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WO2003000929A2 - Polynucleotides, polypeptides et cancer - Google Patents

Polynucleotides, polypeptides et cancer Download PDF

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Publication number
WO2003000929A2
WO2003000929A2 PCT/JP2002/006363 JP0206363W WO03000929A2 WO 2003000929 A2 WO2003000929 A2 WO 2003000929A2 JP 0206363 W JP0206363 W JP 0206363W WO 03000929 A2 WO03000929 A2 WO 03000929A2
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WO
WIPO (PCT)
Prior art keywords
san
polynucleotide
sequence
seq
polypeptide
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PCT/JP2002/006363
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English (en)
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WO2003000929A3 (fr
Inventor
Yohei Ozeki
Takehiro Hirai
Ichiro Kawashima
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Sankyo Company, Limited
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Application filed by Sankyo Company, Limited filed Critical Sankyo Company, Limited
Priority to AU2002314552A priority Critical patent/AU2002314552A1/en
Publication of WO2003000929A2 publication Critical patent/WO2003000929A2/fr
Publication of WO2003000929A3 publication Critical patent/WO2003000929A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to polynucleotides and polypeptides, including the identification of a gene over-expressed in cancer, as well as newly identified polynucleotides, and polypeptides encoded by the polynucleotides. More particularly, the present invention relates to G-protein coupled receptors.
  • proteins participating in signal transduction pathways that involve G-proteins and/ or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351 :353-354).
  • these proteins are referred to as proteins participating in pathways with G-proteins, or PPG proteins.
  • PPG proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl, Acad. Sci., U.S.A., 1987, 84:46-50; Kobilka, B.K., et al, Science, 1987,
  • G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al, Science, 1991, 252:802-8).
  • effector proteins e.g., phospholipase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kinase A and protein kinase C (Simon, M. I., et al, Science, 1991, 252:802-8).
  • the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell.
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP.
  • GTP also influences hormone binding.
  • a G-protein connects the hormone receptor to adenylate cyclase.
  • G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor.
  • the GTP-carrying form then binds to the activated adenylate cyclase.
  • Hydrolysis of GTP to GDP, catalyzed by the G-protein itself returns the G-protein to its basal inactive form.
  • the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
  • the G-protein coupled receptor family includes dopamine receptors, which bind to neuroleptic drugs used for treating psychotic and neurological disorders.
  • Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
  • G-protein coupled receptors are found in numerous sites within a mammalian host.
  • the membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane alpha - helices connected by extracellular or cytoplasmic loops.
  • G-protein coupled receptors (otherwise known as 7 TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops forming disulfide bonds that are believed to stabilize functional protein structure.
  • the seven transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated in signal transduction.
  • G-protein coupled receptors Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors.
  • Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and /or the carboxy terminus.
  • G-protein coupled receptors such as the beta-adrenoreceptor, phosphorylation by protein kinase A and /or specific receptor kinases mediates receptor desensitization.
  • the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protein coupled receptors.
  • the hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form polar ligand binding site.
  • TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue.
  • Serine residues in TM5, asparagines residue in TM6 and phenylalanine or tyrosines residues in TM6 or TM7 are also implicated in ligand binding.
  • G-protein coupled receptors can be intracellularly coupled y heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al, Endoc. Rev., 1989, 10:317-331). Different G-protein alpha- subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors.
  • WO 0127158 relates to human olfactory receptors, and provides isolated polynucleotide comprising a sequence encoding a polypeptide which is involved in olfactory sensation.
  • the olfactory receptor polypeptides are to be found within sequences depicted in given polynucleotide sequences SEQ ID NO: l through SEQ ID NO:73 and SEQ ID NOr l l l through SEQ ID NO: 152. Sequences are given for isolated and purified olfactory receptor polypeptides.
  • the present invention relates to: the SAN_O787_l polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 1 and encodes a SAN_O787_l polypeptide of SEQ ID NO: 1
  • the SAN_O399_3 polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 3 and encodes a SAN_O399_3 polypeptide of SEQ ID NO: 4; the SAN_O437_l polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 5 and encodes a SAN_O437_l polypeptide of SEQ ID NO: 6; the SAN_O437_4 polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 7 and encodes a SAN_O437_4 polypeptide of SEQ ID NO: 8; and the SAN_O817_l polynucleotide which has the nucleotide sequence set forth in SEQ ID NO: 9 and encodes a SAN_O817_l polypeptide of SEQ ID NO: 10.
  • the sequence SAN_0787_1 is known from WO 0127158. We have found that the presence of such a sequence can be a marker for cancer, in that the expression levels of this gene are enhanced in tumor hepatocytes. Accordingly, the present invention provides methods of diagnosing cancer, especially liver cancer.
  • the present invention comprises detecting cancer by investigating the gene SAN_0787_1.
  • Investigation can involve assessing expression levels of the gene, typically by detecting an increased level of transcription or translation.
  • the invention involves detecting variant forms of the gene, or detecting RNA transcribed from such forms, or detecting polypeptides translated from such RNA.
  • Suitable test samples for use in the diagnostic methods can be obtained using cells such as from blood, urine, saliva, sperm, tissue biopsy or autopsy material, and preparations made from such cells, preferably cells from biopsy specimens and prepared RNA samples, more preferably hepatocytes and prepared RNA from mammalian liver.
  • Acording to the present invention there is provided in one aspect a method to detect the expression level in a test sample of a target sequence which is an mRNA which hybridizes with a polynucleotide that is complementary to a polynucleotide having the sequence shown in SEQ ID NO: l or complementary to a variant thereof.
  • the test sample is ordinarily obtained from a subject suspected of having liver cancer.
  • a preferred detection method involves detecting hybridization of the target sequence with a probe having a complementary sequence.
  • An alternative preferred detection method involves detecting amplification of the target sequence with primers using a polymerase chain reaction.
  • Hybridization methods of this invention typically comprise providing a probe complementary to the target sequence, and contacting the sample with the probe under conditions to allow formation of a hybridization complex between the probe and the target sequence.
  • the probe suitably comprises a fragement of a sequence complementary to the target sequence, and ordinarily comprises at least 15, 20, 25, 30, 35 or more nucleotides. Preferred probes have 30 to 50 nucleotides.
  • the conditions are preferably stringent conditions. The formation of any complex is then qualitatively or quantitatively assessed.
  • Amplification methods of this invention typically comprise providing forward and reverse primers.
  • the forward primer amplifies at least a part of the target sequence, and hybridizes with at least a part of a polynucleotide sequence complimentary to the target sequence.
