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WO2000065090A2 - Sequence de genes recepteurs associes au recepteur de l'insuline pour le diagnostic de l'obesite humaine et des troubles d'origine diabetique - Google Patents

Sequence de genes recepteurs associes au recepteur de l'insuline pour le diagnostic de l'obesite humaine et des troubles d'origine diabetique Download PDF

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WO2000065090A2
WO2000065090A2 PCT/US2000/010644 US0010644W WO0065090A2 WO 2000065090 A2 WO2000065090 A2 WO 2000065090A2 US 0010644 W US0010644 W US 0010644W WO 0065090 A2 WO0065090 A2 WO 0065090A2
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irrr
nucleic acid
seq
amino acid
sequence
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PCT/US2000/010644
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WO2000065090A3 (fr
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Si Lok
Theodore E. Whitmore
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Zymogenetics, Inc.
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Priority to AU46498/00A priority Critical patent/AU4649800A/en
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Publication of WO2000065090A3 publication Critical patent/WO2000065090A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • 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

Definitions

  • the present invention relates generally to nucleic acid molecules and antibodies useful as probes for the detection of a metabolic disease.
  • the present invention relates to methods for detecting a metabolic disease using nucleic acid and antibody probes for the insulin receptor-related receptor.
  • Non-insulin dependent diabetes mellitus is the most common of all metabolic disorders (for a review, see Kahn et al, Annu. Rev. Med. 47:509 (1996); Patti and Kahn, Diabetes Reviews 5:149 (1997); Lowe, "Diabetes Mellitus," Principles of Molecular Medicine, (Jameson, ed.), pages 433- 442 (Humana Press Inc. 1998)).
  • Type II diabetes patients and their first degree relatives demonstrate insulin resistance at the level of skeletal muscle and adipose tissue. This suggests a possible primary role for a defect in the insulin signal transduction cascade that results in stimulation of glucose transport and glycogen synthesis.
  • the signaling defect could involve any protein in the insulin signal transduction pathway, defects in pathways that interface with the insulin signal pathway, or defects in molecules essential for cellular function (DeFronzo, Diabetes Reviews 5:177 (1997)).
  • Type II diabetes mellitus has a substantial genetic component (Barnett et al, Diabetologia 20:87 (1981); Knowler et al, Am. J. Epidemiol. 113:144 (1981); Hanson et al, Am. J. Hum. Genet. 57:160 (1995)).
  • Genes that predispose to certain forms of diabetes have been identified, including several loci for Type I diabetes and for maturity-onset diabetes of the young (Froguel et al, Nature 356:162 (1992); Davies et al, Nature 371:130 (1994); Yamagata et al, Nature 384:455 (1996); Staffers et al, Nat. Genet. 77:138 (1997)).
  • Type II diabetes mellitus is a polygenic disease (DeFronzo, Diabetes Reviews 5:177 (1997); Lowe, "Diabetes Mellitus,” Principles of Molecular Medicine, (Jameson, ed.), pages 433- 442 (Humana Press Inc. 1998)).
  • the present invention provides methods for diagnosing a metabolic disease or susceptibility to a metabolic disease by detecting an alteration in chromosome Iq21-q24. Such methods are effected by examining the insulin receptor-related receptor (1RRR) gene and its gene products.
  • the insulin receptor-related receptor was first identified as human and guinea pig genomic DNA sequences that appeared to encode a novel member of the insulin receptor family (Shier and Watt, J. Biol. Chem 264:14605 (1989)). Subsequently, full-length rat and human cDNA molecules were reported by Kurachi et al, Biochem. Biophys. Res. Commun. 187:934 (1992), and Jui et al, J. Biol. Chem. 269:22446 (1994). An illustrative nucleotide sequence that encodes the human IRRR identified by Shier et al. is available as GenBank accession No. J05046, and the sequence is included herein as SEQ ID NO:l .
  • SEQ ID NO: 11 presents the human IRRR amino acid sequence that includes these 29 added amino acids.
  • SEQ ID NO: 11 includes a signal peptide (amino acids 1 to 26), an extracellular domain (amino acids 27 to 921), a transmembrane domain (amino acids 922 to 943), and an intracellular domain (amino acids 944 to 1297).
  • SEQ ID NO: 2 includes a truncated extracellular domain (amino acids 1 to 892), a transmembrane domain (amino acids 893 to 914), and an intracellular domain (amino acids 915 to 1268). These domains are encoded by nucleotides 1 to 2678, nucleotides 2679 to 2744, and nucleotides 2745 to 3806, respectively, of SEQ ID NO:l . Sequences for guinea pig and rat IRRR proteins are available as
  • IRRR-9 a new nucleotide sequence that encodes the full-length rat IRRR, is provided by SEQ ID NO: 6, and its corresponding amino acid sequence is included as SEQ ID NO:7. Certain structural features of IRRR-9 are summarized in Table 1.
  • IRRR proteins show a significant degree of homology with other members of the insulin receptor family. It is expected that functional IRRR, like the other family members, are heterotetrameric glycoproteins that include extracellular ⁇ - subunits, which contain the ligand binding domain. The extracellular subunits are expected to be disulfide-bonded to ⁇ -subunits that span the cell membrane and contain a cytoplasmic tyrosine kinase, which is activated upon ligand binding. These ⁇ - and ⁇ - subunits are derived by proteolytic cleavage of the proreceptor.
  • sIRR-1 consists of the N-terminal 410 amino acids of the rat IRRR, while “sIRR-2” has an additional 59 amino acid insertion in the C-terminal region. Both truncated forms lack the transmembrane and the cytoplasmic tyrosine kinase domain, indicting that the truncated forms are secreted. sIRR-1 and sIRR-2 are expressed in the stomach and kidneys.
  • IRRR-6 is another variant rat IRRR form.
  • the nucleotide and amino acid sequences are proved as SEQ ID NOs:9 and 10, respectively.
  • IRRR-6 has an insertion of 59 amino acids due to nonsplicing of intron 4
  • IRRR-6 has a different cytoplasmic domain that is short and not likely to signal in the same manner as the full-length receptor.
  • the location in the IRRR-6 protein where the short cytoplasmic domain form deviates from the long form occurs at the exon 16/exon 17 junction, suggesting that the short form is created by alternative splicing.
  • 7RRR mRNA is detected in kidney, stomach and thymus, but not in skeletal muscle, brain, intestine and uterus (Shier and Watt, Mol Endocrinol 6:723 (1992)). Expression of IRRR mRNA and protein are also highly restricted to the forebrain cholinergic neurons (Tsujimoto et al, Neuroscience Letters 188:105 (1995); Tsuji et al, Mol. Brain Res. 41:250 (1996)).
  • 7RRR transcripts are concentrated in the neural crest-derived sensory and sympathetic neurons (Reinhardt et al, Endocrinology 133:3 (1993)). In situ hybridization and immunohistochemistry show expression of 7RRR is localized in the subset of neurons where its appearance is closely associated with the nerve growth factor receptor, TRK A (Reinhardt et al, J. Neuroscience 14:4674 (1994)). These findings are consistent with a functional linkage of IRRR and TRK A in nerve growth factor-sensitive neurons, and that IRRR have neurotrophic and neuromodulatory potential. Recently, expression of IRRR was reported in the islets of Langerhans (Ozaki, Eur. J. Endocrinology 139:244 (1998)).
  • 7RRR mRNA has been detected in human kidney, heart, skeletal muscle, liver and pancreas (Zhang and Roth, J. Biol. Chem. 267: 18320 (1992)). From birth to maturity, 7RRR mRNA is abundant in renal epithelial cells localized in the distal tubules of both rat and human kidney (Reinhardt et al, Endocrinology 133:3 (1993)). 7RRR mRNA has also been detected in the basal third of the oxyntic glands of the fundic stomach.
