WO1996017933A2 - Dna encoding a cell growth inhibiting factor and its product - Google Patents

Dna encoding a cell growth inhibiting factor and its product Download PDF

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Publication number
WO1996017933A2
WO1996017933A2 PCT/JP1995/002488 JP9502488W WO9617933A2 WO 1996017933 A2 WO1996017933 A2 WO 1996017933A2 JP 9502488 W JP9502488 W JP 9502488W WO 9617933 A2 WO9617933 A2 WO 9617933A2
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dna
cell growth
seq
growth inhibiting
inhibiting factor
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PCT/JP1995/002488
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French (fr)
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WO1996017933A3 (en
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Koichi Igarashi
Reiko Sasada
Michiyasu Takeyama
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Takeda Chemical Industries, Ltd.
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Priority to AU39951/95A priority Critical patent/AU3995195A/en
Publication of WO1996017933A2 publication Critical patent/WO1996017933A2/en
Publication of WO1996017933A3 publication Critical patent/WO1996017933A3/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel DNA and a method of screening for such DNA. More specifically, the present invention relates to a method of screening for and/or selecting a human DNA encoding a eukaryotic cell growth inhibiting factor, to novel DNA encoding for the inhibiting factor obtained by the method, to novel eukaryotic cell growth inhibiting factors, method for preparing said inhibiting factor and use thereof.
  • Clarifying the nature of such aging-associated genes is not only important in understanding aging, both at the cellular and individual levels, but is also significant in that the use of these genes or gene products would enable the diagnosis of various agmg- associated diseases and the development of prophylactic/therapeutic drugs for such diseases, or their application as prophylactic/therapeutic drugs for various diseases involving uncontrollable cell growth such as cancer.
  • the present inventors found that human cDNA encoding a cell growth inhibiting factor can be screened and subsequently isolated by introducing a cDNA library into a fission yeast, which is a eukaryotic cell host, wherein the cDNA library is prepared by ligating aged- cell-derived cDNA to the downstream of an inducible promoter.
  • the present inventors conducted further investigations based on this finding, and developed the present invention.
  • the present invention relates to:
  • a method of screening for a DNA encoding a eukaryotic cell growth inhibiting factor comprising :
  • the inducible promoter is PH05 promoter, nmt 1 promoter or hsp promoter,
  • a method of screening for a DNA encoding a eukaryotic cell growth inhibiting factor which comprises introducing a human DNA to be tested into a eukaryotic cell host so as to be controlled by an inducible promoter and selecting the host cell which does not grow under the inducible condition of said promoter but grows under the non-inducible condition,
  • a DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 11,
  • a DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 12,
  • a DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 23,
  • a DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 18,
  • a DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 19,
  • a DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 21,
  • a DNA coding for a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 27,
  • a DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 24,
  • a DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 26,
  • a vector comprising any one of DNAs according to the above paragraph (11) to (32) ,
  • a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 11,
  • a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 12,
  • a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 13,
  • a eukaryotic cell growth inhibiting factor which comprises the am o acid sequence of SEQ ID NO. 14,
  • a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 15,
  • a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 22,
  • a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 23,
  • a method for preparing the eukaryotic cell growth inhibiting factor according to the above paragraph (35) which comprises cultivating a transformant containing a DNA encoding said factor under conditions suitable for expression of the said factor and recovering said factor ,
  • a method for treating for a patient suffering from cancer or infection which comprises administering to said said patient an effective effective amount of the eukaryotic cell growth inhibiting factor according to the above paragraph (35) in the form of a pharmaceutical composition containing said factor as the effective component,
  • a method for inhibiting nucleic acid synthesis in target cell comprising containing said cell with an effective amount of a eukaryotic cell growth inhibiting factor encoded by the DNA according to the above paragraph (11) .
  • Figure 1 shows construction scheme for animal expression plasmid pTB1698.
  • the screening method of the present invention can be carried out by:
  • yeasts examples include yeasts, fungi and animal cells.
  • yeasts which involve little background influence under non-mduc g conditions in an expression system using an inducible promoter, with greater preference given to fission yeasts (Schizosaccharomyces) .
  • fission yeasts Schizosaccharomyces pombe is preferable.
  • any inducible promoter can be used, as long as it functions as a promoter in the eukaryotic host cell used, and as long as its activity can be regulated under culturing conditions that do not affect the growth of the host cell; an appropriate inducible promoter is selected according to the eukaryotic host cell used.
  • the host is a yeast, for instance, the PH05 promoter, nmt1 promoter etc. are preferred.
  • the host is an animal cell, the hsp promoter, metallothionein promoter etc. are preferred.
  • the preferred human DNA used as a sample is cDNA; it can be obtained by a common gene engineering procedure using mRNA prepared from human cells as template.
  • the human cell used to prepare the subject cDNA may be any one, as long as it is of human origin, it is preferable to use normal diploid cells, such as normal human fibroblast cells MRC-5, TIG-1 [Experimental Gerontology, Vol. 15, pp. 121-133 (1980)] and TIG-3 [Journal of Gerontology, Vol. 37, pp. 33-37 (1982)] , in the aging phase. It is also preferable to use aged cells prepared by subculturing relatively young cells until growth reaches a plateau.
  • RNA can be prepared from aged cells by the guanidine thiocyanate method [Chirgwin, J.M., et al . , Biochemistry, Vol. 18, p. 5294 (1979)] .
  • cDNA is synthesized by, for example, the method of Okayama, H. et al. [Molecular Cell Biology, Vol. 2, p. 161 (1982) ; ibid., Vol. 3, p. 280 (1983)] or the method of Gubler, U. and Hoffman, B.J. [Gene, Vol. 25, p. 263 (1983)] ; the obtained cDNA is introduced into a vector such as a plasmid or phage to yield a cDNA library.
  • Examples of the plasmid for cDNA insertion include plasmids derived from Escherichia coli such as pBR322 [Gene, Vol. 2, p. 95 (1977)] , pBR325 [Gene, Vol. 4, p. 121 (1978)] , pUC12 [Gene, Vol. 19, p. 259 (1982)] and pUC13 [Gene, Vol. 19, p. 259 (1982)] and those derived from Bacillus subtilis such as pUBIIO [Biochemical and Biophysical Research Communications, Vol. 112, p. 678 (1983)] , but any other can be used for this purpose, as long as it is replicable in the host.
  • cDNA is ligated to the downstream of an inducible promoter of a eukaryotic host cell, using a plasmid into which the promoter is inserted in advance.
  • Example methods for inserting a cDNA into the plasmid include that described by T. Maniatis et al. in Molecular Cloning, Cold Spring Harbor Laboratory, page 239 (1982) .
  • Example methods for inserting a cDNA into the phage vector include the method of Hyunh, T.V. et al. [DNA Cloning, A Practical Approach, Vol. 1, p. 49 (1985)] .
  • the thus-obtained plasmid or phage vector is introduced into an appropriate host, such as Escherichia coli, and stored.
  • Example strains of Escherichia coli include Escherichia coli K12 DH1 [Proceedings of the National Academy of Sciences, USA, Vol. 60, p. 160 (1968)] , JM103 [Nucleic Acids Research, Vol. 9, p. 309 (1981)] , JA221 [Journal of Molecular Biology, Vol. 120, p. 517 (1978)] , HB101 [Journal of Molecular Biology, Vol. 41, p. 459 (1969)] and C600 [Genetics, Vol. 39, p. 440 (1954)] .
  • Example strains of Bacillus subtilis include Bacillus subtilis MI 114 [Gene, Vol. 24, p. 255 (1983)] and 207-21 [Journal of Biochemistry, Vol. 95, p. 87 (1984) ] .
  • Example methods for transforming a host cell with a plasmid include the calcium chloride method described by T. Maniatis et al. [ibid., p. 249 (1982)] , the calcium chloride/rubidium chloride method and the electroporation method.
  • the phage vector can, for example, be introduced into cultured Escherichia coli by the in vitro packaging method.
  • the plasmid or phage vector is isolated from the transformant microorganism thus obtained to prepare subject cDNA for the present selection method.
  • the isolation method is exemplified by the alkali-SDS method [Birmboim, H.C., et al., Nucleic Acids Research, Vol. 1, p. 1513 (1979)] .
  • the subject cDNA as such or, if desired, after digestion with restriction enzymes, can be ligated to the downstream of the inducible promoter, to yield an expression vector.
  • Yeasts can be transformed in accordance with the method described in the Proceedings of the National Academy of Science, USA, Vol. 75, p. 1929 (1978) , for instance.
  • Animal cells can be transformed in accordance with the method described in Virology, Vol. 52, 456 (1973) , for instance.
  • trans ormants carrying the subject cDNA are cultured in accordance with the culturing method for the eukaryotic host cell used, with medium composition (metal ions, nitrogen sources, inorganic or organic acids, bases and other components) , pH, culturing temperature and other culturing conditions changed as appropriate.
  • medium composition metal ions, nitrogen sources, inorganic or organic acids, bases and other components
  • pH culturing temperature
  • other culturing conditions changed as appropriate.
  • the promoter can be induced in the absence of phosphate ions in the medium, using the PH05 promoter under the culturing conditions described later, and promoter activity can be adjusted by changing between the presence and absence of thiamme in the medium as described in Examples below, using the nmtl promoter.
  • promoter activity can be induced by incubation at a temperature (41 - 42t) slightly higher than ordinary culturing temperature (36 - 37T) for a given period of time.
  • a transformant is thus selected whose cell growth is inhibited under the promoter activity-inducing conditions, and which shows normal growth, according to the host cell used, under non-promoter-activity-induc g conditions .
  • cell growth is inhibited means that the cell growth under promoter-inducible-conditions is reduced as comparing with the cell growth under non- promoter-inducible-conditions (control) .
  • the cell growth rate inhibition is at least 25%, preferably 50%, more preferably 75%.
  • “cell does not grow” means that the cell hardly grow and the cell growth inhibition is about 75 - 100%.
  • the cell growth inhibition it is preferable to do when the cell growth reaches plateau.
  • the host cell is a fission yeast
  • it is preferable to observe the cell growth after cultivation is carried at about 20 to 40t for about 12 to 144 hours, more preferably at 20 to 35 t for about 24 to 72 hours.
  • a solid medium For observing cell growth inhibition, it is preferable to use a solid medium. Cells undergoing growth inhibition can easily be selected from the subject cell group by comparing the sizes of colonies formed. In confirming the growth inhibitory activity of the cells thus screened for, it is also effective to culture the cells in a liquid medium, in addition to the assay system using a solid medium, and determine growth inhibition rate by measuring a culture broth turbidity or uptake of tritium thymidine by cultured cell as an index.
  • the sizes of colonies formed are observed by naked eys or using a microscope.
  • the culture broth turbidity is determined by measuring the transmittance of visible radiation of culture broth.
  • the uptake of tritium thymidine by cultured cell is measured by known method.
  • a eukaryotic cell growth inhibiting factor is defined as a peptide or protein that suppresses or terminates the growth of eukaryotic cells such as yeasts and animal cells, under ordinary culturing conditions as shown below, and may be any one, as long as it is capable of reversibly or irreversibly inhibiting the growth of at least one kind of eukaryotic cell.
  • Such inhibiting factors include peptides or proteins having a partial or full-length portion of the amino acid sequence of SEQUENCE ID NOS. 11, 12, 13, 14, 15, 22, 23 or 27, including peptides or proteins whose sequences lack the N-terminal methionine residue.
  • DNA encoding the above-described eukaryotic cell growth inhibitor can be obtained by the method of the present invention for screening DNA and so on.
  • the DNA of the present invention is exemplified by DNA containing the nucleotide sequences shown by NUCLEIC ACID RESIDUE NOS. 279-752 of SEQUENCE ID NO. 8 encoding the amino acid sequenceof SEQ ID No. 12, NUCLEIC ACID RESIDUE NOS. 201-377 of SEQUENCE ID NO. 9 encoding the amino acid sequence of SEQ ID No. 13 , NUCLEIC ACID RESIDUE NOS. 296-1000 of SEQUENCE ID NO. 10 encoding the am o acid sequence of SEQ ID No. 14, NUCLEIC ACID RESIDUE NOS.
  • SEQUENCE ID NO. 7 encoding the amino acid sequence of SEQ ID No. 11, NUCLEIC ACID RESIDUE NOS. 51-740 of SEQUENCE ID NO. 16 encoding the ammo acid sequence of SEQ ID No. 15, NUCLEIC ACID RESIDUE NOS. 1062-1736 of SEQUENCE ID NO. 17 encoding the amino acid sequence of SEQ ID No. 22, NUCLEIC ACID RESIDUE NOS. 55-1488 of SEQUENCE ID NO. 20 encoding the amino acid sequence of SEQ ID No. 23, NUCLEIC ACID RESIDUE NOS. 150-1004 of SEQUENCE ID NO. 25 encoding the amino acid sequence of SEQ ID No. 27 and SEQUENCE ID NOS. 18 19, 21, 24 and 26.