  • the reverse primer amplifies at least a part of a polynucleotide sequence complimentary to the target sequence, and hybridizes with at least the target sequence.
  • the sample is contacted with the primers under conditions for a polymerase chain reaction.
  • the primers suitably comprises at least 15, 20, 25, 30, 35 or more nucleotides.
  • Preferred primers have 20 to 30 nucleotides.
  • the formation of any amplified polynucleotide is then qualitatively or quantitatively assessed.
  • the invention also provides a method to detect a polypeptide which is specifically recognized by a detection agent which specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, or a fragment of the sequence or variant.
  • a typical detection agent is an antibody, especially an antibody specific for the amino acid sequence shown in SEQ ID NO:2.
  • Such antibodies can be employed in assay methods which include radioimmunoassays, competitive-binding assays, western blot analysis, ELISA assays or protein tip technology, especially an ELISA or western blot method.
  • a diagnostic method of cancer, especially liver cancer is also a subject of this invention and comprises the steps of: a) detecting the expression level in a test sample of a target sequence which is an mRNA which hybridizes with a polynucleotide having a sequence complimentary to that of SEQ ID NO: 1 or a variant thereof; and b) comparing the expression level of said mRNA in said test sample with that for a control sample.
  • the diagnostic method can suitably be carried out by a hybridization method using a probe which hybridizes under stringent conditions with the target sequence.
  • Hybridization assays can be performed on the test sample and the control sample to give data for the expression levels in the test and control samples which can then be compared. Cancer is diagnosed when the expression level of said mRNA in the test sample is indicatively higher than that in the control sample.
  • the diagnostic method can suitably be carried out by amplification of the target sequence with primers using a polymerase chain reaction.
  • Amplification assays can be performed on the test sample and the control sample to give data for the expression levels in the test and control samples which can then be compared. Cancer is diagnosed when the expression level of the mRNA in the test sample is indicatively higher than that in the control sample.
  • the present invention further provides a diagnostic method for cancer, especially liver cancer, comprising the steps of: a) detecting the expression level in a test sample of a polypeptide which is specifically recognized by a detection agent which specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or a variant thereof, or a fragment of the sequence or variant; b) comparing the expression level of said polypeptide in the test sample with that for a control sample.
  • the diagnostic method can suitably be carried out using a detection agent such as an antibody which binds to the polypeptide. Binding assays can be performed on the test sample and the control sample to give data for the expression levels in the test and control samples which can then be compared. Cancer is diagnosed when the expression level of said polypeptide in the test sample is indicatively higher than that in the control sample.
  • a detection agent such as an antibody which binds to the polypeptide.
  • the invention further provides the polymerase chain reaction primers, which comprise fragment polynucleotides of this invention.
  • Appropriate pairs of forward and reverse primers are provided, either for DNA amplification or more preferably for RNA amplification.
  • Typical primers of this invention include a forward primer which has a length of say 20 - 30 nucleotides, is suited for specifically amplifying the target sequence and hybridizes with a polynucleotide having a sequence complementary to the target sequence.
  • the target sequence is a part or the full length of the SEQ ID NO: l, or a sequence having at least 70% identity thereto.
  • Typical primers of this invention also include a reverse primer which has a length of say 20 - 30 nucleotides, is suited for specifically amplifying a sequence complimentary to the target sequence and hybridizes with the target sequence.
  • the target sequence is a sequence complimentary to a part or the full length of the SEQ
  • ID NO: l or a sequence having at least 70% identity thereto.
  • a preferred microarray component comprises a support carrying at least one single stranded polynucleotide which hybridizes under the stringent condition with a target sequence which is mRNA hybridizing with a polynucleotide having a sequence complementary to the sequence shown in SEQ ID NO: l or a varinat thereof.
  • the invention also provides antibodies which specifically recognize a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, or a fragment of the sequence or variant.
  • a preferred antibody is specific for the amino acid sequence shown in SEQ ID NO:2.
  • Kits are also provided by the present invention for diagnosis of liver cancer.
  • Such kits can include appropriate reagents selected from a pair of forward and reverse PCR primers; a hybridization probe which is an oligonucloeotide which hybridizes with a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant; the microarray component comprising the support carrying the single stranded polynucleotide; or an antibody which specifically recognizes a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or a variant thereof, or a fragment of the sequence or variant.
  • kits can typically include instructions for using the reagents, along with further components selected from additional reagents and/ or equipment.
  • a further aspect of this invention resides in a screening method for identifying a test compound as an anti-cancer agent.
  • Such a method can include the steps of: a) detecting the expression level of a gene comprizing polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof in the absence of the test compound; b) detecting the expression level of a gene comprizing polynucleotide having sequence shown in SEQ ID NO: 1 or a variant thereof in the presence of the test compound; and c) comparing the expression levels of said gene in the absence of said test compound with that in presence of said test compound.
  • test compounds can be selected for which the expression level of said gene decreased in the presence of the compound. Such compounds are then candidates for investigation as agents against cancer.
  • the screening method allows identification of compounds of use in treating liver cancer.
  • Pharmaceutical compositions of the compound and a pharmaceutically acceptable carrier are provided by the invention.
  • An alternative method for detecting cancer comprises assessing the presence of at least one polymorphism or mutation in a gene selected from the gene SAN_O787_l having the sequence SEQ ID NO: 1 and variants thereof.
  • references to variants of the gene SAN_O787_l having the sequence SEQ ID NO: 1 and to the polypeptide having the sequence SEQ ID NO: 2 include allelic forms and other modifications.
  • the polynucleotide sequence SAN_0399_3 differs from a polynucleotide sequence in WO 0127158 at positions 183, 256 and 716.
  • the nucleotides are respectively C, T and T, in place of A, C and C at the corresponding positions in the sequence of WO 0127158.
  • this invention provides polynucleotide sequences based on SAN_0399_3, provided that the polynucleotide sequence has one or more of C at position 183, T at position 256 or T at position 716.
  • the present invention also provides polypeptide sequences based on SAN_0399_3, provided that the polypeptide has one or more of phenylalanine at postion 63, phenylalanine at position 86 and methionine at position 239.