  • 7RRR mRNA is found in the enterochromaffm-like cells, which produce and store histamine, an important physiological stimulant of acid secretion (Reinhardt et al, Endocrinology 133:3 (1993); Tsujimoto et al, Endocrinology 136:55% (1995)).
  • the 7RRR locus is about 1.5 Mb proximal to APOA2, a region implicated for a major recessive diabetes locus identified from a genome-wide search of susceptibility genes in multigenerational families of Northern European ancestry (Elbien et al, Diabetes 48:1175 (1999)).
  • nucleotide sequences that encode the 7RRR gene can be used in the diagnosis or prognosis of metabolic disease, such as diabetes. These methods are also suitable for diagnosis or prognosis of diabetes in Pima Indians.
  • the present invention provides methods for using 7RRR polynucleotides, anti-IRRR antibodies, and IRRR polypeptides to diagnose disorders associated with abnormal expression of the IRRR protein.
  • the present invention provides methods for identifying abnormalities in expression that are a factor in causing, or predisposing, a person to have a metabolic disease, such as obesity, dyslipidemia, and diabetes, especially Type II diabetes.
  • the present invention provides methods of using polynucleotides, or portions thereof, which encode for the IRRR polypeptide to diagnose mutations in the 7RRR gene.
  • the methods of the present invention provide a way to detect mutations in the 7RRR gene that are associated or linked with a disease, or susceptibility to a disease, which results from abnormally low or high expression or altered expression of the gene.
  • the present invention provides methods for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, comprising: (a) amplifying nucleic acid molecules that encode insulin receptor-related receptor (IRRR) from RNA isolated from a biological sample of the individual, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates metabolic disease or susceptibility to a metabolic disease.
  • IRRR insulin receptor-related receptor
  • methods of detecting a chromosome Iq21-q24 abnormality in a subject comprise: (a) amplifying nucleic acid molecules that encode insulin receptor-related receptor (IRRR) from RNA isolated from a biological sample of the subject, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates a chromosome Iq21-q24 abnormality.
  • the detecting step is performed by comparing the nucleotide sequence of the amplified nucleic acid molecules to the nucleotide sequence of SEQ ID NO: 1.
  • the detecting step can be performed by fractionating the amplified nucleic acid molecules and control nucleic acid molecules that encode the amino acid sequence of SEQ ID NO: 11, and comparing the lengths of the fractionated amplified and control nucleic acid molecules.
  • Exemplary methods for amplification include polymerase chain reaction or reverse transcriptase-polymerase chain reaction.
  • the present invention also includes methods for detecting a chromosome Iq21-q24 abnormality in a subject comprising: (a) amplifying nucleic acid molecules that encode insulin receptor-related receptor (IRRR) from RNA isolated from a biological sample of the subject, (b) transcribing the amplified nucleic acid molecules to express 7RRR mRNA, (c) translating 7RRR mRNA to produce IRRR polypeptides, and (d) detecting a mutation in the IRRR polypeptides, wherein the presence of a mutation indicates a chromosome Iq21-q24 abnormality.
  • IRRR insulin receptor-related receptor
  • the detection step can be performed by fractionating, under denaturing conditions, the IRRR polypeptides and control polypeptides that encode the amino acid sequence of SEQ ID NO:l 1, and comparing the sizes of the fractionated amplified and control polypeptides.
  • Similar methods can be used to diagnose a metabolic disease or susceptibility to a metabolic disease in an individual, in which the presence of a mutation in the IRRR polypeptides indicates metabolic disease or susceptibility to a metabolic disease.
  • the present invention also contemplates methods for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, comprising:
  • nucleic acid molecules that either (i) comprise a nucleotide sequence that encodes at least one of insulin receptor-related receptor (IRRR) exons 2 to 22, or that (ii) comprise a nucleotide sequence that is the complement of (i), and
  • a chromosome Iq21-q24 abnormality can be detected in a subject by (a) amplifying, from genomic DNA isolated from a biological sample of the subject, nucleic acid molecules that either (i) comprise a nucleotide sequence that encodes at least one of insulin receptor-related receptor (IRRR) exons 2 to 22, or that (ii) comprise a nucleotide sequence that is the complement of (i), and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates a chromosome Iq21-q24 abnormality.
  • the detecting step is performed by comparing the nucleotide sequence of the amplified nucleic acid molecules to the nucleotide sequence of SEQ ID NO: 1.
  • the present invention also contemplates methods for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, comprising: (a) amplifying, from genomic DNA isolated from a biological sample of the individual, a segment of the insulin receptor-related receptor (IRRR) gene that comprises either the nucleotide sequence of any one of introns 1 to 21, or the complementary nucleotide sequence of any one of introns 1 to 21, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates metabolic disease or susceptibility to a metabolic disease.
  • IRRR insulin receptor-related receptor
  • the present invention provides methods for detecting a chromosome Iq21-q24 abnormality in a subject comprising: (a) amplifying, from genomic DNA isolated from a biological sample of the subject, a segment of the insulin receptor-related receptor (IRRR) gene that comprises either the nucleotide sequence of any one of introns 1 to 21, or the complementary nucleotide sequence of any one of introns 1 to 21, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates a chromosome Iq21-q24 abnormality.
  • IRRR insulin receptor-related receptor
  • the detecting step is performed by binding the amplified 7RRR gene segments to a membrane, and contacting the membrane with a nucleic acid probe under hybridizing conditions of high stringency, wherein the absence of hybrids indicates metabolic disease or susceptibility to a metabolic disease, or a mutation in chromosome Iq21-q24.
  • the 7RRR gene segment can comprise the complementary nucleotide sequence of any one of introns 1 to 21
  • the nucleic acid probe can comprise the nucleotide sequence of any one of SEQ ID NOs: 12 to 53.
  • the present invention further provides methods for diagnosing a metabolic disease or susceptibility to a metabolic disease in an individual, wherein the disease is related to the expression or activity of an insulin receptor-related receptor (IRRR) polypeptide having the amino acid sequence of SEQ ID NO: 11 in that individual, comprising the step of determining the presence of an alteration in the nucleotide sequence encoding IRRR polypeptide in the genome of the individual, wherein the presence of an alteration in the 7RRR gene indicates metabolic disease or susceptibility to a metabolic disease.
  • IRRR insulin receptor-related receptor
  • Examples of mutations or alterations of the 7RRR gene or its gene products include point mutations, deletions, insertions, and rearrangements. Another example of an 7RRR gene mutation is aneuploidy.
  • Illustrative metabolic diseases include obesity and diabetes, such as Type II diabetes.
  • the diagnostic methods described herein can be used to detect the presence of Type II diabetes or susceptibility to Type II diabetes in a Pima Indian.
  • the present invention also provides isolated polypeptides comprising an extracellular domain, wherein the extracellular domain comprises amino acid residues 27 to 921 of SEQ ID NO:7.
  • Such polypeptides may further comprise a transmembrane domain that resides in a carboxyl-terminal position relative to the extracellular domain, wherein the transmembrane domain comprises amino acid residues 922 to 943 of SEQ ID NO:7.
  • polypeptides may also comprise an intracellular domain that resides in a carboxyl-terminal position relative to the transmembrane domain, wherein the intracellular domain comprises amino acid residues 944 to 1300 of SEQ ID NO:7, and optionally, a signal secretory sequence that resides in an amino-terminal position relative to the extracellular domain, wherein the signal secretory sequence comprises either amino acid residues 1 to 26 of the amino acid sequence of SEQ ID NO:7.
