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WO1996037513A1 - CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES η DE LA PROTEINE G HUMAINE - Google Patents

CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES η DE LA PROTEINE G HUMAINE Download PDF

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Publication number
WO1996037513A1
WO1996037513A1 PCT/US1995/006406 US9506406W WO9637513A1 WO 1996037513 A1 WO1996037513 A1 WO 1996037513A1 US 9506406 W US9506406 W US 9506406W WO 9637513 A1 WO9637513 A1 WO 9637513A1
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WO
WIPO (PCT)
Prior art keywords
subunit
subunits
human
seq
sequence
Prior art date
Application number
PCT/US1995/006406
Other languages
English (en)
Inventor
Janet D. Robishaw
Charles A. Kunsch
Original Assignee
Smithkline Beecham Corporation
Human Genome Sciences, Inc.
Weis Center For Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation, Human Genome Sciences, Inc., Weis Center For Research filed Critical Smithkline Beecham Corporation
Priority to JP8535609A priority Critical patent/JPH11505715A/ja
Priority to PCT/US1995/006406 priority patent/WO1996037513A1/fr
Priority to EP95920580A priority patent/EP0837879A1/fr
Priority to CA002221783A priority patent/CA2221783A1/fr
Priority to AU25987/95A priority patent/AU2598795A/en
Publication of WO1996037513A1 publication Critical patent/WO1996037513A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human ⁇ 2 , ⁇ 3 , ⁇ 4 , ⁇ 5 , ⁇ 7 , ⁇ 10 and ⁇ n subunit sequences.
  • the sense primer is S'-CTATCCAGCACTCCGATGGC-S' (SEQ ID NO: 12) and the antisense primer is 5'-AGACTTAAAGGATGGCACAG-3' (SEQ ID NO: 13); for the ⁇ 3 subunit the sense primer is 5 , -TGTGGCTTCAGGATGAAAGG-3 , (SEQ ID NO: 14) and the antisense primer is 5'-GAGCTCAGAGGAGAGCACAG- 3' (SEQ ID NO: 15); for the ⁇ 5 subunit the sense primer is 5'- GTGCACCATGTCTGGCTCCT-3' (SEQ ID NO: 16) and the antisense primer is 5'- CACTGGATCATAAGGAGTGG-3' (SEQ ID NO: 17); and, for the ⁇ 7 subunit the sense primer is 5'-GATGGCAGACAATGTCAGCC-3' (SEQ ID NO: 18) and the antisense primer is S'-AGTTATAAAATAATACAAGG-S'
  • the cDNA for the ⁇ ⁇ subunit is 654 bp in length, including 106 and 326 bp of 5'- and 3'-UTR sequences, respectively (SEQ ID NO: 11).
  • the 3'-UTR contains a polyadenylation signal and a poly(A) tail towards the 3'-end.
  • the present invention further relates to polypeptides having the deduced amino acid sequences SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8 or those encoded by the cDNA clones of the human ⁇ 2 , ⁇ 3 , ⁇ 4 , ⁇ 5 , ⁇ 7 , ⁇ 10 or ⁇ ⁇ subunits as well as fragments, analogs and derivatives of such polypeptides.
  • the ⁇ 10 subunit may represent a new subclass that is only distantly related to the other ⁇ subunits.
  • the homology ranged from a low of 33 to 44% for the ⁇ 2> ⁇ 3 » 7s and ⁇ 7 subunits to a high of 76% for the x subunit.
  • Analysis of the amino acid sequence conservation suggests that the ⁇ subunit family can be divided into four distinct subclasses, one containing ⁇ j and ⁇ n subunits, a second containing the ⁇ 2 , ⁇ 3 , ⁇ 4 and ⁇ 7 subunits, a third containing the ⁇ 5 subunit, and a fourth containing the ⁇ 10 subunit.
  • the vector containing the appropriate cDNA clone as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate hosts there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc.
  • bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • the gene is attached to a segment of DNA that carries a selectable marker and transfected into the cells, or are cotransfected into the cells. Sublines are then selected in which the number of copies of the gene are greatly amplified.
  • selectable markers available in the art. For example, the dhfr gene is extensively used for coamplification. After several months of growth in progressively increasing concentrations of methotrexate, cell lines can be obtained that carry up to 1000 copies of the dhfr gene.
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue. There are marked differences in the tissue distribution of members of the ⁇ subunit family.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled ⁇ 2 , ⁇ 3 , ⁇ 4 , ⁇ 5 , ⁇ 7 , ⁇ 10 or ⁇ ⁇ subunit RNA or alternatively, radiolabeled ⁇ 2 , ⁇ 3 , ⁇ 4 , ⁇ 5 , ⁇ 7 , ⁇ 10 or ⁇ n subunit antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific- cDNA libraries.
  • the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
  • the amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • Potential antagonists may also be identified by competitive inhibition assays wherein a potential antagonist and a particular ⁇ subunit are combined with membrane bound ⁇ subunit receptor or recombinant ⁇ subunit receptor under appropriate assay conditions. Such appropriate assay conditions can be routinely determined by those of skill in the art.
  • the ⁇ subunit is labeled, preferably radiolabeled, so that the number of ⁇ subunits bound to the receptor can determine the effectiveness of the potential antagonist.
  • GGTTT CGGGA TCTCG GTGCT GCAGA CGGCG AGACC TCCTG CACAG GGTGT 100
  • GGCCC AGAGC TGATG GCAGA CAATG TCAGC CACTA ACAAC ATAGC CCAGG 200 CCCGG AAGCT GGTGG AACAG CTACG CATAG AAGCC GGGAT TGAGC GCATC 250