  • N-terminal Met may be added to the inhibitor polypeptide; the cell growth factor may be a glycoprotein (sugar chain added) or a fused protein with another polypeptide.
  • An expression vector containing the DNA of the present invention that encodes eukaryotic cell growth inhibiting factor can be produced by, for example, 1) preparing cell growth inhibiting factor-encoding mRNA from an aged cell, 2) synthesizing cDNA and then double- stranded DNA from the mRNA to yield a cDNA library, 3) selecting cDNA encoding a polypeptide that inhibits host cell growth from the cDNA library, and 4) ligating the cDNA to the downstream of the promoter m the vector.
  • the cDNA library obtained from an aged cell in accordance with the above-described method may be treated to concentrate the desired cDNA by known methods, e.g., the differential hybridization method [Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, (1989)] , the subtraction method [Molecular Cloning, ibid.] and the cell suicide selection method.
  • the screening method of the present invention is also preferred. In such a case, the cell suicide selection method [Stetten, G. et al., Experimental Cell Research, Vol. 108, p.
  • Brdurd-Hoechst may be used to concentrate cells whose growth has been suppressed by the cDNA introduced, followed by selection of the desired cDNA.
  • the desired cDNA can also be selected by the present selection method using an inducible promoter .
  • the plasmid or phage vector is isolated from the microorganism by a known method such as the above- described alkali-SDS method.
  • the obtained plasmid or phage vector, harboring DNA containing a base sequence encoding a cell growth inhibitor can be used as such or, if desired, after digestion with restriction enzymes, according to the purpose of use.
  • the cloned gene is ligated to the downstream of the promoter, in a vector suitable for its expression, to yield an expression vector.
  • the gene may have ATG as a translational initiation codon at its 5 '-terminal and TAA, TGA or TAG as a translational termination codon at its 3' -terminal.
  • a promoter is ligated to its upstream. Any promoter can be used for the present invention, as long as it is appropriate for the host used to express the gene.
  • Example vectors include the above-mentioned plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13, ptrp 781) , plasmids derived from Bacillus subtilis (e.g., pUBIIO, pTM5 , pC194) , yeast-derived plasmids (e.g., pSH19, pSH15) , bacteriophages such as ⁇ phage, and animal viruses such as retrovirus and vaccinia virus.
  • Escherichia coli e.g., pBR322, pBR325, pUC12, pUC13, ptrp 781
  • Bacillus subtilis e.g., pUBIIO, pTM5 , pC194
  • yeast-derived plasmids e.g., pSH19, pSH15
  • Examples of preferred promoters include the T7 promoter, trp promoter, lac promoter, rec promoter, ⁇ PL promoter and lpp promoter when the transformation host is Escherichia coli , the SP01 promoter, SP02 promoter and pen P promoter when the host is Bacillus subtilis , and the PH05 promoter, PGK promoter, GAP (GLD) promoter, ADH promoter and nmtl promoter when the host is a yeast. Preference is given to the case in which Escherichia coli is used as host in combination with the T7 promoter, trp promoter or ⁇ PL promoter.
  • promoters include the SV40-derived promoter, retrovirus promoter, metallothionein promoter and hsp promoter, with preference given to the SV40-derived promoter.
  • the thus-constructed vector harboring DNA containing a nucleotide sequence such as one of the sequences of SEQUENCE ID NOS. 7-10, 16-21 and 24-26 is used to produce a transformant.
  • the host include prokaryotes such as Escherichia coli, Bacillus subtilis and actinomycetes , and eukaryotes such as yeasts, fungi and animal cells.
  • strains of Escherichia coli and Bacillus subtilis are the same as those mentioned above.
  • yeasts examples include Saccharomyces cerevisiae AH22, AH22R " , NA87-11A, DKD-5D, Schizosaccharomyces po be and mutants thereof.
  • animal cells include simian cells C0S-7, Vero, Chinese hamster cells CHO and mouse L cells.
  • the strains of Escherichia coli can be transformed in accordance with the method described in the Proceedings of the National Academy of Sciences, USA, Vol. 69, p. 2110 (1972) , Gene, Vol. 17, p. 107 (1982) and other publications, for instance.
  • Bacillus subtilis can be transformed in accordance with the method described in Molecular and General Genetics, Vol. 168, p. 111 (1979) and other publications, for instance.
  • Yeasts can be transformed in accordance with the method described in the Proceedings of the National Academy of Sciences, USA, Vol. 75, p. 1929 (1978) , for instance.
  • Animal cells can be transformed in accordance with the method described m Virology, Vol. 52, p. 456 (1973) , for instance.
  • a transformant as transformed with a vector harboring the desired DNA is thus obtained.
  • the host is a eukaryotic cell
  • the transformant is subcultured under non-promoter-mducing conditions using an inducible promoter.
  • liquid medium for culturing a transformant whose host is Escherichia coli , Bacillus subtilis , an actinornycete, yeast or fungus
  • carbon sources include glucose, dextrin, soluble starch and sucrose
  • nitrogen sources include organic or inorganic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extracts, soybean cake and potato extracts
  • minerals include calcium chloride, sodium dihydrogen phosphate and magnesium chloride.
  • the pH of the medium is preferably about 5 to 8.
  • Examples of media preferably used to culture Escherichia coli include the M9 medium containing glucose and casamino acid [Miller, Journal of Experimental Molecular Genetics, p. 431, Cold Spring Harbor Laboratory, New York (1972)] . Cultivation is normally carried out at about 14 to 43t for about 3 to 24 hours, with aeration and/or stirring as necessary.
  • cultivation is normally carried out at about 30 to 40t for about 6 to 24 hours, with aeration and/or stirring as necessary.
  • Examples of media for culturing a transformant whose host is a yeast include Burkholder's minimal medium [Bostian, K.L. et al., Proceedings of the National Academy of Sciences, USA, Vol. 77, p. 4505 (1980)] , preferably adjusted to a pH of about 5 to 8.
  • the transformant For culturing a transformant to express the desired gene using an inducible promoter, when an nmt1 promoter, for instance, is used, the transformant is cultured under promoter-inducing conditions, e.g., in a thiamme- free medium.
  • cultivation is normally carried out at about 20 to 35T for 24 to 72 hours, with aeration and/or stirring as necessary, until cell growth reaches a plateau.
  • Example media for culturing a transformant whose host is an animal cell include MEM containing about 5 to 20% fetal bovine serum [Science, Vol. 122, p. 501 (1952)] , DMEM [Virology, Vol. 8, p. 396 (1959)] , RPMI 1640 medium [Journal of the American Medical Association, Vol. 199, p. 519 (1967)] and 199 medium [Proceedings of the Society of Experimental Biological Medicine, Vol. 73, p. 1 (1950)] .
  • the transformant is cultured under promoter-inducing conditions, e.g.
  • m a medium supplemented with heavy metal ions when a metallothionein promoter is used.
  • the pH is preferably about 6 to 8. Cultivation is normally carried out at about 30 to 40T for 15 to 60 hours. When an inducible promoter is used, aeration and/or stirring is conducted as necessary, until cell growth reaches a plateau.
  • the eukaryotic cell growth inhibiting factor of the present invention is produced and accumulated tracellularly or extracellularly.
  • cultured cells collected by a known method are suspended in a buffer containing a protein denaturant such as guanidme hydrochloride or urea, or a surfactant such as Triton X-100, and then centrifuged to obtain a supernatant containing the cell growth inhibitor, or cells are disrupted by ultrasonication, treatment with an enzyme such as lysozyme, or freeze-thawing, followed by centrifugation to obtain a supernatant containing the cell growth inhibiting factor.
  • a protein denaturant such as guanidme hydrochloride or urea
  • a surfactant such as Triton X-100
  • known methods of separation and purification can be used in combination, as appropriate.
  • Such known methods of separation and purification include those based on solubility differences, such as salting-out and solvent precipitation, those based mainly on molecular weight differences, such as dialysis, ultraflltration, gel filtration and SDS-polyacrylamide gel electrophoresis, those based on charge differences, such as ion exchange chromatography, those based on specific affinity, such as affinity chromatography, those based on hydrophobicity differences, such as reverse-phase high performance liquid chromatography, and those based on isoelectric point differences, such as isoelectric focusing.
  • a eukaryotic cell growth inhibiting factor containing substantially no pyrogen or endotoxin is thus obtained in substantially pure form.
  • the substantially pure cell growth inhibiting factor of the present invention contains the cell growth inhibiting factor protein at not lower than 95% (w/w) , preferably not lower than 98% (w/w) .
  • "containing substantially no pyrogen or endotoxin” means that the cell growth inhibiting factor is negative in, for example, the known limulus test or pyrogen test.
  • the DNA of the present invention that encodes a eukaryotic cell growth inhibiting factor can be used as a probe for examining individual aging at the RNA level.
  • the DNA of the present invention can be used as a diagnostic reagent for various aging- associated diseases.
  • the gene of the present invention may also be introduced into cells of a target tissue, such as skm or vascular endothelium, to establish an in vitro aged cell line of the target tissue. Such a line is useful as a screening system for clarifying the mechanisms of onset and action of various agmg- associated diseases, or for seeking therapeutic drugs for these diseases.
  • the eukaryotic cell growth inhibiting factor encoded by the DNA of the present invention can be used as a pharmaceutical, such as an anticancer agent or infection remedy, as described later.
  • the eukaryotic cell growth inhibiting factor can be safely administered parenterally or orally, preferably topically, in the form of powder as such, or in the form of pharmaceutical compositions (e.g. , injections, tablets, capsules, solutions, ointments) together with pharmacologically acceptable carriers, excipients and diluents, to warm-blooded animals (e.g., humans, mice, rats, hamsters, rabbits, dogs, cats) .
  • the cell growth inhibiting factor can also be used as a skm drug.
  • An in ectable preparation is prepared in accordance with a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliaries.
  • Other pharmaceutical compositions such as tablets and capsules, can also be prepared in accordance with conventional methods.
  • the cell growth inhibiting factor of the present invention When using the cell growth inhibiting factor of the present invention as a pharmaceutical as described above in mammals, it is administered at daily doses of about 0.2 ⁇ g/kg to 20 mg/kg, preferably about 2 ⁇ g/kg to 0.2 mg/kg.
  • the cell growth inhibiting factor obtained according to the present invention is thought of as terminating cell division, and can therefore be used as a reagent for terminating the cell cycle of cultured cells at a given time point, e.g., a reagent for synchronizing cell division.
  • a reagent for synchronizing cell division By making constant the cell cycle of cells in an m_ vitro experimental system, it is possible to improve assay precision or establish an experimental system of a particular cell cycle.
  • the cell growth inhibiting factor of the present invention when using the cell growth inhibiting factor of the present invention as such a reagent, it is preferable to add it to the medium to a final concentration of 1 ng/ml to 1 mg/ml, more preferably 1 ng/ml to 10 ⁇ g/ml.
  • the factor encoded by the DNA of the present invention acts on young cells capable of division, or infinitely growing cancer cells, to prevent their growth.
  • the DNA of the present invention can therefore be used for gene therapy for cancer patients or as a probe for the diagnosis of aging-associated diseases.
  • the factor encoded by the DNA of the present invention can also be used as an anticancer agent. It is also effective against fungal infections (e.g. , cutaneous mycosis, deep mycosis) .
  • the DNA of the present invention can be used as a system for clarifying the mechanism of onset of aging-associated diseases or seeking therapeutic drugs, to establish an _m vitro aged cell line of the target tissue.
  • Substances that inhibit the cell growth inhibiting factor appear to be applicable as prophylactic/therapeutic drugs for aging and various aging-associated diseases, such as dementia and arteriosclerosis. Accordingly, the DNA of the present invention and the factor encoded thereby can be used to seek such drugs. The factor can also be used as a reagent for terminating the cell cycle of cultured cells at a given time point.
  • Antibodies or antiserum to the eukaryotic cell growth inhibiting factor or the partial peptides thereof of the present invention can be produced by the methods known per se in the art, using the factor or the partial peptides thereof as antigens.
  • the antibodies or antiserum can be used for inhibiting the activity of the eukaryotic cell growth inhibiting factor to rejuvenate the aged cell or tissues.