  • the present invention also contemplates isolated polypeptides having an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to the amino acid sequence of SEQ ID NO: 7, wherein such isolated polypeptides can specifically bind with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:7.
  • An illustrative polypeptide is a polypeptide that comprises amino acid residues 27 to 1300 of the amino acid sequence of SEQ ID NO:7.
  • the present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides. Exemplary antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • Illustrative antibody fragments include F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and minimal recognition units.
  • the present invention further contemplates anti-idiotype antibodies that bind with such antibodies or antibody fragments.
  • the present invention also provides isolated nucleic acid molecules that encode an IRRR polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:6, (b) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 7, and (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule comprising the nucleotide sequence of nucleotides 125-3946 of SEQ ID NO:6, or the complement of nucleotides 125-3946 of SEQ ID NO:6.
  • Illustrative nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO: 7 is due to a conservative amino acid substitution.
  • the present invention further contemplates isolated nucleic acid molecules that comprise a nucleotide sequence of nucleotides 125-3946 of SEQ ID NO:6.
  • an isolated nucleic acid molecule can comprise the nucleotide sequence of nucleotides 47-3946 of SEQ ID NO:6.
  • the present invention also includes vectors and expression vectors comprising such nucleic acid molecules.
  • Such expression vectors may comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
  • the present invention further includes recombinant host cells comprising these vectors and expression vectors.
  • Illustrative host cells include bacterial, yeast, fungal, avian, insect, mammalian, and plant cells.
  • Recombinant host cells comprising such expression vectors can be used to prepare IRRR polypeptides by culturing such recombinant host cells that comprise the expression vector and that produce the IRRR protein, and, optionally, isolating the IRRR protein from the cultured recombinant host cells.
  • the present invention also contemplates methods for detecting the presence of IRRR RNA in a biological sample, comprising the steps of (a) contacting an IRRR nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of nucleotides 125-3946 of SEQ ID NO:6, or its complement, and (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of 7RRR RNA in the biological sample.
  • the biological sample can be a human biological sample.
  • the present invention further provides methods for detecting the presence of IRRR polypeptide in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or an antibody fragment that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:7, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
  • an antibody or antibody fragment may further comprise a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold.
  • An exemplary biological sample is a human biological sample.
  • kits for performing these detection methods may comprise a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 125-3946 of SEQ ID NO:6, (b) a nucleic acid molecule comprising the complement of nucleotides 125-3946 of SEQ ID NO:6, (c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, and (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides.
  • kit may also comprise a second container that comprises one or more reagents capable of indicating the presence of the nucleic acid molecule.
  • a kit for detection of IRRR protein may comprise a container that comprises an antibody, or an antibody fragment, that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:7.
  • the present invention also contemplates isolated nucleic acid molecules comprising a nucleotide sequence that encodes an IRRR secretion signal sequence and a nucleotide sequence that encodes a biologically active polypeptide, wherein the IRRR secretion signal sequence comprises an amino acid sequence of residues 1 to 26 of SEQ ID NO:7.
  • exemplary biologically active polypeptide include Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukin, colony stimulating factor, interferon, erythropoietin, and thrombopoietin.
  • the present invention also provides fusion proteins comprising an IRRR secretion signal sequence and a polypeptide, wherein the IRRR secretion signal sequence comprises an amino acid sequence of residues 1 to 26 of SEQ ID NO:7.
  • the present invention further includes isolated nucleic acid molecules that encode an extracellular IRRR domain, wherein the extracellular domain comprises the amino acid sequence of amino acid residues 27-921 of SEQ ID NO:7.
  • the present invention also contemplates isolated polypeptides encodes by such nucleic acid molecules, antibodies that specifically bind with such isolated polypeptides, and anti- idiotype antibodies that specifically bind with such antibodies.
  • the present invention also contemplates methods for detecting a ligand of the IRRR within a test sample, comprising the steps of (a) contacting the test sample with a polypeptide that comprises amino acids 27 to 921 of SEQ ID NO:7, and (b) detecting the binding of the polypeptide to ligand in the sample.
  • the polypeptide further comprises a transmembrane domain and an intracellular domain.
  • a polypeptide can be membrane bound within a cultured cell, and the detecting step would comprise measuring a biological response in the cultured cell.
  • polypeptide comprising an extracellular domain further comprises an immunoglobulin domain, and the immunoglobulin domain of the polypeptide is immobilized on a solid support.
  • the present invention further includes isolated polypeptides, comprising the amino acid sequence of SEQ ID NO: 10, as well as isolated nucleic acid molecules, comprising nucleotides 47 to 3253 of SEQ ID NO:9.
  • the present invention further provides fusion proteins an IRRR polypeptide and an immunoglobulin moiety.
  • the IRRR polypeptide can be an IRRR extracellular domain, such as amino acid residues 1 to 892 of SEQ ID NO:2, 27 to 921 of SEQ ID NO:7, or 27 to 921 of SEQ ID NO:l l .
  • the immunoglobulin moiety may be an immunoglobulin heavy chain constant region, such as a human F c fragment.
  • the present invention further includes isolated nucleic acid molecules that encode such fusion proteins.
  • the present methods can also be used to detect a chromosome Iq21-q23 abnormality in a subject.
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • nucleic acid molecule also includes so- called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • complement of a nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid molecule that encodes a polypeptide.
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • structural gene refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • nucleic acid molecule construct is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear DNA denotes non-circular DNA molecules having free 5' and
  • Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • Codon DNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA refers to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • cDNA also refers to a clone of a cDNA molecule synthesized from an RNA template.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al, Mol. Endocrinol 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol.
  • DSEs differentiation-specific elements
  • CREs cyclic AMP response elements
  • SREs serum response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 2(57:19938 (1992)), AP2 (Ye et al, J. Biol. Chem. 269:2572% (1994)), SPl, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al, eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J.
  • a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • Repressible promoters are also known.
  • a “core promoter” contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • a “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific,” “tissue-specific,” or “organelle-specific” manner.
  • Heterologous DNA refers to a DNA molecule, or a population of
  • DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
  • a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
  • a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
  • a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.”
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
  • integrated genetic element is a segment of DNA that has been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation.
  • integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, which has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • an “expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to” the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell that produces IRRR from an expression vector.
  • IRRR can be produced by a cell that is a "natural source" of IRRR, and that lacks an expression vector.
  • “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
  • “episomal transformants” are stably transformed recombinant host cells that contain an expression vector that persists extrachromosomally (see, for example, Tsui et al, Cell 30:499 (1982); Tsui and Breitman, Somat. Cell. Mol. Genet. 11: 67 (1985)).
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a fusion protein can comprise at least part of an IRRR polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of IRRR using affinity chromatography.
  • Receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • secretory signal sequence denotes a DNA sequence that encodes a peptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • secretory peptide a DNA sequence that encodes a peptide that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • isolated polypeptide is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99%o pure.
  • SDS sodium dodecyl sulfate
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody /antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity less than 10 9 M '1 .
  • an "anti-idiotype antibody” is an antibody that binds with the variable region domain of an immunoglobulin.
  • an anti-idiotype antibody binds with the variable region of an anti-IRRR antibody, and thus, an anti-idiotype antibody mimics an epitope of IRRR.
  • an “antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-IRRR monoclonal antibody fragment binds with an epitope of IRRR.
  • antibody fragment also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • scFv proteins peptide linker
  • a “chimeric antibody” is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a “detectable label” is a molecule or atom, which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al, EMBO J. 4: 1075 (1985); Nilsson et al, Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al, Proc.
