EP0981616A1 - Polypeptid-ligand des g-protein-gekoppelten rezeptors der menschlichen hirnanhangdrüse - Google Patents

Polypeptid-ligand des g-protein-gekoppelten rezeptors der menschlichen hirnanhangdrüse

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Publication number
EP0981616A1
EP0981616A1 EP98917693A EP98917693A EP0981616A1 EP 0981616 A1 EP0981616 A1 EP 0981616A1 EP 98917693 A EP98917693 A EP 98917693A EP 98917693 A EP98917693 A EP 98917693A EP 0981616 A1 EP0981616 A1 EP 0981616A1
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European Patent Office
Prior art keywords
dna
protein
seq
sequence
polypeptide
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EP98917693A
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English (en)
French (fr)
Inventor
Shuji Hinuma
Shoji Fukusumi
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Publication of EP0981616A1 publication Critical patent/EP0981616A1/de
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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
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel ligand polypeptide for the G protein-coupled receptor protein and a DNA comprising a DNA encoding the ligand polypeptide.
  • G protein-binding protein-binding protein (hereinafter sometimes referred to briefly as G protein) and have the common structure comprising 7 transmembrane domains. Therefore, these receptors are collectively referred to as G protein-
  • 25 pituitary hormones from the hypophysis is controlled by hypothalamic hormones (pituitatropic releasing factor) and the functions of the target cells or organs are regulated through the pituitary hormones released into the circulation. This pathway carries out functional
  • hypothalamic hormones and the peripheral hormone secreted from the target endocrine gland.
  • the various receptor proteins present in the hypophysis are playing a central role in the regulation of the hypothalamus-pituitary system.
  • hypothalamus hormone is functioning as a neurotransmitter or a neuromodulator .
  • peripheral tissues as well and thought to be playing important roles in the respective tissue.
  • pancreas is playing a crucial role in the carbohydrate metabolism by secreting glucagon and insulin as well as digestive juice. While insulin is secreted from the pancreatic ⁇ cells, its secretion is mainly stimulated by glucose. However, it is known that ⁇ cells have a variety of receptors and the secretion of insulin is controlled by a number of factors in addition to glucose as well as peptide hormones, e.g. galanine, somatostatin, gastric inhibitory polypeptide, glucagon, amyrin, etc.; sugars, e.g. mannose etc.; amino acids, and neurotransmitters, among others.
  • peptide hormones e.g. galanine, somatostatin, gastric inhibitory polypeptide, glucagon, amyrin, etc.
  • sugars e.g. mannose etc.
  • amino acids e.g. mannose etc.
  • the ligand would be belonging to the family of opioid peptides.
  • the history of research and development in the realm of substances acting on the living body through the opioid receptor dates back to many years ago and various antagonists and agonists had been developed. Therefore, among the compounds artificially synthesized, an agonist of the receptor was picked out and, using it as a probe, expression of the receptor in the receptor cDNA-transfected cells was verified. Then, a search was made for an activator of the intracellular signal transduction which was similar to the agonist, and the activator so found was purified, and then the structure of the ligand was determined.
  • ligands of orphan G protein-coupled receptor proteins which are considered to be show some physiological functions or others in the living body.
  • ligands binding the orphan G protein-coupled receptor proteins expressed in the pituitary, central nervous system, and pancreatic ⁇ cells are expected to be useful drugs, although their structures and functions remain to be fully known.
  • This technology comprises destroying a given chromosomal gene of the pluripotent murine embryonic stem cell (ES cell) by way of a homologous recombination, microinjecting the resulting ES cell into the murine blastocyst to construct chimera mice, and mating them to produce a knockout mouse.
  • ES cell pluripotent murine embryonic stem cell
  • mice are available as such knockout animal models of various diseases .
  • the mouse genomic DNA sequence of the target gene must be known but, for the orphan G protein- coupled receptor protein pHGR3 (GPR10) or UHR-1, neither the corresponding mouse ligand peptide nor the gene (cDNA or genomic DNA) coding for the ligand peptide is known.
  • the inventors of the present invention performed a screening for a bovine polypeptide which this receptor protein recognizes as its ligand and determined its amino acid sequence and DNA sequence. Then, using the DNA coding for said bovine polypeptide as a primer, the inventors isolated the bovine, human, and rat cDNAs coding for said polypeptide by polymerase chain reaction (PCR), synthesized primers based on the uncleotide sequence of the rat cDNA, and succeeded in isolating the cDNA and genomic DNA coding for the mouse polypeptide .
  • PCR polymerase chain reaction
  • the inventors established a screening method for a compound which modifies the binding of the ligand to said receptor protein and further by using the genomic DNA of said mouse ligand polypeptide, constructed a non- human transgenic animal (particularly a useful knockout mouse) to thereby may it possible to analyze the function of the gene.
  • the present invention therefore, relates to
  • polypeptide comprising an amino acid sequence represented by SEQ ID NO:l, or a substantial equivalent thereto, or its amide or ester, or a salt thereof,
  • DNA of the DNA according to the above item (2) (7) a non-human transgenic animal having the DNA according to the above item (2) or its mutein, or the recombinant vector according to the above item (4),
  • a pharmaceutical composition which comprises the polipeptide according to the above item (1) or its amide or ester, or the salt thereof, and
  • this invention relates to an agent for treating or preventing dementia, depression (melancholia), hyperkinetic (microencephalo-pathy) syndrome, disturbance of consciousness, anxiety syndrome, schizophrenia, horror, growth hormone secretory disease, hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia, hyperlipemia, hyperprolactinemia, diabetes, cancer, pancreatitis, renal disease, Turner's syndrome, neurosis, rheumatoid arthritis, spinal injury, transient brain ischemia, amyotrophic lateral sclerosis, acute myocardial infarction, spinocerebellar degeneration, bone fracture, trauma, atopic dermatitis, osteoporosis, asthma, epilepsy, infertility and/or oligogalactia .
  • substantially equivalent means that the activity of the protein, e.g., nature of the binding activity of the ligand and the receptor and physical characteristics are substantially the same. Substitutions, deletions, additions or insertions of amino acids often do not produce radical changes in the physical and chemical characteristics of a polypeptide, in which case polypeptides containing the substitutions, deletions, additions or insertions would be considered to be substantially equivalent to polypeptides lacking the substitutions, deletions, additions or insertions. Substantially equivalent substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the non- polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine .
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine .
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • a mutated polypeptide which is obtained by a mutation (such as substitution, deletion, addition or insertion etc.) of the non- mutated polypeptide is a polypeptide substantially the same as the non-mutated polypeptide, wherein the physiological characteristics and chemical characteristics of the non-mutated polypeptide is not effected by the mutation.
  • the polypeptide of the present invention represents a precursor polypeptide of a matured ligand polypeptide (e.g. a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 or a substantial equivalent thereof) which can bind to the G protein-coupled receptors .
  • the polypeptide of the present invention represents a polypeptide which comprises an amino acid sequence represented by SEQ ID NO:l or its substantial equivalent thereto, or its amide or ester, or a salt thereof (hereinafter, sometimes referred to briefly as ligand polypeptide or polypeptide) .
  • the polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 4 is excluded from the polypeptide of the present invention.
  • Fig. 1 shows the nucleotide sequence of the human pituitary-derived G protein-coupled receptor protein cDNA fragment harbored in cDNA clone pl9P2 isolated by PCR using human pituitary-derived cDNA and the amino acid encoded by the nucleotide sequence.
  • the primer used for sequencing was -21M13.
  • the underscored region corresponds to the synthetic primer.
  • Fig. 2 shows the nucleotide sequence of the human pituitary-derived G protein-coupled receptor protein cDNA fragment harbored in cDNA clone pl9P2 isolated by PCR using human pituitary-derived cDNA and the amino acid sequence encoded thereby.
  • the primer used for sequencing was M13RV-N (Takara) .
  • the underscored region corresponds to the synthetic primer.
  • Fig. 3 shows a partial hydrophobic plot of the protein encoded by the human pituitary-derived G protein-coupled receptor protein cDNA fragment harbored in pl9P2 constructed according to the amino acid sequence shown in Fig. 1.
  • Fig. 4 shows a partial hydrophobic plot of the protein encoded by the human pituitary-derived G protein-coupled receptor protein cDNA fragment harbored in pl9P2 constructed according to the amino acid sequence shown in Fig. 2.
  • Fig. 5 is a diagram comparing the partial amino acid sequence of the protein encoded by the human pituitary-derived G protein-coupled receptor protein cDNA fragment harbored in pl9P2 as shown in Figs. 1 and 2 with the known G protein-coupled receptor protein S12863.
  • the shadowed region represents the region of agreement.
  • the 1st to 9th amino acid sequence of pl9P2 corresponds to the 1st to 99th amino acid sequence of Fig. 1 and the 156th to 230th amino acid sequence corresponds to the 1st to 68th amino acid sequence of Fig. 2.
  • Fig. 6 shows the nucleotide sequence of the MIN6- derived G protein-coupled receptor protein cDNA fragment based on the nucleotide sequences of the MIN6- derived G protein-coupled receptor protein cDNA fragments harbored in the cDNA clones pG3-2 and pGl-10 isolated by PCR using MIN6-derived cDNA and the amino acid sequence encoded by the nucleotide sequence.
  • the underscored region corresponds to the synthetic primer.
  • Fig. 7 is a diagram comparing the partial amino acid sequence encoded by pG3-2/pGl-10 of the MIN6- derived G protein-coupled receptor protein shown in Fig. 6 with the partial amino acid sequence of the protein encoded by pl9P2 shown in Figs. 1 and 2.
  • the shadowed region corresponds to the region of agreement.
  • the 1st to 99th amino acid sequence of the protein encoded by pl9P2 corresponds to the 1st to 99th amino acid sequence of Fig. 1
  • the 156th to 223rd amino acid sequence corresponds to the 1st to 68th amino acid sequence of Fig. 2.
  • the 1st to 223rd amino acid sequence of the protein encoded by pG3-2/pGl-10 corresponds to the 1st to 223rd amino acid sequence of Fig. 6.
  • Fig. 8 is a partial hydrophobic plot of the MIN6- derived G protein-coupled receptor protein constructed according to the partial amino acid sequence shown in Fig. 6.
  • Fig. 9 shows the entire nucleotide sequence of the human pituitary-derived G protein-coupled receptor protein cDNA harbored in the cDNA clone phGR3 isolated from a human pituitary-derived cDNA library by the plaque hybridization method using the DNA fragment inserted in pl9P2 as a probe and the amino acid sequence encoded by the nucleotide sequence.
  • Fig. 10 shows the results of Northern blotting of human pituitary mRNA hybridized with radioisotope- labeled human pituitary cDNA clone phGR3.
  • Fig. 11 shows a hydrophobic plot of the protein encoded by the human pituitary-derived G protein- coupled receptor protein cDNA harbored in the phGR3 as constructed according to the amino acid sequence shown in Fig. 9.
  • Fig. 12 shows the nucleotide sequence of the MIN6- derived G protein-coupled receptor protein cDNA fragment harbored in the cDNA clone p5S38 isolated by PCR using MIN6-derived cDNA and the amino acid sequence encoded by the nucleotide sequence. The underscored region corresponds to the synthetic primer.
  • Fig. 13 shows a diagram comparing the partial amino acid sequence of MIN6-derived G protein-coupled receptor protein encoded by p5S38 shown in Fig. 12 with the partial amino acid sequence of G protein-coupled receptor protein encoded by the cDNA fragment harbored in pl9P2 as shown in Figs.
  • the 1st to 144th amino acid sequence of the protein encoded by p5S38 corresponds to the 1st to 144th amino acid sequence of Fig. 12
  • the 1st to 99th amino acid sequence of the protein encoded by pl9P2 corresponds to the 1st to 99th amino acid sequence of Fig. 1
  • the 156th to 223rd amino acid sequence corresponds to 1st to 68th amino acid sequence of Fig. 2.
  • the 1st to 223rd amino acid sequence of the protein encoded by pG3-2/pGl-10 corresponds to the 1st to 223rd amino acid sequence of Fig. 6.
  • Fig. 14 shows a partial hydrophobic plot of the protein encoded by the MIN6-derived G protein-coupled receptor protein cDNA harbored in p5S38 as constructed according to the partial amino acid sequence shown in Fig. 12.
  • Fig. 15 shows the results of the following analysis.
  • RT-PCR was carried out to confirm the expression of mRNA in CHO cells transfected by pAKKO- 19P2.
  • Lanes 1-7 represent the results obtained by performing PCRs using serial dilutions of pAKKO-19P2 for comparison, i.e.
  • Lanes 8 through 11 are the results obtained by performing PCRs using a 1/10 dilution (lane 8), a 1/100 dilution (lane 9), and a 1/1000 dilution (lane 10) of the cDNA prepared from the CHO-19P2 cell line as templates and subjecting the respective reaction mixtures to electrophoresis.
  • Lane 11 was obtained by performing PCR using a template obtained by carrying out cDNA synthesis without reverse transcriptase and subjecting the PCR reaction product to electrophoresis.
  • Lanes 12 and 13 were obtained by performing PCR using cDNAs prepared from mock CHO cells with and without addition of reverse transcriptase, respectively, as templates and subjecting the respective reaction products to electrophoresis.
  • M represents the DNA size marker.
  • Fig. 16 shows the activity of the crude ligand peptide fraction extracted from rat whole brain to promote release of arachidonic acid metabolites from CHO-19P2 cells. The arachidonic acid metabolite releasing activity was expressed as % of the amount of
  • Fig. 17 shows the activity of the crude ligand polypeptide fraction extracted from bovine hypothalamus to promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the arachidonic acid metabolite release-promoting activity was expressed as % of the amount of [ H] arachidonic acid metabolites released in the presence of the crude ligand polypeptide fraction with the amount of [ H] arachidonic acid metabolites released in the presence of 0.05% BAS-HABB being taken as 100%.
  • the activity to promote release of arachidonic acid metabolites from the CHO-19P2 cell line was detected in a 30% CH 3 CN fraction just as in the crude ligand polypeptide fraction from rat whole brain.
  • Fig. 18 shows the activity of the fraction purified with the reversed-phase column C18 218TP5415 to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the active fraction from RESOURCE S was fractionated on C18 218TP5415.
  • chromatography was carried out at a flow rate of 1 ml/min. on a concentration gradient of 20%-30% CH 3 CN/0.1% TFA/H 2 0, the eluate was collected in 1 ml fractions, and each fraction was lyophilized. Then, the activity of each fraction to specifically promote release of arachidonic acid metabolites from the CHO- 19P2 cell line was determined. As a result, the activity was fractionated into 3 fractions (designated, in the order of elution, as P-l, P-2, and P-3).
  • Fig. 19 shows the activity of the fraction purified with the diphenyl 219TP5415 reversed-phase column to specifically promote arachidonic acid metabolite release from CHO-19P2 cells.
  • the P-3 active fraction from C18 218TP5415 was fractionated on diphenyl 219TP5415.
  • the chromatography was carried out at a flow rate of 1 ml/min. on a concentration gradient of 22%-25% CH 3 CN/0.1% TFA/H 2 0, the eluate was collected in 1 ml fractions, and each fraction was lyophilized. Then, the activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells in each fraction was determined. As a result, the activity converged in a single peak.
  • Fig. 20 shows the activity of the fraction purified by ⁇ RPC C2/C18 SC 2.1/10 reversed-phase column to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the peak active fraction from diphenyl 219TP5415 was fractionated on ⁇ RPC C2/C18 SC 2.1/10.
  • the chromatography was carried out at a flow rate of 100 ⁇ l/min. on a concentration gradient of 22%-23.5% CH 3 CN/0.1% TFA/H 2 0, the eluate was collected in 100 ⁇ l fractions, and each fraction was lyophilized. Then, the activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells in each fraction was determined. As a result, the activity was found as two peaks of apparently a single substance (peptide) .
  • Fig. 21 shows the activity of the P-2 fraction purified by ⁇ RPC C2/C18 SC 2.1/10 reversed-phase column to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the chromatography was carried out at a flow rate of 100 ⁇ l/min. on a concentration gradient of 21.5%-23.0% CH 3 CN/0.1%
  • Fig. 22 shows the nucleotide sequence of bovine hypothalamus ligand polypeptide cDNA fragment as derived from the nucleotide sequence of the bovine hypothalamus-derived ligand polypeptide cDNA fragment which specifically promotes release of arachidonic acid metabolites from CHO-19P2 cells as harbored in a cDNA clone isolated by PCR using bovine hypothalamus-derived cDNA and the amino acid sequence encoded by said nucleotide sequence.
  • the region indicated by the arrow ark corresponds to the synthetic primer.
  • Fig. 23 shows the nucleotide sequence of the bovine hypothalamus-derived ligand polypeptide cDNA fragment generated according to the nucleotide sequence of the bovine hypothalamus-derived ligand polypeptide cDNA fragment which specifically promotes release of arachidonic acid metabolites from CHO-19P2 cells as harbored in a cDNA clone isolated by PCR using bovine hypothalamus-derived cDNA and the amino acid sequence encoded by said nucleotide sequence.
  • the region indicated by the arrowmark corresponds to the synthetic primer.
  • Fig. 24 shows the amino acid sequences (a) and (b) of the bovine hypothalamus-derived ligand polypeptides which specifically promote release of arachidonic acid metabolites from CHO-19P2 cells and the cDNA sequence coding for the full coding region of the ligand polypeptides defined by SEQ ID N0:1 and SEQ ID NO:44.
  • Fig. 25 shows the concentration-dependent activity of synthetic ligand polypeptide (19P2-L31) to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the synthetic peptide was dissolved in degassed dH 2 0 at a final concentration of 10 "3 M and diluted with 0.05% BSA-HBSS to concentrations of 10 "12 M-10 "6 M.
  • the arachidonic acid metabolite releasing activity was expressed in the measured radioactivity of [ H] arachidonic acid metabolites released in the supernatant when the dilution was added to the cells.
  • the activity of 19P2-31 to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells was found in a concentration-dependent manner.
  • Fig. 26 shows the concentration-dependent activity of synthetic ligand polypeptide ( 19P2-L31 (0) ) to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the synthetic ligand peptide was dissolved in degassed dH 2 0 at a final concentration of 10 "3 M and diluted with 0.05% BSA-HBSS to concentrations of 10 "12 M-10 "6 M.
  • the arachidonic acid metabolite releasing activity was expressed in the measured radioactivity of [ H] arachidonic acid metabolites released in the supernatant when the dilution was added to the cells.
  • the activity of 19P2-L31(0) to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells was found in a dose-dependent manner.
  • Fig. 27 shows the activity of synthetic ligand polypeptide 19P2-L20 to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells.
  • the synthetic peptide was dissolved in degassed distilled water at a final concentration of 10 M and diluted with 0.05% BSA-HBSS to concentrations of 10 "12 M-10 "6 M.
  • CHO-19P2 cells was found in a dose-dependent manner.
  • Fig. 28 shows the 1.2% agarose gel electrophoregram of the DNA fragments of the phages cloned from a bovine genomic library as digested with restriction enzymes BamHI(B) and SalI(S).
  • M DNA size marker
  • Styl digests of ⁇ phage DNA were used.
  • lane B two bands derived from the vector were detected in positions between the first (19,329 bp) and second (7.743 bp) marker bands, as well as 3 bands derived from the inserted fragment between the third (6,223 bp) and 5th (3,472 bp) bands.
  • lane S two bands derived from the vector were similarly detected but due to the overlap of the band of the inserted fragment, the upper band is thicker than the band in lane B.
  • Fig. 29 shows the nucleotide sequence around the coding region as decoded from bovine genomic DNA.