  • the antibodies or antiserum can also be used for quantitative analysis or detection of the factor or the partial peptides thereof by methods known per se in the art.
  • Antisense oligonucleotides complementary to the cDNA encoding the eukaryotic cell growth inhibiting factor can be synthesized by the known methods.
  • the oligonucleotides hybridize to the mRNA, inhibit the production of the eukaryotic cell growth inhibiting factor and induce re uvevation of aged cell or tissues.
  • These oligonucleotides can be used for in vivo and x vivo treatment of diseases caused by cellular senescence or agmg-associated diseases, such as arteriosclerosis and dementia.
  • These oligonucleotides are also effective for a normal tissue or cell, such as skin cell, a cell present in wound or burn tissue, lymphocyte, vascular tissue, liver, kidney, heart, bone, spleen, etc.
  • PBS Phosphate-buffered saline
  • DNA Deoxyribonucleic acid
  • cDNA Complementary deoxyribonucleic acid
  • RNA Ribonucleic acid
  • mRNA Messenger ribonucleic acid
  • dATP Deoxyadenosine triphosphate
  • dTTP Deoxythymidme triphosphate
  • dGTP Deoxyguanosme triphosphate
  • dCTP Deoxycytidine triphosphate
  • Trp Tryptophan
  • Plasmid pTB399 (Cell Struct. Funct. , Vol. 12, pp. 205-217) was cleaved with EcoRI and reacted with the Klenow fragment, followed by addition of Bglll linker (CAGATCTG) and cleavage with Bglll, to yield a 3.8 kb DNA fragment deleting the ⁇ nterleuk ⁇ n-2 (IL-2) cDNA region. The fragment was then cyclized using T4 ligase. The MuLV-LTR portion was then replaced with an SR ⁇ promoter derived from pME18S [Maruyama, K. and Takebe, Y., Medical Immunology, Vol. 20, pp. 27-32 (1990)] in accordance with a conventional method, to yield plasmid pTB1695.
  • aged cells aged normal human diploid fibroblasts
  • RNA extraction kit (Dainippon Pharmaceutical) , the cells were treated to extract an RNA fraction using an RNA extraction kit
  • oligo-dT cellulose type 3 (Collaborative Biomedical)
  • TE buffer 10 mM Tris-HCl buffer containing 0.1 M sodium chloride and 1 mM EDTA (pH 7.4)
  • TE buffer 10 mM Tris-HCl buffer containing 0.1 M sodium chloride and 1 mM EDTA (pH 7.4)
  • TE buffer 10 mM Tris-HCl buffer containing 0.1 M sodium chloride and 1 mM EDTA (pH 7.4)
  • TE buffer EC0N0- COLUMN (17 mm in diameter, 15 cm in length)
  • the RNA fraction previously adjusted to a final concentration of 0.5 M sodium chloride, was heated at 65t for 5 minutes then immediately quenched in ice, after which it was applied to the equilibrated column.
  • the coupled RNA fraction was eluted with TE buffer to yield an mRNA fraction (yield 115 ⁇ g) .
  • cDNA was synthesized by the method of Nojima et al. [No ima, H. , Development and Application of New Vector System in an Attempt to Catalog All Human cDNA Banks (Research Subject No. 02557098) , 1992 Grant-m-Aid for Scientific Research from the Ministry of Education (Investigation B(D) Final Report, p. 29 (1993)] .
  • RNA was subjected to reverse transcription with an oligo-dT primer linker having an NotI recognition sequence (GCGGCCGC) as a template, to synthesize a first strand, RNase H and Escherichia coli DNA polymerase I were simultaneously reacted to remove the mRNA region and synthesize a second strand at the same time.
  • oligo-dT primer linker having an NotI recognition sequence (GCGGCCGC) as a template
  • Both ends of the thus-obtained double-stranded DNA were blunted using T4 DNA polymerase, followed by T4 DNA ligase action to bind a dephosphorylation BamHI adapter to both ends; NotI was then reacted to yield a cDNA fragment having a dephosphorylated BamHI site on the 5 '-terminal side and a phosphorylated NotI site on the 3 '-terminal side (yield about 5 ⁇ g) .
  • DNA oligomers (1) , (2) , (4) and (5) were mixed in an amount of 10 ⁇ g each, followed by 5'-termmal phosphorylation by the action of T4 polynucleotide kinase. After the reaction mixture was kept standing at 65T for 15 minutes, DNA oligomers (3) and (6) , 10 ⁇ g each, were added and ligated using T4 DNA ligase. The reaction mixture was subjected to 4% agarose electrophoresis to recover a 73 bp DNA fragment; the 5'- terminal was then phosphorylated by the action of T4 polynucleotide kinase. Next, to the fission yeast expression vector pREPI [Maundrell, K., Gene, Vol.
  • This plasmid is a fission yeast expression vector having a T7 RNA polymerase recognition sequence, Mlul site, BamHI site, B ⁇ tXI site, Sail site, NotI site, T3 RNA polymerase recognition sequence and nmt1 terminator in that order, and an LEU2 gene as a selection marker, downstream of the nmtl promoter [Maundrell, K. , Journal of Biological Chemistry, Vol. 265, p. 10857 (1989)] .
  • the fission yeast expression vector prepared in Example 2 (pTB1589) was digested with NotI, treated with alkaline phosphatase, and further digested with BamHI, followed by 0.7% agarose gel electrophoresis to recover a vector fraction.
  • the cDNA library thus obtained comprised 1.7 x 10 6 independent transformant cells of 1.5 kbp mean cDNA length.
  • the plasmid was purified from the transformant, diluted with TE buffer to a concentration of 1 ⁇ g per 15 ⁇ l, and stored at -20t until use.
  • Fission yeast cells (Schizosaccharomyces pombe h " leul " ) growing on YEA plate were inoculated to 100 ml of MB medium containing 0.25% (w/v) L-leucme at a density of 10 6 cells/ml, and cultured at 30T until the cell density reached 5 x 10 6 to 1 x 10 7 cells/ml.
  • Cells were harvested at room temperature and washed with sterile water, after which they were suspended in a 0.1 M lithium acetate solution (pH 4.9-5.0) to 10 9 cells/ml. This suspension was dispensed to Eppendorf tubes at lOO ⁇ l per tube, and kept standing at 30t for 1 hour. To each tube, 1 ⁇ g (15 ⁇ l) of the plasmid prepared in Example 5 and 290 ⁇ l of a 50% (w/v) polyethylene glycol 4000 solution were added. After thorough mixing, the mixture was kept standing at 30T for 50 minutes, then heated at 43T for 15 minutes, then kept standing at room temperature for 10 minutes, followed by cell harvest.
  • a 50% (w/v) polyethylene glycol 4000 solution 50% (w/v) polyethylene glycol 4000 solution
  • the cells were suspended in 1 ml of 1/2 YEL medium containing 0.25% (w/v) L-leucine; the suspension was then shaken at 30T for 1 to 2 hours. After dilution with 9 ml of 1/2 YEL medium, the suspension was applied to MMA plates containing 2 ⁇ M thiamme (MMAT plates of medium that inhibits nmtl promoter transcription) at lOO ⁇ l per plate, and cultured at 30T for 2 days. The MMAT plates on which minute colonies appeared were replicated to new MMA plates (medium that promotes the same promoter transcription as above) and MMAT plates, followed by culturing at 30T for 3 to 4 days. As a result of screening of 8.25 x 10 4 transformant cells by the above-described method, 18 transformants (candidate strains) that grow on MMAT plates but not on MMA plates were found (cell growth rate inhibiting is more than about 75%) .
  • Each candidate strain was inoculated over the entire surface of an MMAT plate and cultured at 30t for 2 to 3 days.
  • the cells on the plate were recovered into an Eppendorf tube using 1.5 ml of TES solution (TE buffer containing 10 mM sodium sulfite) , followed by cell harvest.
  • the cells were then suspended in 1 ml of a sorbitol solution (1M sorbitol, 100 mM EDTA, 10 mM sodium sulfite, 100 mM lithium acetate) ; the suspension was kept standing at 30t for 1 hour in the presence of 20 units of Lyticase (Boehringer) .
  • the crude DNA fraction was dissolved in 50 ⁇ l of TE buffer; after addition of lOO ⁇ l of a sodium iodide solution (GENECLEAN II Kit, BI0101 Company) and 5 ⁇ l of a glass milk suspension (provided with the kit) , the mixture was kept standing at room temperature for 5 minutes.
  • the glass milk fraction was centrifugally recovered, and washed with 3 portions of 400 ⁇ l of an ice-cooled NEW solution (provided with the kit) .
  • the washed precipitate was treated with lO ⁇ l of TE buffer at 551 for 3 minutes; this operation was repeated in two cycles to yield a purified DNA fraction.
  • Escherichia coli MC1061 was transformed by the electroporation method described in Example 3; the plasmid having cDNA was recovered from the resulting transformant.
  • the cDNA plasmids recovered from the 20 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 10 clones showing cDNA expression-dependent growth inhibition were found.
  • base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd. , US70777) with [ 35 S]dCTPS, in accordance with the kit protocol. The thus-obtained base sequences were examined for homology on the current DNA data base (GeneBank Release 84.0; 196703 entries) ; 4 new clones were found (Table 1) .
  • the plasmids harbored by the respective clones were designated pTB1617, pTB1668, pTB1671 and pTB1673, respectively; the entire base sequences of these cDNAs are shown in SEQUENCE ID NOS. 7, 8, 9 and 10, respectively.
  • ammo acid sequences of the polypeptides or proteins encoded by the cDNAs harbored by pTB1617, PTB1668, pTB1671 and pTB1673 are shown in SEQUENCE ID NOS. 11, 12, 13 and 14, respectively.
  • the expression plasmid of sd ⁇ -1 gene [W09312251; Noda, A et. al., Experimental Cell Research, Vol. 211 p. 90-98 (1994)] was also constructed, in which sd ⁇ -1 gene was ligated downstream of the nmt1 promoter as shown in Example 3, and introduced into fission yeast.
  • the yeast transformant thus obtained could form colonies on MMA plate as same as on MMAT plate (cell growth rate inhibiting is about 0%) , indicating that sd ⁇ -1 type gene can not be obtained by the screening method using fission yeast as described.
  • the cDNA plasmids recovered from 10 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 3 clones showing cDNA expression-dependent growth inhibition were found.
  • base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd., US70777) with [ S5 S]dCTPS, in accordance with the kit protocol. The thus-obtamed base sequences were examined on the current DNA data base (GeneBank Release 86.0; 237775 entries) ; 1 new clone was found.
  • the plasmid having the clone was designated pTB1848; the entire base sequence of its cDNA is shown in SEQUENCE ID NO. 16.
  • the amino acid sequence of the protein encoded by the cDNA harbored by plasmid pTB1848 is shown in SEQUENCE ID NO. 15.
  • plasmid pTB1697 obtained by introducing an E. coli lacZ gene into the Hmdlll site of pRc/CMV (Invitrogen, USA) , a 4.9 kbp Nrul-Nael fragment was cut out and inserted into the Sail site of the plasmid pTB1695 prepared in Reference Example 1 to yield the plasmid pTB1698 ( Figure 1) .
  • the plasmids pTB1668, pTB1671, pTB1673 and pTB1848, obtained in Examples 6 and 7, were each cleaved with BamHI-NotI; the resulting cDNA portions were each inserted into the Bglll site of pTB1698, located downstream of the SR ⁇ promoter, to yield animal cell expression plasmids.
  • the animal cells used to determine DNA synthesis inhibitory activity were normal diploid fibroblasts (purchased from Cell System; defined primary human dermal fibroblast cell system, hereinafter Fb cells) at the growth stage, subcultured m 20-35 generations under the same culturing conditions as Example 1. After being sown over Lab-Tek chamber slides (Nunc, USA) at 5 x 10 4 cells per plate and cultured at 37T for 1 day, Fb cells were transfected with the pTBI848-der ⁇ ved cDNA expression plasmid prepared in Example 8, by the calcium phosphate method [Chen, C. and Okaya a, H. , Molecular Cell Biology, Vol. 7, pp. 2745-2752 (1987)] .
  • the medium was replaced with fresh one, followed by cultivation for 1 more day.
  • 37 KBq/ml (925 GBq/mmol) tritiated thymidine ( s H-thymidine) was added, followed by 48 hours of cultivation to label the cells.
  • glutaraldehyde fixation the cells were stained with X-gal.
  • an emulsion was applied; the plate was kept standing in a dark room for 4-5 days, followed by development.