  • the polynucleotide sequence SAN_0437_1 differs from a polynucleotide sequence in WO 0127158 by the presence of coding at positions 1 to 63°. Such additional coding will provide an N-terminal region, compared to the polypeptide encoded by the polynucleotide sequence in WO
  • polynucleotide sequence SAN_0437_1 differs from that polynucleotide sequence in WO 0127158 by the presence of coding at positions 999 to 1008. Such additional coding will provide a C-terminal region, compared to the polypeptide encoded by the polynucleotide sequence in WO 0127158. The presence of a C-terminal region in a G-protein coupled receptor is important for signaling through the G-protein. Furthermore, the polynucleotide sequence SAN_0437_1 differs from that polynucleotide sequence in WO 0127158 at positions 360 and 362. In SAN_0437_1, the nucleotides are respectively G and T, in place of A and A at the corresponding position in the sequence of WO 0127158.
  • the polynucleotide sequence SAN_0437_4 differs from a polynucleotide sequence in WO 0127158 at positions 139 and 720.
  • the nucleotides are respectively A and T, in place of G and G at the corresponding positions in the sequence of WO 0127158.
  • this invention provides polynucleotide sequences based on SAN_0437_4 > provided that the polynucleotide sequence has one or more of A at position 139 or T at position 720.
  • the nucleotide differences in SAN_0437_4 compared with the sequence in WO 0127158 lead to a change in the encoded amino acid sequence.
  • the present invention also provides polypeptide sequences based on SAN_0437_4, provided that the polypeptide has one or more of isoleucine at position 47.
  • the polynucleotide sequence SAN_0817_1 differs from a polynucleotide sequence in WO 0127158 by the absence of coding beyond position 939. Such a change in the coding length will provide a shortened C-terminal region, compared to the polypeptide encoded by the polynucleotide sequence in WO 0127158.
  • the correct C-terminal region in a G-protein coupled receptor is important for signaling through the G-protein.
  • the invention relates to SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_0437_4 and SAN_O817_l polypeptides and recombinant materials and methods for their production.
  • One aspect of the invention relates to methods for using such SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides and polynucleotides.
  • SAN_O787_l SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides and polynucleotides.
  • SAN_O437_4 and SAN_O817_l such uses include the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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's syndrome, among others.
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l imbalance with the identified compounds.
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l activities or levels.
  • the invention exttends to the sequences of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides which do not encode a polypeptide which is involved in olfactory sensation, and to SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides which are not olfactory receptor polypeptides. DESCRIPTION OF THE INVENTION
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_ l refers, among others, to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or an allelic or other variant thereof
  • Receptor Activity or "Biological Activity of the Receptor” refers to the metabolic or physiologic function of said SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_l.
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l gene refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or allelic or other variants thereof and/or their complements.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the . same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques, which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching.
  • Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains one or more essential properties, especially a biological function of the parent polynucleotide or polypeptide.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by synthesis.
  • a variant polynucleotide is typically a polynucleotide with a sequence having at least 80%, 85%, 90% or 95% identity with the parent sequence.
  • a variant polypeptide is typically a polypeptide with a sequence having at least 80%, 85%, 90% or 95% identity with the parent sequence.
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York,
  • identity is known well to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988)
  • Methods to determine identity and similarity are codified in computer programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to,
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9.
  • nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or
  • polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2, 4, 6, 8, 10.
  • the polypeptide sequence having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention relates to SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides.
  • the SAN_0787_1, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides include the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO: 2, 4, 6, 8, 10, 16; over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2, 4, 6, 8, 10.
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2, 4, 6, 8, 10. Furthermore, those with at least 97-99%) are highly preferred.
  • SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides exhibit at least one biological activity of the receptor.
  • SAN_O437_4 and SAN_O817_l polypeptides may be in the form of the mature protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part; but not all, of the amino acid sequence of the aforementioned SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides.
  • SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides, fragments may be "free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
  • polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides.
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of SAN_O787_l, SAN_O399_3, SAN_O437_l,
  • SAN_O437_4 and SAN_O817_l polypeptides except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • Also preferred fragments are characterized by structural or functional attributes comprising alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • polypeptide fragments retain the biological activity of the receptor, including antigenic activity.
  • polypeptide fragments of this invention comprise at least 10, 15, 18, 20, 25, 30, 35, 40 or more amino acids.
  • Variants of the defined sequence and fragments also form part of the present invention.
  • Preferred variants are those that vary from the referents by conservative amino acid substitutions, that is, those that substitute a residue for another residue of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr, among acidic residues
  • Asp and Glu among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated Naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • SAN_O787_l SAN_O399_3, SAN_O437_l , SAN_O437_4 and SAN_O817_1 polynucleotides.
  • SAN_O787_l , SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides include isolated polynucleotides which encode the SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides and fragments, and polynucleotides closely related thereto.
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotide of the invention include polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9 encoding a SAN_O787_l, SAN_O399_3,
  • SAN_O437_l polypeptide of SEQ ID NO: 2, 4,
  • SAN_O817_l polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and
  • polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred.
  • those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
  • SAN_O787_l also included under SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1, 3, 5, 7, 9 to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides complementary to such SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides.
  • SAN_O787_l , SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l of the invention is structurally related to other proteins of the G-protein coupled receptor family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1, 3, 5, 7, 9) encoding human SAN_O787_l, SAN_O399_3, SAN meaningO437_l, SAN_O437_4 and SAN_O817_l .
  • the cDNA sequence of SEQ ID NO: l contains an open reading frame (nucleotide numbers 1 to 927) encoding a polypeptide of 309 amino acids of SEQ ID NO:2.
  • the cDNA sequence of SEQ ID NO:3 contains an open reading frame (nucleotide numbers 1 to 948) encoding a polypeptide of 316 amino acids of SEQ ID NO:4.
  • the cDNA sequence of SEQ ID NO: 5 contains an open reading frame (nucleotide numbers 1 to 1005) encoding a polypeptide of 335 amino acids of SEQ ID NO:6.
  • the cDNA sequence of SEQ ID NO:7 contains an open reading frame (nucleotide numbers 1 to 960) encoding a polypeptide of 320 amino acids of SEQ ID NO:8.
  • the cDNA sequence of SEQ ID NO:9 contains an open reading frame (nucleotide numbers 1 to 936) encoding a polypeptide of 312 amino acids of SEQ ID NO: 10.
  • gagcaactac atatctggct gtccatcc ⁇ c ttctgcatca tgta ⁇ at ⁇ gc tgccctggaa
  • Polynucleotides of the present invention encoding SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides may be obtained using standard cloning and screening from a cDNA library derived from mRNA in cells of human tissues. Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
  • nucleotide sequences encoding SAN_O787_l, SAN_0399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_ polypeptides of SEQ ID NO: 2, 4, 6, 8, 10 may be identical to the polynucleotides encoding sequences contained in Table 1 , or they may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptides of SEQ ID NO:2, 4, 6, 8, 10.