  • the 1st to 3rd bases (ATG) correspond to the translation start codon and the 767th to 769th bases (TAA) correspond to the translation end codon.
  • Fig. 30 shows a comparison between the nucleotide sequence (genome) around the coding region as deduced from bovine genomic DNA and the nucleotide sequence (cDNA) of bovine cDNA cloned by PCR. The sequence region of agreement is indicated by shading. As to the 101st to 572nd region, there is no corresponding region in the nucleotide sequence of cDNA, indicating that it is an intron.
  • Fig. 31 shows the translation of the amino acid sequence encoded after elimination of the intron from the nucleotide sequence around the coding region as decoded from bovine genomic DNA.
  • Fig. 32 shows the full-length amino acid sequence and the cDNA sequence coding for the full coding region of rat ligand polypeptide.
  • Fig. 33 shows the amino acid sequence of bovine ligand polypeptide and the nucleotide sequences of DNAs coding for bovine polypeptide and rat polypeptide.
  • the arrowmark indicates the region corresponding to the synthetic primer.
  • Fig. 34 shows the full-length amino acid sequence and the sequence of cDNA coding for the full coding region of human ligand polypeptide.
  • Fig. 35 shows a comparison of the amino acid sequences in the translation region of bovine ligand polypeptide, rat ligand polypeptide, and human ligand polypeptide .
  • Fig. 36 shows the nucleotide sequence of the inserted fragment of plasmid pmGB3.
  • the arrowmark - indicates the sequence corresponding to the primer.
  • Fig. 37 shows the cDNA predicted from nucleotide sequence of plasmid pmGB3 and the predicted translated protein.
  • the arrowmark ⁇ indicates the sequence corresponding to the primer.
  • the sequence between the marks ii is the sequence predicted to be the intron.
  • Fig. 38 shows (i) the nucleotide sequence coding the ligand polypeptide of the present invention and its non-coding region, and (ii) the amino acid sequence of the ligand polypeptide of the present invention, which obtained in Example 34.
  • Fig. 39 shows the restriction enzyme map of the ligand polypeptide of the present invention.
  • Fig. 40 shows the construction figure for the targeting vector pmGFEN28 obtained in Example 35.
  • Fig. 41 shows the result of the agarose gel electrophoresis described in Example 36, and the comparative gene map between the wild type and the recombinant (knock out) type.
  • polypeptide its amide or ester, or a salt thereof (hereinafter sometimes referred to briefly as the ligand polypeptide or the polypeptide), processes for their production, and uses for the polypeptide are now described in detail.
  • the above ligand polypeptide of the present invention includes any polypeptides derived from any tissues, e.g. pituitary gland, pancreas, brain, kidney, liver, gonad, thyroid gland, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive canal, blood vessel, heart, etc.; or cells of man and other warm-blooded animals, e.g. guinea pig, rat, mouse, swine, sheep, bovine, monkey, etc. and comprising an amino acid sequence represented by SEQ ID NO:l or a substantial equivalent thereto.
  • tissues e.g. pituitary gland, pancreas, brain, kidney, liver, gonad, thyroid gland, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive canal, blood vessel, heart, etc.
  • cells of man and other warm-blooded animals e.g. guinea pig, rat, mouse, swine, sheep, bovine, monkey, etc. and
  • the ligand polypeptide of the present invention includes the protein comprising an amino acid sequence having a homology of about 50- 99.9%, preferably 70-99.9%, more preferably 80-99.9% and especially preferably 90-99.9% to the amino acid sequence of SEQ ID NO:l and having qualitatively substantially equivalent activity to the protein comprising the amino acid sequence of SEQ ID NO:l.
  • substantially equivalent means the nature of the receptor-binding activity, signal transduction activity and the like is equivalent. Thus, it is allowable that even differences among grades such as the strength of receptor binding activity and the molecular weight of the polypeptide are present.
  • the ligand polypeptide of the present invention includes the polypeptide derived from mouse and comprising the amino acid sequence of SEQ ID N0:1.
  • the ligand polypeptide of the present invention includes the polypeptides which comprises substantially equivalent polypeptides such as (i) polypeptides wherein 1 to 15, preferably 1 to 10, and more preferably 1 to 5 amino acid residues are deleted from the amino acid sequence of SEQ ID N0:1, (ii) polypeptides wherein 1 to 80, preferably 1 to 50, more preferably 1 to 10 amino acid residues are added to the amino acid sequence of SEQ ID N0:1, or polypeptides wherein 1 to 15, preferably 1 to 10, more preferably 1 to 5 amino acid residues are substituted with other amino acid residues.
  • the ligand polypeptide of the present invention includes the polypeptides wherein its constructive amino acid (especially its side chain) is modified, or its amide or ester, or a salt thereof.
  • the polypeptide of the present invention includes those wherein Gin of the constitutive amino acid at the N-terminal side is cleaved in vivo to form pyroglutamyl group .
  • the peptides described in this specification the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of the peptide indication. While the C-terminus of the polypeptide of SEQ ID NO:l is usually carboxyl (-COOH) or carboxylato (-COO " ), it may be amide (-C0NH 2 ) or ester (-COOR) form.
  • the ester residue R includes a C ⁇ _ 6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl , etc., a C 3 .
  • cycloalkyl group such as cyclopentyl, cyclohexyl, etc., a C 6 . 12 aryl group such as phenyl, ot-naphthyl, etc., and a C 7 . 1A aralkyl group such as a phenyl-C ⁇ alkyl group, e.g. benzyl, phenethyl, benzhydryl, etc. or an ot-naphthyl-C ⁇ .. 2 alkyl, e.g. -naphthylmethyl etc.
  • the ester may be a pivaloyloxymethyl ester which is broadly used for oral administration.
  • the polypeptide of SEQ ID NO:l has a carboxyl or carboxylato group in any position other than the C- terminus
  • the corresponding amide or ester are also included in the concept of the polypeptide of the present invention.
  • the ester mentioned just above includes the esters mentioned for the C-terminus.
  • the salt of polypeptide of the present invention includes salts with physiologically acceptable bases, e.g. alkali metals or acids such as organic or inorganic acids, and is preferably a physiologically acceptable acid addition salt.
  • physiologically acceptable bases e.g. alkali metals or acids such as organic or inorganic acids
  • examples of such salts are salts thereof with inorganic acids, e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid, etc. and salts thereof with organic acids, e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid, etc .
  • the ligand polypeptide or amide or ester, or a salt thereof of the present invention may be (i) manufactured from the tissues or cells of warm-blooded animals inclusive of human by purifying techniques or
  • the ligand polypeptide can be purified and isolated by a process which comprises homogenizing the tissue or cells of human or other warm-blooded animal, extracting the homogenate with an acid, for instance, and subjecting the extract to a combination of chromatographic procedures such as reversed-phase chromatography, ion- exchange chromatography, affinity chromatography, etc.
  • the ligand polypeptide in the present invention can be produced by per se known procedures for peptide synthesis.
  • the methods for peptide synthesis may be any of a solid-phase synthesis and a liquid-phase synthesis.
  • the objective peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein with the residual part thereof and, when the product has a protective group, the protective group is removed whereupon a desired peptide can be manufactured.
  • the known methods for condensation and deprotection includes the procedures described in the following literature (l)-(5) .
  • the protein can be purified and isolated by a combination of conventional purification techniques such as solvent extraction, column chromatography, liquid chromatography, and recrystallization.
  • the protein isolated as above is in a free form, it can be converted to a suitable salt by the known method.
  • the isolated product is a salt, it can be converted to the free peptide by the known method.
  • the amide of polypeptide can be obtained by using a resin for peptide synthesis which is suited for amidation.
  • the resin includes chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenz- hydrylamine resin, PAM resin, 4- hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- ( 2 ' , 4 ' -dimethoxyphenyl- hydroxymethyl ) phenoxy resin, 4- ( 2 ' , 4 ' -dimethoxyphenyl- Fmoc aminoethyl) phenoxy resin, and so on.
  • amino acids whose ⁇ -amino groups and functional groups of side-chain have been suitably protected are condensed on the resin according to the sequence of the objective peptide by various condensation techniques which are known per se .
  • the peptide or the protected peptide is sepasated from the resin and the protective groups are removed to obtain the objective polypeptide.
  • a variety of activating reagents for peptide synthesis can be used but a carbodiimide compound is particularly suitable.
  • the carbodiimide includes DCC, N,N' -diisopropylcarbodiimide, and N- ethyl- ' - ( 3-dimethylaminoprolyl ) carbodiimide .
  • a racemization inhibitor additive e.g. HOBt and the protected amino acid are directly added to the resin or the protected amino acid pre-activated as symmetric acid anhydride, HOBt ester, or HOOBt ester is added to the resin.
  • the solvent for the activation of protected amino acids or condensation with the resin can be properly selected from among those solvents which are known to be useful for peptide condensation reactions.
  • solvents which are known to be useful for peptide condensation reactions.
  • N,N- di ethylformamide, N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide, DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran, acetonitrile, ethyl acetate, or suitable mixtures of them can be mentioned.
  • the reaction temperature can be selected from the range hitherto-known to be useful for peptide bond formation and is usually selected from the range of about -20°C - 50°C.
  • the activated amino acid derivative is generally used in a proportion of 1.5-4 fold excess. If the condensation is found to be insufficient by a test utilizing the ninhydrin reaction, the condensation reaction can be repeated to achieve a sufficient condensation without removing the protective group. If repeated condensation still fails to provide a sufficient degree of condensation, the unreacted amino group can be acetylated with acetic anhydride or acetylimidazole .
  • the protecting group of amino group for the starting material amino acid includes Z, Boc, tertiary- amyloxycarbonyl, isobornyloxycarbonyl, 4- ethoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl , diphenylphosphinothioyl, or Fmoc .
  • the carboxy-protecting group that can be used includes but is not limited to the above-mentioned C ⁇ _ 6 alkyl, C 3 _ 8 cycloalkyl and C 7 .
  • 1A aralkyl as well as 2- adamantyl, 4-nitrobenzyl , 4-methoxybenzyl , 4- chlorobenzyl, phenacyl, benzyloxycarbonylhydrazido, tertiary-butoxycarbonylhydrazido, and tritylhydrazido .
  • the hydroxy group of serine and threonine can be protected by esterification or etherification .
  • the group suited for said esterification includes carbon- derived groups such as lower alkanoyl groups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc., benzyloxycarbonyl, and ethoxycarbonyl .
  • the group suited for said etherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.
  • the protective group for the phenolic hydroxyl group of tyrosine includes Bzl, Cl 2 -Bzl, 2-nitrobenzyl, Br-Z, and tertiary-butyl.
  • the protecting group for imidazole moiety of histidine includes Tos, 4-methoxy-2 , 3 , 6- trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc .
  • the activated carboxyl group of the starting amino acid includes the corresponding acid anhydride, azide, and active esters, e.g. esters with alcohols such as pentachlorophenol, 2 , 4 , 5-trichlorophenol, 2,4- dinitrophenol, cyanomethyl alcohol, p-nitrophenol ,
  • the activated amino group of the starting amino acid includes the corresponding phosphoramide .
  • the method for elimination of protective groups includes catalytic reduction using hydrogen gas in the presence of a catalyst such as palladium black or palladium-on-carbon, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture of such acids, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, reduction with sodium metal in liquid ammonia.
  • a catalyst such as palladium black or palladium-on-carbon
  • acid treatment with anhydrous hydrogen fluoride methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture of such acids
  • base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine reduction with sodium metal in liquid ammonia.
  • the elimination reaction by the above- mentioned acid treatment is generally carried out at a temperature of -20°C - 40°C and can be conducted advantageously with addition of a cation acceptor such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethyl sulfide, 1 , 4-butanedithiol, 1 , 2-ethanedithiol .
  • a cation acceptor such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethyl sulfide, 1 , 4-butanedithiol, 1 , 2-ethanedithiol .
  • the 2 , 4-dinitrophenyl group used for protecting the imidazole group of histidine can be eliminated by treatment with thiophenol, while the formyl group used for protecting the indole group of tryptophan can be eliminated by alkali treatment with dilute sodium hydroxide solution or dilute aqueous ammonia as well as the above-mentioned acid treatment in the presence of 1 , 2-ethanedithiol , 1 , 4-butanedithiol .
  • the method for protecting functional groups which should not take part in the reaction of the starting material, the protective groups that can be used, the method of removing the protective groups, and the method of activating the functional groups that are to take part in the reaction can all be selected from among the known groups and methods .
  • An another method for obtaining the amide form of the polypeptide comprises amidating the ot-carboxyl group of the C-terminal amino acid at first, then extending the peptide chain to the N-side until the desired chain length, and then selectively deprotecting the ct-amino group of the C-terminal peptide and the - carboxy group of the amino acid or peptide that is to form the remainder of the objective polypeptide and condensing the two fragments whose -amino group and side-chain functional groups have been protected with suitable protective groups mentioned above in a mixed solvent such as that mentioned hereinbefore.
  • the parameters of this condensation reaction can be the same as described hereinbefore.
  • the ot- carboxyl group of the C-terminal amino acid is condensed with a desired alcohol to give an amino acid ester and then, the procedure described above for production of the amide is followed.
  • the ligand polypeptide of the present invention can be any peptide that has the same activities, e.g. pituitary function modulating activity, central nervous system function modulating activity, or pancreatic function modulating activity as the polypeptide which has an amino acid sequence of SEQ ID N0:1 or substantial equivalent thereto.
  • the ligand polypeptide or partial peptide thereof can be used as antigen for preparation of anti-ligand polypeptide antibody.
  • the preferable polypeptide as antigen includes (1) Ser-Arg-Ala-His-Gln-His-Ser-Met-Glu (SEQ ID NO:7),
  • the ligand peptide may be a peptide containing each of the domains which can act as an antigenic determinant or a peptide containing a plurality of the domains within the molecule.
  • the ligand peptide mentioned in this specification may be one ending with an amide bond (-CONH 2 ) or an ester bond (-COOR) at the C-terminus.
  • the ester here includes the same one of the above polypeptide.
  • the ester here may be of the same one as the above-mentioned ester at the C-terminus .
  • the ligand polypeptide or its partial peptide of the present invention may be in the form of a fused protein which is fused with a protein whose functions or properties are already known.
  • the salt of such partial peptide of the ligand polypeptide of present invention may be of the same one as the above-mentioned salt of the polypeptide.
  • the partial peptide of the ligand polypeptide of the invention, its amide or ester, or a salt thereof can be produced by the same synthetic processes as mentioned for the polypeptide or by cleaving the polypeptide of the present invention with a suitable peptidase .
  • the DNA coding for the ligand polypeptide or a partial peptide thereof of the present invention may be any DNA comprising the nucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO: 1
  • the DNA may be any of genomic DNA, genomic DNA library, tissue- or cell-derived cDNA, tissue- or cell-derived cDNA library, and synthetic DNA.
  • the vector for such a library may be any of bacteriophage, plasmid, cosmid, and phagimid.
  • it can be directly amplified by the RT-PCR (reverse transcription polymerase chain reaction) method by using an RNA fraction may be prepared from a tissue or cells.
  • the DNA coding for a polypeptide comprising the amino acid sequence of SEQ ID N0:1 the cDNA comprising the nucleotide sequence of SEQ ID NO: 2 or the genomic DNA comprising the nucleotide sequence of SEQ ID NO: 3 can be exemplified.
  • DNAs coding for the mouse-derived polypeptide comprising the amino acid sequence of SEQ ID N0:1 DNA fragments comprising partial nucleotide sequences of 6 to 90, preferably 6 to 60, more preferably 9 to 30, and especially preferably 12 to 30 can be advantageously used as DNA probes as well.
  • the DNA coding for the ligand polypeptide of the present invention can be produced by the following genetic engineering procedures .
  • the DNA fully encoding the polypeptide or partial peptide of the present invention can be cloned either by PCR amplification using synthetic DNA primers having a partial nucleotide sequence of the polypeptide or partial peptide or by hybridization using the DNA inserted in a suitable vector and labeled with a DNA fragment comprising a part or full region of a murine- derived polypeptide or a synthetic DNA.
  • the hybridization can be carried out typically by the procedure described in Molecular Cloning (2nd ed . , J. Sambrook et al . , Cold Spring Harbor Lab. Press, 1989). When a commercial library is used, the instructions given in the accompanying manual can be followed.
  • the cloned DNA coding for the polypeptide or partial peptide can be used directly or after digestion with a restriction enzyme or addition of a linker depending on purposes .
  • This DNA has ATG as the translation initiation codon at the 5' end and may have TAA, TGA, or TAG as the termination codon at the 3' end.
  • the translation initiation and termination codons can be added by means of suitable DNA adapters.
  • An expression vector for the polypeptide or partial peptide can be produced by, for example (a) cutting out a target DNA fragment from the DNA for the polypeptide or partial peptide of the present invention and (b) ligating the target DNA fragment with the downstream side of a promoter in a suitable expression vector.
  • the vector may include plasmids derived from Escherichia coli, e.g., pBR322, pBR325, pUC12, pUC13, etc.; plasmids derived from Bacillus subtilis , e.g., pUBHO, pTP5, pC194, etc.; plasmids derived from yeasts e.g., pSH19, pSH15, etc.; bacteriophages such as 1 - phage, and animal viruses such as retrovirus, vaccinia virus and baculovirus .
  • any promoter can be used as long as it is compatible with the host cell which is used for expressing a gene.
  • the promoters are preferably trp promoters, lac promoters, recA promoters, ⁇ PL promoters, lpp promoters, etc.
  • the promoters are preferably SP01 promoters, SP02 promoters, penP promoters, etc.
  • the promoters are preferably PH05 promoters, PGK promoters, GAP promoters, ADH promoters, etc.
  • the promoters include SV40-derived promoters, retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus (CMV) promoters, SR ⁇ promoters, etc.
  • An enhancer can be effectively utilized for expression.
  • a host-compatible signal sequence is added to the N-terminal side of the polypeptide or partial peptide thereof.
  • the utilizable signal sequences may include alkaline phosphatase signal sequence, OmpA signal sequence, etc.
  • the host is Bacillus , they may include ⁇ -amylase signal sequence, subtilisin signal sequence, etc.
  • the host is a yeast, they may include mating factor ⁇ signal sequence, invertase signal sequence, etc.
  • the host is an animal cell, they may include insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc.
  • a transformant or transfeetant is produced by using the vector thus constructed, which carries the polypeptide- or partial peptide-encoding DNA of the present invention.
  • the host may be, for example, Escherichia microorganisms, Bacillus microorganisms, yeasts, insect cells, animal cells, etc.
  • Escherichia and Bacillus microorganisms include Escherichia coli K12OH1 [Proc. Natl. Acad. Sci. USA, Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], JA221 [Journal of Molecular Biology, Vol.
  • Bacillus microorganism examples include Bacillus subtilis MI114 [Gene, Vol. 24, 255 (1983)], 207-21 [Journal of Biochemistry, Vol. 95, 76 (1984)], etc.
  • the yeast may be, for example, Saccharomyces cerevisiae AH22, AH22R " , NA87-11A, DKD-5D, 20B-12, etc.
  • the insect may include a silkworm ( Bombyx mori larva) , [Maeda et al, Nature, Vol. 315, 592 (1985)] etc.
  • the host animal cell may be, for example, monkey-derived cell line, COS-7, Vero, Chinese hamster ovary cell line (CHO cell), DHFR gene-deficient Chinese hamster cell line (dhfr " CHO cell), mouse L cell, mouse myeloma cell, human FL, etc. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells.