  • the blue-stained-galactosidase expression cells were counted under a microscope; the ratio of cells showing black particles in their nuclei due to s H-thym ⁇ d ⁇ ne uptake was determined.
  • DNA synthesis inhibitory rates were calculated with the labeling index, taking plasmid pTB1698 as 0%.
  • Fb cells were used which had been transformed with the expression plasmid pTB1699 constructed by introducing the sd ⁇ -1 gene [Noda, A. et al. , Experimental Cell Research, Vol. 211, pp. 90-98 (1994)] into the Bglll site of pTB1698 in accordance with the method described in Example 8. The results are shown in Table 2.
  • the plasmids having cDNA recovered from 25 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 10 clones showing cDNA expression-dependent growth inhibition were found.
  • base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd. , US70777) with [ s 5 S]dCTPS, in accordance with the kit protocol. The thus-obtained base sequences were examined on the current DNA data base (GeneBank Release 86.0; 237775 entries) ; 5 new clones were found (Table 3) .
  • the plasmids having the clones were designated as pTB1618, pTB1689, pTB1756, pTB1761 and pTB1721; the entire base sequences of their cDNAs are shown in SEQUENCE ID NOS. 17, 18, 19, 20 and 21, respectively.
  • the amino acid sequences of the polypeptides or proteins encoded by the cDNAs harbored by the plasmids pTB1618 and pTB1761 are shown in SEQUENCE ID NOS. 22 and 23, respectively.
  • Expression plasmids for animal cell were constructed by cleaving the plasmids pTB1618, pTB1689, pTB1721, pTB1756 or pTB1761 obtained in Example 10 with BamHI-NotI, and introducing into the Bglll site of pTB1698 each of the obtained cDNA portions by the method described in Example 8. Using these cDNA expression plasmids, DNA synthesis inhibitory activity was determined by the method described in Example 9. As shown in Table 4, the cells incorporating the pTB1689- de ⁇ ved cDNA underwent thymidine uptake inhibition in 3 experiments, as with the positive control. Similarly, all cells incorporating pTB1618, pTB1721, pTB1756 or pTB1761 underwent thymidine uptake inhibition in 3 experiments, as shown in Table 5.
  • the plasmids having cDNA recovered from 22 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 9 clones showing cDNA expression-dependent growth inhibition were found.
  • base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd., US70777) with [ S 5 S]dCTPS, in accordance with the kit protocol. The thus-obtained base sequences were examined on the current DNA data base (GeneBank Release 86.0; 237775 entries) ; 3 new clones were found (Table 6) .
  • the plasmids having the clones were designated as pTB1786, pTB1810 and pTB1819; the entire base sequences of their cDNAs are shown in SEQUENCE ID NOS. 24, 25 and 26, respectively.
  • the amino acid sequence of the polypeptide or protein encoded by the cDNA harbored by the plasmid pTB1810 is shown in SEQUENCE ID NO. 27.
  • Expression plasmids for animal cell were constructed by cleaving the plasmids pTB1786, pTB1810 or pTB1819 obtained in Example 12 with BamHI-NotI, and introducing into the Bglll site of pTB1698 each of the obtained cDNA portions by the method described in Example 8. Using these cDNA expression plasmids, DNA synthesis inhibitory activity was determined by the method described in Example 9. As shown in Tables 7 and 8, the cells incorporating the pTB1689-der ⁇ ved cDNA underwent thymidine uptake inhibition in more than 2 experiments, as with the positive control.
  • TTTTTTTCCT CAGGAATAAT TTTCTCTTTT GGAAAGCACT TTCCCCGTCT CAGTAGAAAA 540
  • ACATCAGTCC CACCTGCACG AATAAGGAGC TTCGAGCCAA GTTTGAGGAG TATGGTCCGG 36
  • CAGTGGAGGC CATCAGGGGC CTTGATAACA CAGAGTTTCA AGGCAAACGA ATGCACGTGC 48
  • CTTTGCCATC TTGGTCCAGT CTGGCTGCAG CAAAAAGCAG GCGATGCGTC TGCAACTACT 360
  • TTGCAATCAC TTCTGGAGGG TGCTGGGGCT GCTGGGGGGA ATTATCATGA TGGTGCTGAT 600 TCCCCACCTT GAGTGAGGGG TGGATAAACT ACCCCTCCCC AAACCTCTAC CCCTAACTCC 660
  • CTGCCGCTCA CCTACCAGCC TGACATCTCC ACAGCTGCCC TGGCCCACCC ACAAGGGGCC 480
  • CAGACACTAC ATGGGTAGCT CAGGGGAGGA GGTGGGGGTC
  • TTCTCCCCGG CAGAATTTTT TTTCCTTGTT AGATATCAGG GATATAGGCC GGGTGCGGTG 1680

Abstract

Disclosed are (1) a method of screening a DNA encoding a human cell growth inhibiting factor, by introducing a human DNA into a eukaryotic cell host under the control of an inducible promoter, and selecting DNA whose host cell does not grow under promoter-inducing conditions, but grows under non-promoter-inducing conditions, (2) new DNA encoding a human cell growth inhibiting factor selectable by this method, (3) a vector containing said DNA, (4) a transformant as transformed with said vector, (5) new human cell growth inhibiting factor, (6) a method for preparing said factor and a pharmaceutical composition containing said factor. According to the above screening method, a DNA encoding a human cell growth inhibiting factor can be selectively and conveniently obtained. The thus obtained DNA is useful as a probe for investigating aging, or as a reagent for diagnosing various aging-associated diseases. The human cell growth inhibiting factor can be useful as an anticancer agent, infection remedy and so on.

Description

DESCRIPTION DNA ENCODING A CELL GROWTH INHIBITING FACTOR AND ITS PRODUCT
Technical Field
The present invention relates to novel DNA and a method of screening for such DNA. More specifically, the present invention relates to a method of screening for and/or selecting a human DNA encoding a eukaryotic cell growth inhibiting factor, to novel DNA encoding for the inhibiting factor obtained by the method, to novel eukaryotic cell growth inhibiting factors, method for preparing said inhibiting factor and use thereof.
Background Art
In higher multicellular organisms, there are various aging associated diseases such as dementia and arteriosclerosis. To basically clarify the causes of these diseases, the mechanism of aging must be understood. However, the mechanism of individual aging is extremely complex; there is no clue to an understanding of the mechanism. Against this background, the aging of individual-constituting cells may be analyzed as a first step toward the understanding of individual aging.
Animal tissue cells in culture lose their growth capability as the number of subculturing generations increases, eventually terminating growth and dying, although showing good growth initially. This phenomenon is called cell aging [Hayflick, L. and Moorhead, P.S., Experimental Cell Research, Vol. 25, p. 585 (1961) ; Hayflick, L., ibid., Vol. 37, p. 614 (1965)] . Very limited portions of such cells may become immortal (immortalized cells) .
From the following experimental results, it is evident that aging at the cell level is closely associated with individual aging. (1) The maximum possible number of divisions (division life span) of cultured cells is inversely proportional to individual age [Martin, G.M. et al.. Laboratory Investigation, Vol. 23, p. 86 (1970) ; Schneider, E.L. and Mitsui, Y., Proceedings of the National Academy of Sciences, USA, Vol. 73, p. 3584 (1976) ; Goldstein, S. et al . , Science, Vol. 199, p. 781 (1978)] . (2) Cells derived from patients with hereditary progeria are short in division life span while in culture [Martin, G.M. et al., ibid. ; Goldstein, S. , Lancet, Vol. 1, p. 424 (1969) ; Goldstein, S., Journal of Investigative Dermatology, Vol. 73, p. 19 (1979) ; Norwood, T.H. et al . , ibid., Vol. 73, p. 92 (1979)] . (3) There is a correlation between the maximum life span of various animal species and the division life span of cultured cells derived therefrom [Roeme , D., Proceedings of the National Academy of Sciences, USA, Vol. 78, p. 5009 (1981)] . To summarize, cell aging is not assumed to be unique to
Figure imgf000004_0001
vitro systems.
Even if an aged cell is fused with a young or immortalized cell, DNA synthesis does not occur again in the aged cell; on the contrary, DNA synthesis in the young and immortalized cell is suppressed [Norwood, T.H. et al., Proceedings of the National Academy of Sciences, USA, Vol. 71, p. 2231 (1974) ; Yanishevsky, R.M. and Stein, G.H., Experimental Cell Research, Vol. 126, p. 469 (1980) ; Stein, G.H. and Yanishevsky, R.M., Proceedings of the National Academy of Sciences, USA, Vol. 78, p. 3025 (1981)] . This demonstrates that the phenotypes related to cellular senescence are dominant, and that the aged cell does not lack substances essential to its growth but has a substance that suppresses DNA synthesis therein. In fact, mιcroιn]ectιon of mRNA prepared from an aged cell into a young cell is known to inhibit DNA synthesis in the latter [Lumpkm, C.K. et al . , Science, Vol. 232, p. 393 (1986)] . It can therefore be held that there are some genes whose expression occurs newly or increases with cell age, and that such genes play an important role in cell aging, directly or indirectly.
Smith et al. tested the complementation of a large number of immortalized human cells in fused pairs, demonstrating the presence of 4 groups of human aging genes [Pereira-Smith, O.M. and Smith, J., Science, Vol. 221, p. 964 (1983) ; Pereira-Smith, O.M. and Smith, J., Proceedings of the National Academy of Sciences, USA, Vol. 85, p. 6042 (1988)] . Also, they have recently found DNA that encodes a DNA synthesis-inhibiting protein (SDI) [W09312251] .
Clarifying the nature of such aging-associated genes is not only important in understanding aging, both at the cellular and individual levels, but is also significant in that the use of these genes or gene products would enable the diagnosis of various agmg- associated diseases and the development of prophylactic/therapeutic drugs for such diseases, or their application as prophylactic/therapeutic drugs for various diseases involving uncontrollable cell growth such as cancer.
Disclosure of Invention
With the expectation that there are genes involved in the suppression of aged cell growth and showing little or no expression in young cells, there have been many attempts to clone such aging-associated genes in the form of cDNA. However, due to marked increase in the mRNA of extracellular substrates (e.g., collagen, fibronectm) , which are not directly associated with aged cell growth suppression, the desired mRNA or cDNA is difficult to select and obtain. It is also known that the obtained cDNA is expressed to a considerable extent even in young cells. For these reasons, no one has succeeded in cloning cDNA specific to aged cells.
The present inventors found that human cDNA encoding a cell growth inhibiting factor can be screened and subsequently isolated by introducing a cDNA library into a fission yeast, which is a eukaryotic cell host, wherein the cDNA library is prepared by ligating aged- cell-derived cDNA to the downstream of an inducible promoter. The present inventors conducted further investigations based on this finding, and developed the present invention.