  • naked antibody is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies. such as chimeric and humanized antibodies.
  • antibody component includes both an entire antibody and an antibody fragment.
  • an “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
  • antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein") and toxins ("antibody-toxin fusion protein”).
  • a “target polypeptide” or a “target peptide” is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen.
  • T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
  • antigenic peptide is a peptide, which will bind a major histocompatibility complex molecule to form an MHC-peptide complex which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell.
  • antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell, which binds or expresses the antigen.
  • the antigenic peptide can be bound in the context of a class I or class II major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
  • RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA.
  • a nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an "anti- sense RNA” and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene.”
  • Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • an “anti-sense oligonucleotide specific for IRRR” or an “IRRR anti- sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the 7RRR gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the 7RRR gene.
  • a "ribozyme” is a nucleic acid molecule that contains a catalytic center.
  • the term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene.”
  • an “external guide sequence” is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene.”
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • Nucleic acid molecules encoding a human 7RRR gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ID NO:l, or sequences disclosed in references described above. These techniques are standard and well-established. See, for example, White (ed.), Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 r " Edition (John Wiley & Sons 1995) ["Ausubel (1995)”], and Wu et al, Methods in Gene Biotechnology (CRC Press, Inc. 1997) ["Wu (1997)”]. Similar methods can be used to obtain nucleotide sequences that encode the rat IRRR protein, which is exemplified by the amino acid sequence of SEQ ID NO:7.
  • an 7RRR gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).
  • Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al, Plant Molec. Biol. 27:1131 (1993), Bambot et al, PCR Methods and Applications 2:266 (1993), Dillon et al, "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al, PCR Methods Appl 4:299 (1995)).
  • the nucleic acid molecules of the present invention can also be synthesized with "gene machines” using protocols such as the phosphoramidite method. If chemically-synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 base pairs is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 base pairs), however, special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • SEQ ID NO:l represents a single allele of human 7RRR, and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO:l, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are 7RRR allelic variants. cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the IRRR polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • a human IRRR splice variant may include a shortened cytoplasmic domain, in which the deviation occurs at the exon 16/exon 17 junction.
  • IRRR polypeptides can be produced in recombinant host cells following conventional techniques.
  • a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene, which is suitable for selection of cells that carry the expression vector.
  • Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • an 7RRR expression vector may comprise an 7RRR gene and a secretory sequence derived from any secreted gene.
  • IRRR proteins of the present invention can be expressed in prokaryotic or eukaryotic cells.
  • Illustrative prokaryotic cells include E. coli and Bacillus subtilus.
  • Illustrative eukaryotic cells include mammalian cells, fungal cells, avian cells, yeast cells, insect cells, and plant cells. Methods for expressing proteins in recombinant host cells are well-known to those of skill in the art (see, for example, Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991), Ausubel (1995), Williams et al, "Expression of foreign proteins in E.
  • IRRR polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well-known to those of skill in the art. For example, standard solid phase peptide synthesis is described by Merrifield, J. Am. Chem. Soc. 55:2149 (1963). Variations in total chemical synthesis strategies, such as "native chemical ligation” and “expressed protein ligation” are also standard (see, for example, Dawson et al, Science 266:776 (1994), Ralpheng et al, Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson, Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol Chem. 273:16205 (1998)).
  • Peptides and polypeptides of the present invention can comprise at least six, at least nine, or at least 15 contiguous amino acid residues of SEQ ID NO:7 or 10.
  • the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:7 or 10.
  • polypeptides can comprise 15, 20, 30, 40, or 50 contiguous amino acids of amino acid residues 27 to 921 of SEQ ID NO:7.
  • Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • SEQ ID NO:8 is a degenerate nucleotide sequence that encompasses all nucleic acid molecules that encode the rat IRRR-9 polypeptide of SEQ ID NO:7.
  • the degenerate sequence of SEQ ID NO: 8 also provides all RNA sequences encoding SEQ ID NO:7, by substituting U for T.
  • the present invention contemplates rat IRRR polypeptide-encoding nucleic acid molecules comprising nucleotide 47 to nucleotide 3946 of SEQ ID NO:6, and their RNA equivalents.
  • Table 2 sets forth the one-letter codes used within SEQ ID NO: 8 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO:8, encompassing all possible codons for a given amino acid, are set forth in Table 3.
  • ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding an amino acid.
  • WSN degenerate codon for serine
  • MGN degenerate codon for arginine
  • AGY serine
  • a similar relationship exists between codons encoding phenylalanine and leucine.
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:7.
  • Fusion proteins of IRRR can be used to express 7RRR in a recombinant host, and to isolate the produced IRRR. As described below, particular IRRR fusion proteins also have uses in diagnosis and therapy.
  • fusion protein comprises a peptide that guides an IRRR polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be derived from IRRR, a suitable signal sequence may also be derived from another secreted protein or synthesized de novo.
  • the secretory signal sequence is operably linked to an IRRR- encoding sequence such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide sequence of interest (see, e.g., Welch et ⁇ l, U.S. Patent No. 5,037,743; Holland et ⁇ l, U.S. Patent No. 5,143,830).
  • yeast signal sequence is preferred for expression in yeast cells.
  • suitable yeast signal sequences are those derived from yeast mating phermone ⁇ -factor (encoded by the MF ⁇ l gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene).
  • IRRR can be produced as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • an IRRR fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose.
  • Peptide tags that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al, Arch. Biochem. Biophys. 329:215 (1996), Morganti et al, Biotechnol. Appl Biochem. 23:67 (1996), and Zheng et al, Gene 186:55 (1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO).
  • fusion protein comprises an IRRR polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two or three constant region domains and a hinge region but lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • F c fragment an immunoglobulin heavy chain constant region
  • Chang et al, U.S. Patent No. 5,723,125 describe a fusion protein comprising a human interferon and a human immunoglobulin Fc fragment.
  • the C-terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety.
  • An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert.
  • An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO:3).
  • a preferred Fc moiety is a human ⁇ 4 chain, which is stable in solution and has little or no complement activating activity.
  • the present invention contemplates an IRRR fusion protein that comprises an IRRR moiety and a human Fc fragment, wherein the C-terminus of the IRRR moiety is attached to the N-terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO:3.
  • the IRRR moiety can be an IRRR molecule or a fragment thereof, such as an extracellular IRRR domain.
  • an IRRR fusion protein comprises an IgG sequence, an IRRR moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the IRRR moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH 2 domain, and a CH 3 domain. Accordingly, the IgG sequence lacks a CH, domain.
  • the IRRR moiety displays an IRRR activity, as described herein, such as the ability to bind with an IRRR ligand.
  • Fusion proteins comprising an IRRR moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of an IRRR ligand in a biological sample can be detected using an IRRR-immunoglobulin fusion protein, in which the IRRR moiety is used to target the cognate ligand, and a macromolecule, such as Protein A or anti-Fc antibody, is used to detect the bound fusion protein-receptor complex.
  • fusion proteins can be used to identify agonists and antagonists that interfere with the binding of an IRRR ligand to its receptor.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16- 19 to 16-25. IRRR receptor polypeptides can be used to identify and to isolate IRRR ligands.
  • Fragments of rat IRRR such as an IRRR extracellular domain (e.g., amino acids 27 to 921 of SEQ ID NO:7) and other forms of a soluble IRRR receptor, are particularly useful for these methods.
  • proteins and peptides of the present invention can be immobilized on a column and used to bind ligands from a biological sample that is run over the column (Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)).