  • Transformation of Escherichia microorganisms can be carried out in accordance with the methods as disclosed in, for example, Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972), Gene, Vol. 17, 107 (1982), etc. Transformation of Bacillus microorganisms can be carried out in accordance with the methods as disclosed in, for example, Molecular & General Genetics, Vol. 168, 111 (1979), etc. Transformation of the yeast can be carried out in accordance with the methods as disclosed in, for example, Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978), etc. The insect cells can be transformed in accordance with the methods as disclosed in, for example, Bio/Technology, 6, 47-55, 1988.
  • the animal cells can be transformed by the methods as disclosed in, for example. Virology, Vol. 52, 456, 1973, etc.
  • the transformants or transfectants which harbor the expression vector carrying DNA encoding a polypeptide or partial peptide thereof are produced according to the aforementioned techniques .
  • Cultivation of the transformant ( transfectant ) in which the host is Escherichia or Bacillus microorganism can be carried out suitably in a liquid culture medium.
  • the culture medium may contains carbon sources, nitrogen sources, minerals, etc. necessary for growing the transformant.
  • the carbon source may include glucose, dextrin, soluble starch, sucrose, etc.
  • the nitrogen source may include organic or inorganic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extracts, bean-cakes, potato extracts, etc.
  • the minerals may include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc. It is further allowable to add yeast extracts, vitamines , growth-promoting factors, etc. It is desired that the culture medium is pH from about 5 to about 8.
  • the Escherichia microorganism culture medium is preferably an M9 medium containing, for example, glucose and casa ino acids (Miller, Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York, 1972. Depending on necessity, the medium may be supplemented with drugs such as 3 ⁇ -indolyl acrylic acid in order to improve efficiency of the promoter.
  • the cultivation is carried out usually at about 15 to 43°C for about 3 to 24 hours.
  • the cultivation is carried out usually at about 30 to 40°C for about 6 to 24 hours.
  • aeration and stirring may be also applied.
  • the culture medium used may include, for example, a Burkholder minimum medium [Bostian, K.L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)], an SD medium containing 0.5% casamino acids [Bitter, G.A. et al . , Proc. Natl. Acad. Sci. USA, Vol. 81, 5330 (1984)], etc.
  • the pH of the culture medium is adjusted to be from about 5 to about 8.
  • the cultivation is carried out usually at about 20 to 35°C for about 24 to 72 hours.
  • aeration and stirring may be applied.
  • the culture medium used may include those obtained by suitably adding additives such as passivated (or immobilized) 10% bovine serum and the like to the Grace's insect medium (Grace, T.C.C., Nature, 195, 788 (1962)).
  • the pH of the culture medium is adjusted to be about 6.2 to 6.4.
  • the cultivation is usually carried out at about 27°C for about 3 to 5 days. As desired, aeration and stirring may be applied.
  • the culture medium used may include MEM medium [Science, Vol. 122, 501 (1952)], DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association, Vol. 199, 519 (1967)], 199 medium [Proceedings of the Society of the Biological Medicine, Vol. 73, 1 (1950)], etc. which are containing, for example, about 5 to 20% of fetal calf serum. It is preferable that the pH is from about 6 to about 8.
  • the cultivation is usually carried out at about 30 to 40°C for about 15 to 60 hours. As required, medium exchange, aeration and stirring may be applied.
  • the microorganisms or cells are collected by known methods after the cultivation, suspended in a suitable buffer solution, disrupted by ultrasonic waves, lysoz me and/or freezing and thawing, etc. and, then, a crude extract of the polypeptide or partial peptide is obtained by centrifugation or filtration.
  • a suitable buffer solution may contain a protein- denaturing agent such as urea or guanidine hydrochloride or a surfactant such as Triton X-100 (registered trademark, hereinafter often referred to as "TM”) .
  • TM Triton X-100
  • the polypeptide or partial peptide is secreted into culture media
  • supernatant liquid is separated from the microorganisms or cells after the cultivation is finished and the resulting supernatant liquid is collected by widely known methods .
  • the culture supernatant liquid and extract containing the polypeptide or partial peptide can be purified by a suitable combination of widely known methods for separation, isolation and purification.
  • the widely known methods of separation, isolation and purification may include methods which utilizes solubility, such as salting out or sedimentation with solvents, methods which utilizes chiefly a difference in the molecular size or weight, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in the electric charge, such as ion-exchange chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing a difference in the hydrophobic property, such as reverse-phase high-performance liquid chromatography, and methods utilizing a difference in the isoelectric point such as isoelectric electrophoresis, or chromatofocusing, etc.
  • solubility such as salting out or sedimentation with solvents
  • methods which utilizes chiefly a difference in the molecular size or weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis
  • methods utilizing a difference in the electric charge such as ion-exchange chromat
  • the free protein can be converted into a salt thereof by known methods or method analogous thereto.
  • the protein salt can be converted into a free form or into any other salt thereof by known methods or method analogous thereto .
  • the polypeptide or partial peptide produced by the transformant can be arbitrarily modified or a polypeptide can be partly removed therefrom, by the action of a suitable protein-modifying enzyme before or after the purification.
  • the protein-modifying enzyme may include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase, etc.
  • the activity of the polypeptide or partial peptide thus formed can be measured by experimenting the coupling (or binding) with receptor or by enzyme immunoassays (enzyme linked immunoassays) using specific antibodies.
  • the DNA coding for the ligand polypeptide of the present invention, the ligand polypeptide or a partial peptide thereof can be used for (1) synthesis of a part or the full length of the ligand for G protein-coupled receptor protein, (2) search for the physiological activities of the ligand polypeptide or partial peptide thereof of the present invention, (3) preparation of a synthetic oligonucleotide probe or a PCR primer, (4) acquisition of DNAs coding for ligands of G protein- coupled receptor proteins and precursor proteins, (5) development of receptor-binding assay systems using the expression of recombinant receptor proteins and screening of candidates for medicinally active compounds, (6) acquisition of antibodies and antisera, (7) development of diagnostic agents utilizing said antibodies or antisera, (8) development of drugs such as pituitary
  • the DNA comprising the DNA cording the ligand polypeptide of this invention is useful for a production of a non-human transgenic animal or a knock out mouse and for analyzing a physiological function or a functional mechanism of the ligand polypeptide by using the non-human transgenic animal or the knock out mouse.
  • the ligand polypeptide, or the DNA encording either of them of the present invention is useful as a safe pharmaceutical composition of low toxic potential because it is recognized as a ligand by the G protein-coupled receptor protein expressed in the hypophysis, central nervous system and pancreatic ⁇ cells.
  • the ligand polypeptide, a partial peptide thereof, or the DNA encoding either of them of the present invention is associated with the modulation of pituitary function, central nervous system function, and pancreatic function and, therefore, can be used as a pharmaceutical composition for treatment or prevention of dementia such as senile dementia, cerebrovascular dementia (dementia due to cerebrovascular disorder), dementia associated with phylodegenerative retroplastic diseases (e.g.
  • Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, etc. dementia due to infectious diseases (e.g. delayed viral infections such as Creutzfelt-Jakob disease), dementia associated with endocrine, metabolic, and toxic diseases (e.g. hypothyroidism, vitamin B12 deficiency, alcoholism, and poisoning due to various drugs, metals, or organic compounds), dementia associated with oncogenous diseases (e.g. brain tumor), dementia due to traumatic diseases (e.g. chronic subdural hematoma):, depression (melancholia), hyperkinetic (microencephalo- pathy) syndrome, disturbance of consciousness, anxiety syndrome, schizophrenia, horror, growth hormone secretory disease (e.g.
  • infectious diseases e.g. delayed viral infections such as Creutzfelt-Jakob disease
  • dementia associated with endocrine e.g. hypothyroidism, vitamin B12 deficiency, alcoholism, and poisoning due to various drugs, metals, or organic compounds
  • gigantism acromegalic gigantism etc.
  • hyperphagia polyphagia
  • hypercholesterolemia hyperglyceridemia
  • hyperlipemia hyperprolactinemia
  • diabetes e.g. diabetic complications, diabetic nephropathy, diabetic neurophathy, diabetic retinopathy etc.
  • cancer e.g. mammary cancer, lymphatic leukemia, cystic cancer, ovary cancer, prostatic cancer etc.
  • pancreatitis e.g.
  • chromic renal failure nephritis etc.
  • Turner's syndrome neurosis, rheumatoid arthritis, spinal injury, transient brain ischemia, amyotrophic lateral sclerosis, acute myocardial infarction, spinocerebellar degeneration, bone fracture, trauma, atopic dermatitis, osteoporosis, asthma, epilepsy, infertility or oligogalactia .
  • they can be also used as the agent for improvement in postoperative nutritional status and/or vasopressor .
  • the polypeptide, or the DNA encoding either of them of the present invention can be used by conventional methods.
  • it can be used orally in the form of tablets which may be sugar coated as necessary, capsules, elixirs, microcapsules etc., or non-orally in the form of injectable preparations such as aseptic solutions and suspensions in water or other pharmaceutically acceptable liquids.
  • injectable preparations such as aseptic solutions and suspensions in water or other pharmaceutically acceptable liquids.
  • These preparations can be produced by mixing the polypeptide, a partial peptide thereof, or the DNA encoding either of them with physiologically acceptable carriers, flavoring agents, excipients, vehicles, antiseptics, stabilizers, binders etc. in unit dosage forms required for generally accepted manners of pharmaceutical manufacture.
  • Additives which can be mixed in tablets, capsules etc. include binders such as gelatin, corn starch, tragacanth and gum arabic , excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose and saccharin, and flavoring agents such as peppermint, akamono oil and cherry.
  • the unit dosage form is a capsule, the above-mentioned materials may further incorporate liquid carriers such as oils and fats .
  • Sterile compositions for injection can be formulated by ordinary methods of pharmaceutical manufacture, for example, by dissolving or suspending active ingredients, naturally occuring vegetable oils such as sesame oil and coconut oil, etc. in vehicles such as water for injection.
  • Aqueous liquids for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents, e.g., D-sorbitol, D-mannitol and sodium chloride, and may be used in combination with appropriate dissolution aids such as alcohols, e.g., ethanol, polyalcohols , e.g., propylene glycol and polyethylene glycol, nonionic surfactants, e.g., polysorbate 80 (TM) and HCO-50 etc.
  • Dissolution aids such as alcohols, e.g., ethanol, polyalcohols , e.g., propylene glycol and polyethylene glycol, nonionic surfactants, e.g., polysorbate 80 (TM) and HCO-50 etc.
  • Oily liquids include sesame oil and soybean oil, and may be used in combination with dissolution aids such as benzyl benzoate and benzyl alcohol .
  • the above- mentioned materials may also be formulated with buffers, e.g., phosphate buffer and sodium acetate buffer; soothing agents, e.g., benzalkonium chloride, procaine hydrochloride; stabilizers, e.g., human serum albumin, polyethylene glycol; preservatives, e.g., benzyl alcohol, phenol; antioxidants etc.
  • buffers e.g., phosphate buffer and sodium acetate buffer
  • soothing agents e.g., benzalkonium chloride, procaine hydrochloride
  • stabilizers e.g., human serum albumin, polyethylene glycol
  • preservatives e.g., benzyl alcohol, phenol
  • antioxidants e.g., antioxidants etc.
  • the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or warm-blooded mammals, e.g., mouse, rats, guinea pig, rabbits, chicken, sheep, pigs, bovines , cats, dogs, monkeys, baboons, chimpanzees, for instance .
  • warm-blooded mammals e.g., mouse, rats, guinea pig, rabbits, chicken, sheep, pigs, bovines , cats, dogs, monkeys, baboons, chimpanzees, for instance .
  • the dose of said polypeptide, or the DNA encoding either of them is normally about 0.1-100 mg, preferably 1.0-50 mg, and more preferably 1.0-20 mg per day for an adult (weighing 60 kg) in oral administration, depending on symptoms etc.
  • the G protein-coupled receptor protein for the above ligand polypeptide of the present invention may be any of G protein-coupled receptor proteins derived from various tissues, e.g. hypophysis, pancreas, brain, kidney, liver, gonad, thyroid gland, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, alimentary canal, blood vessel, heart, etc. of human and other warm-blooded animals, e.g. guinea pig, rat, mouse, swine, sheep, bovine, monkey, etc.; and comprising an amino acid sequence of SEQ ID NO: 10, 11, 12, 18 or 14, or a substantial equivalent thereto.
  • the G protein-coupled receptor protein of the present invention includes, in addition to a protein comprising the SEQ ID NO: 10, 11, 12, 13 or 14, those proteins comprising amino acid sequences of about 90-99.9% homology to the amino acid sequence of SEQ ID NO: 10, 11, 12, 13 or 14 and having qualitatively substantially equivalent activity to proteins comprising the amino acid sequence of SEQ ID NO: 10, 11, 12, 13, or 14.
  • the activities which these proteins are possessed may include ligand binding activity and signal transduction activity.
  • the term "substantially equivalent" means that the nature of the ligand binding activity and the like is equivalent. Therefore, it is allowable that even differences among grades such as the strength of ligand binding activity and the molecular weight of receptor protein are present.
  • the G protein-coupled receptor proteins include human pituitary-derived G protein-coupled receptor proteins which comprises the amino acid sequence of SEQ ID NO: 10 or/and SEQ ID NO: 11, mouse pancreas-derived G protein-coupled receptor proteins which comprises the amino acid sequence of SEQ ID NO: 13, and mouse pancreas-derived G protein-coupled receptor proteins which comprises the amino acid sequence of SEQ ID NO: 14.
  • human pituitary-derived G protein-coupled receptor proteins which comprises the amino acid sequence of SEQ ID NO: 10 and/or SEQ ID NO: 11 include the human pituitary-derived G protein-coupled receptor protein which comprises the amino acid sequence of SEQ ID NO: 12.
  • the protein which comprises an amino acid sequence of SEQ ID NO: 12 or a substantial equivalent thereto contains the full-length of the amino acid sequence for human pituitary-derived G protein-coupled receptor protein.
  • the protein which comprises an amino acid sequence of SEQ ID NO: 10 or/and SEQ ID NO: 11 or a substantial equivalent thereto may be a partial peptide of the protein which comprises an amino acid sequence of SEQ ID NO: 12 or a substantial equivalent thereto.
  • the protein which comprises an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 or a substantial equivalent thereto is a G protein-coupled receptor protein which is derived from mouse pancreas but, since its amino acid sequence is quite similar to the amino acid sequence of SEQ ID NO: 10 or/and SEQ ID NO: 11, the protein which comprises an amino acid sequence of SEQ ID NO: 13 or 14 or a substantial equivalent thereto is also, subsumed in the category of said partial peptide of the protein which comprises an amino acid sequence of SEQ ID NO: 12 or a substantial equivalent thereto.
  • the above-mentioned protein comprising an amino acid sequence of SEQ ID NO: 12 or a substantial equivalent thereto or a partial peptide of the protein or a salt thereof, which will be described below, includes the protein comprising an amino acid sequence of SEQ ID NO: 10, 11, 12, or 13 or a substantial equivalent thereto, or a salt thereof.
  • the G protein-coupled receptor protein includes the protein in which the N-terminal Met has been protected with a protective group, e.g.
  • C ⁇ _ 6 acyl such as formyl or acetyl
  • the protein in which the N-terminal side of Gin has been cleaved in vivo to form pyroglutamyl the protein in which the side chain of any relevant constituent amino acid has been protected with a suitable protective group, e.g. C ⁇ _ 6 acyl such as formyl or acetyl
  • the complex protein such as glycoproteins available upon attachment of sugar chains.
  • the salt of G protein-coupled receptor protein includes the same kinds of salts as mentioned for the ligand polypeptide.
  • the G protein-coupled receptor protein or a salt thereof or a partial peptide thereof can be produced from the tissues or cells of human or other warm- blooded animals by per se known purification techniques or, as described above, by culturing a transformant carrying a DNA coding for the G protein-coupled receptor protein. It can also be produced in accordance with the procedures for peptide synthesis which are described above. The production method is described in, for example, Examples 3, 4, 6 and 17 of WO96/05302, in detail.
  • the DNA coding for the G protein-coupled receptor protein may be any DNA comprising a nucleotide sequence encoding the G protein-coupled receptor protein which comprises an amino acid sequence of SEQ ID NO: 10, 11, 12, 13, or 14 or a substantial equivalent thereto. It may also be any one of genomic DNA, genomic DNA library, tissue- or cell-derived cDNA, tissue- or cell- derived cDNA library, and synthetic DNA.
  • the vector for such a library may include bacteriophage, plasmid, cosmid, and phargimid.
  • a direct amplification can be carried out by the RT-PCR method.
  • the DNA encoding the human pituitary-derived G protein-coupled receptor protein which comprises the amino acid sequence of SEQ ID NO: 10 include a DNA which comprises the nucleotide sequence of SEQ ID NO: 15.
  • the DNA encoding the human pituitary- derived G protein-coupled receptor protein which comprises the amino acid sequence of SEQ ID NO: 11 include a DNA which comprises the nucleotide sequence of SEQ ID NO: 16.
  • the DNA encoding the human pituitary- derived G protein-coupled receptor protein which comprises the amino acid sequence of SEQ ID NO: 12 include a DNA which comprises the nucleotide sequence of SEQ ID NO: 17.
  • the DNA encoding the mouse pancreas- derived G protein-coupled receptor protein which comprises the amino acid sequence of SEQ ID NO: 13 include a DNA which comprises the nucleotide sequence of SEQ ID NO: 18.
  • the DNA encoding the mouse pancreas- derived G protein-coupled receptor protein which comprises the amino acid sequence of SEQ ID NO: 14 include a DNA comprising the nucleotide sequence of SEQ ID NO: 19.
  • a method for cloning the DNA completely coding for the G protein-coupled receptor protein, vector, promoter, host cell, a method for transformation, a method for culturing the transformant or a method for separation and purification of the G protein-coupled receptor protein may include the same one as mentioned for the ligand polypeptide.
  • the G protein-coupled receptor protein-encoding DNA can be used as a prophylactic and/or therapeutic agent for treating said ligand polypeptide deficiency diseases depending upon the action that said ligand exerts .
  • the amount of the G protein-coupled receptor protein or ligand polypeptide in the brain cells of said patient can be increased whereby the action of the ligand can be fully achieved by:
  • the G protein-coupled receptor protein or ligand polypeptide-encoding DNA can be used as a safe and less toxic preventive and therapeutic agent for the G protein- coupled receptor protein or ligand polypeptide deficiency diseases .
  • said DNA may be used alone or after inserting it into a suitable vector such as retrovirus vector, adenovirus vector, adenovirus-associated virus vector, etc. followed by subjecting the product vector to a conventional means which is the same means as using the DNA coding for the ligand polypeptide or partial peptide thereof as the pharmaceutical composition.
  • a suitable vector such as retrovirus vector, adenovirus vector, adenovirus-associated virus vector, etc.
  • the ligand polypeptide that has a binding property for a G protein-coupled receptor protein or a partial peptide thereof, or a salt thereof is capable of determining quantitatively an amount of a G protein- coupled receptor protein or a partial peptide thereof, or a salt thereof in vivo with good sensitivity.
  • This quantitative determination may be carried out by, for example, combining with a competitive analysis.
  • a sample to be determined is contacted with the ligand polypeptide so that the concentration of a G protein-coupled receptor protein or a partial peptide thereof in said sample can be determined.
  • the protocols described in the following 1) and 2) or methods similar thereto may be used:
  • Ligand Polypeptide or salt thereof (hereinafter sometimes referred to briefly as ligand or ligand polypeptide) with the G protein- coupled receptor Protein
  • G protein-coupled receptor proteins or partial peptide or salt thereof can be used.
  • expression systems for recombinant G protein-coupled receptor proteins are constructed and receptor binding assay systems using said expression system are used.
  • assay systems it is possible to screen compounds, e.g. peptides, proteins, nonpeptidic compounds, synthetic compounds, formentation products, cell extracts, animal tissue extracts, etc.; or salts thereof which change the binding activity of a ligand polypeptide with the G protein-coupled receptor protein.