Namely, the present invention relates to:
(1) A method of screening for a DNA encoding a eukaryotic cell growth inhibiting factor comprising :
(a) introducing a human DNA operably linked to an inducible promoter into a eukaryotic host cell ;
(b) testing said host cell for the presence of said DNA by measuring host cell growth rate under conditions in which the promoter is induced and not induced ; and
(c) determmg the differential growth of these two groups at selected times whereby a host cell showing at least about 25% growth rate inhibition under the inducible condition as compared with the cell growth rate under the non-mducible condition is identified as containing the DNA encoding a eukaryotic cell growth inhibiting factor,
(2) The method according to the above paragraph (1) , wherein a host cell showing at least about 50% growth rate inhibition is identified as containing the DNA,
(3) The method according to the above paragraph (1) , wherein a host cell that has at least about 75% growth rate inhibition is identified as containing the DNA,
(4) The method according to the above paragraph (1) , further comprising the step of isolating the DNA encoding the eukaryotic cell growth inhibiting factor from the host identified as containing the DNA,
(5) The method according to the above paragraph (1) , the inducible promoter is PH05 promoter, nmt 1 promoter or hsp promoter,
(6) The method according to the above paragraph (1) , the selected times is about 12 to 144 hours after culture, (7) A method of screening for a DNA encoding a eukaryotic cell growth inhibiting factor, which comprises introducing a human DNA to be tested into a eukaryotic cell host so as to be controlled by an inducible promoter and selecting the host cell which does not grow under the inducible condition of said promoter but grows under the non-inducible condition,
(8) The method according to the above paragraph (1) or (7) , wherein said eukaryotic cell host is a yeast,
(9) The method according to the above paragraph (8) , wherein said yeast is a fission yeast,
(10) The method according to the above paragraph (9) , wherein said fission yeast is a Shizosaccharomyces pombe,
(11) An isolated DNA encoding a eukaryotic cell growth inhibiting factor, which is screened by the method according to the above paragraph (1) ,
(12) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 11,
(13) The DNA according to the above paragraph (12) , wherein said DNA comprises a nucleotide sequence at least from the 248th to the 448th residues of the nucleotide sequence represented by SEQ ID NO. 7,
(14) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 12,
(15) The DNA according to the above paragraph (14) , wherein said DNA comprises a nucleotide sequence at least from the 279th to the 752nd residues of the nucleotide sequence represented by SEQ ID NO. 8,
(16) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 13,
(17) The DNA according to the above paragraph (16) , wherein said DNA comprises a nucleotide sequence at least from the 201st to the 377th residues of the
-D- nucleotide sequence represented by SEQ ID NO. 9,
(18) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 14,
(19) The DNA according to the above paragraph (18) , wherein said DNA comprises a nucleotide sequence at least from the 296th to the 1000th residues of the nucleotide sequence represented by SEQ ID NO. 10,
(20) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 15,
(21) The DNA according to the above paragraph (20) , wherein said DNA comprises a nucleotide sequence at least from the 51st to the 740th residues of the nucleotide sequence represented by SEQ ID NO. 16,
(22) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 22,
(23) The DNA according to the above paragraph (22) , wherein said DNA comprises a nucleotide sequence at least from the 1062nd to the 1736th residues of the nucleotide sequence represented by SEQ ID NO. 17,
(24) A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 23,
(25) The DNA according to the above paragraph (24) , wherein said DNA comprises a nucleotide sequence at least from the 55th to the 1488th residues of the nucleotide sequence represented by SEQ ID NO. 20,
(26) A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 18,
(27) A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 19,
(28) A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 21,
(29) A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 27,
(30) The DNA according to the above paragraph (29) , wherein said DNA comprises a nucleotide sequence at least from the 150th to the 1004th residues of the nucleotide sequence represented by SEQ ID NO. 25,
(31) A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 24,
(32) A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 26,
(33) A vector comprising any one of DNAs according to the above paragraph (11) to (32) ,
(34) A transformant harboring the vector according to the above paragraph (33) ,
(35) A eukaryotic cell growth inhibiting factor which is coded by the DNA obtained by the method according to the above paragraph (1) ,
(36) A eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 11,
(37) A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 12,
(38) A eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 13,
(39) A eukaryotic cell growth inhibiting factor which comprises the am o acid sequence of SEQ ID NO. 14,
(40) A eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 15,
(41) A eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 22,
(42) A eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 23,
(43) A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 27, (44) A eukaryotic cell growth inhibiting factor which is encoded by the DNA according to the above paragraph
(26) ,
(45) A eukaryotic cell growth inhibiting factor which is encoded by the DNA according to the above paragraph
(27) ,
(46) A eukaryotic cell growth inhibiting factor which is encoded by the DNA according to the above paragraph
(28) ,
(47) A eukaryotic cell growth inhibiting factor which is encoded by the DNA according to the above paragraph
(31) ,
(48) A eukaryotic cell growth inhibiting factor which is encoded by the DNA according to the above paragraph
(32) ,
(49) A method for preparing the eukaryotic cell growth inhibiting factor according to the above paragraph (35) which comprises cultivating a transformant containing a DNA encoding said factor under conditions suitable for expression of the said factor and recovering said factor ,
(50) A pharmaceutical composition which comprises an effective amount of any one of eukaryotic cell growth inhibiting factors according to the above paragraph
(35) ,
(51) Use of the eukaryotic cell growth inhibiting factor according to the above paragraph (35) for preparing an anticancer agent or infection remedy,
(52) A method for treating for a patient suffering from cancer or infection which comprises administering to said said patient an effective effective amount of the eukaryotic cell growth inhibiting factor according to the above paragraph (35) in the form of a pharmaceutical composition containing said factor as the effective component,
(53) A method for inhibiting nucleic acid synthesis in target cell comprising containing said cell with an effective amount of a eukaryotic cell growth inhibiting factor encoded by the DNA according to the above paragraph (11) .
Brief Description of Drawings
Figure 1 shows construction scheme for animal expression plasmid pTB1698.
Best Mode for Carrying Out the Invention
The screening method of the present invention can be carried out by:
1) synthesizing cDNA using mRNA prepared from a human aged cell as template,
2) ligating the cDNA to the downstream of an inducible promoter to prepare an expression cDNA library,
3) introducing the library into a eukaryotic host cell,
4) culturing the obtained transformant eukaryotic host cell under inducible promoter-inducing conditions and non-promoter-inducing conditions, and
5) screening cells for those having reduced growth rates under inducible promoter-inducing conditions as compared with growth under non-promoter-inducing conditions.
While the combination of these steps is novel, commonly known techniques are applicable to these processes 1) through 5) .
Examples of the eukaryotic cell host for the present method include yeasts, fungi and animal cells. Preferred are yeasts which involve little background influence under non-mduc g conditions in an expression system using an inducible promoter, with greater preference given to fission yeasts (Schizosaccharomyces) . Of the fission yeasts, Schizosaccharomyces pombe is preferable.
Any inducible promoter can be used, as long as it functions as a promoter in the eukaryotic host cell used, and as long as its activity can be regulated under culturing conditions that do not affect the growth of the host cell; an appropriate inducible promoter is selected according to the eukaryotic host cell used. When the host is a yeast, for instance, the PH05 promoter, nmt1 promoter etc. are preferred. When the host is an animal cell, the hsp promoter, metallothionein promoter etc. are preferred.
In the present screening method, the preferred human DNA used as a sample is cDNA; it can be obtained by a common gene engineering procedure using mRNA prepared from human cells as template. Although the human cell used to prepare the subject cDNA may be any one, as long as it is of human origin, it is preferable to use normal diploid cells, such as normal human fibroblast cells MRC-5, TIG-1 [Experimental Gerontology, Vol. 15, pp. 121-133 (1980)] and TIG-3 [Journal of Gerontology, Vol. 37, pp. 33-37 (1982)] , in the aging phase. It is also preferable to use aged cells prepared by subculturing relatively young cells until growth reaches a plateau. For example, RNA can be prepared from aged cells by the guanidine thiocyanate method [Chirgwin, J.M., et al . , Biochemistry, Vol. 18, p. 5294 (1979)] .
Using the thus-obtained RNA as template, in combination with reverse transcriptase , cDNA is synthesized by, for example, the method of Okayama, H. et al. [Molecular Cell Biology, Vol. 2, p. 161 (1982) ; ibid., Vol. 3, p. 280 (1983)] or the method of Gubler, U. and Hoffman, B.J. [Gene, Vol. 25, p. 263 (1983)] ; the obtained cDNA is introduced into a vector such as a plasmid or phage to yield a cDNA library.
Examples of the plasmid for cDNA insertion include plasmids derived from Escherichia coli such as pBR322 [Gene, Vol. 2, p. 95 (1977)] , pBR325 [Gene, Vol. 4, p. 121 (1978)] , pUC12 [Gene, Vol. 19, p. 259 (1982)] and pUC13 [Gene, Vol. 19, p. 259 (1982)] and those derived from Bacillus subtilis such as pUBIIO [Biochemical and Biophysical Research Communications, Vol. 112, p. 678 (1983)] , but any other can be used for this purpose, as long as it is replicable in the host. Also included is λ gt 11 [Young, R. and Davis. R., Proceedings of the National Academy of Sciences, USA, Vol. 80, p. 1194 (1983)] , but any other can be used, as long as it is capable of growing in the host. From the viewpoint of procedural simplicity, it is particularly preferable that cDNA is ligated to the downstream of an inducible promoter of a eukaryotic host cell, using a plasmid into which the promoter is inserted in advance.
Example methods for inserting a cDNA into the plasmid include that described by T. Maniatis et al. in Molecular Cloning, Cold Spring Harbor Laboratory, page 239 (1982) . Example methods for inserting a cDNA into the phage vector include the method of Hyunh, T.V. et al. [DNA Cloning, A Practical Approach, Vol. 1, p. 49 (1985)] . The thus-obtained plasmid or phage vector is introduced into an appropriate host, such as Escherichia coli, and stored.
Example strains of Escherichia coli include Escherichia coli K12 DH1 [Proceedings of the National Academy of Sciences, USA, Vol. 60, p. 160 (1968)] , JM103 [Nucleic Acids Research, Vol. 9, p. 309 (1981)] , JA221 [Journal of Molecular Biology, Vol. 120, p. 517 (1978)] , HB101 [Journal of Molecular Biology, Vol. 41, p. 459 (1969)] and C600 [Genetics, Vol. 39, p. 440 (1954)] .
Example strains of Bacillus subtilis include Bacillus subtilis MI 114 [Gene, Vol. 24, p. 255 (1983)] and 207-21 [Journal of Biochemistry, Vol. 95, p. 87 (1984) ] .
Example methods for transforming a host cell with a plasmid include the calcium chloride method described by T. Maniatis et al. [ibid., p. 249 (1982)] , the calcium chloride/rubidium chloride method and the electroporation method. The phage vector can, for example, be introduced into cultured Escherichia coli by the in vitro packaging method. Next, the plasmid or phage vector is isolated from the transformant microorganism thus obtained to prepare subject cDNA for the present selection method. The isolation method is exemplified by the alkali-SDS method [Birmboim, H.C., et al., Nucleic Acids Research, Vol. 1, p. 1513 (1979)] . The subject cDNA, as such or, if desired, after digestion with restriction enzymes, can be ligated to the downstream of the inducible promoter, to yield an expression vector.
The above-described eukaryotic host cells can be transformed with the thus-obtained vector containing the desired DNA as follows: Yeasts can be transformed in accordance with the method described in the Proceedings of the National Academy of Science, USA, Vol. 75, p. 1929 (1978) , for instance. Animal cells can be transformed in accordance with the method described in Virology, Vol. 52, 456 (1973) , for instance.
In the present selection method, trans ormants carrying the subject cDNA are cultured in accordance with the culturing method for the eukaryotic host cell used, with medium composition (metal ions, nitrogen sources, inorganic or organic acids, bases and other components) , pH, culturing temperature and other culturing conditions changed as appropriate. When the host is a yeast, for instance, the promoter can be induced in the absence of phosphate ions in the medium, using the PH05 promoter under the culturing conditions described later, and promoter activity can be adjusted by changing between the presence and absence of thiamme in the medium as described in Examples below, using the nmtl promoter. For observing cell growth in an animal host system using the hsp promoter, promoter activity can be induced by incubation at a temperature (41 - 42t) slightly higher than ordinary culturing temperature (36 - 37T) for a given period of time.
A transformant is thus selected whose cell growth is inhibited under the promoter activity-inducing conditions, and which shows normal growth, according to the host cell used, under non-promoter-activity-induc g conditions .
Here, "cell growth is inhibited" means that the cell growth under promoter-inducible-conditions is reduced as comparing with the cell growth under non- promoter-inducible-conditions (control) . For example, the cell growth rate inhibition is at least 25%, preferably 50%, more preferably 75%. And "cell does not grow" means that the cell hardly grow and the cell growth inhibition is about 75 - 100%.
For observing cell growth inhibition, it is preferable to do when the cell growth reaches plateau. For example, when the host cell is a fission yeast, it is preferable to observe the cell growth after cultivation is carried at about 20 to 40t for about 12 to 144 hours, more preferably at 20 to 35 t for about 24 to 72 hours.
For observing cell growth inhibition, it is preferable to use a solid medium. Cells undergoing growth inhibition can easily be selected from the subject cell group by comparing the sizes of colonies formed. In confirming the growth inhibitory activity of the cells thus screened for, it is also effective to culture the cells in a liquid medium, in addition to the assay system using a solid medium, and determine growth inhibition rate by measuring a culture broth turbidity or uptake of tritium thymidine by cultured cell as an index.
The sizes of colonies formed are observed by naked eys or using a microscope. The culture broth turbidity is determined by measuring the transmittance of visible radiation of culture broth. The uptake of tritium thymidine by cultured cell is measured by known method.
In the present invention, a eukaryotic cell growth inhibiting factor is defined as a peptide or protein that suppresses or terminates the growth of eukaryotic cells such as yeasts and animal cells, under ordinary culturing conditions as shown below, and may be any one, as long as it is capable of reversibly or irreversibly inhibiting the growth of at least one kind of eukaryotic cell. Such inhibiting factors include peptides or proteins having a partial or full-length portion of the amino acid sequence of SEQUENCE ID NOS. 11, 12, 13, 14, 15, 22, 23 or 27, including peptides or proteins whose sequences lack the N-terminal methionine residue.