  • an IRRR polypeptide can be observed by a silicon- based biosensor microphysiometer, which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CYTOSENSOR Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method (see, for example, McConnell et al, Science 257:1906 (1992), Pitchford et al, Meth. Enzymol. 228:84 (1997), Arimilli et al, J. Immunol Meth.
  • the microphysiometer can be used for assaying eukaryotic, prokaryotic, adherent, or non-adherent cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including agonists, ligands, or antagonists of IRRR.
  • the microphysiometer can be used to measure responses of an IRRR- expressing eukaryotic cell, compared to a control eukaryotic cell that does not express IRRR polypeptide.
  • Suitable cells responsive to IRRR-modulating stimuli include recombinant host cells comprising an IRRR expression vector, and cells that naturally express IRRR, as discussed above. Extracellular acidification provides one measure for an IRRR-modulated cellular response.
  • this approach can be used to identify ligands, agonists, and antagonists of IRRR.
  • a compound can be identified as an agonist of IRRR by providing cells that express an IRRR polypeptide, culturing a first portion of the cells in the absence of the test compound, culturing a second portion of the cells in the presence of the test compound, and determining whether the second portion exhibits a cellular response, in comparison with the first portion.
  • a solid phase system can be used to identify a ligand, agonist, or antagonist of IRRR.
  • an IRRR polypeptide or IRRR fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden). The use of this instrument is disclosed, for example, by Karlsson,_7mmw «o/. Methods 145:229 (1991), and Cunningham and Wells, J. Mol. Biol. 234:554 (1993).
  • an IRRR polypeptide or fusion protein is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell.
  • a test sample is then passed through the cell. If a ligand is present in the sample, it will bind to the immobilized polypeptide or fusion protein, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. This system can also be used to examine antibody-antigen interactions, and the interactions of other complement/anti-complement pairs.
  • IRRR binding domains can be further characterized by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids of IRRR ligand agonists. See, for example, de Vos et al, Science 255:306 (1992), Smith et al, J. Mol. Biol. 224:899 (1992), and Wlodaver et al, FEBS Lett. 309:59 (1992).
  • IRRR receptor extracellular domains can also be administered to a subject as a pharmaceutical composition. These soluble IRRR receptors are used to treat conditions characterized by an excess of IRRR ligand.
  • Antibodies to IRRR can be obtained, for example, using the product of an 7RRR expression vector or IRRR isolated from a natural source as an antigen. Particularly useful anti-IRRR antibodies "bind specifically" with IRRR. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to IRRR with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to IRRR.
  • antibodies specifically bind if they bind to an IRRR polypeptide, peptide or epitope with a binding affinity (K ) of 10 6 M “ ' or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “ ' or greater, and most preferably 10 9 M “1 or greater.
  • K binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 57:660 (1949)).
  • antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect IRRR, but not presently known polypeptides using a standard Western blot analysis.
  • Anti-IRRR antibodies can be produced using antigenic IRRR epitope- bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides can contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids.
  • Such epitope-bearing peptides and polypeptides can be produced by fragmenting an IRRR polypeptide, or by chemical peptide synthesis, as described herein.
  • epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of an IRRR polypeptide, also are useful for inducing antibodies that bind with IRRR.
  • suitable antibodies include antibodies that bind with the extracellular domain, represented by amino acids 27 to 921 of SEQ ID NO:7.
  • Preferred anti-extracellular domain antibodies bind with IRRR in the region of amino acids 411 to 921.
  • Suitable anti-rat IRRR antibodies bind with the transmembrane domain (e.g., amino acids 922 to 943 of SEQ ID NO:7) or with the intracellular domain (e.g., amino acids 944 to 1300 of SEQ ID NO:7).
  • Polyclonal antibodies to recombinant IRRR protein or to IRRR isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al, "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al, "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995).
  • the immunogenicity of an IRRR polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of IRRR or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like,” such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • an anti- IRRR antibody of the present invention may also be derived from a subhuman primate antibody.
  • General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465, and in Losman et al, Int. J. Cancer 46:3 0 (1990).
  • monoclonal anti-IRRR antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al, Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1- 2.6.7 (John Wiley & Sons 1991) ["Coligan”], Picksley et al, "Production of monoclonal antibodies against proteins expressed in E coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an 7RRR gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B- lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • an anti-IRRR antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al, Nature Genet.
  • Human antibodies can also be obtained using phage display methods (see, for example, Griffiths et al, U.S. patent No.5, 885,793). Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques.
  • isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).
  • antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al, Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 75: 119 (1959), Edelman et al, in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
  • Fv fragments comprise an association of V H and V L chains. This association can be noncovalent, as described by Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 72:437 (1992)).
  • the Fv fragments may comprise V H and V L chains, which are connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • scFvs Methods for producing scFvs are described, for example, by Whitlow et al, Methods: A Companion to Methods in Enzymology 2:97 (1991) (also see, Bird et al, Science 242:423 (1988), Ladner et al, U.S. Patent No. 4,946,778, Pack et al, Bio/Technology 77:1271 (1993), and Sandhu, supra).
  • a scFV can be obtained by exposing lymphocytes to lymphocytes.
  • IRRR polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled IRRR protein or peptide).
  • Genes encoding polypeptides having potential IRRR polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • Random peptide display libraries can be screened using the IRRR sequences disclosed herein to identify proteins, which bind to IRRR.
  • Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR).
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest.
  • Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al, Methods: A Companion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al (eds.), page 166 (Cambridge University Press 1995), and Ward et al, "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al, (eds.), page 137 (Wiley-Liss, Inc.
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-IRRR antibodies or antibody fragments, using standard techniques. See, for example, Green et al, "Production of Polyclonal Antisera,” in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7.
  • monoclonal anti- idiotype antibodies can be prepared using anti-IRRR antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.
  • IRRR-encoding domain can be obtained from various species, such as human, rat and guinea pig, the human gene is a preferred source for diagnostic methods. Accordingly, the nucleic acids, which comprise the sequence of IRRR, illustrated by the nucleotide sequence of SEQ ID NO: 1, or a portion thereof, or their complements, can be used as the basis of preparing a nucleic acid probe.
  • the nucleic acids tested for the presence of, or abnormality in, the nucleotide sequence can be obtained from a variety of cells, tissues or bodily fluids, such as urine, saliva or blood. The cell or tissue sample is processed in a manner that will result in rendering nucleic acid acceptable for nucleic acid detection techniques.
  • Suitable probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • portion refers to at least eight nucleotides to at least 20 or more nucleotides.
  • Suitable probes bind with regions of the 7RRR gene that have a low sequence similarity to comparable regions in other proteins of the insulin receptor family.
  • RNA isolated from a biological sample
  • RNA isolated from a biological sample
  • RNA detection includes northern analysis and dot/slot blot hybridization (see, for example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997)).
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S.
  • 7RRR RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Humana Press, Inc. 1993)).
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • nucleic acid molecules comprising at least a portion of the nucleotide sequence of SEQ ID NO:l, or a sequence complementary thereto, are hybridized under "stringent conditions" to a test nucleic acid sample.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a test nucleic acid sample can be hybridized with a nucleic acid molecule comprising a portion of the nucleotide sequence of SEQ ID NO:l (or its complement) at 42°C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution lOOx Denhardt's solution:
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • a higher temperature such as about 65°C
  • premixed hybridization solutions are available (e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.
  • a test nucleic acid sample can be hybridized with a nucleic acid molecule comprising a portion of the nucleotide sequence of SEQ ID NO:l (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • stringent washing conditions in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • SSPE for SSC in the wash solution.
  • Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • a test nucleic acid sample can be hybridized with a nucleic acid molecule comprising a portion of the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2xSSC with 0.1% SDS at 65°C.
  • PCR polymerase chain reaction
  • Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
  • PCR primers can be designed to amplify a portion of the IRRR gene that has a low sequence similarity to a comparable region in other proteins of the insulin receptor family.
  • RT-PCR reverse transcriptase- PCR
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with IRRR primers (see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from biological sample using, for example, the guanidinium-thiocyanate cell lysis procedure described above.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or 7RRR anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • 7RRR sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.
  • PCR amplification products can be detected using a variety of approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled 7RRR probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • IRRR sequences can utilize approaches such as nucleic acid sequence-based amplification, cooperative amplification of templates by cross-hybridization, and the ligase chain reaction (see, for example, Marshall et al, U.S. Patent No. 5,686,272 (1997), Dyer et al, J. Virol. Methods 60: 16 (1996), Ehricht et al, Eur. J. Biochem. 243:358 (1997), and Chadwick et al, J. Virol. Methods 70:59 (1998)).
  • Other standard methods are known to those of skill in the art.
  • 7RRR probes and primers can also be used to detect and to localize
  • truncated IRRR refers to an IRRR protein that lacks a portion of the IRRR protein that is normally present. The truncation may occur throughout the protein and ranges from about a few amino acids to several hundred amino acids. According to the protein truncation test, RNA is isolated from a biological sample, and used to synthesize cDNA.
  • PCR is then used to amplify the 7RRR target sequence and to introduce an RNA polymerase promoter, a translation initiation sequence, and an in-frame ATG triplet.
  • PCR products are transcribed using an RNA polymerase, and the transcripts are translated in vitro with a T7-coupled reticulocyte lysate system.
  • the translation products are then fractionated by SDS- polyacrylamide gel electrophoresis to determine the lengths of the translation products.
  • the protein truncation test is described, for example, by Dracopoli et al. (eds.), Current Protocols in Human Genetics, pages 9.11.1 - 9.11.18 (John Wiley & Sons 1998).
  • IRRR protein is isolated from a subject, the molecular weight of the isolated IRRR protein is determined, and then compared with the molecular weight a normal IRRR protein, such as a protein having the amino acid sequence of SEQ ID NOs:2 or 11.
  • a substantially lower molecular weight for the isolated IRRR protein is indicative that the protein is truncated.
  • substantially lower molecular weight refers to at least about 10 percent lower, and preferably, at least about 25 percent lower.
  • the IRRR protein may be isolated by various procedures known in the art including immunoprecipitation, solid phase radioimmunoassay, enzyme-linked immunosorbent assay, or Western blotting.
  • the molecular weight of the isolated IRRR protein can be determined using standard techniques, such as SDS- polyacrylamide gel electrophoresis.
  • a truncation can reflect a mutation at any of the exon intron junctions.
  • truncations can occur in the extracellular domain (e.g., amino acids 27 to
  • Nucleic acid molecules comprising 7RRR nucleotide sequences can also be used to examine a subject's genomic DNA for a mutation in the 7RRR gene.
  • Detectable chromosomal aberrations at the 7RRR gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Of particular interest are genetic alterations that inactivate the 7RRR gene, or that result in changes to transcription rate, transcription processing, or mRNA stability.
  • Aberrations associated with the 7RRR locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted
  • Mutations can also be detected by hybridizing an oligonucleotide probe comprising a normal 7RRR sequence to a Southern blot or to membrane-bound PCR products. Discrimination is achieved by hybridizing under conditions of high stringency, or by washing under varying conditions of stringency. This analysis can be targeted to a particular coding sequence. Alternatively, this approach is used to examine splice-donor or splice-acceptor sites in the immediate flanking intron sequences, where disease-causing mutations are often located. Suitable oligonucleotides can be designed using the sequences of the coding strand, provided in Table 5 (Watt et al, Adv. Exp. Biol. Med. 343:125 (1993)). Longer oligonucleotides can be designed by extending the sequence into an exon of choice, using the information of Table 4 and SEQ ID NO:l
  • duplication of all or part of a gene can cause a disorder when the insertion of the duplicated material is inserted into the reading frame of a gene and causes premature termination of translation.
  • the effect of such duplication can be detected with the protein truncation assay described above.
  • Duplication and insertion can be examined directly by analyzing a subject's genomic DNA with standard methods, such as Southern hybridization, fluorescence in situ hybridization, pulsed- field gel analysis, or PCR.
  • a point mutation can lead to a nonconservative change resulting in the alteration of IRRR function or a change of an amino acid codon to a stop codon. If a point mutation occurs within an intron, the mutation may affect the fidelity of splicing.
  • a point mutation can be detected using standard techniques, such as Southern hybridization analysis, PCR analysis, sequencing, ligation chain reaction, and other approaches. In single-strand conformation polymorphism analysis, for example, fragments amplified by PCR are separated into single strands and fractionated by polyacrylamide gel electrophoresis under denaturing conditions. The rate of migration through the gel is a function of conformation, which depends upon the base sequence. A mutation can alter the rate of migration of one or both single strands.
  • test and control DNA e.g., DNA that encodes the amino acid sequence of SEQ ID NO: 11.
  • Sites of base pair mismatch due to a mutation will be mispaired, and the strands will be susceptible to chemical cleavage at these sites.
  • a mutation can be detected using ribonuclease A, which will cleave the RNA strand of an RNA-DNA hybrid at the site of a sequence mismatch.
  • ribonuclease A a PCR-amplified sequence of an 7RRR gene or cDNA of a subject is hybridized with in vitro transcribed labeled RNA probes prepared from the DNA of a normal, healthy individual chosen from the general population.
  • the RNA-DNA hybrids are digested with ribonuclease A and analyzed using denaturing gel electrophoresis.
  • Sequence mismatches between the two strands will cause cleavage of the protected fragment, and small additional fragments will be detected in the samples derived from a subject who has a mutated 7RRR gene.
  • the site of mutation can be deduced from the sizes of the cleavage products.
  • the methods of the present invention provide a method of detecting a chromosome Iq21-q24 abnormality in a sample from an individual comprising: (a) obtaining 7RRR RNA from the sample, (b) generating 7RRR cDNA by polymerase chain reaction, and (c) comparing the nucleotide sequence of the 7RRR cDNA to the nucleic acid sequence as shown in SEQ ID NO:l.
  • the difference between the sequence of the 7RRR cDNA or 7RRR gene in the sample and the 7RRR sequence as shown in SEQ ID NO:l is indicative of chromosome Iq21-q24 abnormality.
  • the present invention provides methods for detecting in a sample from an individual, a chromosome Iq21-q24 abnormality associated with a disease, comprising the steps of: (a) contacting nucleic acid molecules of the sample with a nucleic acid probe that hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, its complements or fragments, under stringent conditions, and (b) detecting the presence or absence of hybridization of the probe with nucleic acid molecules in the sample, wherein the absence of hybridization is indicative of a chromosome Iq21-q24 abnormality, such as an abnormality that causes a defective glucose metabolism.
  • the present invention also provides methods of detecting in a sample from an individual, an 7RRR gene abnormality associated with a disease, comprising: (a) isolating nucleic acid molecules that encode IRRR from the sample, and (b) comparing the nucleotide sequence of the isolated IRRR-encoding sequence with the nucleotide sequence of SEQ ID NO:l, wherein the difference between the sequence of the isolated IRRR-encoding sequence or a polynucleotide encoding the IRRR polypeptide generated from the isolated IRRR-encoding sequence and the nucleotide sequence of SEQ ID NO:l is indicative of an 7RRR gene abnormality associated with disease or susceptibility to a disease in an individual, such as a defective glucose metabolism or diabetes.