  • Such a compound includes a compound exhibiting a G protein-coupled receptor-mediated cell stimulating activity, e.g.
  • the present invention provides a method of screening for a compound which changes the binding activity of a ligand with a G protein-coupled receptor protein or a salt thereof, characterized by comparing the following two cases: (i) the case wherein the ligand is contacted with the G protein-coupled receptor protein or salt thereof, or the partial peptide thereof or a salt thereof; and (ii) the case wherein the ligand is contacted with a mixture of the G protein-coupled receptor protein or salt thereof or the partial peptide or salt thereof and said test compound.
  • one characteristic feature of the present invention resides in that the amount of the ligand bonded with said G protein-coupled receptor protein or the partial peptide thereof, the cell stimulating activity of the ligand, etc. are measured in both the case where (i) the ligand polypeptide is contacted with G protein-coupled receptor proteins or partial peptide thereof and in the case where (ii) the ligand polypeptide and the test compound are contacted with the G protein-coupled receptor protein or the partial peptide thereof, respectively and then compared therebetween.
  • a method of screening for a compound or a salt thereof which changes the binding activity of a ligand polypeptide with a G protein-coupled receptor protein characterized in that, when a labeled ligand polypeptide is contacted with a G protein-coupled receptor protein or a partial peptide thereof and when a labeled ligand polypeptide and a test compound are contacted with a G protein-coupled receptor protein or a partial peptide thereof, the amounts of the labeled ligand polypeptide bonded with said protein or a partial peptide thereof or a salt thereof are measured and compared;
  • a ligand polypeptide of the present invention, etc. is contacted with cells containing G protein-coupled receptor proteins and when the G protein-coupled receptor protein-activating compound and a test compound are contacted with cells containing G protein-coupled receptor proteins, the resulting G protein-coupled receptor protein-mediated cell stimulting activities, e.g.
  • activities of promoting or activities of inhibiting physiological responses including liberation of arachidonic acid, liberation of acetylcholine, intracellular Ca 2+ liberation, intracellular cAMP production, intracellular cGMP production, production of inositol phosphate, changes in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, lowering of pH, activation of G protein, cell promulgation, etc.; are measured and compared; and
  • a method of screening for a compound or a salt thereof which changes the binding activity of a ligand polypeptide with a G protein-coupled receptor protein characterized in that, when a G protein-coupled receptor protein-activating compound, e.g. a ligand polypeptide of the present invention, etc.
  • G protein-coupled receptor protein-mediated cell stimulating activities e.g.
  • activities of promoting or activities of inhibiting physiological responses such as liberation of arachidonic acid, liberation of acetylcholine, intracellular Ca liberation, intracellular cAMP production, intracellular cGMP production, production of inositol phosphate, changes in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, lowering of pH, activation of G protein, and cell promulgation, etc.; are measured and compared.
  • the G protein-coupled receptor agonist or antagonist can be screened by, first, obtaining a candidate compound by using G protein-coupled receptor protein-containing cells, tissues or cell membrane fractions derived from mouse, rat or the like (primary screening) , then, making sure whether the candidate compound really inhibits the binding between human G protein-coupled receptor proteins and ligands (secondary screening).
  • primary screening G protein-coupled receptor protein-containing cells, tissues or cell membrane fractions derived from mouse, rat or the like
  • secondary screening Other receptor proteins inevitably exist and when the cells, the tissues or the cell membrane fractions are used, they intrinsically make it difficult to screen agonists or antagonists to the desired receptor proteins.
  • the human- derived G protein-coupled receptor protein By using the human- derived G protein-coupled receptor protein, however, there is no need of effecting the primary screening, whereby it is possible to efficiently screen a compound that changes the binding activity between a ligand and a G protein-coupled receptor. Additionally, it is possible to evaluate whether the compound that is screened is a G protein-coupled receptor agonist or a G protein-coupled receptor antagonist.
  • any product may be used so far as it contains G protein-coupled receptor proteins or partial peptides thereof although the use of a membrane fraction of mammalian organs is preferable.
  • human organs can be extremely scarce and, accordingly, G protein-coupled receptor proteins which are expressed in a large amount using a recombinant technique are suitable for the screening.
  • the above-mentioned method can be used.
  • the G protein-coupled receptor protein-containing cells or cell membrane fractions are used in the screening method, the above-mentioned method can be used.
  • a suitable G protein-coupled receptor fraction and a labeled ligand polypeptide are necessary.
  • the G protein-coupled receptor fraction it is preferred to use naturally occurring G protein-coupled receptors (natural type G protein-coupled receptors) or recombinant type G protein-coupled receptor fractions with the activity equivalent to that of the natural type G protein coupled reaction.
  • activity equivalent to means the same ligand binding activity, or the substantially equivalent ligand binding activity.
  • labeled ligand it is possible to use labeled ligands, labeled ligand amalogized compounds, etc.
  • labeled ligands labeled with [ 3 H], [ 123 I], [ C], [ 35 S], etc. and other labeled substances may be utilized.
  • G protein-coupled receptor protein- containing cells or cell membrane fractions are first suspended in a buffer which is suitable for the determining method to prepare the receptor sample in conducting the screening for a compound which changes the binding activity of the ligand with the G protein- coupled receptor protein.
  • a buffer which is suitable for the determining method to prepare the receptor sample in conducting the screening for a compound which changes the binding activity of the ligand with the G protein- coupled receptor protein.
  • any buffer such as Tris-HCl buffer or phosphate buffer of pH 4-10, preferably, pH 6-8 which does not inhibit the binding of the ligand with the receptor may be used.
  • a surface-active agent such as CHAPS
  • Tween 80 (Kao-Atlas, Japan), digitonin, deoxycholate, etc. and/or various proteins such as bovine serum albumin (BSA), gelatin, etc. may be added to the buffer with an object of decreasing the nonspecific binding.
  • BSA bovine serum albumin
  • a protease inhibitor such as PMSF, leupeptin, E-64 manufactured by Peptide Laboratory, Japan, pepstatin, etc. may be added with an object of inhibiting the decomposition of the receptor and the ligand by protease.
  • a labeled ligand in a certain amount (5,000 cpm to 500,000 cpm) is added to 0.01 ml to 10 ml of said receptor solution and, at the same time, 10 " M to 10 "10 M of a test compound coexists.
  • NBS nonspecific binding amount
  • a reaction tube to which a great excessive amount of an unlabeled test compounds is added is prepared as well.
  • the reaction is carried out at 0-50°C, preferably at 4-37 °C for 20 minutes to 24 hours, preferably 30 minutes to three hours. After the reaction, it is filtered through a glass fiber filter, a filter paper, or the like, washed with a suitable amount of the same buffer and the radioactivity retained in the glass fiber filter, etc.
  • a test compound in which the specific binding amount (B - NSB) obtained by subtracting the nonspecific binding amount (NSB) from the total binding amount (B) is, for example, less than 50% may be selected as a candidate ligand to the G protein-coupled receptor protein of the present invention .
  • the G protein-coupled receptor protein-mediated cell stimulating activity e.g. activities of promoting or activities of inhibiting physiological responses such as release of arachidonic acid, release of acetylcholine, intracellular Ca + increase, intracellular cAMP production, production of inositol phosphate, changes in the cell membrane potential, phosphorylation of intracullular proteins, activation of c-fos, lowering of pH, activation of G protein and cell proliferation, etc.; may be measured by known methods or by the use of commercially available measuring kits.
  • G protein-coupled receptor protein-containing cells are at first cultured in a multiwell plate or the like. In conducting the screening, it is substituted with a suitable buffer which does not show toxicity to fresh media or cells in advance, incubated under appropriate conditions and for a specified time after additing a test compound, etc. thereto. The resultant cells are extracted or the supernatant liquid is recovered and the resulting product is determined, preferably quantitatively, by each of the methods.
  • an assay may be carried out by adding an inhibitor against said decomposing enzyme. With respect to the activities such as an inhibitory action against cAMP production, it may be detected as an inhibitory action against the cAMP production in the cells whose fundamental production has been increased by forskolin or the like.
  • G protein-coupled receptor protein-expressing cells are naturally occurring G protein-coupled receptor protein (natural type G protein-coupled receptor protein) -containing cell lines or strains, e.g. mouse pancreatic ⁇ cell line, MIN6 , etc . , the above-mentioned recombinant type G protein- coupled receptor protein-expressing cell lines or strains, etc.
  • test compound examples include peptide, proteins, non-peptidic compounds, synthesized compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, serum, blood, body fluid, etc. Those compounds may be novel or known.
  • a kit for screening the compound which changes the binding activity of the ligand with the G protein- coupled receptor protein or a salt thereof comprises a G protein-coupled receptor protein or a partial peptide thereof, or G protein-coupled receptor protein- containing cells or cell membrane fraction thereof.
  • screening kit examples include as follows: 1. Reagent for Determining Ligand.
  • Hanks' Balanced Salt Solution manufactured by Gibco
  • This may be sterilized by filtration through a membrane filter with a 0.45 ⁇ m pore size, and stored at 4°C or may be prepared upon use.
  • CHO cells in which a G protein-coupled receptor protein is expressed are subcultured at the rate of 5 x 10 5 cells/well in a 12-well plate and cultured at 37 °C with a 5% C0 2 and 95% air atmosphere for two days to prepare the sample.
  • the product in a state of an aqueous solution is stored at 4°C or at -20°C and, upon use, diluted to 1 ⁇ M with a buffer for the measurement.
  • Ligand is dissolved in PBS containing 0.1% of bovine serum albumin (manufactured by Sigma) to make 1 mM and stored at -20°C.
  • CHO cells are cultured in a 12-well tissue culture plate to express G protein-coupled receptor proteins.
  • the G protein-coupled receptor protein-expressing CHO cells are washed with 1 ml of buffer for the measurement twice. Then 490 ⁇ l of buffer for the measurement is added to each well .
  • Five ⁇ l of a test compound solution of 10 3 to 1010 M is added, then 5 ⁇ l of a labeled ligand is added and subjected to reaction at room temperature for one hour. For knowing the non-specific binding amount, 5 ⁇ l of
  • the compound or a salt thereof obtained by the screening method or by the screening kit is a compound which changes the binding activity of a ligand polypeptide with a G protein-coupled receptor protein, wherein the compound inhibits or promotes the binding, and, more particularly, it is a compound having a cell stimulating activity mediated via a G protein-coupled receptor or a salt thereof, so-called "G protein- coupled receptor agonist” or a compound having no said stimulating activity, so-called “G protein-coupled receptor antagonist".
  • G protein-coupled receptor agonist a compound having no said stimulating activity
  • G protein-coupled receptor antagonist examples of said compound are peptides, proteins, non-peptidic compounds, synthesized compounds, fermentation products, etc. and the compound may be novel or known.
  • Said G protein coupled receptor agonist has the same physiological action as the ligand to the G protein-coupled receptor protein and, therefore, it is useful as a safe and less toxic pharmaceutical composition depending upon said ligand activity.
  • said G protein-coupled receptor antagonist is capable of inhibiting the physiological activity of the ligand to the G protein-coupled receptor protein and, therefore, it is useful as a safe and less toxic pharmaceutical composition for inhibiting said ligand activity.
  • the ligand polypeptide of the present invention relates to the pituitary function modulating action, central nervous system function modulating action or pancreatic function modulating action. Therefore, the above-mentioned agonist or antagonist can be used as a therapeutic and/or prophylactic agent for dementia such as senile dementia, cerebrovascular dementia (dementia due to cerebrovascular disorder) , dementia associated with phylodegenerative retroplastic diseases (e.g. Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, etc.), dementia due to infectious diseases (e.g. delayed viral infections such as Creutzfelt-Jakob disease) , dementia associated with endocrine, metabolic, and toxic diseases (e.g.
  • dementia such as senile dementia, cerebrovascular dementia (dementia due to cerebrovascular disorder) , dementia associated with phylodegenerative retroplastic diseases (e.g. Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, etc.), dementia due
  • hypothyroidism e.g., hypothyroidism, vitamin B12 deficiency, alcoholism, and poisoning due to various drugs, metals, or organic compounds
  • dementia associated with oncogenous diseases e.g. brain tumor
  • dementia due to traumatic diseases e.g. chronic subdural hematoma:, depression (melancholia) , hyperkinetic (microencephalo-pathy) syndrome, disturbance of consciousness, anxiety syndrome, schizophrenia, horror, growth hormone secretory disease (e.g.
  • gigantism acromegalic gigantism etc.
  • hyperphagia polyphagia
  • hypercholesterolemia hyperglyceridemia, hyperlipemia, hyperprolactinemia, hypoglycemia, pituitarism, pituitary drawfism
  • diabetes e.g. diabetic complications, diabetic nephropathy, diabetic neurophathy, diabetic retinopathy etc.
  • cancer e.g. mammary cancer, lymphatic leukemia, cystic cancer, ovary cancer, prostatic cancer etc.
  • pancreatitis e.g.
  • chromic renal failure nephritis etc.
  • Turner's syndrome neurosis, rheumatoid arthritis, spinal injury, transient brain ischemia, amyotrophic lateral sclerosis, acute myocardial infarction, spinocerebellar degeneration, bone fracture, trauma, atopic dermatitis, osteoporosis, asthma, epilepsy, infertility or oligogalactia .
  • the agonist or antagonist can be also used as hypnotic-sedative agent for improvement in postoperative nutritional status, vasopressor or depressor.
  • Antibodies e.g. polyclonal antibody, monoclonal antibody, and antisera against the ligand polypeptide may be manufactured by antibody- or antiserum- manufacturing methods per se known to those of skill in the art or methods similar thereto, using the ligand polypeptide as antigen.
  • polyclonal antibodies can be manufactured by the method as given below.
  • the above-mentioned polypeptide or protein as the antigen is coupled to a carrier protein.
  • the carrier protein may for example be bovine thyroglobulin, bovine serum albumin, bovine gamma-globulin, hemocyanine, or Freund's complete adjuvant (Difco) .
  • the coupling reaction between the antigen protein and the carrier protein can be carried out by the known procedure.
  • the reagent for use in the coupling reaction includes but is not limited to glutaraldehyde and water-soluble carbodiimide.
  • the suitable ratio of the antigen protein to the carrier protein is about 1:1 through about 1:10 and as to the reaction pH, satisfactory results are obtained in many cases when the reaction is carried out around neutral, particularly in the range of pH about 6-8.
  • the reaction time is preferably about 1 to 12 hours in many cases and more preferably about 2 to 6 hours .
  • the conjugate thus obtained is dialyzed against water at about 0 to 18 °C in the routine manner and stored frozen or optionally lyophilized and stored.
  • a warm-blooded animal is inoculated with the immunogen produced in the manner described hereinbefore.
  • the warm-blooded animal that can be used for this purpose includes mammalian warm-blooded animals, e.g. rabbit, sheep, goat, rat, mouse, guinea pig, bovine, equine, swine, etc.; and avian species, e.g. chicken, dove, duck, goose, quail, etc.
  • the inoculum size of the immunogen may be just sufficient for antibody production.
  • the desired antibody can be produced in many instances by emulsifying 1 mg of the immunogen in 1 ml of saline with Freund's complete adjuvant and injecting the emulsion subcutaneously at the back and hind-limb footpad of rabbits 5 times at 4-week intervals.
  • the blood is withdrawn from the auricular vein usually during day 7 through day 12 after the last inoculation dose and centrifuged to recover an antiserum.
  • the antiserum is generally subjected to affinity chromatography using a carrier to which each antigen peptide has been conjugated and the adsorbed fraction is recovered to provide a polyclonal antibody.
  • the monoclonal antibody can be produced by the following method.
  • the ligand polypeptide is administered to warmblooded animals either solely or together with carriers or diluents to the site where the production of antibody is possible by the administration.
  • complete Freund's adjuvants or incomplete Freund's adjuvants may be administered.
  • the administration is usually carried out once every two to six weeks and two to ten times in total. Examples of the applicable warm-blooded animals are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens and the use of mice and rats is preferred.
  • an animal wherein the antibody titer is noted is selected from warm-blooded animals (e.g. mice) immunized with antigens, then spleen or lymph node is collected after two to five days from the final immunization and antibody-producing cells contained therein are fused with myeloma cells to give monoclonal antibody-producing hybridomas.
  • Measurement of the antibody titer in antisera may, for example, be carried out by reacting a labeled ligand polypeptide or a labeled G protein-coupled receptor protein (which will be mentioned later) with the antiserum followed by measuring the binding activity of the labeling agent with the antibody.
  • the operation for fusing may be carried out, for example, by the method of Koehler and Milstein (Nature, 256 , 495, 1975).
  • the fusion accelerator are polyethylene glycol (PEG), Sendai virus, etc. and the use of PEG is preferred.
  • PEG polyethylene glycol
  • Sendai virus etc.
  • the myeloma cells are NS-1, P3U1, SP2/0, AP-1, etc. and the use of P3U1 is preferred.
  • the preferred fusion ratio of the numbers of antibody- producing cells used (spleen cells) to the numbers of myeloma cells is within a range of about 1:1 to 20:1.
  • PEG preferably, PEG 1000 to PEG 6000
  • concentration of about 10-80% followed by incubating at 20-40°C (preferably, at 30-37°C) for one to ten minutes, an efficient cell fusion can be carried out.
  • a supernatant liquid of hybridoma culture is added to a solid phase (e.g. microplate) to which the ligand polypeptide antigen is adsorbed either directly or with a carrier, then anti- immunoglobulin antibody (anti-mouse immunoglobulin antibody is used when the cells used for the cell fusion are those of mouse) which is labeled with a radioactive substance, an enzyme or the like, or protein A is added thereto and then anti-ligand polypeptide monoclonal antibodies bound on the solid phase are detected; or a supernatant liquid of the hybridoma culture is added to the solid phase to which anti-immunoglobulin or protein A is adsorbed, then the ligand polypeptide labeled with a radioactive substance or an enzyme is added and anti-ligand polypeptide monoclonal antibodies bonded with the solid phase is detected.
  • a supernatant liquid of the hybridoma culture is added to the solid phase to which anti-immunoglobulin or protein A is
  • Selection and cloning of the anti-ligand polypeptide monoclonal antibody-producing hybridoma may be carried out by methods per se known to those of skill in the art or methods similar thereto. Usually, it is carried out in a medium for animal cells, containing HAT (hypoxanthine, aminopterin and thymidine) . With respect to a medium for the selection, for the cloning and for the growth, any medium may be used so far as hybridoma is able to grow therein.
  • HAT hyperxanthine, aminopterin and thymidine
  • the medium examples include an RPMI 1640 medium (Dainippon Pharmaceutical Co., Ltd., Japan) containing 1-20% (preferably 10-20%) of fetal calf serum (FCS), a GIT medium (Wako Pure Chemical, Japan) containing 1-20% of fetal calf serum and a serum-free medium for hybridoma culturing (SFM-101; Nissui
  • the culturing temperature is usually 20-40°C and, preferably, about 37 °C.
  • the culturing time is usually from five days to three weeks and, preferably, one to two weeks.
  • the culturing is usually carried out in 5% carbon dioxide gas.
  • the antibody titer of the supernatant liquid of the hybridoma culture may be measured in the same manner as the above-mentioned measurement of the antibody titer of the anti-ligand polypeptide in the antiserum. (b) Purification of the Monoclonal Antibody.
  • the separation/purification of the anti-ligand polypeptide monoclonal antibody may be carried out by methods for separating/purifying immunoglobulin such as salting- out, precipitation with an alcohol, isoelectric precipitation, electrophoresis, adsorption/deadsorption using ion exchangers such as DEAE, ultracentrifugation, gel filtration, specific purifying methods in which only an antibody is collected by treatment with an active adsorbent such as an antigen-binding solid phase, protein A or protein G and the bond is dissociated whereupon the antibody is obtained.