DNA encoding the above-described eukaryotic cell growth inhibitor can be obtained by the method of the present invention for screening DNA and so on. The DNA of the present invention is exemplified by DNA containing the nucleotide sequences shown by NUCLEIC ACID RESIDUE NOS. 279-752 of SEQUENCE ID NO. 8 encoding the amino acid sequenceof SEQ ID No. 12, NUCLEIC ACID RESIDUE NOS. 201-377 of SEQUENCE ID NO. 9 encoding the amino acid sequence of SEQ ID No. 13 , NUCLEIC ACID RESIDUE NOS. 296-1000 of SEQUENCE ID NO. 10 encoding the am o acid sequence of SEQ ID No. 14, NUCLEIC ACID RESIDUE NOS. 248-448 of SEQUENCE ID NO. 7 encoding the amino acid sequence of SEQ ID No. 11, NUCLEIC ACID RESIDUE NOS. 51-740 of SEQUENCE ID NO. 16 encoding the ammo acid sequence of SEQ ID No. 15, NUCLEIC ACID RESIDUE NOS. 1062-1736 of SEQUENCE ID NO. 17 encoding the amino acid sequence of SEQ ID No. 22, NUCLEIC ACID RESIDUE NOS. 55-1488 of SEQUENCE ID NO. 20 encoding the amino acid sequence of SEQ ID No. 23, NUCLEIC ACID RESIDUE NOS. 150-1004 of SEQUENCE ID NO. 25 encoding the amino acid sequence of SEQ ID No. 27 and SEQUENCE ID NOS. 18 19, 21, 24 and 26. For obtaining eukaryotic cell growth inhibiting factor encoded by the DNA of the present invention using that DNA, N-terminal Met may be added to the inhibitor polypeptide; the cell growth factor may be a glycoprotein (sugar chain added) or a fused protein with another polypeptide.
An expression vector containing the DNA of the present invention that encodes eukaryotic cell growth inhibiting factor can be produced by, for example, 1) preparing cell growth inhibiting factor-encoding mRNA from an aged cell, 2) synthesizing cDNA and then double- stranded DNA from the mRNA to yield a cDNA library, 3) selecting cDNA encoding a polypeptide that inhibits host cell growth from the cDNA library, and 4) ligating the cDNA to the downstream of the promoter m the vector.
The cDNA library obtained from an aged cell in accordance with the above-described method may be treated to concentrate the desired cDNA by known methods, e.g., the differential hybridization method [Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, (1989)] , the subtraction method [Molecular Cloning, ibid.] and the cell suicide selection method. The screening method of the present invention is also preferred. In such a case, the cell suicide selection method [Stetten, G. et al., Experimental Cell Research, Vol. 108, p. 447 (1977) , Brdurd-Hoechst] may be used to concentrate cells whose growth has been suppressed by the cDNA introduced, followed by selection of the desired cDNA. The desired cDNA can also be selected by the present selection method using an inducible promoter .
Next, the plasmid or phage vector is isolated from the microorganism by a known method such as the above- described alkali-SDS method. The obtained plasmid or phage vector, harboring DNA containing a base sequence encoding a cell growth inhibitor, can be used as such or, if desired, after digestion with restriction enzymes, according to the purpose of use.
The cloned gene is ligated to the downstream of the promoter, in a vector suitable for its expression, to yield an expression vector. The gene may have ATG as a translational initiation codon at its 5 '-terminal and TAA, TGA or TAG as a translational termination codon at its 3' -terminal. To express the gene, a promoter is ligated to its upstream. Any promoter can be used for the present invention, as long as it is appropriate for the host used to express the gene. Example vectors include the above-mentioned plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13, ptrp 781) , plasmids derived from Bacillus subtilis (e.g., pUBIIO, pTM5 , pC194) , yeast-derived plasmids (e.g., pSH19, pSH15) , bacteriophages such as λ phage, and animal viruses such as retrovirus and vaccinia virus.
Examples of preferred promoters include the T7 promoter, trp promoter, lac promoter, rec promoter, λ PL promoter and lpp promoter when the transformation host is Escherichia coli , the SP01 promoter, SP02 promoter and pen P promoter when the host is Bacillus subtilis , and the PH05 promoter, PGK promoter, GAP (GLD) promoter, ADH promoter and nmtl promoter when the host is a yeast. Preference is given to the case in which Escherichia coli is used as host in combination with the T7 promoter, trp promoter or λ PL promoter.
When the host is an animal cell, preferable promoters include the SV40-derived promoter, retrovirus promoter, metallothionein promoter and hsp promoter, with preference given to the SV40-derived promoter.
The thus-constructed vector, harboring DNA containing a nucleotide sequence such as one of the sequences of SEQUENCE ID NOS. 7-10, 16-21 and 24-26 is used to produce a transformant. Examples of the host include prokaryotes such as Escherichia coli, Bacillus subtilis and actinomycetes , and eukaryotes such as yeasts, fungi and animal cells.
Examples of the strains of Escherichia coli and Bacillus subtilis are the same as those mentioned above.
Examples of the yeasts include Saccharomyces cerevisiae AH22, AH22R" , NA87-11A, DKD-5D, Schizosaccharomyces po be and mutants thereof. Examples of the animal cells include simian cells C0S-7, Vero, Chinese hamster cells CHO and mouse L cells. The strains of Escherichia coli can be transformed in accordance with the method described in the Proceedings of the National Academy of Sciences, USA, Vol. 69, p. 2110 (1972) , Gene, Vol. 17, p. 107 (1982) and other publications, for instance.
Strains of Bacillus subtilis can be transformed in accordance with the method described in Molecular and General Genetics, Vol. 168, p. 111 (1979) and other publications, for instance.
Yeasts can be transformed in accordance with the method described in the Proceedings of the National Academy of Sciences, USA, Vol. 75, p. 1929 (1978) , for instance.
Animal cells can be transformed in accordance with the method described m Virology, Vol. 52, p. 456 (1973) , for instance.
A transformant as transformed with a vector harboring the desired DNA is thus obtained. When the host is a eukaryotic cell, the transformant is subcultured under non-promoter-mducing conditions using an inducible promoter.
For culturing a transformant whose host is Escherichia coli , Bacillus subtilis , an actinornycete, yeast or fungus, it is appropriate to use liquid medium supplemented with carbon sources, nitrogen sources, minerals and other substances necessary for the growth of the transformant. Examples of carbon sources include glucose, dextrin, soluble starch and sucrose; examples of nitrogen sources include organic or inorganic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extracts, soybean cake and potato extracts; examples of minerals include calcium chloride, sodium dihydrogen phosphate and magnesium chloride. The pH of the medium is preferably about 5 to 8. Examples of media preferably used to culture Escherichia coli include the M9 medium containing glucose and casamino acid [Miller, Journal of Experimental Molecular Genetics, p. 431, Cold Spring Harbor Laboratory, New York (1972)] . Cultivation is normally carried out at about 14 to 43t for about 3 to 24 hours, with aeration and/or stirring as necessary.
When the host is Bacillus subtilis , cultivation is normally carried out at about 30 to 40t for about 6 to 24 hours, with aeration and/or stirring as necessary.
Examples of media for culturing a transformant whose host is a yeast include Burkholder's minimal medium [Bostian, K.L. et al., Proceedings of the National Academy of Sciences, USA, Vol. 77, p. 4505 (1980)] , preferably adjusted to a pH of about 5 to 8.
For culturing a transformant to express the desired gene using an inducible promoter, when an nmt1 promoter, for instance, is used, the transformant is cultured under promoter-inducing conditions, e.g., in a thiamme- free medium.
When an inducible promoter is used, cultivation is normally carried out at about 20 to 35T for 24 to 72 hours, with aeration and/or stirring as necessary, until cell growth reaches a plateau.
Example media for culturing a transformant whose host is an animal cell include MEM containing about 5 to 20% fetal bovine serum [Science, Vol. 122, p. 501 (1952)] , DMEM [Virology, Vol. 8, p. 396 (1959)] , RPMI 1640 medium [Journal of the American Medical Association, Vol. 199, p. 519 (1967)] and 199 medium [Proceedings of the Society of Experimental Biological Medicine, Vol. 73, p. 1 (1950)] . For culturing a transformant to express the desired gene using an inducible promoter, the transformant is cultured under promoter-inducing conditions, e.g. , m a medium supplemented with heavy metal ions when a metallothionein promoter is used. The pH is preferably about 6 to 8. Cultivation is normally carried out at about 30 to 40T for 15 to 60 hours. When an inducible promoter is used, aeration and/or stirring is conducted as necessary, until cell growth reaches a plateau.
The eukaryotic cell growth inhibiting factor of the present invention is produced and accumulated tracellularly or extracellularly. For extracting the intracellular cell growth inhibitor from the culture, cultured cells collected by a known method are suspended in a buffer containing a protein denaturant such as guanidme hydrochloride or urea, or a surfactant such as Triton X-100, and then centrifuged to obtain a supernatant containing the cell growth inhibitor, or cells are disrupted by ultrasonication, treatment with an enzyme such as lysozyme, or freeze-thawing, followed by centrifugation to obtain a supernatant containing the cell growth inhibiting factor.
For separating and purifying the cell growth inhibiting factor produced and accumulated in the supernatant or extracellularly, known methods of separation and purification can be used in combination, as appropriate. Such known methods of separation and purification include those based on solubility differences, such as salting-out and solvent precipitation, those based mainly on molecular weight differences, such as dialysis, ultraflltration, gel filtration and SDS-polyacrylamide gel electrophoresis, those based on charge differences, such as ion exchange chromatography, those based on specific affinity, such as affinity chromatography, those based on hydrophobicity differences, such as reverse-phase high performance liquid chromatography, and those based on isoelectric point differences, such as isoelectric focusing.
A eukaryotic cell growth inhibiting factor containing substantially no pyrogen or endotoxin is thus obtained in substantially pure form. The substantially pure cell growth inhibiting factor of the present invention contains the cell growth inhibiting factor protein at not lower than 95% (w/w) , preferably not lower than 98% (w/w) . Here, "containing substantially no pyrogen or endotoxin" means that the cell growth inhibiting factor is negative in, for example, the known limulus test or pyrogen test.
The DNA of the present invention that encodes a eukaryotic cell growth inhibiting factor can be used as a probe for examining individual aging at the RNA level. Specifically, the DNA of the present invention can be used as a diagnostic reagent for various aging- associated diseases. The gene of the present invention may also be introduced into cells of a target tissue, such as skm or vascular endothelium, to establish an in vitro aged cell line of the target tissue. Such a line is useful as a screening system for clarifying the mechanisms of onset and action of various agmg- associated diseases, or for seeking therapeutic drugs for these diseases.
The eukaryotic cell growth inhibiting factor encoded by the DNA of the present invention can be used as a pharmaceutical, such as an anticancer agent or infection remedy, as described later. In such case, the eukaryotic cell growth inhibiting factor can be safely administered parenterally or orally, preferably topically, in the form of powder as such, or in the form of pharmaceutical compositions (e.g. , injections, tablets, capsules, solutions, ointments) together with pharmacologically acceptable carriers, excipients and diluents, to warm-blooded animals (e.g., humans, mice, rats, hamsters, rabbits, dogs, cats) . The cell growth inhibiting factor can also be used as a skm drug.
An in ectable preparation is prepared in accordance with a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliaries. Other pharmaceutical compositions, such as tablets and capsules, can also be prepared in accordance with conventional methods.
When using the cell growth inhibiting factor of the present invention as a pharmaceutical as described above in mammals, it is administered at daily doses of about 0.2 μg/kg to 20 mg/kg, preferably about 2 μg/kg to 0.2 mg/kg. The cell growth inhibiting factor obtained according to the present invention is thought of as terminating cell division, and can therefore be used as a reagent for terminating the cell cycle of cultured cells at a given time point, e.g., a reagent for synchronizing cell division. By making constant the cell cycle of cells in an m_ vitro experimental system, it is possible to improve assay precision or establish an experimental system of a particular cell cycle. When using the cell growth inhibiting factor of the present invention as such a reagent, it is preferable to add it to the medium to a final concentration of 1 ng/ml to 1 mg/ml, more preferably 1 ng/ml to 10 μg/ml.
