (see, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • P ⁇ As of ⁇ OVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to P ⁇ A, by the formation of P ⁇ A-D ⁇ A chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • P ⁇ A-D ⁇ A chimeras of ⁇ OVX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra.
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaifre, et al, 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaifre,
  • oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the co ⁇ esponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
  • an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • One aspect ofthe invention pertains to isolated NOVX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies.
  • native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • NOVX proteins are produced by recombinant DNA techniques.
  • an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of NOVX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly-produced.
  • the language "substantially free of cellular material” includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also refe ⁇ ed to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins.
  • non-NOVX proteins also refe ⁇ ed to herein as a "contaminating protein”
  • the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume of the NOVX protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
  • Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe NOVX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein.
  • biologically-active portions comprise a domain or motif with at least one activity ofthe NOVX protein.
  • a biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
  • biologically-active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
  • the NOVX protein has an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • the NOVX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31, and retains the functional activity ofthe protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
  • the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31, and retains the functional activity ofthe NOVX proteins of SEQ ID NOS.2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31.
  • the sequences are aligned for optimal comparison pu ⁇ oses (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at co ⁇ esponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the co ⁇ esponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453.
  • the coding region ofthe analogous nucleic acid sequences refe ⁇ ed to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison ( .e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • an NOVX "chimeric protein” or “fusion protein” comprises an NOVX polypeptide operatively- linked to a non-NOVX polypeptide.
  • An "NOVX polypeptide” refers to a polypeptide having- an amino acid sequence co ⁇ esponding to an NOVX protein (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31), whereas a “non-NOVX polypeptide” refers to a polypeptide having an amino acid sequence co ⁇ esponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism.
  • an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein.
  • an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein.
  • an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein.
  • the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
  • the non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
  • the fusion protein is a GST-NO VX fusion protein in which the NOVX sequences are fused to the C-terminus ofthe GST (glutathione S-transferase) sequences.
  • Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
  • the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an NOVX-immunogloDulin fusion protein in which the NOVX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the NOVX-immunoglobulin fusion proteins ofthe invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo.
  • the NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand.
  • NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of _ NOVX with an NOVX ligand.
  • An NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
  • NOVX Agonists and Antagonists The invention also pertains to variants ofthe NOVX proteins that function as either
  • NOVX agonists i.e., mimetics
  • variants ofthe NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation ofthe NOVX protein).
  • An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein.
  • An a tagonist of the NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.
  • Variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) ofthe NOVX proteins for NOVX protein agonist or antagonist activity.
  • a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of NOVX variants can be produced by, for example, enzymatically li gating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences.
  • Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11 : 477.
  • libraries of fragments ofthe NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector.
  • expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX proteins.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
  • Anti-NOVX Antibodies encompasses antibodies and antibody fragments, such as F ab or (F ab ) 2, that bind immunospecifically to any ofthe NOVX polypeptides of said invention.
  • An isolated NOVX protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind to NOVX polypeptides using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length NOVX proteins can be used or, alternatively, the invention provides antigenic peptide fragments of NOVX proteins for use as immunogens.
  • the antigenic NOVX peptides comprises at least 4 amino acid residues ofthe amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31 and encompasses an epitope of NOVX such that an antibody raised against the peptide forms a specific immune complex with NOVX.
  • the antigenic peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to someone skilled in the art.
  • At least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface ofthe protein (e.g., a hydrophilic region).
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation (see, e.g., Hopp and Woods, 1981. Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle, 1982. J. Mol. Biol. 157: 105-142, each inco ⁇ orated herein by reference in their entirety).
  • NOVX protein sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31, or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components.
  • antibody refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically-binds (immunoreacts with) an antigen, such as NOVX.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a b and F( a b- ⁇ fragments, and an F ab expression library.
  • antibodies to human NOVX proteins are disclosed.
  • Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to an NOVX protein sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 29, and 31, or a derivative, fragment, analog or homolog thereof.
  • an appropriate immunogenic preparation can contain, for example, recombinantly-expressed NOVX protein or a chemically-synthesized NOVX polypeptide.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • the antibody molecules directed against NOVX can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of NOVX.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular NOVX protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975.
  • techniques can be adapted for the production of single-chain antibodies specific to an NOVX protein (see, e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F a b expression libraries (see, e.g., Huse, et al, 1989. Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for an NOVX protein or derivatives, fragments, analogs or homologs thereof.
  • Non-human antibodies can be "humanized” by techniques well known in the art. See, e.g., U.S. Patent No. 5,225,539.
  • Antibody fragments that contain the idiotypes to an NOVX protein may be produced by techniques known in the art including, but not limited to: (i) an F ⁇ b' ⁇ fragment produced by pepsin digestion of an antibody molecule; (it) an F a b fragment generated by reducing the disulfide bridges of an F( ab ') 2 fragment; (HI) an F ab fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent; and ( v) F v fragments.
  • recombinant anti-NOVX antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Application No. PCT7US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No.
  • methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme-linked immunosorbent assay
  • selection of antibodies that are specific to a particular domain of an NOVX protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX protein possessing such a domain.
  • antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • Anti-NOVX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an NOVX protein (e.g., for use in measuring levels ofthe NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies for NOVX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
  • An anti-NOVX antibody (e.g., monoclonal antibody) can be used to isolate an NOVX polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-NOVX antibody can facilitate the purification of natural NOVX polypeptide from cells and of recombinantly-produced NOVX polypeptide expressed in host cells.
  • an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe NOVX protein.
  • Anti-NOVX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling ( .e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, lucife ⁇ n, and aequo ⁇ n, and examples of suitable radioactive material include I,
  • vectors preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or - homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are refe ⁇ ed to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid ofthe invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
  • the recombinant expression vectors ofthe invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells.
  • NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: (i) to increase expression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (HI) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
  • GST glutathione S-transferase
  • Suitable inducible non- fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118).
  • the NOVX expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Co ⁇ oration, San Diego, Calif), and picZ (InVitrogen Co ⁇ , San Diego, Calif).
  • NOVX can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO J.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • the recombinant mammalian expression vector Is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • the invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to NOVX mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. , cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die). -
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells ofthe invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
  • a transgenic animal ofthe invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the human NOVX cDNA sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human homologue ofthe human NOVX gene such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells.
  • transgenic founder animal can be identified based upon the presence ofthe NONX transgene in its genome and/or expression of ⁇ ONX mR ⁇ A in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene- encoding ⁇ OVX protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of an ⁇ OVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the ⁇ OVX gene.
  • the ⁇ OVX gene can be a human gene (e.g., the cD ⁇ A of SEQ ID ⁇ OS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30), but more preferably, is a non-human homologue of a human NOVX gene.
  • a mouse homologue of human NOVX gene of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also refe ⁇ ed to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein).
  • the altered portion ofthe NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid ofthe NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell.
  • flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5'- and 3'-termini
  • the vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission ofthe transgene.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: _ 6232-6236.
  • Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O 'Gorman, et al, 1991. Science 251 :1351-1355.
  • cre/loxP recombinase system is used to regulate expression ofthe transgene
  • animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transfe ⁇ ed to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone ofthe animal from which the cell (e.g., the somatic cell) is isolated.
  • compositions The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also refe ⁇ ed to herein as "active compounds") ofthe invention, and derivatives, fragments, analogs and homologs thereof, can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is inco ⁇ orated herein by reference.
  • Prefe ⁇ ed examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion ofthe injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent- with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., an NOVX protein or anti-NOVX antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect- the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 9T : 3054-3057).
  • the pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the isolated nucleic acid molecules ofthe invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications) ⁇ to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below.
  • the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or abe ⁇ ant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias.
  • the anti-NOVX antibodies ofthe invention can be used to detect and isolate NOVX proteins and modulate NOVX activity.
  • the invention can be used in methods to influence appetite, abso ⁇ tion of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
  • the invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
  • the invention provides a method (also refe ⁇ ed to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity.
  • the invention also includes compounds identified in the screening assays described herein.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an NOVX protein or polypeptide or biologically-active portion thereof.
  • test compounds ofthe invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145.
  • a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
  • Small _ molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
  • Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any ofthe assays ofthe invention. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to an NOVX protein determined.
  • the cell for example, can of mammalian origin or a yeast cell. Determining the ability ofthe test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein- determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule.
  • a "target molecule” is a molecule with which an NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide ofthe invention.
  • an NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
  • Determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity ofthe target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e.
  • an assay ofthe invention is a cell-free assay comprising - contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the NOVX protein or biologically- active portion thereof.
  • Binding ofthe test compound to the NOVX protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to modulate (e.g. stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability ofthe NOVX protein to bind to an NOVX target molecule by one ofthe methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described, supra.
  • the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe NOVX protein to preferentially bind to or modulate the activity of an NOVX target molecule.
  • the cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein.
  • solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton ® X-100, Triton ® X-l 14, Thesit ® , Isotridecypoly(ethylene glycol ether) n , N-dodecyl ⁇ N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-
  • binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
  • NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with NOVX protein or target molecules can be derivatized to the wells ofthe plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
  • modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression.
  • the candidate compound when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression.
  • the level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
  • the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Cell 72: 223-232; Madura, et al, 1993. J. Biol Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993.
  • NOVX-binding proteins proteins that bind to or interact with NOVX
  • NOVX-binding proteins proteins that bind to or interact with NOVX
  • NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements ofthe NOVX pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constmcts.
  • the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait” and the “prey” proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g.
  • LacZ that is operably linked to a transcriptional regulatory site responsive to the transcription factor.
  • Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
  • the invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
  • Detection Assays Portions or fragments ofthe cDNA sequences identified herein (and the co ⁇ esponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
  • this sequence can be used to map the location ofthe gene on a chromosome.
  • This process is called chromosome mapping.
  • portions or fragments ofthe NOVX sequences SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, or fragments or derivatives thereof, can be used to map the location ofthe NOVX genes, respectively, on a chromosome.
  • the mapping ofthe NOVX sequences to chromosomes is an important first step in co ⁇ elating these sequences with genes associated with disease.
  • NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis ofthe NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene co ⁇ esponding to the NOVX sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes.
  • mammals e.g., human and mouse cells.
  • Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that dismpts the mitotic spindle.
  • FISH Fluorescence in situ hybridization
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1 ,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents co ⁇ esponding to noncoding regions ofthe genes actually are prefe ⁇ ed for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for stmctural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the NOVX sequences ofthe invention can also be used to identify individuals from minute biological samples.
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • the sequences ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymo ⁇ hisms," described in U.S. Patent No. 5,272,057).
  • sequences ofthe invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of co ⁇ esponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences ofthe invention can be used to obtain such identification sequences from individuals and from tissue.
  • the NOVX sequences ofthe invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymo ⁇ hisms (SNPs), which include restriction fragment length polymo ⁇ hism& (RFLPs).
  • SNPs single nucleotide polymo ⁇ hisms
  • RFLPs restriction fragment length polymo ⁇ hism&
  • each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses. Because greater numbers of polymo ⁇ hisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • the invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abe ⁇ ant NOVX expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
  • Another aspect ofthe invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refe ⁇ ed to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype ofthe individual (e.g., the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.)
  • Yet another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample.
  • a compound or an agent capable of detecting NOVX protein or nucleic acid e.g., mRNA, genomic DNA
  • An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 22, 24, 26, 28, and 30, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA.
  • oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
  • An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently- labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method ofthe invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of NOVX protein include enzyme linked _ immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a prefe ⁇ ed biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of NOVX in a biological sample can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determimng the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect NONX protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with abe ⁇ ant ⁇ OVX expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with ⁇ OVX protein, nucleic acid expression or activity.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the invention provides a method for identifying a disease or disorder associated with abe ⁇ ant ⁇ OVX expression or activity in which a test sample is obtained from a subject and ⁇ OVX protein or nucleic acid (e.g., mR ⁇ A, genomic D ⁇ A) is detected, wherein the presence of ⁇ OVX protein or nucleic acid is diagnostic for a _ subject having or at risk of developing a disease or disorder associated with abe ⁇ ant ⁇ OVX expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., semm), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abe ⁇ ant NOVX expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abe ⁇ ant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent fo treat a disorder associated with abe ⁇ ant NOVX expression or activity).
  • the methods ofthe invention can also be used to detect genetic lesions in an NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by abe ⁇ ant cell proliferation and/or differentiation.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression ofthe NOVX gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of; (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (in) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rea ⁇ angement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) abe ⁇ ant modification of an NOVX gene, such as ofthe methylation pattern ofthe genomic DNA, (vii) the presence of a non- wild- type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non- wild-type level of an NOVX protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein.
  • a prefe ⁇ ed biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • detection ofthe lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al, 1988. Science 241: 1077-1080; and Nakazawa, et al, 1994. Proc. Natl. Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al, 1995. Nucl.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification ofthe NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the, amplification product and comparing the length to a control sample. It is anticipated that PCR and or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al, 1990. Proc. Natl Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Q ⁇ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, e.g., U.S. Patent No. 5,493,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density a ⁇ ays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al, 1996. Human Mutation 7: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759.
  • genetic mutations in NOVX can be identified in two dimensional a ⁇ ays containing light-generated - DNA probes as described in Cronin, et al, supra.
  • a first hybridization a ⁇ ay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear a ⁇ ays of sequential overlapping probes. This step allows the identification of point mutations.
  • a second hybridization a ⁇ ay that allows the characterization of specific mutations by using smaller, specialized probe a ⁇ ays complementary to all variants or mutations detected.
  • Each mutation a ⁇ ay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence ofthe sample NOVX with the co ⁇ esponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995.
  • Biotechniques 19: 448 including sequencing by mass specfromefry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl. Biochem. Biotechnol. 38: 147-159).
  • Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242.
  • RNA/DNA duplexes can be treated with RNase and DNA DNA hybrids treated with Sj nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol. 217: 286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662.

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Abstract

L'invention se rapporte à des séquences d'acides nucléiques qui codent pour des polypeptides associés à des récepteurs-protéines couplés à une protéine G. L'invention se rapporte à des polypeptides codés par ces séquences d'acides nucléiques, et à des anticorps qui se lient de manière immuno-spécifique audit polypeptide, ainsi qu'à des dérivés, variants, mutants ou fragments du polypeptide, polynucléotide ou anticorps ci-dessus mentionné. L'invention se rapporte en outre à des méthodes thérapeutiques, diagnostiques et de recherche permettant le traitement, le diagnostic et la prévention de troubles impliquant n'importe lequel de ces nouveaux acides nucléiques ou n'importe laquelle de ces nouvelles protéines d'origine humaine.
PCT/US2001/007116 2000-03-03 2001-03-05 Nouvelles proteines et acides nucleiques codant pour ces proteines Ceased WO2001066746A2 (fr)

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JP2001565900A JP2003525633A (ja) 2000-03-03 2001-03-05 Ｇタンパク質結合レセプター関連ポリペプチド
AU4344401A AU4344401A (en) 2000-03-03 2001-03-06 Novel proteins and nucleic acids encoding same

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