  • an active adsorbent such as an antigen-binding solid phase, protein A or protein G
  • the ligand polypeptide antibody which is manufactured by the aforementioned method (a) or (b) is capable of specifically recognizing ligand polypeptide, accordingly, it can be used for a quantitative determination of the ligand polypeptide in test liquid samples and particularly for a quantitative determination by sandwich immunoassays.
  • the present invention provides, for example, the following methods:
  • a quantitative determination of a ligand polypeptide in a test liquid sample which comprises (a) competitively reacting the test liquid sample and a labeled ligand polypeptide with an antibody which reacts with the ligand polypeptide, and (b) measuring the ratio of the labeled ligand polypeptide binding with said antibody; and (ii) a quantitative determination of a ligand polypeptide in a test liquid sample, which comprises (a) reacting the test liquid sample with an antibody immobilized on an insoluble carrier and a labeled antibody simultaneously or continuously, and (b) measuring the activity of the labeling agent on the insoluble carrier wherein one antibody is capable of recognizing the N- terminal region of the ligand polypeptide while another antibody is capable of recognizing the C-terminal region of the ligand polypeptide.
  • anti-ligand polypeptide antibody (hereinafter, may be referred to as "anti-ligand polypeptide antibody”) is used, ligand polypeptide can be measued and, moreover, can be detected by means of a tissue staining, etc. as well.
  • antibody molecules per se may be used or F(ab') 2 .
  • Fab' or Fab fractions of the antibody molecule may be used too.
  • any measuring method may be used so far as it relates to a method in which the amount of antibody, antigen or antibody-antigen complex, depending on or corresponding to the amount of antigen, e.g. the amount of ligand polypeptide, etc.
  • Examples of the labeling agent used in the measuring method using the labeling substance are radioisotopes, enzymes, fluorescent substances, luminescent substances, colloids, magnetic substances, etc.
  • Examples of the radioisotope are [ I], [ I], [ H] and [ C]; preferred examples of the enzyme are those which are stable and with big specific activity, such as ⁇ -galactosidase, ⁇ -glucosidase, alkali phosphatase, peroxidase and malate dehydrogenase;
  • examples of the fluorescent substance are fluorescamine, fluorescein isothiocyanate, etc.;
  • examples of the luminescent substance are luminol, luminol derivatives, luciferin, lucigenin, etc.
  • a biotin-avidin system may also be used for binding an antibody or antigen with a labeling agent.
  • an insolubilization (immobilization) of antigens or antibodies a physical adsorption may be used or a chemical binding which is usually used for insolubilization or immobilization of proteins or enzymes may be used as well.
  • the carrier are insoluble polysaccharides such as agarose, dextran and cellulose; synthetic resins such as polystyrene, polyacrylamide and silicone; glass; etc.
  • the test liquid is subjected to reaction with an insolubilized anti-ligand polypeptide antibody (the first reaction), then it is subjected to reaction with a labeled anti- ligand polypeptide antibody (the second reaction) and the activity of the labeling agent on the insoluble carrier is measured whereupon the amount of the ligand polypeptide in the test liquid can be determined.
  • the first reaction and the second reaction may be conducted reversely or simultaneously or they may be conducted with an interval.
  • the type of the labeling agent and the method of insolubilization (immobilization) may be the same as those mentioned already herein.
  • the antibody used for the labeled antibody and the antibody for the solid phase is one type or one species but, with an object of improving the measuring sensitivity, etc., a mixture of two or more antibodies may be used too.
  • the preferred anti-ligand polypeptide antibodies used for the first and the second reactions are antibodies wherein their sites binding to the ligand polypeptide are different each other.
  • the antibodies used in the first and the second reactions are those wherein, when the antibody used in the second reaction recognizes the C-terminal region of the ligand polypeptide, then the antibody recognizing the site other than C-terminal regions, e.g. recognizing the N- terminal region, is preferably used in the first reaction.
  • the anti-ligand polypeptide antibody of the present invention may be used in a measuring system other than the sandwich method such as a competitive method, an immunometric method and a naphrometry.
  • a competitive method an antigen in the test solution and a labeled antigen are subjected to reaction with an antibody in a competitive manner, then an unreacted labeled antigen (F) and a labeled antigen binding with an antibody (B) are separated (i.e. B/F separation) and the labeled amount of any of B and F is measured whereupon the amount of the antigen in the test solution is determined.
  • liquid phase method in which a soluble antibody is used as the antibody and the B/F separation is conducted by polyethylene glycol, a second antibody to the above-mentioned antibody, etc.
  • solid phase method in which an immobilized antibody is used as the first antibody or a soluble antibody is used as the first antibody while an immobilized antibody is used as the second antibody.
  • an antigen in the test solution and an immobilized antigen are subjected to a competitive reaction with a certain amount of a labeled antibody followed by separating into solid and liquid phases; or the antigen in the test solution and an excess amount of labeled antibody are reacted, then a immobilized antigen is added to bind an unreacted labeled antibody with the solid phase and separated into solid and liquid phases. After that, the labeled amount of any of the phases is measured to determine the antigen amount in the test solution.
  • the amount of insoluble sediment which is produced as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the antigen amount in the test solution is small and only a small amount of the sediment is obtained, a laser nephrometry wherein scattering of laser is utilized can be suitably used.
  • a measuring system for ligand polypeptide may be constructed taking the technical consideration of the persons skilled in the art into consideration in the conventional conditions and operations for each of the methods. With details of those conventional technical means, a variety of reviews, reference books, etc. may be referred to. They are, for example, Hiroshi Irie (ed) : “Radioimmunoassay” (Kodansha, Japan, 1974); Hiroshi Irie (ed): "Radioimmunoassay; Second
  • the present invention further provides a non-human mammal harboring a foreign DNA coding for the ligand polypeptide of the present invention (hereinafter referred to briefly as foreign DNA) or a mutant thereof (sometimes referred to briefly as a foreign mutant DNA) .
  • foreign DNA a foreign DNA coding for the ligand polypeptide of the present invention
  • mutant thereof sometimes referred to briefly as a foreign mutant DNA
  • Examples of the DNA coding for the ligand polypeptide of the present invention is a DNA comprises the DNA having a nucleotide sequence encoding the polypeptide comprising the amino acid sequence represented by SEQ ID N0:1, or a substantial equivalent thereof (hereinafter, may simply referred as the DNA of the present invention) , such as the DNA comprising the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the non-human mammal harboring the foreign DNA of the present invention or a foreign mutant DNA thereof can be constructed by transferring the objective DNA to a germinal cell such as an unfertilized egg cell, fertilized egg cell, or sperm cell or its primordial cell, preferably in the period of embryogenesis in the ontogenesis of a non- human mammal (more preferably in the stage of a single cell or a fertilized egg cell and generally at the 8- cell stage or earlier) , by the calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, or DEAE-dextran method.
  • a germinal cell such as an unfertilized egg cell, fertilized egg cell, or sperm cell or its primordial cell, preferably in the period of embryogenesis in the ontogenesis of a non- human mammal (more preferably in the stage of a single cell or a fertilized egg cell and generally at the 8- cell stage or earlier)
  • a non- human mammal
  • the DNA of the present invention can be introduced into somatic cells, various organs in the body, tissue cells and can be used for cell cultivation, tissue cultivation, etc. Moreover, these cells can be hybridized with above-mentioned germinal cell to produce the non-human transgenic mammal.
  • the non-human mammal used includes bovine, swine, sheep, goat, rabbit, canine, feline, guinea pig, hamster, murine, rat, and so on. From the standpoint of construction of a diseased animal model, rodents which have comparatively short ontogenesis and life cycles and can be easily bred, particularly mice (e.g. pure strains such as C57BL/6, DBA2 , etc. and hybrid strains such as B6C3F,, BDF,, B6D2F,, BALB/c, ICR, etc.) or rats (e.g. Wistar, SD, etc.) are preferred.
  • mice e.g. pure strains such as C57BL/6, DBA2 , etc. and hybrid strains such as B6C3F,, BDF,, B6D2F,, BALB/c, ICR, etc.
  • rats e.g. Wistar, SD, etc.
  • the "mammal" as mentioned with reference to the recombinant vector capable of being expressed in a mammal includes the same non-human mammals as those mentioned above and humans .
  • the foreign DNA of the present invention may be one derived from a mammal of the same species as the host animal or a mammal of a different species.
  • a DNA construct prepared by linking the DNA at downstream of a promoter capable of being expressed in animal cells For example, in transferring the human-derived DNA of the present invention, this human DNA of the present invention can be linked at downstream of a promoter capable of causing expression of DNAs derived from various animals (e.g.
  • a DNA construct e.g. a vector
  • a fertilized egg cell of a host mammal such as a fertilized murine egg cell
  • a transgenic mammal showing a high expression of the DNA of the present invention can be provided.
  • Examples of the expression vector used for the protein of the present invention are plasmids derived from E_. coli, plasmids derived from B. subtilis . plasmids of the yeast origin, ⁇ phage and other bacteriophages , retroviruses such as Molony leukemia virus, and animal viruses such as vaccinia virus and vaculovirus .
  • Preferable examples are plasmids of the
  • E_. coli origin plasmids of the B. subtilis origin, and yeast-derived plasmids .
  • the promoter for the regulation of the expression of the DNA are (1) promoters for DNAs derived from viruses (e.g. simian virus, cytomegalovirus , Molony leukemia virus, JC virus, papilloma virus, poliovirus, etc.), (2) promoters derived from mammals (e.g.
  • albumin insulin II, uroprakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S- transferase, platelet-derived growth factor ⁇ , keratin Kl, K10, and K14, collagen type I and type II, cyclic AMP-dependent protein kinase ⁇ l subunit, dystrophin, tartaric acid-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2 ) , sodium/potassium-exchanging adenosinetriphosphatase (Na , K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloprot
  • promoters are promoters conducive to high expression in the whole body, such as cytomegalovirus promoter, human polypeptide chain elongation factor l ⁇ (EF-l ⁇ ) promoter, and human and chicken ⁇ -actin promoters .
  • the vector preferably has a sequence for terminating the transcription of the objective mRNA (generally called terminator) in the transgenic mammal.
  • terminator a sequence for terminating the transcription of the objective mRNA
  • the examples of the sequence are sequences derived from viruses, various mammals. Preferable examples are the SV40 terminator derived from simian virus, and so on.
  • the translation region of the normal protein of the present invention can be obtained, as the whole or part of the genomic DNA, from the DNAs derived from the liver, kidney, or thyroid cells or fibroblasts of various mammals (e.g. rabbit, canine, feline, guinea pig, hamster, rat, murine, man, etc.) or from various commercial genomic DNA libraries, or starting with the complementary DNAs prepared from RNAs derived from the liver, kidney, thyroid cells or fibroblasts by the known technique.
  • the foreign abnormal DNA can be constructed by mutating the translated region of the normal protein obtained from the above-mentioned cells or tissues by the mutagenesis method.
  • the translated region can be prepared as a DNA construct which can be expressed in a transgenic animal, by the routine recombinant DNA technique, i.e. by coupling it at downstream of the promoter and, if desired, at upstream of the transcription termination site.
  • the transfer of the DNA of the present invention at the fertilized egg cell stage insures that the DNA will be ubiquitous in all the germ cells and somatic cells of the host mammal.
  • the presence of the DNA of the present invention in the germ cells of the transgenic animal following DNA transfer means that all the germ cells and somatic cells of all the progeny of the transgenic animal harbor the DNA of the present invention.
  • the offspring of animals of this line to which DNA is passed down have the DNA of the present invention in their germ cells and somatic cells.
  • the non-human mammal to which the foreign normal DNA of the present invention has been transferred can be verified by mating to retain the DNA stably and then bred as a strain harboring the transferred DNA from generation to generation under the usual breeding conditions.
  • the transfer of the DNA of the present invention in the fertilized egg cell stage is carried out in such a manner that the transferred DNA will be present in excess in all the germ cells and somatic cells of the transgenic animal.
  • the presence of an excess of the DNA of the present invention in the germ cells of the transgenic animal means that all the progeny of this line harbor an excess of the DNA of the present invention in their germ cells and somatic cells .
  • the non-human mammal harboring the normal DNA of the present invention features a high expression of the DNA and may eventually develop a hyperergasia of the protein of the present invention through activation of the function of the endogenous normal DNA and, therefore, can be utilized as an animal model of the disease.
  • the transgenic animal harboring the normal DNA of the present invention it is possible to study the hyperergasia of the protein of the present invention to elucidate the mechanisms of diseases with which the protein of the present invention is associated, and explore therapeutic modalities for the diseases.
  • the mammal to which the foreign normal DNA of the present invention has been transferred develops symptoms due to an increase in the free protein of the present invention and, therefore, can also be used in the screening of therapeutic drugs for diseases with which the protein of the present invention is associated.
  • the non-human mammal harboring the foreign abnormal DNA of the present invention can be verified by mating to retain the DNA stably and then bred as a line harboring the DNA from generation to generation under the usual breeding conditions .
  • the DNA construct with the promoter can be prepared by the routine recombinant DNA technique. Transfer of the abnormal DNA of the present invention in the fertilized egg cell stage insures that the transferred DNA will be ubiquitous in all the germ cells and somatic cells of the host mammal.
  • the presence of the abnormal DNA of the present invention in the germ cells of the transgenic animal means that all the offspring of this transgenic animal harbor the abnormal DNA of the present invention in all of their germ cells and somatic cells.
  • the progeny of this animal harbor the abnormal DNA of the present invention in all of their germ cells and somatic cells .
  • the non-human mammal harboring the abnormal DNA of the present invention features a high expression of the abnormal DNA and, therefore, may eventually develop adiaphoria associated with functional inactivation of the protein of the present invention through inhibition of the function of the endogenous normal DNA and, therefore, can be utilized as an animal model of the disease.
  • adiaphoria associated with functional inactivation of the protein of the present invention through inhibition of the function of the endogenous normal DNA and, therefore, can be utilized as an animal model of the disease.
  • analysis of the mechanism of this functional inactivation adiaphoria due to the protein of the present invention and therapeutic modalities for the disease can be explored.
  • the transgenic animal with a high expression of the abnormal DNA of the present invention can be used as a model for elucidating the functional inhibition of the normal protein by the abnormal protein of the present invention (dominant negative effect) in adiaphoria of functional inactivation type due to the protein of the present invention.
  • the transgenic mammal harboring the foreign abnormal DNA of the present invention develops symptoms due to an decrease in the protein of the present invention and, therefore, can be utilized in the screening of therapeutic compounds for adiaphoria due to functional inactivation of the protein of the present invention.
  • transgenic animal of the present invention clinical symptoms of diseases associated with the protein of the present invention, inclusive of said adiaphoria associated with functional inactivation of the protein of the present invention, can be investigated.
  • more detailed pathological findings can be generated in various organs of this model of diseases associated with the protein of the present invention, thus contributing to the development of new therapies and the study and treatment of secondary diseases arising from such diseases .
  • the present invention further provides a non-human mammalian embryonic stem cell wherein the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention wherein the DNA is inactivated.
  • the present invention therefore, provides:
  • the non-human mammalian embryonic stem cell according to (1) which is neomycin-resistant (3) the non-human mammalian embryonic stem cell according to (1) which is neomycin-resistant ; (4) the non-human mammalian embryonic stem cell according to (1) wherein the non-human mammal is a rodent; (5) the non-human mammalian embryonic stem cell according to (4) wherein the rodent is a mouse; (6) a non-human mammal deficient in expression of the
  • DNA of the present invention wherein the DNA is inactivated; (7) the non-human mammal according to (6) wherein the
  • DNA is inactivated by introduction of a reporter gene (e.g. a ⁇ -galactosidase gene of E.. coli origin) and the reporter gene can be expressed under the control of the promoter against the DNA of the present invention;
  • a reporter gene e.g. a ⁇ -galactosidase gene of E.. coli origin
  • (10) a method for screening for a compound or a salt thereof which enhances or inhibits an activity of the promoter against the DNA of the present invention, which comprises administering a test compound to the non-human mammal according to (7) and detecting an expression of the reporter gene.
  • non-human mammalian embryonic stem cell wherein the DNA of the present invention is inactivated means the embryonic stem cell (hereinafter referred to briefly as ES cell) of a non-human mammal in which the DNA has been deprived of the capacity to express the protein of the present invention (hereinafter referred to sometimes as the knockout DNA of the present invention) through introduction of an artificial mutation to the DNA of the present invention possessed by the non-human mammal to thereby inhibit expression of the DNA of the present invention or through substantial deprivation of the activity of the protein of the present invention which is encoded by the DNA.
  • ES cell embryonic stem cell
  • the knockout DNA of the present invention through introduction of an artificial mutation to the DNA of the present invention possessed by the non-human mammal to thereby inhibit expression of the DNA of the present invention or through substantial deprivation of the activity of the protein of the present invention which is encoded by the DNA.
  • the non-human mammal includes the same animals mentioned hereinbefore.
  • Examples of the method for introducing an artificial mutation to the DNA of the present invention are a deletion of some or all of the DNA sequence, or an insertion or substitution of a different DNA by the genetic engineering technology. By such a mutation, the codon reading frame can be shifted or the function of the promoter or exon can be disrupted to provide the knockout DNA of the present invention.
  • the non-human mammalian embryonic stem cell wherein the DNA of the present invention is inactivated can be prepared by, for example, a procedure which comprises isolating the DNA of the present invention from an objective non-human mammal, inserting a drug-resistance gene, typically the neoraycin- resistance gene or hygromycin-resistance gene, or a reporter gene such as lacZ ( ⁇ -galactosidase gene) or cat (chloramphenicol acetyltransferase gene) in its exon region to disrupt the function of the exon or inserting a DNA sequence for terminating gene transcription (e.g.
  • the targeting vector a DNA sequence adapted to eventually disrupt the gene
  • the targeting vector introducing the thus-constructed DNA chain having a DNA sequence adapted to eventually disrupt the gene into the chromosomes of the host animal by homologous recombination, subjecting the resulting ES cell to Southern hybridization analysis using the DNA sequence of the DNA of the present invention or in its vicinity as the probe or a PCR procedure using the DNA sequence of the targeting vector and a DNA sequence in the vicinity but not including the DNA of the present invention used in the construction of the targeting vector as primers, and selecting the knockout ES cell of the present invention.
  • the original ES cell used for inactivation of the DNA of the present invention by the homologous recombination technique or the like may be an already established cell line such as those mentioned hereinbefore or a new cell line established de novo by the known method of Evans and Kaufma.
  • C57BL/6 and DBA/2) for preparing pure-line ES cells with an immunologically defined genetic background can be used with advantage.
  • BDF mice have the background of C57BL/6 mice so that in the construction of a disease model with ES cells obtained, the genetic background of the model mice can be converted to that of C57BL/6 mice by back-crossing with C57BL/6.
  • blastocytes 3.5 days following fertilization but, aside from them, a large number of early embryos can be prepared with high efficiency by harvesting the embryos at the 8-cell stage and culturing them into blastocytes.
  • ES cells from both male and female animals can be employed, generally ES cells of a male animal are more convenient for the construction of reproduction line chimeras. Moreover, for the purpose of reducing the burden of the complicated cultural procedure, it is preferable to carry out sexing as early as possible.
  • ES cells As a typical method for sexing ES cells, there can be mentioned the method in which the gene in the sex determination region on the Y chromosome is amplified and detected by PCR. Whereas the conventional karyotype analysis requires about 10 cells, the above method requires only about one colony equivalent of ES cells (about 50 cells). Therefore, the primary selection of ES cells in an early stage can be made by this sexing method. Since male cells can thus be selected in the early stage, the trouble in the initial stage of culture can be drastically reduced.