The factor encoded by the DNA of the present invention acts on young cells capable of division, or infinitely growing cancer cells, to prevent their growth. The DNA of the present invention can therefore be used for gene therapy for cancer patients or as a probe for the diagnosis of aging-associated diseases. The factor encoded by the DNA of the present invention can also be used as an anticancer agent. It is also effective against fungal infections (e.g. , cutaneous mycosis, deep mycosis) . Moreover, the DNA of the present invention can be used as a system for clarifying the mechanism of onset of aging-associated diseases or seeking therapeutic drugs, to establish an _m vitro aged cell line of the target tissue. Substances that inhibit the cell growth inhibiting factor appear to be applicable as prophylactic/therapeutic drugs for aging and various aging-associated diseases, such as dementia and arteriosclerosis. Accordingly, the DNA of the present invention and the factor encoded thereby can be used to seek such drugs. The factor can also be used as a reagent for terminating the cell cycle of cultured cells at a given time point.
Antibodies or antiserum to the eukaryotic cell growth inhibiting factor or the partial peptides thereof of the present invention can be produced by the methods known per se in the art, using the factor or the partial peptides thereof as antigens. The antibodies or antiserum can be used for inhibiting the activity of the eukaryotic cell growth inhibiting factor to rejuvenate the aged cell or tissues. The antibodies or antiserum can also be used for quantitative analysis or detection of the factor or the partial peptides thereof by methods known per se in the art.
Antisense oligonucleotides complementary to the cDNA encoding the eukaryotic cell growth inhibiting factor can be synthesized by the known methods. The oligonucleotides hybridize to the mRNA, inhibit the production of the eukaryotic cell growth inhibiting factor and induce re uvevation of aged cell or tissues. These oligonucleotides can be used for in vivo and x vivo treatment of diseases caused by cellular senescence or agmg-associated diseases, such as arteriosclerosis and dementia. These oligonucleotides are also effective for a normal tissue or cell, such as skin cell, a cell present in wound or burn tissue, lymphocyte, vascular tissue, liver, kidney, heart, bone, spleen, etc.
Abbreviations for bases, amino acids and others used in the present speci ication and attached drawings are based on abbreviations specified by the IUPAC-IUB Commission on Biochemical Nomenclature or abbreviations in common use in relevant fields. Some examples are given below. When an optical isomer may be present in amino acid, it is of the L-configuration, unless otherwise stated. PPCCTI/JP95/02488
PBS : Phosphate-buffered saline
DNA : Deoxyribonucleic acid cDNA: Complementary deoxyribonucleic acid
A : Adenine
T : Thymme
G : Guanine
C : Cytosine
RNA : Ribonucleic acid mRNA: Messenger ribonucleic acid dATP: Deoxyadenosine triphosphate dTTP: Deoxythymidme triphosphate dGTP: Deoxyguanosme triphosphate dCTP: Deoxycytidine triphosphate
ATP : Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS : Sodium dodecyl sulfate
Gly : Glycine
Ala : Alanine
Val : Valine
Leu : Leucine
He : Isoleucme
Ser : Serine
Thr : Threonine
Cys : Cysteine
Met : Methionine
Glu : Glutamic acid
Asp : Aspartic acid
Lys : Lysine
Arg : Arginine
His : Histidme
Phe : Phenylalanine
Tyr : Tyrosine
Trp : Tryptophan
Pro : Proline
Asn : Asparagine
Gin : Glutamine Examp l e s
The present invention is hereinafter described in more detail by means of the following reference example and working examples, which are not to be construed as limitative to the present invention.
The transformants obtained in the following examples, that carry the DNA of the present invention, have been deposited as follows:
Transformant IFO NIBH (IFO No.) (FERM No.)
E. coli 15627 BP-4551 MC1061/pTB1617 January 21, 1994 February 7, 1994
E. coli 15628 BP-4552 MC1061/pTB1618 January 21 , 1994 February 7, 1994
E. coli 15756 BP-4890 DH1/pTB1668 October 27, 1994 November 14, 1994
E. coli 15757 BP-4891 DH1/pTB1671 October 27, 1994 November 14, 1994
E. coli 15758 BP-4892 DH1/pTB1673 October 27, 1994 November 14, 1994
E. coli 15807 BP-5126 DH1/pTB1848 March 16, 1995 June 9, 1995
E. coli 15821 BP-5127 MC1061/pTB1689 May 30, 1995 June 9, 1995
E. coli 15822 BP-5128 MC1061/pTB1721 May 30, 1995 June 9, 1995
E. coli 15823 BP-5129 MC1061/pTB1756 May 30, 1995 June 9, 1995
E. coli 15824 BP-5130 MC1756/pTB1761 May 30, 1995 June 9, 1995
E. coli BP-5268 MC1061/pTB1786 October 27, 1995
E. coli BP-5269 MC1061/pTB1810 October 27, 1995
E. coli BP-5270 MC1061/pTB1819 October 27, 1995
IFO : Institute for Fermentation, Osaka (foundation) NIBH: National Institute for Bioscience and Human- Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry Dates in parentheses are dates of accession. Reference Example 1 Construction of vector for animal cells
Plasmid pTB399 (Cell Struct. Funct. , Vol. 12, pp. 205-217) was cleaved with EcoRI and reacted with the Klenow fragment, followed by addition of Bglll linker (CAGATCTG) and cleavage with Bglll, to yield a 3.8 kb DNA fragment deleting the ιnterleukιn-2 (IL-2) cDNA region. The fragment was then cyclized using T4 ligase. The MuLV-LTR portion was then replaced with an SRα promoter derived from pME18S [Maruyama, K. and Takebe, Y., Medical Immunology, Vol. 20, pp. 27-32 (1990)] in accordance with a conventional method, to yield plasmid pTB1695.
Example 1 Preparation of cDNA from aged normal human diploid fibroblast
A relatively young line (22PDL) of normal diploid fibroblast MRC-5 of human male fetal lung origin
[Jacobs, J.P. et al . , Nature, Vol. 227, p. 168 (1970)] was purchased from 3the Institute for Fermentation,
Osaka (IFO 50073) . The cell line was subcultured in
Eagle MEM medium (produced by Nissui Pharmaceutical Co. ,
Ltd.) containing 10% (v/v) fetal bovine serum (FBS) until its growth terminated (cell aging) at 44.5 PDL, to yield aged normal human diploid fibroblasts (hereinafter aged cells) . Aged cells growing on 25 petπ dishes (10cm in diameter) at confluences of 75% or more were scraped using a cell scraper. After washing with Dulbecco's PBS
(Dainippon Pharmaceutical) , the cells were treated to extract an RNA fraction using an RNA extraction kit
(Pharmacia-LKB) , as directed in the kit protocol.
After 0.2 g of oligo-dT cellulose (type 3) (Collaborative Biomedical) , previously swollen with 10 mM Tris-HCl buffer containing 0.1 M sodium chloride and 1 mM EDTA (pH 7.4) (TE buffer) , was packed in a EC0N0- COLUMN (17 mm in diameter, 15 cm in length) (Nippon Bio- Rad Laboratories) , the column was equilibrated with TE buffer containing 0.5 M sodium chloride. The RNA fraction, previously adjusted to a final concentration of 0.5 M sodium chloride, was heated at 65t for 5 minutes then immediately quenched in ice, after which it was applied to the equilibrated column. The coupled RNA fraction was eluted with TE buffer to yield an mRNA fraction (yield 115 μg) .
Using a ZAP-cDNA synthesis kit (Stratagene) , cDNA was synthesized by the method of Nojima et al. [No ima, H. , Development and Application of New Vector System in an Attempt to Catalog All Human cDNA Banks (Research Subject No. 02557098) , 1992 Grant-m-Aid for Scientific Research from the Ministry of Education (Investigation B(D) Final Report, p. 29 (1993)] . After 5 μg of mRNA was subjected to reverse transcription with an oligo-dT primer linker having an NotI recognition sequence (GCGGCCGC) as a template, to synthesize a first strand, RNase H and Escherichia coli DNA polymerase I were simultaneously reacted to remove the mRNA region and synthesize a second strand at the same time. Both ends of the thus-obtained double-stranded DNA were blunted using T4 DNA polymerase, followed by T4 DNA ligase action to bind a dephosphorylation BamHI adapter to both ends; NotI was then reacted to yield a cDNA fragment having a dephosphorylated BamHI site on the 5 '-terminal side and a phosphorylated NotI site on the 3 '-terminal side (yield about 5 μg) .
Example 2 Modification of fission yeast expression vector
The following six DNA oligomers (SEQUENCE ID NOS. (1) to (6) ) were synthesized.
(1) 5'-ACGCGTCCAGGATCCTGGTCGACGC-3' (SEQUENCE ID N0: 1)
(2) 5 '-GGCCGCCCTTTAGTGAGGGTTAA-3 ' (SEQUENCE ID NO: 2)
(3) 5 '-CGCGTCCCTATAGTGAGTCGTATTAC-3 ' (SEQUENCE ID NO: 3)
(4) 5' -GGCGGCCGCGTCGACCAGGATCCTGGA-3' (SEQUENCE ID NO: 4)
(5) 5 '-GATCTTAACCCTCACTAAAG-3 ' (SEQUENCE ID NO: 5) (6) 5'-TCGAGTAATACGACTCACTATAGGG-3' (SEQUENCE ID NO: 6)
DNA oligomers (1) , (2) , (4) and (5) were mixed in an amount of 10 μg each, followed by 5'-termmal phosphorylation by the action of T4 polynucleotide kinase. After the reaction mixture was kept standing at 65T for 15 minutes, DNA oligomers (3) and (6) , 10 μg each, were added and ligated using T4 DNA ligase. The reaction mixture was subjected to 4% agarose electrophoresis to recover a 73 bp DNA fragment; the 5'- terminal was then phosphorylated by the action of T4 polynucleotide kinase. Next, to the fission yeast expression vector pREPI [Maundrell, K., Gene, Vol. 123, p. 127 (1993)] , previously cleaved at the Sall-BamHI site, the above 73 bp DNA fragment was ligated by the action of T4 DNA ligase to yield pTB1589. This plasmid is a fission yeast expression vector having a T7 RNA polymerase recognition sequence, Mlul site, BamHI site, BεtXI site, Sail site, NotI site, T3 RNA polymerase recognition sequence and nmt1 terminator in that order, and an LEU2 gene as a selection marker, downstream of the nmtl promoter [Maundrell, K. , Journal of Biological Chemistry, Vol. 265, p. 10857 (1989)] .
Example 3 cDNA insersion into fission yeast expression vector pTB1589
The fission yeast expression vector prepared in Example 2 (pTB1589) was digested with NotI, treated with alkaline phosphatase, and further digested with BamHI, followed by 0.7% agarose gel electrophoresis to recover a vector fraction. 1 μg of the cDNA of aged normal human diploid fibroblast origin prepared in Example 1, and 100 ng of the above-described linearized vector, were ligated by the action of T4 DNA ligase; the ligation product was introduced into Escherichia col MC1061
(electro-competent cell MC1061, Nippon Bio-Rad Laboratories) by electroporation using a Gene Pulser
(Nippon Bio-Rad Laboratories) . The cDNA library thus obtained comprised 1.7 x 106 independent transformant cells of 1.5 kbp mean cDNA length. The plasmid was purified from the transformant, diluted with TE buffer to a concentration of 1μ g per 15μl, and stored at -20t until use.
Example 4 Transformation of fission yeast and screening for trans ormants showing cDNA-dependent growth inhibition
The reagents used to transform a fission yeast were prepared in accordance with the formulation of Moreno, S. et al. [Methods in Enzymology, Vol. 194, p. 795 (1991)] unless otherwise stated, with the same designations. Fission yeast cells (Schizosaccharomyces pombe h" leul") growing on YEA plate were inoculated to 100 ml of MB medium containing 0.25% (w/v) L-leucme at a density of 106 cells/ml, and cultured at 30T until the cell density reached 5 x 106 to 1 x 107 cells/ml. Cells were harvested at room temperature and washed with sterile water, after which they were suspended in a 0.1 M lithium acetate solution (pH 4.9-5.0) to 109 cells/ml. This suspension was dispensed to Eppendorf tubes at lOOμl per tube, and kept standing at 30t for 1 hour. To each tube, 1μg (15μl) of the plasmid prepared in Example 5 and 290μl of a 50% (w/v) polyethylene glycol 4000 solution were added. After thorough mixing, the mixture was kept standing at 30T for 50 minutes, then heated at 43T for 15 minutes, then kept standing at room temperature for 10 minutes, followed by cell harvest. The cells were suspended in 1 ml of 1/2 YEL medium containing 0.25% (w/v) L-leucine; the suspension was then shaken at 30T for 1 to 2 hours. After dilution with 9 ml of 1/2 YEL medium, the suspension was applied to MMA plates containing 2μM thiamme (MMAT plates of medium that inhibits nmtl promoter transcription) at lOOμl per plate, and cultured at 30T for 2 days. The MMAT plates on which minute colonies appeared were replicated to new MMA plates (medium that promotes the same promoter transcription as above) and MMAT plates, followed by culturing at 30T for 3 to 4 days. As a result of screening of 8.25 x 104 transformant cells by the above-described method, 18 transformants (candidate strains) that grow on MMAT plates but not on MMA plates were found (cell growth rate inhibiting is more than about 75%) .