  • the polynucleotides of the invention may include the coding sequence for the mature polypeptides or a fragment thereof by itself in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence that facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86: 821-824, or is an
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, 3, 5, 7, 9 or a fragment thereon may be used as hybridization probes for cDNA and genomic
  • SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the SAN_p787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and
  • SAN_O817_1 gene Such hybridization techniques are known to those of skill in the art. Typically these nucleotide sequences are 80% identical, preferably
  • the probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides.
  • Particularly preferred probes will range between 30 and 50 nucleotides.
  • to obtain polynucleotides encoding the SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the SEQ ID NO: 1, 3, 5, 7, 9 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Such hybridization techniques are well known to those of skill in the art.
  • Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 °C in a solution comprising: 50% formamide, ⁇ .times; SSC (150mM NaCl, 15mM sodium citrate), 5. times; Denhardt's solution, and 0.5% SDS, followed by washing the filters in 0.1. times SSC at about 65° C.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS,
  • Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual (supra).
  • SAN_0787_1, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides are to be expressed for use in screening assays, generally, it is preferred that the polypeptide be localized at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay.
  • SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides are secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • This invention also relates to the use of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN _0437_4 and SAN_O817_l polynucleotides for use as diagnostic reagents.
  • SAN_O437_l, SAN_O437_4 and SAN_O817_l gene associated with dysfunction can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and
  • SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l genes may be detected by a variety of techniques.
  • Nucleotides for diagnosis of liver cancer in respect of SAN_O787_l are most suitably obtained from hepatocytes.
  • Nucleotides acids for diagnosis in respect of SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l may be obtained from a subject's cells, such as from blood, urine, saliva, sperm, tissue biopsy or autopsy material. Hepatocytes and liver tissue are preferred sources for use in detecting liver cancer using diagnosis based on SAN_787_ 1.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion.
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labeled SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of
  • DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al, Science (1985) 230: 1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method.
  • an array of oligonucleotides probes comprising SAN_O787_l,
  • SAN_0399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l nucleotide sequences or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M. Chee et al, Science, Vol 274, pp
  • the diagnostic assays in respect of SAN_O399_3, SAN_O437_l, SAN_0437_4 and SAN_O817_l offer a process for diagnosing or determining a susceptibility to infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; asthma; allergies; benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe
  • SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l genes by the methods described.
  • infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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 G
  • SAN_O437_4 and SAN_O817_l can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, Rnase protection, Northern blotting, other hybridization methods and DNA microarray technology. Assay techniques that can be used to determine levels of a protein, such as
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l in a sample derived from a host are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays,
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).
  • genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • linkage analysis coinheritance of physically adjacent genes.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • polypeptides of the invention or their fragments or analogs thereof or cells expressing them can also be used as immunogens to produce antibodies immuno specific for the SAN_O399_3, SAN_O437_l, SAN_O437_4; SAN_O787_l and SAN_O817_l polypeptides.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • Antibodies generated against the SAN_0787_1, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols.
  • any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G.
  • Antibodies against SAN_O787_l may be used to treat liver cancer.
  • SAN_O437_4 and SAN_O817_l polypeptides may be employed to treat infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non- insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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's syndrome, among others.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l, polypeptides, or a fragments thereof, adequate to produce antibody and/ or T cell immune response to protect said animal.
  • the protection can be from infections such as bacterial, fungal, protozoan and viral infections, particular infections caused by HIV-1 or HIV-2; pain; cancers; non- insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary t rombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides via a vector directing expression of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polynucleotides in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • composition which, when introduced into a mammalian host, induces an immunological response in that mammal to SAN_O787_l, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l polypeptides
  • the composition comprises SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides or SAN_0787__1, SAN_O399_3, SAN_0437_1, SAN_O437_4 and SAN_O817_l genes.
  • the vaccine formulation may further comprise a suitable carrier. Since SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • polypeptides of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention.
  • polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).
  • SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4, and SAN_O817_l polypeptides are responsible for many biological functions, including various pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l on the one hand and which can inhibit the function of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_0817_1 on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV- 1 or HIV-2; pain; cancers; non-insulin-dependent diabetes
  • NIDDM insulin-dependent diabetes
  • IDDM insulin-dependent diabetes
  • asthma anorexia
  • bulimia asthma
  • Parkinson's disease arteriosclerosis
  • coronary thrombosis acute heart failure
  • hypotension hypertension
  • urinary retention articular rheumatism
  • osteoarthritis osteoporosis
  • renal disease cardiac hypertrophy ; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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
  • Antagonists including inverse agonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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's syndrome.
  • such screening procedures involve producing appropriate cells, which express the receptor polypeptide of the present invention on the surface thereof.
  • Such cells include cells from mammals, baculovirus, adenovirus, yeast, Drosophila or E. coli.
  • Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding or stimulation or inhibition of a functional response.
  • One screening technique includes the use of cells which express receptor of this invention (for example, transfected CHO cells) in a system which measures GTP binding activity of coupled G proteins, extracellular pH or intracellular calcium changes caused by receptor activation.
  • compounds may be contacted with cells expressing the receptor polypeptide of the present invention.
  • a second messenger response e.g., signal transduction, changes of GTP binding activity, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
  • Another method involves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/ or adenylate cyclase accumulation.
  • Such a method involves rransfecting a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to potential antagonists in the presence of the receptor of this invention. The amount of cAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased.
  • Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described in U.S. Pat. No. 5,482,835.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Standard methods for conducting such screening assays are well understood in the art.
  • Examples of potential SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the SAN_O787_l, SAN_O399_ 3, SAN_O437_l, SAN_O437_4 and SAN_O817_l e.g., a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
  • Prophylactic and Therapeutic Methods include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the SAN_O787_l, SAN_O399_ 3, SAN_O437_l, SAN_O437_4 and SAN_O817_l e.g., a fragment
  • This invention provides methods of treating abnormal conditions such as, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; non-insulin-dependent diabetes (NIDDM); insulin-dependent diabetes (IDDM); obesity; anorexia; bulimia; asthma; Parkinson's disease; arteriosclerosis; coronary thrombosis; acute heart failure; hypotension; hypertension; urinary retention; articular rheumatism; osteoarthritis; osteoporosis; renal disease; cardiac hypertrophy; angina pectoris; myocardial infarction; ulcers; atopic dermatitis; psoriasis arthropathy; 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's syndrome, related to both an excess of and insufficient amounts of SAN_O399
  • One approach comprises administering to a subject an inhibitor compound (antagonist including inverse agonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the SAN _O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_0817_1 or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • SAN_O437_l, SAN_O437_4 and SAN_O817_l may be administered.