  • the secondary selection can be carried out by G-banding for the number of chromosomes .
  • the embryonic stem cell line thus established is generally very satisfactory in proliferation characteristic but since it is liable to lose its ontogenic ability, it must be subcultured with sufficient care.
  • this cell line should be cultured on suitable feeder cells such as STO fibroblasts in the presence of LIF (1-10000 U/ml) in a carbon dioxide incubator (preferably 5% C0 2 -95% air or 5% oxygen-5% CO 2 -90% air) at about 37°C and, in subculture, it should be treated with trypsin/EDTA solution (generally 0.001-0.5% trypsin/0.1-5 mM EDTA, preferably about 0.1% trypsin/1 mM EDTA) to provide single cells and seed them on freshly prepared feeder cells.
  • suitable feeder cells such as STO fibroblasts in the presence of LIF (1-10000 U/ml) in a carbon dioxide incubator (preferably 5% C0 2 -95% air or 5% oxygen-5% CO 2 -90% air) at about 37°C and, in subculture, it should be treated with tryp
  • ES cells can be allowed to differentiate into various types of cells, such as head long muscle cells, visceral muscle cells, heart muscle cells, etc. by conducting monolayer culture to a high density under suitable conditions or suspension culture until a mass of cells is formed (M. J. Evans & M. H. Kaufman,
  • the non-human mammal deficient in expression of the DNA of the present invention can be differentiated from the normal animal by assaying the mRNA in the animals by the known method and comparing the amounts of expression indirectly.
  • the non-human mammal used for this purpose includes the animals mentioned hereinbefore.
  • the DNA of the present invention can be knocked out by introducing the targeting vector constructed as above into, for example, a murine embryonic stem cell or a murine egg cell and thereby causing the DNA sequence of the targeting vector harboring the inactivated DNA of the present invention to undergo homologous recombination with, and accordingly replacing, the DNA of the present invention on the murine embryonic stem cell or egg cell chromosomes.
  • the cell with the DNA of the present invention thus knocked out can be obtained by Southern hybridization analysis using a DNA sequence of the DNA of the present invention or in its vicinity as a probe or by PCR using a DNA sequence of the targeting vector or a murine-derived DNA sequence in a region adjacent to but not including the DNA of the present invention used in the targeting vector as primers .
  • a cell line with the DNA of the present invention knocked out by the homologous recombination technique is cloned and injected into the non-human mammalian embryo or blastocyte at a suitable stage of embryogenesis , for example at the 8-cell stage, and the resulting chimera embryo is transplanted in the pseudopregnant uterus of the non-human mammal .
  • the animal thus obtained is a chimera animal constituted by both the cells harboring the normal DNA of the present invention and the cells harboring the artificially mutated DNA of the present invention.
  • an individual of which the entire tissues are constituted by cells harboring the mutated DNA of the present invention can be screened from the colony of animals obtained by crossing such a chimera animal with a normal animal, for example by coat color discrimination.
  • the individuals thus selected are usually animals deficient in hetero-expression of the protein of the present invention and by mating such individuals deficient in hetero-expression of the protein of the present invention with each other, animals deficient in homo-expression of the protein of the present invention can be acquired.
  • a transgenic non-human mammal with the targeting vector having been introduced into its chromosomes can be prepared by injecting the DNA solution into the egg cell nucleus by the microinjection technique and selecting animals expressing a mutation of the DNA of the present invention by homologous recombination.
  • the individuals with the DNA of the present invention knocked out are mated to verify that the animals obtained by mating also have the DNA knocked out and they can be sub-bred under the usual breeding conditions . Preparation and maintenance of the reproduction line can also be carried out in the routine manner.
  • homozygotes having the inactivated DNA in both homologous chromosomes can be obtained.
  • the homozygotes thus obtained are bred under such conditions that, with regard to the dam, the number of homozygotes is plural per normal individual.
  • homozygotes and heterozygotes both harboring the inactivated DNA can be sub-bread .
  • the non-human mammalian embryonic stem cell harboring the inactivated DNA of the present invention is very useful for the construction of non-human mammals deficient in expression of the DNA of the present invention.
  • the mouse deficient in expression of the protein of the present invention lacks the various biological activities inducible by the protein of the present invention and can, there- fore, be of use as an animal model of diseases arising from inactivation of the biological activities of the protein of the present invention, thus being of use in the etiological studies of diseases and development of therapeutics .
  • the reporter gene is under the control of the promoter for the DNA of the present invention and, therefore, the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene .
  • ⁇ -galactosidase when part of the DNA region coding for the protein of the present present invention has been inactivated by Escherichia coli-derived ⁇ - galactosidase gene (lacZ), ⁇ -galactosidase is expressed in those tissues in which, the protein of the present invention would have been expressed. Therefore, the status of expression of the protein of the present invention in a living animal body can be traced, easily and expediently, for example, by the staining method using a reagent serving as a substrate for ⁇ - galactosidase, such as 5-bromo-4-chloro-3-indolyl- ⁇ - galactopyranoside (X-gal).
  • a reagent serving as a substrate for ⁇ - galactosidase such as 5-bromo-4-chloro-3-indolyl- ⁇ - galactopyranoside (X-gal).
  • a tissue section of a mouse defective in the protein of the present invention is fixed with glutaraldehyde or the like, washed with Dulbecco's phosphate-buffered saline (PBS), and reacted with a staining solution containing X-gal at room temperature or around 37 °C for about 30 minutes to 1 hour.
  • the tissue sample is then washed with 1 mM EDTA/PBS solution to terminate the ⁇ - galactosidase reaction and observed for color development.
  • the mRNA coding for lacZ may be detected by a conventional method.
  • the non-human mammals deficient in expression of the DNA of the present invention is very useful for screening the compounds which activate or inactivate the promoter of the polypeptide of the present invention, and can contribute to find the mechanism of the diseases derived from the deficiency of producing the polypeptide of the present invention or to develop the drug for treating such diseases .
  • Cytosine RNA Ribonucleic acid mRNA : Messenger ribonucleic acid
  • dATP Deoxyadenosine triphosphate
  • dTTP Deoxythymidine triphosphate
  • dGTP Deoxyguanosine triphosphate
  • dCTP Deoxycytidine triphosphate
  • A, Ala Alanine (or Alanyl)
  • I lie: Isoleucine (or Isoleucyl) S, Ser: Serine (or Seryl)
  • Threonine or Threonyl
  • Cys Cysteine (or Cysteinyl)
  • Trp Tryptophan (or Tryptophanyl)
  • HONB N-hydroxy-5-norbornene-2 , 3-dicarboxyimide
  • OcHex cyclohexyl ester
  • Bzl benzyl
  • Bom benzyloxymethyl
  • SEQ ID NO:l is an entire amino acid sequence of the murine pituitary-derived ligand polypeptide encoded by the cDNA included in pBOV3.
  • [SEQ ID NO: 2] is an entire nucleotide sequence of the murine pituitary-derived ligand polypeptide cDNA.
  • [SEQ ID NO: 3] is a genomic nucleotide sequence of the murine pituitary-derived ligand polypeptide cDNA.
  • [SEQ ID NO: 4] is an entire amino acid sequence of the matured murine pituitary-derived ligand polypeptide encoded by the cDNA included in pB0V3.
  • [SEQ ID NO: 5] is an amino acid sequence of the antigen which can be used for preparation of the anti-ligand polypeptide antibody.
  • [SEQ ID NO: 6] is an amino acid sequence of the antigen which can be used for preparation of the anti-ligand polypeptide antibody.
  • [SEQ ID NO: 7] is an amino acid sequence of the antigen which can be used for preparation of the anti-ligand polypeptide antibody.
  • [SEQ ID NO: 8] is an amino acid sequence of the antigen which can be used for preparation of the anti-ligand polypeptide antibody.
  • [SEQ ID NO: 9] is an amino acid sequence of the antigen which can be used for preparation of the anti-ligand polypeptide antibody.
  • [SEQ ID NO: 10] is a partial amino acid sequence of the human pituitary-derived G protein-coupled receptor protein encoded by the human pituitary-derived G protein-coupled receptor protein cDNA fragment included in pl9P2.
  • [SEQ ID NO: 11] is a partial amino acid sequence of the human pituitary-derived G protein-coupled receptor protein encoded by the human pituitary-derived G protein-coupled receptor protein cDNA fragment include in pl9P2.
  • [SEQ ID NO: 12] is an entire amino acid sequence of the human pituitary-derived G protein-coupled receptor protein encoded by the human pituitary-derived G protein-coupled receptor protein cDNA include in phGR3.
  • [SEQ ID NO: 13] is a partial amino acid sequence of the mouse pancreatic ⁇ -cell line, MIN6-derived G protein- coupled receptor protein encoded by the mouse pancreatic ⁇ -cell line, MIN6-derived G protein-coupled receptor protein cDNA fragment having a nucleotide sequence (SEQ ID NO:18), derived based upon the nucleotide sequences of the mouse pancreatic ⁇ -cell line, MIN6-derived G protein-coupled receptor protein cDNA fragments each included in pG3-2 and pGl-10.
  • [SEQ ID NO: 14] is a partial amino acid sequence of the mouse pancreatic ⁇ -cell line, MIN6-derived G protein- coupled receptor protein encoded by p5S38.
  • [SEQ ID NO: 15] is a nucleotide sequence of the human pituitary-derived G protein-coupled receptor protein cDNA fragment include in pl9P2.
  • [SEQ ID NO: 16] is a nucleotide sequence of the human pituitary-derived G protein-coupled receptor protein cDNA fragment include in pl9P2.
  • [SEQ ID NO: 17] is an entire nucleotide sequence of the human pituitary-derived G protein-coupled receptor protein cDNa include in phGR3.
  • [SEQ ID NO: 18] is a nucleotide sequence of the mouse pancreatic ⁇ -cell line, MIN6-derived G protein-coupled receptor protein cDNA, derived based upon the nucleotide sequences of the mouse pancreatic ⁇ -cell line, MIN6-derived G protein-coupled receptor protein cDNA fragments each included in pG3-2 and pGl-10.
  • [SEQ ID NO: 19] is a nucleotide sequence of the mouse pancreatic ⁇ -cell line, MIN6-derived G protein-coupled receptor protein cDNA include in p5S38.
  • [SEQ ID NO: 20] is a synthetic DNA primer for screening of cDNA coding for the G protein-coupled receptor protein.
  • [SEQ ID NO: 21] is a synthetic DNA primer for screening of cDNA coding for the G protein-coupled receptor protein.
  • [SEQ ID NO: 22] is a synthetic DNA primer for screening of cDNA coding for the G protein-coupled receptor protein.
  • [SEQ ID NO: 23] is a synthetic DNA primer for screening of cDNA coding for the G protein-coupled receptor protein .
  • [SEQ ID NO: 24] is a synthetic DNA primer for screening of cDNA coding for the G protein-coupled receptor protein.
  • [SEQ ID NO: 25] is a synthetic DNA primer for screening of cDNA coding for the G protein-coupled receptor protein.
  • [SEQ ID NO:26] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide which was obtained by purification and analysis of N-terminal sequence for P-3 fraction. The amino acid sequence corresponds to 23rd to 51st positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO: 27] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide which was obtained by purification and analysis of N-terminal sequence for P-2 fraction. The amino acid sequence corresponds to 34th to 52nd positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO: 28] is entire amino acid sequence of the murine pituitary-derived ligand polypeptide encoded by the cDNA included in pBOV3.
  • [SEQ ID NO: 29] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by P5-1.
  • [SEQ ID NO: 30] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by P3-1.
  • [SEQ ID NO: 31] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by P3-2.
  • [SEQ ID NO: 32] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by PE .
  • [SEQ ID NO: 33] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by PDN.
  • [SEQ ID NO: 34] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by FB.
  • [SEQ ID NO: 35] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by FC .
  • [SEQ ID NO: 36] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by BOVF .
  • [SEQ ID NO: 37] is a synthetic DNA primer for screening of cDNA coding for the bovine pituitary-derived ligand polypeptide, wherein the primer is represented by BOVR.
  • [SEQ ID NO: 38] is an entire amino acid sequence of the bovine genome-derived ligand polypeptide.
  • [SEQ ID NO: 39] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide. The amino acid sequence corresponds to 23rd to 53rd positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO:40] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide. The amino acid sequence corresponds to 23rd to 54th positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO: 41] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide. The amino acid sequence corresponds to 23rd to 55th positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO: 42] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide.
  • the amino acid sequence corresponds to 34th to 53rd positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO:43] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide.
  • the amino acid sequence corresponds to 34th to 54th positions of the amino acid sequence of SEQ ID NO:l.
  • [SEQ ID NO: 44] is an amino acid sequence of the bovine pituitary-derived ligand polypeptide.
  • the amino acid sequence corresponds to 34th to 55th positions of the amino acid sequence of SEQ ID N0:1.
  • [SEQ ID NO: 45] is a synthetic DNA primer for screening of cDNA coding for the murine-derived ligand polypeptide, wherein the primer is represented by RA.
  • [SEQ ID NO: 46] is a synthetic DNA primer for screening of cDNA coding for the murine-derived ligand polypeptide, wherein the primer is represented by RC .
  • [SEQ ID NO: 47] is a synthetic DNA primer for screening of cDNA coding for the murine-derived ligand polypeptide, wherein the primer is represented by rF .
  • [SEQ ID NO:48] is a synthetic DNA primer for screening of cDNA coding for the murine-derived ligand polypeptide, wherein the primer is represented by rR.
  • [SEQ ID NO: 49] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by RI .
  • [SEQ ID NO: 50] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by R3.
  • [SEQ ID NO: 51] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by R4.
  • [SEQ ID NO: 52] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by HA.
  • [SEQ ID NO: 53] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by HB .
  • [SEQ ID NO: 54] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by HE.
  • [SEQ ID NO: 55] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by HF .
  • [SEQ ID NO: 56] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by 5H.
  • [SEQ ID NO: 57] is a synthetic DNA primer for screening of cDNA coding for the human-derived ligand polypeptide, wherein the primer is represented by 3HN.
  • [SEQ ID NO: 58] is an entire nucleotide sequence of the bovine pituitary-derived ligand polypeptide cDNA.
  • [SEQ ID NO: 59] is an entire nucleotide sequence of the murine-derived ligand polypeptide cDNA.
  • [SEQ ID NO: 60] is an entire nucleotide sequence of the human-derived ligand polypeptide cDNA.
  • [SEQ ID NO: 61] is a synthetic DNA primer for screening of cDNA coding for the murine-derived ligand polypeptide, wherein the primer is represented by rFBG.
  • [SEQ ID NO: 62] is a synthetic DNA primer for screening of cDNA coding for the murine-derived ligand polypeptide, wherein the primer is represented by rRSA.
  • the transformant Escherichia coli designated INV ⁇ F'/pl9P2, which is obtained in the Example 2 mentioned herein below, is on deposit under the terms of the Budapest Treaty from August 9, 1994, with the National Institute of Bioscience and Human-Technology (NIBH) , Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan and has been assigned the Accession Number FERM BP-4776. It is also on deposit from August 22, 1994 with the Institute for Fermentation, Osaka, Japan (IFO) and has been assigned the Accession Number IFO 15739.
  • NIBH National Institute of Bioscience and Human-Technology
  • IFO Institute for Fermentation, Osaka, Japan
  • the transformant Escherichia coli designated INV ⁇ F' /pG3-2, which is obtained in the Example 4 mentioned herein below, is on deposit under the terms of the Budapest Treaty from August 9, 1994, with NIBH and has been assigned the Accession Number FERM BP- 4775. It is also on deposit from August 22, 1994 with IFO and has been assigned the Accession Number IFO 15740 .
  • the transformant Escherichia coli designated JM109/phGR3, which is obtained in the Example 5 mentioned herein below, is on deposit under the terms of the Budapest Treaty from September 27, 1994, with NIBH and has been assigned the Accession Number FERM BP-4807. It is also on deposit from September 22, 1994 with IFO and has been assigned the Accession Number IFO 15748.
  • the transformant Escherichia coli, designated JM109/p5S38, which is obtained in the Example 8 mentioned herein below, is on deposit under the terms of the Budapest Treaty from October 27, 1994, with NIBH and has been assigned the Accession Number FERM BP- 4856. It is also on deposit from October 25, 1994 with IFO and has been assigned the Accession Number IFO 15754.
  • JM109/pBOV3 The transformant Escherichia coli, designated JM109/pBOV3, which is obtained in the Example 20 mentioned herein below, is on deposit under the terms of the Budapest Treaty from February 13, 1996, with NIBH and has been assigned the Accession Number FERM BP-5391. It is also on deposit from January 25, 1996 with IFO and has been assigned the Accession Number IFO 15910.
  • JM109/pRAV3 The transformant Escherichia coli, designated JM109/pRAV3, which is obtained in the Example 29 mentioned herein below, is on deposit under the terms of the Budapest Treaty from September 12, 1996, with NIBH and has been assigned the Accession Number FERM
  • JM109/pHOV7 The transformant Escherichia coli, designated JM109/pHOV7, which is obtained in the Example 32 mentioned herein below, is on deposit under the terms of the Budapest Treaty from September 12, 1996, with NIBH and has been assigned the Accession Number FERM BP-5666. It is also on deposit from September 5, 1996 with IFO and has been assigned the Accession Number IFO 16013.
  • JM109/pmGB3 The transformant Escherichia coli, designated JM109/pmGB3, which is obtained in the Example 33 mentioned herein below, is on deposit under the terms of the Budapest Treaty from March 3, 1997, with NIBH and has been assigned the Accession Number FERM BP- 5666. It is also on deposit from February 19, 1997 with IFO and has been assigned the Accession Number IFO 16059.
  • the bioactive substance of the present invention namely the ligand polypeptide or its amide or ester thereof, or a salt thereof, a partial peptide thereof, or the DNA coding for said ligand polypeptide, has function modulating activity for various tissues or internal organs, e.g. heart, lung, liver, spleen, thymus, kidney, adrenal glands, skeltal muscle, testis etc., besides pituitary, central nervous system or pancreas, and are useful as medicines.
  • the substance also is useful for the screening of agonists or antagonists of G protein-coupled receptor proteins.
  • the compounds which can be obtained by such screening also have function modulating activity for above- described tissues or internal organs, and are useful as medicines.
  • the substance is useful for producing a non-human transgenic animal or a non-human knockout animal for analyzing the mechanism of the gene .
  • the parentheses indicate the incorporation of a plurality of bases, leading to multiple oligonucleotides in the primer preparation.
  • nucleotide resides in parentheses of the aforementioned DNAs were incorporated in the presence of a mixture of plural bases at the time of synthesis.
  • Amplification of Receptor cDNA by PCR Using Human Pituitary Gland-Derived cDNA By using human pituitary gland-derived cDNA (QuickClone, CLONTECH Laboratories, Inc.) as a template, PCR amplification using the DNA primers synthesized in Reference Example 1 was carried out.
  • the composition of the reaction solution consisted of the synthetic DNA primers (SEQ: 5' primer sequence and 3' primer sequence) each in an amount of 1 ⁇ M, 1 ng of the template cDNA, 0.25 mM dNTPs, 1 ⁇ l of Taq DNA polymerase and a buffer attached to the enzyme kit, and the total amount of the reaction solution was made to be 100 ⁇ l .
  • PCR products were separated by using a 0.8% low-melting temperature agarose gel, the band parts were excised from the gel with a razor blade, and were heat-melted, extracted with phenol and precipitated in ethanol to recover DNAs .
  • the recovered DNAs were subcloned into a plasmid vector,
  • TM pCR II (TM represents registered trademark) .