  • the present invention also provides methods of detecting in a sample from a individual, an abnormality in expression of the 7RRR gene associated with disease or susceptibility to disease, comprising: (a) obtaining 7RRR RNA from the sample, (b) generating 7RRR cDNA by polymerase chain reaction from the 7RRR RNA, and (c) comparing the nucleotide sequence of the 7RRR cDNA to the nucleotide sequence of SEQ ID NO:l, wherein a difference between the sequence of the 7RRR cDNA and the nucleotide sequence of SEQ ID NO:l is indicative of an abnormality in expression of the 7RRR gene associated with disease or susceptibility to disease.
  • the disease is defective glucose metabolism or diabetes.
  • the present invention provides methods for detecting in a sample from an individual, an 7RRR gene abnormality associated with a disease, comprising: (a) contacting sample nucleic acid molecules with a nucleic acid probe, wherein the probe hybridizes to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, its complements or fragments, under stringent conditions, and (b) detecting the presence or absence of hybridization is indicative of an 7RRR abnormality. The absence of hybridization of the probe is associated with defective glucose metabolism.
  • an 7RRR probe is labeled with a detectable marker by any method known in the art.
  • the probe can be directly labeled by random priming, end labeling, PCR, or nick translation.
  • Suitable direct labels include radioactive labels such as 32 P, 3 H, and 35 S and non-radioactive labels such as fluorescent markers (e.g., fluorescein, Texas Red, AMCA blue (7- amino-4-methyl-coumanine-3-acetate), lucifer yellow, rhodamine, etc.), cyanin dyes, which are detectable with visible light, enzymes, and the like.
  • Probes labeled with an enzyme can be detected through a colorimetric reaction by providing a substrate for the enzyme. In the presence of various substrates, different colors are produced by the reaction, and these colors can be visualized to separately detect multiple probes if desired.
  • Suitable substrates for alkaline phosphatase include 5-bromo-4-chloro-3- indolylphosphate and nitro blue tetrazolium.
  • One suitable substrate for horseradish peroxidase is diaminobenzoate.
  • a method of performing in situ hybridization with an 7RRR probe to detect a chromosome structural abnormality in a cell from a fixed tissue sample obtained from a patient suspected of having a metabolic disease can comprise the steps of: (1) obtaining a fixed tissue sample from the patient, (2) pretreating the fixed tissue sample obtained in step (1) with a bisulfite ion composition, (3) digesting the fixed tissue sample with proteinase, (4) performing in situ hybridization on cells obtained from the digested fixed tissue sample of step (3) with a probe which specifically hybridizes to the 7RRR gene, wherein a signal pattern of hybridized probes is obtained, (5) comparing the signal pattern of the hybridized probe in step (4) to a predetermined signal pattern of the hybridized probe obtained when performing in situ hybridization on cells having a normal critical chromosome region of interest, and (6) detecting a chromosome structural abnormality in the patient's cells, by detecting a difference between the signal pattern obtained in step (4) and the predetermined signal pattern.
  • kits for performing a diagnostic assay for 7RRR gene expression or to detect mutations in the 7RRR gene comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO: 1 , or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • Kits can comprise nucleic acid primers for performing PCR or oligonucleotides for performing the ligase chain reaction.
  • kits can contain all the necessary elements to perform a nucleic acid diagnostic assay described above.
  • a kit will comprise at least one container comprising an 7RRR probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of 7RRR sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like.
  • a kit may also comprise a means for conveying to the user that the 7RRR probes and primers are used to detect 7RRR gene expression, or to diagnose the presence or incipience of a disease of glucose metabolism, such as type II diabetes.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes IRRR, or a nucleic acid molecule having a nucleotide sequence that is complementary to an IRRR-encoding nucleotide sequence.
  • the written instructions may state that the enclosed nucleic acid molecules can be used for the diagnosis or prognosis of a disease of glucose metabolism, such as type II diabetes.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the present invention contemplates the use of anti-IRRR antibodies to screen biological samples in vitro for the presence of IRRR.
  • anti-IRRR antibodies are used in liquid phase.
  • the presence of IRRR in a biological sample can be tested by mixing the biological sample with a trace amount of labeled IRRR and an anti-IRRR antibody under conditions that promote binding between IRRR and its antibody.
  • Complexes of IRRR and anti-IRRR in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or Staphylococcus protein A.
  • the concentration of IRRR in the biological sample will be inversely proportional to the amount of labeled IRRR bound to the antibody and directly related to the amount of free labeled IRRR.
  • rat or human anti-IRRR antibodies can be used to detect IRRR, human anti-IRRR antibodies are preferred for human diagnostic assays.
  • In vitro assays can also be performed in which anti-IRRR antibody is bound to a solid-phase carrier.
  • antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube.
  • polymer such as aminodextran
  • anti-IRRR antibodies can be used to detect IRRR in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of IRRR and to determine the distribution of IRRR in the examined tissue.
  • General immunochemistry techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application,” in Mammalian Development: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press, Inc. 1992)).
  • Immunochemical detection can be performed by contacting a biological sample with an anti-IRRR antibody, and then contacting the biological sample with a detectably labeled molecule, which binds to the antibody.
  • the detectably labeled molecule can comprise an antibody moiety that binds to anti-IRRR antibody.
  • the anti-IRRR antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin). Numerous variations of this basic technique are well-known to those of skill in the art.
  • an anti-IRRR antibody can be conjugated with a detectable label to form an anti-IRRR immunoconjugate.
  • Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography.
  • Isotopes that are particularly useful for the purpose of the present invention are ⁇ , 125 1, 131 1, 35 S, 14 C, and the like.
  • Anti-IRRR immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
  • Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-IRRR immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • a bioluminescent compound can be used to label anti-IRRR immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-IRRR immunoconjugates can be detectably labeled by linking an anti-IRRR antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galac- tosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • the convenience and versatility of immunochemical detection can be enhanced by using anti-IRRR antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al, "Immunochemical Applications of Avidin-Biotin Technology," in Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992). Methods for performing immunoassays are well-established.
  • Suitable biological samples for detection of IRRR protein include cells, tissues or bodily fluids, such as urine, saliva or blood.
  • biotin- or FITC-labeled anti-IRRR antibodies can be used to identify cells that bind IRRR. Such can binding can be detected, for example, using flow cytometry.
  • kits for performing an immunological diagnostic assay for 7RRR gene expression comprise at least one container comprising an anti-IRRR antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of IRRR antibody or antibody fragments.
  • indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like.
  • a kit may also comprise a means for conveying to the user that IRRR antibodies or antibody fragments are used to detect IRRR protein. For example, written instructions may state that the enclosed antibody or antibody fragment can be used to detect IRRR.
  • the written instructions may state that the enclosed anti-IRRR antibodies, or antibody fragments, can be used for the diagnosis or prognosis of a disease of glucose metabolism, such as type II diabetes.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • Transgenic mice can be engineered to over-express the 7RRR gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers of IRRR can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human disease caused by excess IRRR. Transgenic mice that over-express 7RRR also provide model bioreactors for production of IRRR in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses an 7RRR gene can begin with adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl , 2-4 months, (Taconic Farms)).
  • the donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) I. P. to induce superovulation.
  • Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating. Fertilized eggs are collected under a surgical scope. The oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma).
  • Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (described, for example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated with 5% CO 2 , 5% O 2 , and 90% N 2 at 37°C.
  • the eggs are then stored in a 37°C/5% CO 2 incubator until microinjection.