  • Typical embodiments of such competitors comprise fragments of the SAN_O787_l,
  • expression of the gene encoding endogenous SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56: 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression CRC Press, Boca Raton, Fla. (1988).
  • oligonucleotides which form triple helices with the gene can be supplied.
  • oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound which activates SAN_O787_l, SAN_O399_3, SAN_O437_l,
  • SAN_O437_4 and SAN_O817_l i.e. an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of SAN_O787_l, SAN_O399_3,
  • SAN_O437_l SAN_O437_4 and SAN_O817_l by the relevant cells in the subject.
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
  • SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides in combination with a suitable pharmaceutical carrier.
  • Peptides such as the soluble form of SAN_O787_l, SAN_O399_3, SAN_O437_l, SAN_O437_4 and SAN_O817_l polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible.
  • Administration of these compounds may also be topical and/ or localized, in the form of salves, pastes, gels and the like. The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner.
  • Suitable dosages are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection.
  • Polypeptides used in treatment can also be generated endogenously in the subject in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • FIG. 1 Expression of the 5 GPCR-like gene transcripts in human tissues.
  • Reverse transcription (RT) - PCR amplification was performed to detect the GPCR-like gene transcripts.
  • a mixture of the cDNA templates synthesized from 18 human tissue poly(A) + RNAs was used as a template. Aliquots of the RT PCR products were electrophoresed through a 1.5% agarose gel.
  • M indicates a 100-bp DNA ladder used as a molecular weight standard. The 600 - bp band appears as a brighter band than the other ladder bands.
  • FIG. 1 The expression pattern of SAN_0399_3 in 24 human tissues.
  • Reverse transcription (RT) - PCR amplification was performed to detect SAN_0399_3 transcripts.
  • 24 human tissue poly(A) + RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel.
  • the "marker” indicates a 100-bp DNA ladder used as a molecular weight standard. The 600 - bp band appears as a brighter band than the other ladder bands.
  • FIG. 3 The expression pattern of SAN_0437_1 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0437_1 transcripts. 24 human tissue poly (A) + RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a
  • the "marker” indicates a 100-bp DNA ladder used as a molecular weight standard.
  • the 600- bp band appears as a brighter band than the other ladder bands.
  • FIG. 4 The expression pattern of SAN_0437_4 in 24 human tissues.
  • Reverse transcription (RT) - PCR amplification was performed to detect SAN_0437_4 transcripts.
  • 24 human tissue poly(A) + RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel.
  • the "marker” indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
  • FIG. 5 The expression pattern of SAN_0787_1 in 24 human tissues. Reverse transcription (RT) - PCR amplification was performed to detect SAN_0787_1 transcripts. 24 human tissue poly(A) + RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel. The "marker" indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
  • RT Reverse transcription
  • FIG. 6 The expression pattern of SAN_0817_1 in 24 human tissues.
  • Reverse transcription (RT) - PCR amplification was performed to detect SAN_0817_1 transcripts.
  • 24 human tissue poly(A) + RNAs were used as a template. Aliquots of the RT-PCR products were electrophoresed through a 1.5% agarose gel.
  • the "marker” indicates a 100-bp DNA ladder used as a molecular weight standard. The 600- bp band appears as a brighter band than the other ladder bands.
  • FIG. 7 Differential expression of SAN_0787_1 mRNA in normal and tumor liver.
  • SAN_ 0787_1 mRNA in total RNA of human normal and tumor liver was detected by analytical PCR method.
  • the selected ESTs were mapped into the HTG sequences as listed in Table 3.
  • the coding regions embedded in these HTG sequences were obtained as described below.
  • the individual HTG sequences were dissected into fragments of 5000 bases.
  • the resulted fragments were submitted to BLASTX search against GenPept and GeneSeqp(Derwent). 28 single exon regions encoding novel GPCR-like genes including those given in Table 1 and 2 were identified (Table 3), along with some other gene sequences not listed in the Tables.
  • first strand cDNAs were generated by reverse transcription of 18 human tissue poly(A) + RNAs, and the mixture of the 18 tissue cDNAs was used as a template for polymerase chain reaction (PCR).
  • 13 human adult tissue poly(A) + RNAs were obtained from Clontech Laboratories, Inc. (Palo Alto, CA): adrenal gland, bone marrow, brain, heart, liver, lung, pancreas, placenta, prostate, skeletal muscle, small intestine, spleen and thyroid.
  • Four human fetal tissue poly(A) + RNAs were also obtained from Clontech Laboratories: brain, kidney, liver and lung.
  • Human colon poly(A) + RNA was obtained from BioChain Institute, Inc. (Hayward, CA).
  • Reverse transcription reaction was carried out in a nuclease-free microcentrifuge tube for each poly(A) + RNA of the 18 tissues. Two micrograms of poly(A) + RNA and 1 ⁇ g of oligo(dT) ⁇ s (Roche Diagnostics Corp., Indianapolis, IN) were added to the tube and filled up to 15 ⁇ l with water. The mixture was heated to 70°C for 10 minutes and then chilled on ice for 5 minutes.
  • PCR primers were designed for the GPCR-like genes and 2 known human olfactory receptor genes (OR1A2 and OR3A3 genes), and custom - sythesized by Genset Co., Ltd. (Kyoto, Japan).
  • GenBank accession numbers of OR1A2 and OR3A3 genes are G 7144635 and G 5081803, respectively (Glusman, G. et al Genomics 63:227-245, 2000).
  • Synthesized DNA was diluted to 10 ⁇ M with water. When human genomic DNA was used as a PCR template, all the primer sets gave rise to single bands which corresponded with the expected sizes of the PCR products.
  • Each tube contained a 50 ⁇ l reaction mixture which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM
  • PCR (Takara Shuzo Co., Ltd., Kyoto, Japan). Forty cycles of PCR was performed under the following conditions: initial denaturation at 94 °C for 5 min; then 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; followed by a final extension of 72°C for 10 minutes. Aliquots of the PCR products were analyzed by agarose gel electrophoresis.