  • the recombinant vectors were introduced into E_i_ coli INV ⁇ F' competent cells (Invitrogen Co.) to produce transformants. Then, transformant clones having a cDNA-inserted fragment were selected in an LB agar culture medium containing ampicillin and X-gal. Only transformant clones exhibiting white color were picked with a sterilized toothpick to obtain transformant Escherichia coli INV ⁇ F ' /pl9P2.
  • the individual clones were cultured overnight in an LB culture medium containing ampicillin and treated with an automatic plasmid extracting machine (Kurabo Co., Japan) to prepare plasmid DNAs.
  • An aliquot of the DNA thus prepared was cut by EcoRI to confirm the size of the cDNA fragment that was inserted.
  • An aliquot of the remaining DNA was further processed with RNase, extracted with phenol/chloroform, and precipitated in ethanol so as to be condensed.
  • Sequencing was carried out by using a DyeDeoxy terminator cycle sequencing kit (ABI Co.), and the DNAs were decoded by using a fluorescent automatic sequencer, and then the data of the nucleotide sequences obtained were read by using DNASIS (Hitachi System Engineering Co., Japan).
  • the underlined portions of Figure 1 and Figure 2 represent regions corresponding to the synthetic primers . Homology retrieval was carried out based upon the determined nucleotide sequences [SEQ ID NO: 15 and 16 (Here, the determined nucleotide sequence is the nucleotide sequence which the underlined portion is deleted from the sequence of Figure 1 or Figure 2 respectively) ] . As a result, it was learned that a novel G protein-coupled receptor protein was encoded by the cDNA fragment insert in the plasmid, pl9P2, possessed by the transformant Escherichia coli INV ⁇ F ' /pl9P2.
  • RNA was prepared from the mouse pancreatic ⁇ -cell strain, MIN6 (Jun-ichi Miyazaki et al . , Endocrinology, Vol. 127, No. 1, p.126-132) according to the guanidine thiocyanate method (Kaplan B.B. et al . , Biochem. J., 183, 181-184 (1979) and, then, poly(A) + RNA fractions were prepared with a mRNA purifying kit (Pharmacia Co.).
  • PCR amplification using the DNA primers synthesized in Reference Example 1 was carried out under the same condition as in Example 1.
  • the resulting PCR product was subcloned into the plasmid vector, pCR TMII, in the same manner as in Example 2 to obtain a plasmid, pG3-2.
  • the EL. coli INV ⁇ F' was transfected with the plasmid pG3-2 to obtain transformed Escherichia coli INV ⁇ F ' /pG3-2.
  • TM plasmid vector pCR II in the same manner as described in Example 2 to obtain a plasmid, pGl-10.
  • the reaction for determining the nucleotide sequence was carried out with a DyeDeoxy terminator cycle sequencing kit (ABI Co.), and the DNA was decoded with the fluorescent automatic sequencer (ABI Co.), and then the data of the nucleotide sequence obtained were analyzed with DNASIS (Hitachi System Engineering Co., Japan).
  • Figure 6 shows a mouse pancreatic ⁇ -cell strain MIN6-derived G protein-coupled receptor protein- encoding DNA (SEQ ID NO: 18) and an amino acid sequence (SEQ ID NO: 13) encoded by the isolated DNA based upon the nucleotide sequences of plasmids pG3-2 and pGl-10.
  • the underlined portions of Figure 6 represent regions corresponding to the synthetic primers.
  • coli was plated onto a 1.5% agar (Wako-Junyaku Co.) LB plate (containing 50 ⁇ g/ml of ampicillin) .
  • a nitrocellulose filter was placed on the plate on which plaques were formed so that the plaque was transferred onto the filter.
  • the filter was denatured with an alkali and then heated at 80 °C for 3 hours to fix DNAs.
  • the filter was incubated overnight at 42 °C together with the probe mentioned herein below in a buffer containing 50% formamide, 5 x SSPE (20 x SSPE (pH 7.4) is 3 M NaCl, 0.2 M NaH 2 PO ⁇ -H 2 0, 25 mM EDTA), 5 X Denhardt's solution (Nippon Gene, Japan), 0.1% SDS and 100 ⁇ g/ml of salmon sperm DNA for hybridization.
  • the probe used was obtained by cutting the DNA fragment inserted in the plasmid, pl9P2, obtained in Example 2, with EcoRI, followed by recovery and labelling by incorporation of [ 32 P]dCTP (Dupont Co.) with a random prime DNA labelling kit (Amasham Co.).
  • hybridization signals were recognized in three independent plaques .
  • Each DNA was prepared from the three clones.
  • the DNAs digested with EcoRI were subjected to agarose gel electrophoresis and were analyzed by the southern blotting using the same probe as the one used in the screening.
  • Hybridizing bands were identified at about 0.7kb, 0.8kb and 2.0kb, respectively.
  • Okb ⁇ hGR3
  • the ⁇ hGR3-derived EcoRI fragment with a hybridizable size was subcloned to the EcoRI site of the plasmid, pUC18, and L. coli JM109 was transformed with the plasmid to obtain transformant I coli
  • the reaction for determining the nucleotide sequence was carried out with a DyeDeoxy terminator cycle sequencing kit (ABI Co.), and the DNA was decoded with the fluorescent automatic sequencer (ABI Co.), and then the data of the nucleotide sequence obtained were analyzed with DNASIS (Hitachi System Engineering Co., Japan).
  • Figure 9 shows a nucleotide sequence of from just after the EcoRI site up to the Nhel site encoded by ⁇ hGR3.
  • the nucleotide sequence of the human pituitary gland-derived receptor protein-encoding DNA corresponds to the nucleotide sequence (SEQ ID NO: 17) of from 118th to 1227th nucleotides [ Figure 9].
  • An amino acid sequence of the receptor protein that is encoded by the nucleotide sequence is shown in SEQ ID NO: 12.
  • Example 7 Northern Hybridization with Human Pituitary Gland- Derived Receptor Protein-Encoding phGR3 Northern blotting was carried out in order to detect the expression of phGR3-encoded human pituitary gland-derived receptor proteins obtained in Example 5 in the pituitary gland at a mRNA level.
  • Human pituitary gland mRNA (2.5 ⁇ g, Clontech Co.) was used as a template mRNA and the same as the probe used in Example 5 was used as a probe.
  • Nylon membrane (Pall Biodyne, U.S.A.) was used as a filter for northern blotting and migration of the mRNA and adsorption
  • Figure 12 shows a mouse pancreatic ⁇ -cell strain MIN6-derived G protein-coupled receptor protein- encoding DNA (SEQ ID NO: 19) and an amino acid sequence (SEQ ID NO: 14) encoded by the isolated DNA based upon the nucleotide sequence of plasmid, p5S38.
  • the underlined portions represent regions corresponding to the synthetic primers.
  • MIN6-derived G protein-coupled receptor protein encoded by p5S38 would recognize the same ligand as the human pituitary gland-derived G protein-coupled receptor protein encoded by pl9P2 while the animal species from which the receptor protein encoded by p5S38 was derived was different from the species from which the receptor protein encoded by pl9P2 was derived.
  • the plasmid phGR3 (Example 5) containing a cDNA encoding the full-length amino acid sequence of human pituitary receptor protein was digested with the restriction enzyme Nco I and electrophoresed on agarose gel and a fragment of about lkb was recovered. Both ends of the recovered fragment were blunted with a DNA blunting kit (Takara Shuzo Co., Japan) and, after a Sail linker was added, the fragment was treated with Sail and inserted into the Sail site of pUC119 to provide plasmid S10. Then, S10 was treated with Sail and SacII to prepare a fragment of about 700 bp (containing the N-terminal coding region) .
  • a fragment of about 700 bp (containing the C-terminal coding region including initiation and termination codons) was cut out from phGR3 with Sac II and Nhe I. These two fragments were added to the animal cell expression vector plasmid pAKKO-lllH (the vector plasmid identical to the pAKKOl.ll H described in Biochim. Biophys. Acta, Hinuma, S., et al . , 1219 251- 259, 1994) and a ligation reaction was carried out to construct a full-length receptor protein expression plasmid pAKKO-19P2.
  • E_. coli transfected with pAKKO-19P2 was cultured and the pAKKO-19P2 plasmid DNA was mass-produced using QUIAGEN Maxi.
  • a 20 ⁇ g portion of the plasmid DNA was dissolved in 1 ml of sterile PBS, and in a gene transfer vial (Wako Pure Chemical Ind.), the solution was vortexed well for liposome formation.
  • This liposo e, 125 ⁇ l, was added to CHOdhfr " cells previously subcultured at 1 x 10 cells per lOcm-dia. dish 24 hr and placed in fresh medium immediately before addition and overnight culture was carried out.
  • the reaction mixture was incubated at 30 °C for 10 minutes to conduct an amplification reaction to some extent. Then, it was incubated at 42 °C for 30 minutes to let the reverse transcription reaction proceed. The enzyme was inactivated by heating at 99 °C for 5 minutes and the reaction system was cooled at 5°C for 5 minutes .
  • sequences of the primers prepared according to the base sequence of the coding region of the full-length receptor protein were CTGACTTATTTTCTGGGCTGCCGC (SEQ ID NO: 24) for 5' end and AACACCGACACATAGACGGTGACC (SEQ ID NO: 25) for 3' end.
  • the PCR reaction was carried out in a total volume of 100 ⁇ l using 1 ⁇ M each of the primers, 0.5 ⁇ l of Taq DNA polymerase (Takara Shuzo Co., Japan), the reaction buffer and dNTPs accompanying the enzyme, and 10 ⁇ l of template DNA (cDNA or plasmid solution) .
  • First the reaction mixture was heat-treated at 94 °C for 2 minutes for sufficient denaturation of the template DNA and subjected to 25 cycles of 95°C x 30 seconds, 65°C x 30 seconds, and 72 °C x 60 seconds. After completion of the reaction, 10 ⁇ l of the reaction mixture was subjected to agarose gel electrophoresis and the detection and quantitative comparison of amplification products were carried out.
  • a crude peptide fraction was prepared from rat whole brain by the following procedure.
  • the rat whole brain enucleated immediately after sacrifice was frozen in liquefied nitrogen and stored at -80 °C.
  • the frozen rat whole brain 20 g (the equivalent of 10 rats) was finely divided and boiled in 80 ml of distilled water for 10 minutes. After the boiled tissue was quenched on ice, 4.7 ml of acetic acid was added at a final concentration of 1.0 M and the mixture was homogenized using a Polytron (20,000 rpm, 6 min.). The homogenate was stirred overnight and then centrifuged (10,000 rpm, 20 min.) to separate the supernatant.
  • the sediment was homogenized in 40 ml of 1.0 M acetic acid and centrifuged again to recover the supernatant.
  • the supernatants were pooled, diluted in 3 volumes of acetone, allowed to stand on ice for 30 minutes, and centrifuged (10,000 rpm, 20 min.) to recover the supernatant .
  • the recovered supernatant was evaporated to remove acetone.
  • To the resulting acetone-free concentrate was added 2 volumes of 0.05% trifluoroacetic acid(TFA) /H 2 0 and the mixture was applied to a reversed-phase C18 column (Prep C18 125A, Millipore) .
  • the column was washed with 0.05% TFA/H 2 0, and gradient elution was carried out with 10%, 20%, 30%, 40%, 50%, and 60% CH 3 CN/0.05%TFA/H 2 O .
  • the fractions were respectively divided into 10 equal parts and lyophilized.
  • the dried sample derived from one animal equivalent of rat whole brain was dissolved in 20 ⁇ l of dimethyl sulfoxide (DMSO) and suspended in 1 ml of Hank's balanced saline solution (HBSS) supplemented with 0.05% bovine serum albumin (BSA) to provide a crude peptide fraction.
  • DMSO dimethyl sulfoxide
  • HBSS Hank's balanced saline solution
  • BSA bovine serum albumin
  • the full-length receptor protein-expressed CHO cells and mock CHO cells were seeded in a 24-well plate, 0.5 x 10 cells/well, and cultured for 24 hours. Then, [ H] arachidonic acid was added at a final concentration of 0.25 ⁇ Ci/well . Sixteen (16) hours after addition of [ H] arachidonic acid, the cells were rinsed with 0.05% BSA-HBSS and the above-mentioned crude peptide fraction was added, 400 ⁇ l/well.
  • Example 12 Detection of the activity to specifically promote release of arachidonic acid metabolites from CHO- 19P2 cells in a bovine hypothalamus extract A crude peptide fraction was prepared from 360 g (the equivalent of 1 animals) of bovine brain tissue including hypothalamus in the same manner as in Example 11. A dried peptide sample per 0.05 animal was dissolved in 40 ⁇ l of DMSO and suspended in 2 ml of 0.05% BSA-HBSS and the detection of arachidonic acid metabolite-releasing activity was attempted in the same manner as in Example 11.
  • a typical process for harvesting the activity to specifically promote release of arachidonic acid metabolites from the CHO-19P2 cell line by purification from bovine hypothalamus is now described.
  • a frozen bovine brain tissue specimen including hypothalamus, 4.0 kg (the equivalent of 80 animals) was ground and boiled in 8.0 L of distilled water for 20 minutes. After quenching on ice, 540 ml of acetic acid was added at a final concentration of 1.0 M and the mixture was homogenized using a Polytron (10,000 rpm, 12 min.). The homogenate was stirred overnight and then centrifuged (9,500 rpm, 20 min) to recover a supernatant.
  • the sediment was suspended in 4.0 L of 1.0 M acetic acid and homogenized with the Polytron and centrifuged again to recover a further supernatant.
  • the supernatants were pooled and TFA was added at a final concentration of 0.05%.
  • the mixture was applied to reversed-phase C18 (Prep C18 125A, 160 ml; Millipore) packed in a glass column. After addition, the column was washed with 320 ml of 0.05% TFA/H z 0 and 3-gradient elution was carried out with 10%, 30%, and 50% CH 3 CN/0.05% TFA/H 2 0.
  • this fraction was diluted with 3 volumes of acetone, centrifuged for deproteination, and concentrated in an evaporator. To the concentrated fraction was added TFA ( final concentration 0.1%) and the mixture was adjusted to pH 4 with acetic acid and applied to 3 ml of the reversed- phase column RESOURCE RPC (Pharmacia) . Elution was carried out on a concentration gradient of 15%-30% CHjCN. As a result, activity to specifically promote the release of arachidonic acid metabolites from the CHO-19P2 cell line was detected in the 19%-21% CH 3 CN fraction.
  • the active fraction eluted from RESOURCE RPC was lyophilized, dissolved with DMSO, suspended in 50 mM MES pH 5.0/10% CH 3 CN, and added to 1 ml of the cation exchange column RESOURCE S. Elution was carried out on a concentration gradient of 0 M-0.7 M NaCl. As a result, the activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells was detected in the 0.32 M-0.46 M NaCl fraction.
  • the active eluate from RESOURCE S was lyophilized, dissolved with DMSO, suspended in 0.1% TFA/H 2 0, and added to reversed-phase column C18 218TP5415 (Vydac), and elution was carried out on a concentration gradient of 20%-30% CH 3 CN.
  • the activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells was detected in the three fractions 22.5%, 23%, and 23.5% CH 3 CN (these active fractions are designated as P-l, P-2, and P-3) [Fig. 18].
  • the 23.5% CH 3 CN fraction (P-3) was lyophilized, dissolved with DMSO, suspended in 0.1% TFA/H 2 0, and added to the reversed-phase column diphenyl 219TP5415 (Vydac), and elution was carried out on a gradient of 22%-25% CH 3 CN.
  • the activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells was converged by recovery in one elution peak obtained with 23% CH 3 CN [Fig. 19].
  • the active peak fraction from the reverse-phased column diphenyl 219TP5415 was lyophilized, dissolved with DMSO, suspended in 0.1% TFA/H 2 0, and added to the reversed- phase column ⁇ RPC C2/C18 SC 2.1/10 (Pharmacia), and elution was carried out on a gradient of 22%-23.5% CH 3 CN.
  • the activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells was detected in the two peaks eluted with 23.0% and 23.2% CH 3 CN [Fig. 20].
  • the amino acid sequence of the peptide (P-3) having activity to specifically promote release of arachidonic acid metabolites from CHO-19P2 cells as purified in Example 13 was determined.
  • the fraction showing peak activity from the reversed-phase ⁇ RPC C2/C18 SC 2.1/10 was lyophilized and dissolved in 20 ⁇ l of 70% CHjCN and analyzed for amino acid sequence with the peptide sequencer (ABI.491).
  • the sequence defined by SEQ ID NO:26 was obtained.
  • the 7th and 19th amino acids were not determined by only the analysis of amino acid sequence.
  • Example 15 Preparation of the active substance (peptide) which specifically promotes release of arachidonic acid metabolites from CHO-19P2 cells as purified from bovine hypothalamus Of the three active fractions obtained with Vydac C18 218TP5415 in Example 13, the active fraction (P-2) eluted with 23.0% CH 3 CN was further purified. This active fraction was lyophilized, dissolved with DMSO, suspended in 0.1% TFA/dH 2 0, and added to reversed-phase column diphenyl 219TP5415 (Vydac), and elution was carried out on a gradient of 21.0%-24.0% CH 3 CN.
  • RNA was prepared from one animal equivalent of bovine hypothalamus. Then, using Fast Track (Invitrogen), a poly(A) + RNA fraction was prepared. From 1 ⁇ g of this poly(A) RNA fraction, cDNA was synthesized using 3' RACE system (GIBCO BRL) and Marathon cDNA amplification kit (Clontech) according to the manuals and dissolved in 20 and 10 ⁇ l, respectively.
  • Isogen Nippon Gene
  • Example 14 the acquisition of a base sequence coding for SEQ ID NO: 28 was attempted in the first place.
  • primers P5-1 SEQ ID NO: 29
  • P3-1 SEQ ID NO: 30
  • P3-2 SEQ ID NO: 31
  • I represents inosine
  • This reaction mixture was diluted 50-fold with tricin-EDTA buffer and using 2.5 ⁇ l of the dilution as a template and the primer combination of P5-1 and P3-2, the reaction was carried out in otherwise the same manner as described above.
  • Gene Amp 9600 Perkin Elmer
  • the amplification product was subjected to 4% agarose gel electrophoresis and ethidium bromide staining and a band of about 70 bp was cut out and subjected to thermal fusion, phenol extraction, and ethanol precipitation. The recovered
  • Example 18 RACE using the sequence established in Example 18 First, for amplification (5' RACE) of the sequence at 5' end, the two primers PE (SEQ ID NO: 32) and PDN (SEQ ID NO: 33) were synthesized by utilizing the sequence shown in Fig. 22.
  • the cDNA prepared using Marathon cDNA amplification kit in Example 17 was diluted 100-fold with tricin-EDTA buffer. Then, in the same manner as Example 2, a reaction mixture was prepared using 2.5 ⁇ l of the dilution and a combination of the adapter primer API accompanying the kit and the primer PE and after treatmenet for one minute at 94 °C, the cycle of 98°C x 10 seconds and 68°C x 5 minutes was repeated 30 times.
  • This reaction system was further diluted 50-fold with tricin-EDTA buffer and using 2.5 ⁇ l of the dilution as a template and the changed primer combination of API and PDN, the reaction was conducted at 94°C for one minute, followed by 4 cycles of 94°C x 1 minute, 98°C x 10 seconds and 72°C x 5 minutes, 4 cycles of 98°C x 10 seconds and 70°C x 5 minutes, and 26 cycles of 98°C x 10 seconds and 68°C x 5 minutes.
  • the amplification product was electrophoresed on 1.2% agarose gel and stained with ethidium bromide and a band of about 150 bp was cut out and centrifugally filtered through a centrifugal filter tube (Millipore) , extracted with phenol, and precipitated from ethanol.