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur des molécules d'acide nucléique codant des sous-unités humaines η2, η3, η4, η5, η7, η10 et η11 ainsi que sur des polypeptides de sous-unités. Elle concerne, de surcroît, des procédés permettant de déceler des formes mutées de la sous-unité η humaine ainsi que des niveaux modifiés de la sous-unité η humaine. L'invention concerne également des procédés permettant d'identifier des antagonistes et des agonistes de l'interaction d'un ligand βη avec son récepteur.
PCT/US1995/006406 1995-05-22 1995-05-22 CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES η DE LA PROTEINE G HUMAINE WO1996037513A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP8535609A JPH11505715A (ja) 1995-05-22 1995-05-22 ヒトＧ−蛋白質γサブユニットをコードするｃＤＮＡクローン
PCT/US1995/006406 WO1996037513A1 (fr) 1995-05-22 1995-05-22 CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES η DE LA PROTEINE G HUMAINE
EP95920580A EP0837879A1 (fr) 1995-05-22 1995-05-22 CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES $g(g) DE LA PROTEINE G HUMAINE
CA002221783A CA2221783A1 (fr) 1995-05-22 1995-05-22 Clones d'adn complementaire codant des sous-unites .gamma. de la proteine g humaine
AU25987/95A AU2598795A (en) 1995-05-22 1995-05-22 Cdna clones encoding human g protein gamma subunits

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1995/006406 WO1996037513A1 (fr) 1995-05-22 1995-05-22 CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES η DE LA PROTEINE G HUMAINE
CA002221783A CA2221783A1 (fr) 1995-05-22 1995-05-22 Clones d'adn complementaire codant des sous-unites .gamma. de la proteine g humaine

Publications (1)

Publication Number Publication Date
WO1996037513A1 true WO1996037513A1 (fr) 1996-11-28

Family

ID=25679849

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/006406 WO1996037513A1 (fr) 1995-05-22 1995-05-22 CLONES D'ADN COMPLEMENTAIRE CODANT DES SOUS-UNITES η DE LA PROTEINE G HUMAINE

Country Status (4)

Country Link
EP (1) EP0837879A1 (fr)
AU (1) AU2598795A (fr)
CA (1) CA2221783A1 (fr)
WO (1) WO1996037513A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012325A1 (fr) * 1996-09-18 1998-03-26 Incyte Pharmaceuticals, Inc. Nouvelle proteine humaine gamma 3 de fixation de la gtp
WO1999021884A1 (fr) * 1997-10-29 1999-05-06 Shanghai Second Medical University Cblaeh07: sous-unite gamma 5 de proteine a g
WO2002018425A3 (fr) * 2000-09-01 2003-05-30 Millennium Pharm Inc 47324, nouvelle proteine g humaine et ses utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENE, Vol. 149, issued 1994, K. RAY et al., "Cloning and Sequencing of a Rat Heart cDNA Encoding a G-Protein beta Subunit Related to the Human Retinal beta3 Subunit", pages 337-340. *
MOLECULAR AND CELLULAR BIOLOGY, Vol. 12, No. 4, issued April 1992, K.J. FISHER et al., "Characterization of the cDNA and Genomic Sequence of a G Protein gamma Subunit (gamma5)", pages 1585-1591. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 87, issued October 1990, N. GAUTAM et al., "G Protein Diversity is Increased by Associations with a Variety of gamma Subunits", pages 7973-7977. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012325A1 (fr) * 1996-09-18 1998-03-26 Incyte Pharmaceuticals, Inc. Nouvelle proteine humaine gamma 3 de fixation de la gtp
US5935812A (en) * 1996-09-18 1999-08-10 Incyte Pharmaceuticals, Inc. Human GTP binding protein gamma-3
WO1999021884A1 (fr) * 1997-10-29 1999-05-06 Shanghai Second Medical University Cblaeh07: sous-unite gamma 5 de proteine a g
WO2002018425A3 (fr) * 2000-09-01 2003-05-30 Millennium Pharm Inc 47324, nouvelle proteine g humaine et ses utilisations

Also Published As

Publication number Publication date
EP0837879A1 (fr) 1998-04-29
AU2598795A (en) 1996-12-11
CA2221783A1 (fr) 1996-11-28

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