  • Each tube contained a 100 ⁇ l reaction mixture which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM (NH 4 ) 2 SO 4 /2 mM MgCl 2 /6.74 ⁇ M Na 2 EDTA/ 10% DMSO/0.2 mM dNTPs (0.2 mM each dATP, dCTP, dGTP and dTTP)/ 10 mM 2-mercaptoethanol/0.04% 18-mix cDNA template solution /0.5 ⁇ M forward primer/ 0.5 ⁇ M reverse primer/ 0.035 units/ ⁇ l Ex Taq DNA polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan).
  • PCR was performed under the following conditions: initial denaturation at 94°C for 5 min; then 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min; followed by a final extension of 72°C for 10 minutes.
  • RT-PCR products were purified using Millipore MultiScreen FB 96-Well Filtration Plate
  • READY- LOAD 100 bp DNA Ladder (Invitrogen Corporation) was used as a molecular weight standard.
  • RNAs Poly(A) + RNA of human adult adrenal gland, bone marrow, brain, heart, kidney, liver, lung, pancreas, placenta, prostate, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea and uterus and poly(A) + RNA of human fetal brain , kidney and liver were purchased from Clontech
  • RNA was further diluted by 80 times with water. PCR was performed in a 50 ⁇ l reaction which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM
  • SAN_0399_3 mRNA was expressed highly in placenta, prostate, skeletal muscle, spinal cord, stomach, testis, thymus and trachea.
  • SAN_0437_1 mRNA was expressed highly in placenta, prostate, small intestine, spinal cord, spleen, testis, thymus, trachea, uterus and fetal kidney.
  • SAN_0437_4 mRNA was ubiquitously expressed in the human tissues.
  • SAN_0787_1 mRNA was expressed highly in colon, kidney, liver and testis.
  • SAN_0817_1 mRNA was expressed highly in testis.
  • RT-PCR was performed using human liver cDNA as a template.
  • Human liver poly(A) + RNA was purchased from Clontech Laboratories (Cat. #6510-1, lot 0030614, 1 male Caucasian, 35 yr., sudden death).
  • First strand cDNA template was synthesized by reverse transcription with Superscript ⁇ and oligo(dT) primer as described earlier. Nucleotide sequences of PCR primers used were 5'-GCGGCCGCTGTTGAGAATTTACTCCTTGTTG-3' (forward primer) and 5'-GCGGCCGCTAATGGTCTCTTAATGAAATAAT-3' (reverse primer).
  • Both primers contained a linker sequence 5 '-GCGGCC-3' at their 5' ends for cloning into pCR-Blunt IT-TOPO vector (Invitrogen Corp.).
  • PCR was conducted using KOD-plus DNA polymerase (Toyobo Co., Ltd.) and the PCR product was inserted into pCR-Blunt ⁇ -TOPO vector using topoisomerase.
  • the resultant plasmid was introduced into Escherichia coli strain TOP 10. Transformants were picked up and the nucleotide sequences of their cDNAs were analyzed using an ABI 3700 DNA sequencer (Applied Biosystems, Foster City, CA). The nucleotide sequence was identical to the sequence of SEQ ID NO: 1 in the
  • SAN_0787_1 cDNA was also cloned from human kidney poly(A) + RNA (Clontech Laboratories, Cat.#6538-1) and the sequence was identical to that of the cDNA cloned from human liver poly(A) + RNA.
  • SAN_0787_1 DNA was amplified by PCR from human genomic DNA templates that were prepared from two donors, and the PCR products were cloned into pCR-Blunt ⁇ -TOPO and the DNA inserts were sequenced.
  • the DNAs were purchased from Roche Diagnostics Corp. and Promega Corp. (Madison, Wl). The nucleotide sequencing analysis revealed that the DNA from Roche Diagnostics (Cat.
  • SAN_0787_1 cDNA insert was excised with Notl from the plasmid and subcloned into a Notl site of mammalian expression vector pcD ⁇ A3.1(+) (Invitrogen Corp.).
  • the plasmid having the cDNA insert in the same orientation as CMV promoter of the expression vector was selected.
  • the resultant plasmid can express SAN_0787_1 protein in mammalian cells such as CHO cells, COS cells, 293 cells etc. when the cells are transfected with the plasmid by conventional methods. Expression of GPCR protein is confirmed by Western blotting of transfected cell lysates using SAN_0787_1 -protein specific antibody which was prepared as described below.
  • Synthesized peptide was released from the support material by treatment with hydrogen fluoride.
  • the peptide was extracted with 0.1%) trifluoroacetic acid and freeze-dried.
  • the peptide was then purified by high-performance liquid chromatography (HPLC) in an acetonitrile-0.1% trifluoroacetic acid solvent system using HPLC Model LC8A (Shimadzu Corp., Kyoto, Japan). Purity of the peptide obtained was evaluated by HPLC and amino acid composition analysis.
  • N-(6-maleimidocaproyloxy)-succinimide (EMCS, manufactured by Dojindo Laboratories, Kumamoto, Japan). An incorporation ratio of the peptide into the carrier protein was evaluated by amino acid composition analysis.
  • the peptide-carrier conjugate was suspended in saline solution at a concentration of 1 mg/ml and used to immunize rabbits.
  • the conjugate was mixed well with an equal volume of complete Freund's adjuvant and then injected into the back of two rabbits (body weight 2-2.5 kg) hypodermically or intradermically.
  • incomplete Freund's adjuvant was used for the second or later immunization. Immunization was performed every two weeks and after the second immunization test-blood collection was performed and the antibody titer in the serum was measured by a solid phase method of an enzyme-linked innmunosorbent assay (ELISA).
  • ELISA enzyme-linked innmunosorbent assay
  • Antigen peptide was coated on a 96-well plate for ELISA, and a Western horseradish-peroxidase labeled anti-rabbit IgG antibody was used as a second antibody. The exsanguination was conducted 2 months after the first immunization. Purification of Antibody from Immunized Rabbit Antiserum with Peptide
  • the antibody was purified after blood collecting using a peptide affinity column. About 8 mg of the peptide was combined with 5 ml of carrier agarose Affi-Gel 102 (Bio-Rad Laboratories, Inc., Hercules, CA) by conventional methods. About 15 ml of the antiserum was diluted with an equal volume of PBS (containing 0.02 M phosphoric acid buffer solution (pH 7.0), 0.9% sodium chloride) and a precipitate was obtained by the ammonium sulfate precipitation (final concentration 40%) method. This precipitate was dissolved in PBS, and then dialyzed with PBS. The dialyzed antibody solution was used as the semi-purified IgG fraction.
  • PBS containing 0.02 M phosphoric acid buffer solution (pH 7.0), 0.9% sodium chloride
  • Antibody concentration of the eluate of 3.5 M potassium thiocyanate solution was 35.0 ⁇ g/ml for one rabbit and 40.0 ⁇ g/ml for the other rabbit.