  • the recovered DNA was subcloned into plasmid vector
  • PCR was carried out at 94°C for 1 minute, followed by 5 cycles of 98°C x 10 seconds and 72°C x 5 minutes, 5 cycles of 98°C x 10 seconds and 70°C x 5 minutes, and 25 cycles of 98°C x 10 seconds and 68°C x 5 minutes.
  • the reaction was further conducted at 94 °C for one minute, followed by 4 cycles of 98°C x 10 seconds and 72°C x 5 minutes, 4 cycles of 98°C x 10 seconds and 70°C x 5 minutes, and 27 cycles of 98°C x 10 seconds and 68°C x 5 minutes.
  • the amplification product was electrophoresed on 1.2% agarose gel and stained with ethidium bromide and a band of about 400 bp was cut out and the DNA was recovered as in 5 ' -RACE . This DNA
  • TM fragment was subcloned into plasmid vector pCR II and introduced into E.. coli JM109 and the sequence of the inserted cDNA fragment in the resulting transformant was analyzed. From the results of 5' RACE and 3' RACE, the DNA sequence [Fig. 24] coding for the complete coding region of the bioactive polypeptide defined by SEQ ID NO:l was established. Thus, in Fig. 24 (a) and (b), the base is G, the base 18 is T or C, and the base was T or C.
  • the cDNA shown in Fig. 24 was the cDNA encoding a polypeptide consisting of 98 amino acids.
  • the amino acids in 1 - 22-positions comprise a cluster of hydrophobic amino acids taken together with the fact that the N-terminal region of the active peptide begins with Ser in 23-position as shown in Example 14 suggested that the amino acids 1-22 represent a secretion signal sequence.
  • the Gly Arg Arg Arg sequence in 54-57 positions of the polypeptide was found to be a typical amino acid sequence motif which exists in the event of cleavage of a bioactive peptide. As it is the case with this cleavage motif, it is known that because of the presence of Gly, the C-terminus of the product peptide is frequently amidated.
  • Example 14 The P-3 N-terminal sequence data of Example 14 and P-2 N-terminal sequence data in Example 16 coupled with this GlyArgArgArg sequence suggest that at least the same of the bioactive peptides cut out from the polypeptide encoded by this cDNA are defined by SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44. [Example 20]
  • BOVF contains the initiation codon of bioactive polypeptide cDNA and is a sense sequence corresponding to -2 - +22 (A of the initiation codon ATG being reckoned as +1) with restriction enzyme Sail site added.
  • BOVR is an antisense sequence corresponding to +285 - +309 which includes the termination codon of bioactive polypeptide cDNA.
  • the PCR was conducted as follows.
  • the cDNA prepared using Marathon cDNA amplification kit in Example 17 was diluted 100-fold in tricin-EDTA buffer and using 2.5 ⁇ l of the dilution, a reaction mixture was prepared as in Example 2 and subjected to 94 °C x 1 minute, 3 cycles of 98°C x 10 seconds and 72°C x 5 minutes, 3 cycles of 98°C x 10 seconds and 70°C x 5 minutes, and 27 cycles of 98°C x 10 seconds and 68°C x 5 minutes.
  • the amplification product was subjected to 2% agarose electrophoresis and ethidium bromide staining and a band of about 320 bp was cut out.
  • the DNA was recovered and subcloned in plasmid vector
  • Example 21 TM pCR II as in Example 3.
  • the vector was introduced into Escherichia coli JM109 to provide the transformant E_. coli JM109/pBOV3.
  • the sequence of the cDNA fragment inserted in the transformant was then analyzed. As a result, this DNA fragment was confirmed to be a fragment covering the entire coding region of the bioactive polypeptide cDNA.
  • Boc-Gly, Boc-Val, Boc-Pro, Boc- Arg(Tos), Boc-Ile, Boc-Gly, Boc-Arg(Tos) , Boc-Gly, Boc- Ala, Boc-Tyr(Br-Z) was carried out.
  • Boc-Pro Asp(OcHex), Boc-Pro, Boc-Thr( Bzl) , Boc-Arg(Tos ) , Boc- lie, Boc-Glu ( OcHex) , Boc-Met, Boc-Ser(Bzl) , Boc- His(Bom), Boc-Gln, Boc-His (Bom) , Boc-Ala, Boc-Arg(Tos ) , Boc-Ser (Bzl) were serially condensed and recondensed until sufficient condensation was confirmed by ninhydrin test. After introduction of the full sequence of amino acids of 19P2-L31, the resin was treated with 50% TFA/DCM to remove Boc groups on the resin and, then, dried to provide 1.28 g of the peptide resin.
  • the resin obtained in 1) was reacted with 3.8 g of p-cresol, 1 ml of 1,4-butanedithiol, and 10 ml of hydrogen fluoride at 0°C for 60 minutes.
  • the hydrogen fluoride and 1,4- butanedithiol (1 ml) were distilled off under reduced pressure and the residue was diluted with 100 ml of diethyl ether, stirred, filtered through a glass filter, and the fraction on the filter was dried. This fraction was suspended in 50 ml of 50% acetic acid/H 2 0 and stirred to extract the peptide.
  • Example 21-1 To the resin subjected to condensations up to Boc- Tyr(Br-Z) in Example 21-1) was further condensed with Boc-Trp(CHO) , Boc-Ala, Boc-Pro, Boc-Asn, Boc-Ile, Boc- Asp(OcHex), Boc-Pro and Boc-Thr(Bzl) serially in the same manner to provide 1.14 g of Boc-Thr(Bzl) -Pro- Asp (OcHex) -Ile-Asn-Pro-Ala-Trp( CHO) -Tyr (Br-Z ) -Ala-Gly- Arg( Tos ) -Gly-Ile-Arg(Tos ) -Pro-Val-Gly-Arg( Tos ) -Phe- pMBHA-resin.
  • This resin was treated with hydrogen fluoride and purified column chromatography in the same manner as Example 21-2) to provide 60 mg of white powders .
  • the plaques were transferred to a nitrocellulose filter and after alkaline modification and neutralization, heat-treated (80°C, 2 hours) to inactivate the DNA.
  • This filter was incubated with the labeled probe in 50% formamide-Hybri buffer (50% formamide, 5 x Denhardt solution, 4 x SSPE, 0.1 mg/ml heat-denatured salmon sperm DNA, 0.1% SDS) at 42°C overnight for hybridization. After this hybridization, the filter was washed with 2 x SSC, 0.1% SDS at room temperature for 1.5 hours, and further washed in the same buffer at 55°C for 30 minutes.
  • formamide-Hybri buffer 50% formamide, 5 x Denhardt solution, 4 x SSPE, 0.1 mg/ml heat-denatured salmon sperm DNA, 0.1% SDS
  • Detection of the clone hybridizing with the probe was carried out on Kodak X-ray film (X-OMATTMAR) after 4 days of exposure using a sensitization screen at -80°C After development of the film, the film was collated with plate positions and the phages which had hybridized were recovered. Then, plating and hybridization were repeated in the same manner for cloning of the pharges .
  • the cloned phages were prepared on a large scale by the plate lysate method and the phage DNA was extracted.
  • the Sail-digested fragment being considered to harbor the full length and in order to subclone this fragment into a plasmid vector, it was ligated to BAP (£• coli-derived alkaline phosphatase) -treated plasmid vector pUC18 (Pharmacia) and introduced into E. coli
  • a genome-derived Sail fragment-inserted plasmid DNA was prepared on a production scale and the base sequence in the neighborhood of its coding region was analyzed using Perkin Elmer Applied Biosystems 370A fluorecent sequencer and the same manufacturer's kit. As a result, the sequence shown in Fig. 29 was obtained. Comparison with the coding region of cDNA reveals that because of its being derived from genomic DNA, the coding region is divided in two by a 472 bp intron [Fig- 30]. Fig. 31 and SEQ ID NO:44 present the amino acid sequence predicted from this bovine genome coding region (excluding the intron region) . [Example 27]
  • RNA was prepared from the dorsal region of rat medulla oblongata and using FastTrack (Invitrogen), poly(A) + RNA fraction was prepared. To 5 ⁇ g of this poly(A) + RNA was added the primer random DNA hexamer (BRL) and using Moloney mouse leukemia reverse transcriptase (BRL) and the accompanying buffer, complementary DNA was synthesized. The reaction product was precipitated from ethanol and dissolved in 12 ⁇ l of distilled water.
  • PCR was carried out using the same primers P5-1 (SEQ ID NO:29) and P3-1 (SEQ ID NO:30) as used in Example 18 as primers and the complementary DNA synthesized in Example 27 using the primer random DNA hexamer (BRL) and Moloney mouse leukemia reverse transcriptase (BRL) as a template.
  • the reaction system was composed of 1.25 ⁇ l of the template cDNA, 200 ⁇ M of dNTP, 1 ⁇ M each of the primers, ExTaq (Takara Shuzo Co., Japan) as DNA polymerase, and 2.5 ⁇ l of the accompanying buffer, with a sufficient amount of water to make a total of 25 ⁇ l .
  • the reaction was carried out at 94°C for 1 minute, followed by 40 cycles of 98°C x 10 seconds, 50°C x 30 seconds, and 72 °C x 5 seconds, and the reaction mixture was then allowed to stand at 72°C for 20 seconds.
  • the thermal cycler used was GeneAmp2400 (Perkin Elmer) .
  • the amplification product was subjected to 4% agarose gel electrophoresis and ethidium bromide staining and the band of about 80 bp was cut out. Then, in the manner described in Example 19, the DNA was recovered, subcloned into plasmid vector pCR II, and introduced into E_. coli JM109, and the inserted cDNA fragment was sequenced. As a result, a partial sequence of rat bioactive polypeptide could be obtained. Based on this sequence, two primers, namely RA (SEQ ID NO: 45) for 3' RACE and RC (SEQ ID NO: 46) for 5' RACE were synthesized and 5' and 3' RACEs were carried out.
  • RA SEQ ID NO: 45
  • RC SEQ ID NO: 46
  • RA 5 ' -CARCAYTCCATGGAGACAAGAACCCC-3 ' (where R means A or G; Y means T or G) (SEQ ID NO: 45)
  • RC 5 ' -TACCAGGCAGGATTGATACAGGGG-3 '
  • Example 27 As a template, the template synthesized using Marathon cDNA amplification kit (Clontech) in Example 27 was diluted 40-fold with the accompanying tricin- EDTA buffer and 2.5 ⁇ l of the dilution was used. As primers, RA and the adapter primer API accompanying the kit were used for 3' RACE, and RC and API for 5' RACE. The reaction mixture was prepared in otherwise the same manner as above. The reaction conditions were 94 °C x 1 minute, 5 cycles of 98°C x 10 seconds and 72°C x 45 seconds, 3 cycles of 98°C x 10 seconds and 70°C x 45 seconds, and 40 cycles of 98°C x 10 seconds and 68°C x 45 seconds.
  • PCR was carried out by repeating 40 cycles of 95 °C x 30 seconds and 68°C x 60 seconds.
  • the amplification product was subjected to agarose gel electrophoresis and ethidium bromide staining and a band of about 350 bp was cut out.
  • the DNA was recovered, subcloned into plasmid vector pCR TMII, and introduced into E.. coli JM109 as in Example 19.
  • the plasmid was extracted from the transformant and the base sequence was determined.
  • E. coli JM 109/pRAV3 having the full-length cDNA of rat bioactive polypeptide was obtained [Fig. 32] .
  • R3 5'-GCCTGATCCCGCGGCCCGTGTACCA-3' (SEQ ID NO: 50)
  • the cDNA prepared using Marathon cDNA amplification kit (Clontech) in Example 30 was diluted 30-fold with tricin-EDTA buffer and 0.25 ⁇ l of the dilution was used as a template.
  • the reaction mixture was composed of 200 ⁇ M of dNTP, 0.2 ⁇ M each of the primers RI and R4 , a 50:50 mixture of Taq Start Antibody (Clontech) and DNA polymerase ExTaq (Takara Shuzo Co., Japan), 2.5 ⁇ l of the accompanying buffer, and a sufficient amount of water to make a total volume of 25 ⁇ l .
  • the reaction conditions were 94 °C x 1 minute, followed by 42 cycles of 98°C x 10 seconds and 68°C x 40 seconds, and 1 minute of standing at 72°C. Then, using 1 ⁇ l of a 100-fold dilution of the above reaction mixture in tricine-EDTA buffer as a template, the same reaction mixture as above except that the primer combination was changed to RI and R3 was prepared and PCR was carried out in the sequence of 94°C x 1 minute and 25 cycles of 98°C x 10 seconds and 68°C x 40 seconds. The amplification product was subjected to 4% agarose gel electrophoresis and ethidium bromide staining.
  • reaction mixtures were prepared in the same manner as above except that HA and adapter primer API were used for 3' RACE and HE and API for 5' RACE.
  • the reaction sequence was 94 °C x 1 minute, 5 cycles of 98°C x 10 seconds and 72°C for 35 seconds, 5 cycles of 98°C x 10 seconds and 70°C x 35 seconds, and 40 cycles of 98°C x 10 seconds and 68°C x 35 seconds.
  • Example 30 Using 2.5 ⁇ l of the cDNA prepared using Moloney mouse leukemia reverse transcriptase (BRL) in Example 30 as a template and the reaction mixture prepared using Klen Taq DNA polymerase (Clontech), the PCR reaction was conducted in the sequence of 94 °C x 1 minute and 40 cycles of 98°C x 10 seconds and 68°C x 30 seconds. The fragment of about 360 bp obtained was recovered and subcloned (pCRTM 2.1 was used as the vector) in the same manner as Example 29. The plasmid was recovered and its base sequence was determined. As a result, E_. coli JM109/pHOV7 harboring the human bioactive polypeptide full-length cDNA was obtained [Fig- 34].
  • Example 33 Acquisition and sequencing of a DNA including the ligand polypeptide coding region from the murine genomic DNA
  • reaction components used were: 200 nM each synthetic DNA primers, 0.5 ng template DNA, 0.5 ⁇ l of 0.25 mM dNTPs ExTaq polymerase, and enzyme-attached buffer, total volume 50 ⁇ l .
  • a thermal cycler Perkin-Elmer
  • an amplification reaction was carried out in 30 cycles of 30 seconds at 95 °C and 60 seconds at 67 °C.
  • Identification of the amplification product was made by 1.2% agarose gel electrophoresis and ethidium bromide staining, and a band of about 1 kb was recovered and subcloned using TA Cloning Kit (Invitrogen).
  • This ligation mixture was used to transform Escherichia coli JM109 and clones harboring the inserted fragment were selected in LB agar containing ampicillin and X-gal. A white-colored clone was picked out to obtain a transformant Escherichia coli JM109/ ⁇ mGB3. This clone was cultured overnight in ampicillin-containing LB medium and the plasmid DNA was prepared using an automatic plasmid extraction apparatus. Using a portion of the DNA thus prepared, a sequencing reaction was carried out using ABI Dye Terminator Cycle Sequencing Kit (ABI), and after decoding with a fluorescent automated sequencer, the nucleotide sequence data was analyzed with DNASIS ( Hitachi System Engineering) [Fig. 36]. The underlined sequences correspond to the primers .
  • the DNA fragment contained in the plasmid pmGB3 as obtained in Example 33 was prepared and sent to Genome Systems with a request for hybridization screening from Mouse ES129/SuJ BAC library using the fragment as the probe (Catalogue BAC, 4921). Fromt the BAC clone received from Genome Systems, the DNA fragment containing the objective ligand peptide coding region was subcloned in the EcoRI site of pUC18 Vector and JM109/pmGFEI was obtained. Then, the sequence of the region of interest was similarly determined. The determined sequence (Fig. 38) coded for the indicated amino sequence. The sequence in parentheses differs from that obtained in Example 33. While the latter was the PCR primer used, the sequence obtained this time was shorter by 5 residues. [Example 35]
  • the targeting vector was constructed using pmGFEl obtained in Example 34 and pGT-N28 (NEB) .
  • the BamHI- Hind III fragment including Vspl site was inserted between the BamHI site and Hind III site of pGT-N28 (5' upstream region of the peptide gene) .
  • the Hpal- Sall fragment was inserted, after modification to XhoI-NotI by linker ligation, between the Xhol and NotI sites to provide pmGFEN28 (Fig. 40).
  • ES cells purchased from Genome Systems in accordance with the attahed manual .
  • RW-4 purchased from Genome Systems in accordance with the attahed manual .
  • a 13 kbp (approx.) DNA fragment available upon VspI/NotI digestion of the pmGFEN28 obtained in Example 35 was isolated by agarose gel electrophoresis and, using Bio-Rad Gene Pulser, electroporated into RW- 4 cells to obtain recombinant cells.
  • the same DNA fragment was sent to Genome Systems with a request for electroporation and performed an antibiotic selection of ES cells (MK 2/20).
  • MK 2/20 antibiotic selection of ES cells
  • GCCACTGTCA CCTCCCATCC ATATGCTTCC CAAATGCCTT GAGTACCCAG CCCCTGAATG 180
  • GGAGGTTAGC CATCTCCTAA GCCAGTGGTT TCCAACCTTC CTAATACAGA ACTTTTAATA 240 CAGATCCTTA TGTTGTGGTG ACCCCCAGCC AGAAAATTAT TGTGATGCTG TTTTCATAGT 300
  • MOLECULE TYPE Peptide (iV) SEQUENCE DESCRIPTION; SEQ ID NO: 4:
  • MOLECULE TYPE Peptide (iV) SEQUENCE DESCRIPTION; SEQ ID NO: 5: Ser Arg Ala His Gin His Ser Met Glu Thr Arg Thr Pro Asp He Asn
  • MOLECULE TYPE Peptide (iV) SEQUENCE DESCRIPTION; SEQ ID NO: 8: Thr Pro Asp lie Asn Pro Ala Trp Tyr 1 5 10 15
  • GCCTTCGAGC CACGCGGCTG GGTGTTCGGC GGCGGCCTGT GCCACCTGGT CTTCTTCCTG 180 CAGGCGGTCA CCGTCTATGT GTCGGTGTTC ACGCTCACCA CCATCGCAGT GGACCGCTAC 240
  • CAGCGCCAGC TCTACGCCTG GGGGCTGCTG CTGGTCACCT
  • ACCTGCTCCC TCTGCTGGTC 480 ATCCTCCTGT CTTACGCCCG GGTGTCAGTG AAGCTCCGCA ACCGCGTGGT GCCGGGCCGC 540
  • Synthetic DNA (xi) SEQUENCE DESCRIPTION; SEQ ID NO: 32 CCGGCGTACC AGGCAGGGTT 20
  • MOLECULE TYPE Peptide (iV) SEQUENCE DESCRIPTION; SEQ ID NO: 42: Thr Pro Asp He Asn Pro Ala Trp Tyr Ala Gly Arg Gly He Arg Pro 1 5 10 15 Val Gly Arg Phe
  • MOLECULE TYPE Peptide (iV) SEQUENCE DESCRIPTION; SEQ ID NO: 43: Thr Pro Asp He Asn Pro Ala Trp Tyr Ala Gly Arg Gly He Arg Pro

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EP98917693A 1997-04-28 1998-04-27 Polypeptid-ligand des g-protein-gekoppelten rezeptors der menschlichen hirnanhangdrüse Withdrawn EP0981616A1 (de)

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CA2196788A1 (en) * 1994-08-09 1996-02-22 Mohamed E. El Halawani Dna encoding turkey hypothalamic vasoactive intestinal peptide
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AU7011896A (en) * 1995-08-29 1997-03-19 Chiron Corporation Human hypothalmic ("hr") receptor polypeptide compositions, methods and uses thereof
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