  • IRRR encoding sequences can comprise at least a portion of the human IRRR sequence (e.g., SEQ ID NO:l), or guinea pig (e.g., GenBank accession No. J05047), and rat sequences (e.g., SEQ ID NO:6), as described above.
  • Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO -equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
  • Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnig toht in a 37°C/5% CO 2 incubator.
  • two-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
  • a baby serrefine Robot, Rockville, MD
  • the pipette is transferred into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette.
  • the fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in.
  • the peritoneal wall is closed with one suture and the skin closed with a wound clip.
  • the mice recuperate on a 37°C slide warmer for a minimum of four hours.
  • the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
  • the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
  • Genomic DNA is prepared from the tail snips using, for example, a QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify an 7RRR gene or a selectable marker gene that was introduced in the same plasmid. After animals are confirmed to be transgenic, they are back-crossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type female(s). As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping.
  • a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the zyphoid process.
  • a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized.
  • a tie is made around the lower lobe securing it outside the body cavity.
  • An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, NJ) is placed proximal to the first tie.
  • a distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
  • the excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice.
  • the surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hours post operatively.
  • the animal is checked daily post operatively and the wound clips removed 7-10 days after surgery.
  • the expression level of 7RRR mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • transgenic mice that over-express 7RRR, it is useful to engineer transgenic mice with either abnormally low or no expression of the gene.
  • Such transgenic mice provide useful models for diseases associated with a lack of IRRR.
  • Methods for producing transgenic mice that have abnormally low expression of a particular gene are known to those in the art (see, for example, Wu et al, "Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology, pages 205-224 (CRC Press 1997)).
  • inhibitory sequences are targeted to 7RRR mRNA.
  • 7RRR gene expression can be inhibited using anti-sense genes derived from the IRRR-encoding sequences disclosed herein.
  • an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337).
  • ribozymes include nucleotide sequences that bind with IRRR mRNA.
  • expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode an 7RRR gene.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Airman et al, U.S. Patent No. 5,168,053, Yuan et al, Science 263:1269 (1994), Pace et al, international publication No. WO 96/18733, George et al, international publication No.
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary to IRRR mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base-paired region.
  • Another general method for producing transgenic mice that have little or no 7RRR gene expression is to generate mice having at least one normal 7RRR allele replaced by a nonfunctional 7RRR gene.
  • One method of designing a nonfunctional 7RRR gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes IRRR. Standard methods for producing these so-called “knockout mice” are known to those skilled in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al. , “New Strategies for Gene Knockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).
  • the insulin receptor-related receptor was mapped to chromosome 1 using the commercially available version of the "Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc.; Huntsville, AL).
  • the Stanford G3 RH Panel contains DNA molecules from each of 83 radiation hybrid clones of the whole human genome, plus two control DNA molecules (the RM donor and the A3 recipient).
  • An Internet-accessible server http://shgc-www.stanford.edu) allows chromosomal localization of markers.
  • Each of the 85 PCR reactions consisted of 2 ⁇ l lOx KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc.; Palo Alto, CA), 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKTN-ELMER, Foster City, CA), 1 ⁇ l sense primer, ZC 22,391 (5' GGC GAG GAG TGT GCT GAC 3'; SEQ ID NO:4), 1 ⁇ l antisense primer, ZC 22,392 (5' GCC CGC TGA AGG TGG TCT 3'; SEQ ID NO:5), 2 ⁇ l "RediLoad” (Research Genetics, Inc.; Huntsville, AL), 0.4 ⁇ l 50x Advantage KlenTaq Polymerase Mix (CLONTECH Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH2O for a total volume of 20 ⁇ l.
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 70°C, and 1 minute and 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
  • the results showed linkage of the insulin receptor-related receptor to chromosome 1 framework marker SHGC-69054 with a LOD score of 11.39 and at a distance of 0 cR_10000 from the marker.
  • the use of surrounding markers positions the insulin receptor-related receptor in the Iq23-q24 region on the integrated LDB chromosome 1 map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/).
  • comparison with surrounding markers which have been cytogenetically mapped, indicates that the insulin receptor-related receptor gene locus resides in the Iq21-q23 region of chromosome 1.

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Abstract

La présente invention concerne des procédés d'identification d'anomalies, dans le chromosome humain 1q, liées aux troubles du métabolisme du glucose. La présente invention concerne également des procédés d'identification de polymorphismes dans un gène récepteur associé au récepteur de l'insuline se trouvant sur le chromosome 1q21-q24, et plus particulièrement 1q21-q23, un locus lié à une forme de diabète de type II héréditaire.
PCT/US2000/010644 1999-04-22 2000-04-19 Sequence de genes recepteurs associes au recepteur de l'insuline pour le diagnostic de l'obesite humaine et des troubles d'origine diabetique WO2000065090A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2151502A1 (fr) * 2008-07-30 2010-02-10 Lohmann Tierzucht GmbH Variations génétiques associées au comportement de becquetage chez les espèces aviaires

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2000023591A2 (fr) * 1998-10-21 2000-04-27 Zymogenetics, Inc. Proteine secretee zsig49

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Publication number Priority date Publication date Assignee Title
WO2000023591A2 (fr) * 1998-10-21 2000-04-27 Zymogenetics, Inc. Proteine secretee zsig49

Non-Patent Citations (7)

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Title
ELBEIN S C ET AL: "GENOME-WIDE SEARCH FOR TYPE 2 DIABETES SUSCEPTIBILITY GENES IN CAUCASIANS: EVIDENCE FOR A RECESSIVE LOCUS ON CHROMOSOME 1" DIABETES,NEW YORK, NY,US, 13 June 1998 (1998-06-13), page 1278b XP000906839 ISSN: 0012-1797 *
ELBEIN S.C. ET AL.: "A genome-wide search for type 2 diabetes susceptibility genes in Utah Caucasians" DIABETES, vol. 48, no. 5, May 1999 (1999-05), pages 1175-1182, XP000944793 cited in the application *
HANSON R L ET AL: "AN AUTOSOMAL GENOMIC SCAN FOR LOCI LINKED TO TYPE II DIABETES MELLITUS AND BODY-MASS INDEX IN PIMA INDIANS" AMERICAN JOURNAL OF HUMAN GENETICS,US,UNIVERSITY OF CHICAGO PRESS, CHICAGO,, vol. 63, no. 4, 1998, pages 1130-1138, XP000909507 ISSN: 0002-9297 cited in the application *
ITOH N. ET AL.: "Two truncated forms of rat insulin receptor-related receptor" J. BIOL. CHEM., vol. 268, no. 24, 25 August 1993 (1993-08-25), pages 17983-17986, XP002154781 cited in the application *
SHIER P. AND WATT V.M.: "Primary structure of a putative receptor for a ligand of the insulin family" J. BIOL. CHEM., vol. 264, no. 25, 5 September 1989 (1989-09-05), pages 14605-14608, XP002154780 cited in the application *
SHIER P. ET AL.: "Localization of the insulin receptor-related receptor gene on human chromosome 1" CYTOGENET CELL GENET, vol. 54, 1990, pages 80-81, XP000944567 cited in the application *
WHITMORE T.E. ET AL.: "The assignment of the human insulin receptor-related receptor gene (INSRR) tp chromosome 1q21-q23 by the use of radiation hybrid mapping" CYTOGENET CELL GENET, vol. 87, no. 1-2, 1999, pages 93-94, XP000944566 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2151502A1 (fr) * 2008-07-30 2010-02-10 Lohmann Tierzucht GmbH Variations génétiques associées au comportement de becquetage chez les espèces aviaires

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