  • These antibody cotaining eluates were dialyzed to PBS and used in further experiments as an specific antibody to SAN_0787_1 protein. Titers of the antibody solutions were measured using an ELISA system as described above.
  • SAN_0787_1 mRNA expression level was analyzed in human normal and tumor liver tissues using ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). This analysis was conducted according to User Bulletin #2 that was provided by the manufacturer. The method involved preparing a standard curve for each gene of interest by assaying a dilution series prepared from a first-strand cDNA in a reverse transcriptase-5'-exonuclease PCR amplification assay. A threshold cycle (ct) for each member of the reverse transcribed-amplification reaction was determined and plotted versus the log of the amount of a first-strand cDNA in dilution series. The plot was used to determine an RNA equivalent from which the normalized human GAPDH RNA equivalent for
  • SAN_0787_1 was determined. The assay was used to determine the level of expression of SAN_0787_1 mRNA in sample tissues.
  • Human liver total RNAs were purchased from Biochain: human normal liver total RNA (Cat. No.61009, Lot.A503276), human tumor liver total RNA (Cat. No.64003, Lot.A505425).
  • the PCR primer pairs and probes were designed using the Primer Express 1.0 program (PE Applied Biosystems).
  • the oligonucleotide hybridization probe and the primer pairs for SAN_0787_1 gene with the following sequences were synthesized: TaqMan probe 5'-FAM-ATATCCTTGGCAGGCTGCGCAA-TAMRA-3', forward primer
  • First-strand cDNA synthesis was performed using TaqMan Reverse Transcription Reagents Kit (PE Applied Biosystems, Cat. No.N808-0234, Lot No.D02956). One microgram total RNA of each sample was dissolved in 20 ⁇ l of RNase-free water and denatured for 10 minutes at 65 ° C and chilled on ice. First-strand cDNA was synthesized in two steps at 25°C for 10 minutes and at
  • RT-PCR reactions were performed in duplicates with a final volume of 50 ⁇ l.
  • Each RT-PCR reaction includes 25 ⁇ l TaqMan Universal PCR Master mix (PE Applied Biosystems), 200 nM forward primer, 200 nM reverse primer, 50 nM probe and 5 ⁇ l of first-strand cDNA.
  • RT-PCR conditions were 2 minutes at 50°C, 10 minutes at 95°C, and 50 cycles of 15 seconds at 95°C, 1 minute at 60°C.
  • Standard curves were obtained each time a gene assayed using first-strand cDNA prepared from 7 standard dilution series (designated as quantity 2, 1, 0.4, 0.2, 0.1, 0.04, 0.02).
  • Reactions were run in 96-well plates (MicroAmp Optical 96-well Reaction Plate, PE Applied Biosystems) on ABI PRISM 7700 Sequence Detection System. Calculation of Relative RNA Equivalents
  • RNA expression of SAN_0787_1 was higher in tumor liver than in normal liver by about 100 fold ( Figure 7). No amplification signal was detected in analytical PCR of all the samples when the first strand cDNA was synthesized without reverse transcriptase. This showed no contamination of genomic DNA in the total RNA samples used. Therefore SAN_0787_1 gene can be used as a cancer marker gene especially for liver cancer.
  • Probe DNA of SAN_0787_1 gene was prepared for microarray analysis by RT-PCR using SAN_0787_l-gene specific primers which were listed in Table 4. Forty cycles of RT-PCR were carried out in four microcentrifuge tubes. Each tube contained 100- ⁇ l reaction mixture which consisted of 67.4 mM Tris-HCI (pH8.8)/ 16.7 mM (NH 4 ) 2 SO 4 /2 mM MgCl 2 /6.74 ⁇ M Na 2 EDTA/ 10% DMSO/0.2 mM dNTPs (0.2 mM each dATP, dCTP, dGTP and dTTP)/ 10 mM 2-merca ⁇ toethanol/0.04% 18-mix cDNA template solution
  • RT-PCR products were purified using Millipore MultiScreen FB 96-Well
  • Tris-HCI, ⁇ H8.0/0.1 mM Na 2 EDTA were added to each well of the FB plate, and the plate was centrifuged to elute the purified DNA from the filters.
  • GPDH GenBank GL28251
  • Gfyceraldehyde-3- ⁇ hos ⁇ hate dehydrogenase GenBank GL4503912
  • Gene-specific oligonucleotide probes which are about 20 to 80 nucleotides in length can be also used as spot DNAs for detection of expression of SAN_0787_1 gene in normal and tumor tissues.
  • Probe DNAs of human SAN_0787_1 gene, ⁇ -actin gene, GAPDH gene and other human genes including GPCR genes were spotted onto microarray slides Type 7 (Amersham Biosciences, Inc., Uppsala, Sweden; Cat. No.RPK0331) using Generation III Array Spotter (Amersham Biosciences, Inc.) according to a manufacturer's instruction.
  • Microarray SocreCard (Amersham Biosciences, Inc., Cat. No. RPK1161) was also spotted onto the same slides. ScoreCard included controls for dynamic range, retio, and positive and negative hybridization controls. For oligonucleotide microarrays spotting methods are optimized for probe DNA to stick well to microarray slides using coated microscope slides such as poly-L lysine slides.
  • RNA or poly(A) + RNA is isolated from human normal and diseased tissues such as normal liver and tumor liver tissues by conventional methods. For example RNA is extracted from tissue samples using Trizol reagent (Invitrogen Corp., Cat. No.15596) according to the manufacturer's protocol.
  • Poly(A) + RNA is purified from total RNA using commercially available kits such as PolyATtract mRNA Isolation System (Promega Corp., Cat. No. Z5200).
  • cDNA is synthesized by reverse transcription from RNA and fluorescent-dye labeled nucleotides, Cy3- and Cy5-dCTP (Amersham Biosciences, Inc., Cat. No. PA53023 and PA55023, respectively), are incorporated into cDNA during its synthesis reaction.
  • Cy3- and Cy5-dCTP Amersham Biosciences, Inc., Cat. No. PA53023 and PA55023, respectively
  • human normal liver and tumor liver samples are labeled with Cy3- and Cy5-dCTP, respectively.
  • cDNA synthesis, labeling and purification are conducted according to the protocol provided by the manufacturer of microarray slides Type 7.
  • RNA can be amplified using T7 RNA polymerase according to the linear amplification method based on Eberwine protocol (Van Gelder, R. N. et al. Proc. Natl. Acad. Sci. U.S.A. 87(5): 1663-1667, 1990) or using commercially available kits such as RiboAmp RNA Amplification Kit (Takara Shuzo Co., Ltd.). After amplification of RNA, cDNA is synthesized and labeled with Cy3- and Cy-5 dCTP.
  • Hybridization of DNA microarray with labeled target cDNAs is performed manually, or automatically using Automated Slide Processor (Amersham Biosciences, Inc.), in accordance with the protocol provided by the manufacturer of microarray slides Type 7.
  • Automated Slide Processor Amersham Biosciences, Inc.
  • Cy3-labeled normal liver cDNA and Cy5-labeled tumor liver cDNA are co-hybridized with arrayed probe DNAs containing SAN_0787_1 DNA. After washing the slides, they are processed to fluorescent signal detection steps.
  • DNA microarrays containing SAN_0787_1 gene By using DNA microarrays containing SAN_0787_1 gene, diagnosis of liver tumor can be efficiently conducted.
  • DNA microarrays containing SAN_0787_1 gene and other cancer marker genes provide high-throughput diagnosis tools for detecting various tumors in patients' tissues.
  • GPCR GPCR expression and purification of QPCRs are achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are introduced into suitable bacterial hosts, e.g. , Escherichia coli BL21 (DES) .
  • Antibiotic resistant bacteria express GPCRs upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG) .
  • GPCRs in eukaryotic cells are achieved by infecting insect or mammalian cell lines with recombinant Autographica California nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica California nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of the baculovirus is replaced with cDNA encoding GPCRs by either homologous recombination or bacterial-mediated polyhedrin promoter which drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodopter frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to the baculovirus.
  • Sf9 Spodopter frugiperda
  • GPCRs are synthesized as a fusion protein with, e. g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST a 26 kDa enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech) .
  • Fo 1 lowing purification the GST moiety can be proteolytically cleaved from GPCRs at specifically engineered sites.
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN) . Methods for protein expression and purification are discussed in Ausubel (1995, ch. 10 and 16).
  • GPCRs substantially purified using polyacrylamide gel electrophoresis or other purification techniques are used to immunize rabbits and to produce antibodies using standard protocols. Rabbits are immunized with the purified GPCRs suspended in complete Freund's adjuvant. Resulting antisera are tested for anti-GPCRs activity by, for example, binding the GPCRs to a substrate, blocking with 1% BSA, reacting with the rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • SEQ ID NO: 1 is nucleotide sequence of SAN_O787_l
  • SEQ ID NO: 2 is amino acid sequence of SAN_O787_l
  • SEQ ID NO: 3 is nucleotide sequence of SAN_O399_3
  • SEQ ID NO: 4 is amino acid sequence of SAN_O399_3
  • SEQ ID NO: 5 is nucleotide sequence of SAN_O437_l
  • SEQ ID NO: 6 is amino acid sequence of SAN_O437_l
  • SEQ ID NO: 7 is nucleotide sequence of SAN_O437_4
  • SEQ ID NO: 8 is amino acid sequence of SAN_O437_4
  • SEQ ID NO: 9 is nucleotide sequence of SAN_O817_l
  • SEQ ID NO: 10 is amino acid sequence of SAN_O817_l
  • SEQ ID NO: 11 is forward primer of SAN_O399_3
  • SEQ ID NO: 12 is reverse primer of SAN_O399_3
  • Product size is 453
  • SEQ ID NO: 13 is forward primer of SAN_O437_l
  • SEQ ID NO: 14 is reverse primer of SAN_O437_l Product size is 495
  • SEQ ID NO: 15 is forward primer of SAN_O437_4
  • SEQ ID NO: 16 is reverse primer of SAN_O437_4
  • SEQ ID NO: 17 is forward primer of SAN_O787_l
  • SEQ ID NO: 18 is reverse primer of SAN_O787_l
  • Product size is 223
  • SEQ ID NO: 19 is forward primer of SAN_O817_l
  • SEQ ID NO: 20 is reverse primer of SAN_O817_l Product size is 407.

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Abstract

L'invention porte sur des polypeptides et des polynucléotides SAN O787 1, SAN O399 3, SAN O437 1, SAN O437 4, et SAN O817 1 et sur des procédés de fabrication de tels polypeptides grâce à des techniques recombinantes. SAN 0787 1 présente un intérêt particulier puisque des niveaux plus élevés d'expression sont associés avec le cancer du foie. L'invention concerne aussi des procédés de diagnostic du cancer, notamment du cancer du foie, ainsi que leurs aspects correspondants. Elle se rapporte également à des procédés d'utilisation de polypeptides et des polynucléotides SAN O787 1, SAN O399 3, SAN O437 1, SAN O437 4, et SAN O817 1 dans la conception de protocoles et de réactifs pour des traitements thérapeutiques.
PCT/JP2002/006363 2001-06-26 2002-06-25 Polynucleotides, polypeptides et cancer WO2003000929A2 (fr)

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CA2113957A1 (fr) * 1993-01-29 1994-07-30 University Of Guelph Sequences nucleotidiques pour la determination du sexe des bovins
EP1016717A1 (fr) * 1997-06-26 2000-07-05 Sumitomo Electric Industries, Limited Nouvelle proteine ayant une excellente expression dans le cancer du sang, gene codant cette proteine, anticorps dirigee contre cette derniere et procede de detection de l'expression de cette proteine
WO1999005274A1 (fr) * 1997-07-23 1999-02-04 Smithkline Beecham Corporation Procede servant a identifier de nouveaux recepteurs de proteines g et leurs fonctions
WO1999006830A1 (fr) * 1997-07-30 1999-02-11 The Regents Of The University Of California Procede servant a moduler des recepteurs couples a une proteine g
WO1999043811A1 (fr) * 1998-02-25 1999-09-02 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Adn complementaires codant un gene bog (b5t-over-expressed gene) et son produit proteinique
WO1999057269A1 (fr) * 1998-04-30 1999-11-11 Sumitomo Electric Industries, Ltd. Gene hrpi humain
WO2000023625A2 (fr) * 1998-10-22 2000-04-27 Ludwig Institute For Cancer Research Molecules isolees d'acide nucleique codant des proteines scp et leur utilisation
WO2001027158A2 (fr) * 1999-10-08 2001-04-19 Digiscents Sequences de recepteurs olfactifs
WO2002010202A2 (fr) * 2000-07-26 2002-02-07 Curagen Corporation Nouvelles proteines et nouveaux acides nucleiques codant celles-ci
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