Example 5 Recovery of plasmid having cDNA from candidate strains
Each candidate strain was inoculated over the entire surface of an MMAT plate and cultured at 30t for 2 to 3 days. The cells on the plate were recovered into an Eppendorf tube using 1.5 ml of TES solution (TE buffer containing 10 mM sodium sulfite) , followed by cell harvest. The cells were then suspended in 1 ml of a sorbitol solution (1M sorbitol, 100 mM EDTA, 10 mM sodium sulfite, 100 mM lithium acetate) ; the suspension was kept standing at 30t for 1 hour in the presence of 20 units of Lyticase (Boehringer) . After centrifugal recovery, the protoplast was suspended in 300 1 of 100 mM Tris-HCl buffer (pH 7.5) containing 0.5% (w/v) SDS and 50 mM EDTA, and kept standing at 65T for 1 hour. After the suspension was thoroughly mixed with 150μl of a 3 M potassium acetate solution, the mixture was kept standing on ice for 30 minutes. After cent i ugation at 15,000 rpm at room temperature for 5 minutes, the supernatant was extracted with an equal amount of phenol/chloroform mixture (v/v = 1/1) . This extraction was repeated 2 to 3 times; after addition of a 2-fold volume of ethanol, the supernatant was kept standing at -20t for not less than 4 hours, to precipitate a crude DNA fraction.
The crude DNA fraction was dissolved in 50μl of TE buffer; after addition of lOOμl of a sodium iodide solution (GENECLEAN II Kit, BI0101 Company) and 5μl of a glass milk suspension (provided with the kit) , the mixture was kept standing at room temperature for 5 minutes. The glass milk fraction was centrifugally recovered, and washed with 3 portions of 400μl of an ice-cooled NEW solution (provided with the kit) . The washed precipitate was treated with lOμl of TE buffer at 551 for 3 minutes; this operation was repeated in two cycles to yield a purified DNA fraction. Using 3μl of the extracted purified DNA fraction, Escherichia coli MC1061 was transformed by the electroporation method described in Example 3; the plasmid having cDNA was recovered from the resulting transformant.
Example 6 Analysis of candidate strains
The cDNA plasmids recovered from the 20 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 10 clones showing cDNA expression-dependent growth inhibition were found. For the cDNAs in plasmids of these reproducible clones, base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd. , US70777) with [35S]dCTPS, in accordance with the kit protocol. The thus-obtained base sequences were examined for homology on the current DNA data base (GeneBank Release 84.0; 196703 entries) ; 4 new clones were found (Table 1) . The plasmids harbored by the respective clones were designated pTB1617, pTB1668, pTB1671 and pTB1673, respectively; the entire base sequences of these cDNAs are shown in SEQUENCE ID NOS. 7, 8, 9 and 10, respectively.
The ammo acid sequences of the polypeptides or proteins encoded by the cDNAs harbored by pTB1617, PTB1668, pTB1671 and pTB1673 are shown in SEQUENCE ID NOS. 11, 12, 13 and 14, respectively.
The expression plasmid of sdι-1 gene [W09312251; Noda, A et. al., Experimental Cell Research, Vol. 211 p. 90-98 (1994)] was also constructed, in which sdι-1 gene was ligated downstream of the nmt1 promoter as shown in Example 3, and introduced into fission yeast. The yeast transformant thus obtained could form colonies on MMA plate as same as on MMAT plate (cell growth rate inhibiting is about 0%) , indicating that sdι-1 type gene can not be obtained by the screening method using fission yeast as described.
[Table 1]
Plasmid cDNA Length
PTB1617 0.6Kbp pTB1668 0.9Kbp
PTB1671 0.6Kbp
PTB1673 1.6Kbp
Example 7 Analysis of candidate strains
The cDNA plasmids recovered from 10 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 3 clones showing cDNA expression-dependent growth inhibition were found. For the cDNA plasmids of these reproducible clones, base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd., US70777) with [S5S]dCTPS, in accordance with the kit protocol. The thus-obtamed base sequences were examined on the current DNA data base (GeneBank Release 86.0; 237775 entries) ; 1 new clone was found. The plasmid having the clone was designated pTB1848; the entire base sequence of its cDNA is shown in SEQUENCE ID NO. 16. The amino acid sequence of the protein encoded by the cDNA harbored by plasmid pTB1848 is shown in SEQUENCE ID NO. 15.
Example 8 Construction of animal cell expression plasmids
From plasmid pTB1697 obtained by introducing an E. coli lacZ gene into the Hmdlll site of pRc/CMV (Invitrogen, USA) , a 4.9 kbp Nrul-Nael fragment was cut out and inserted into the Sail site of the plasmid pTB1695 prepared in Reference Example 1 to yield the plasmid pTB1698 (Figure 1) . Next, the plasmids pTB1668, pTB1671, pTB1673 and pTB1848, obtained in Examples 6 and 7, were each cleaved with BamHI-NotI; the resulting cDNA portions were each inserted into the Bglll site of pTB1698, located downstream of the SRα promoter, to yield animal cell expression plasmids.
Example 9 Determination of DNA synthesis inhibitory activity
The animal cells used to determine DNA synthesis inhibitory activity were normal diploid fibroblasts (purchased from Cell System; defined primary human dermal fibroblast cell system, hereinafter Fb cells) at the growth stage, subcultured m 20-35 generations under the same culturing conditions as Example 1. After being sown over Lab-Tek chamber slides (Nunc, USA) at 5 x 104 cells per plate and cultured at 37T for 1 day, Fb cells were transfected with the pTBI848-derιved cDNA expression plasmid prepared in Example 8, by the calcium phosphate method [Chen, C. and Okaya a, H. , Molecular Cell Biology, Vol. 7, pp. 2745-2752 (1987)] . After the obtained transformant cells were cultured at 37t for 1 day, the medium was replaced with fresh one, followed by cultivation for 1 more day. Next, 37 KBq/ml (925 GBq/mmol) tritiated thymidine (s H-thymidine) was added, followed by 48 hours of cultivation to label the cells. After glutaraldehyde fixation, the cells were stained with X-gal. After further fixation in methanol, an emulsion was applied; the plate was kept standing in a dark room for 4-5 days, followed by development. The blue-stained-galactosidase expression cells were counted under a microscope; the ratio of cells showing black particles in their nuclei due to sH-thymιdιne uptake was determined.
DNA synthesis inhibitory rates (%) were calculated with the labeling index, taking plasmid pTB1698 as 0%. For positive control, Fb cells were used which had been transformed with the expression plasmid pTB1699 constructed by introducing the sdι-1 gene [Noda, A. et al. , Experimental Cell Research, Vol. 211, pp. 90-98 (1994)] into the Bglll site of pTB1698 in accordance with the method described in Example 8. The results are shown in Table 2.
As shown in Table 2, the cells incorporating the pTBI848-derιved cDNA underwent thymidine uptake inhibition in 3 experiments, as with the positive control . [Tab l e 2 ]
Ex. Clone Number of Labeled Labeling DNA Synthesis
Nuclei per Blue Index Inhibitory strained Cell (%) Rates (%)
1 pTB1698 77/257 56/178 30.8 0 PTB1699 59/229 26/151 21.5 30.1 PTB1848 29/134 26/110 22.6 26.5
2 pTB1698 59/126 63/133 47.1 0 PTB1699 62/225 56/139 34.0 27.9 PTB1848 50/130 32/96 35.9 23.8
3 pTB1698 73/143 89/170 51.7 0 PTB1699 49/161 60/145 35.9 30.6 PTB1848 36/88 29/66 42.4 18.0
Example 10 Analysis of candidate strains
The plasmids having cDNA recovered from 25 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 10 clones showing cDNA expression-dependent growth inhibition were found. For the cDNA m plasmids of these reproducible clones, base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd. , US70777) with [s 5S]dCTPS, in accordance with the kit protocol. The thus-obtained base sequences were examined on the current DNA data base (GeneBank Release 86.0; 237775 entries) ; 5 new clones were found (Table 3) . The plasmids having the clones were designated as pTB1618, pTB1689, pTB1756, pTB1761 and pTB1721; the entire base sequences of their cDNAs are shown in SEQUENCE ID NOS. 17, 18, 19, 20 and 21, respectively. The amino acid sequences of the polypeptides or proteins encoded by the cDNAs harbored by the plasmids pTB1618 and pTB1761 are shown in SEQUENCE ID NOS. 22 and 23, respectively.
[Table 3]
Plasmid cDNA Length
PTB1618 2.5Kbp
PTB1689 1.1Kbp
PTB1756 0.7Kbp
PTB1761 1.9Kbp
PTB1721 0.5Kbp Example 11 Determination of DNA synthesis inhibitory activity
Expression plasmids for animal cell were constructed by cleaving the plasmids pTB1618, pTB1689, pTB1721, pTB1756 or pTB1761 obtained in Example 10 with BamHI-NotI, and introducing into the Bglll site of pTB1698 each of the obtained cDNA portions by the method described in Example 8. Using these cDNA expression plasmids, DNA synthesis inhibitory activity was determined by the method described in Example 9. As shown in Table 4, the cells incorporating the pTB1689- deπved cDNA underwent thymidine uptake inhibition in 3 experiments, as with the positive control. Similarly, all cells incorporating pTB1618, pTB1721, pTB1756 or pTB1761 underwent thymidine uptake inhibition in 3 experiments, as shown in Table 5.
[Table 4]
Ex. Clone Number of Labeled Labeling DNA Synthesis Nuclei per Blue Index Inhibitory strained Cell (%) Rates (%)
1 pTB1698 77/257 56/178 30.8 0 PTB1699 59/229 26/151 21.5 30.1 PTB1689 42/224 44/161 23.1 25.0
2 pTB1698 59/126 63/133 47.1 0 PTB1699 62/225 56/139 34.0 27.9 PTB1689 55/178 71/170 36.4 22.8
3 pTB1698 73/143 89/170 51.7 0 PTB1699 49/161 60/145 35.9 30.6 pTB1689 21/85 31/88 30.0 42.1 [Tab l e 5 ]
Ex. Clone Number of Labeled Labeling DNA Synthesis
Nuclei per Blue Index Inhibitory strained Cell (%) Rates (%)
1 pTB1698 114/219 84/168 51.0 0 PTB1699 101/311 83/237 33.8 34.3 PTB1618 56/157 44/141 33.5 34.3 PTB1721 87/245 62/188 34.3 32.7 PTB1756 64/184 72/195 35.9 29.6 PTB1761 76/239 52/199 29.0 43.1
2 pTB1698 140/300 100/264 42.3 0 PTB1699 83/325 63/274 24.3 42.6 PTB1618 71/197 65/201 34.2 19.1 PTB1721 52/160 51/142 34.2 19.1 PTB1756 74/214 53/145 35.6 15.8 PTB1761 76/225 76/190 36.9 12.8
3 pTB1698 156/386 91/287 36.1 0 PTB1699 69/281 78/290 25.8 28.6 PTB1721 58/181 32.0 11.2 PTB1756 68/221 43/184 27.1 24.8 PTB1761 42/141 27/123 25.9 28.2
4 pTB1698 43/105 54/102 47.0 0 PTB1699 54/177 47/181 28.3 39.8 PTB1618 37/132 29/120 26.1 44.4 Example 12 Analysis of candidate strains
The plasmids having cDNA recovered from 22 candidate strains were used to transform fission yeasts by the method described in Example 4. The resulting transformants were again replicated to MMA plates; 9 clones showing cDNA expression-dependent growth inhibition were found. For the cDNA in plasmids of these reproducible clones, base sequences were determined using the Sequenase Ver. 2.0 DNA sequencing kit (Amersham Medical Ltd., US70777) with [S 5S]dCTPS, in accordance with the kit protocol. The thus-obtained base sequences were examined on the current DNA data base (GeneBank Release 86.0; 237775 entries) ; 3 new clones were found (Table 6) . The plasmids having the clones were designated as pTB1786, pTB1810 and pTB1819; the entire base sequences of their cDNAs are shown in SEQUENCE ID NOS. 24, 25 and 26, respectively. The amino acid sequence of the polypeptide or protein encoded by the cDNA harbored by the plasmid pTB1810 is shown in SEQUENCE ID NO. 27.
[Table 6]
Plasmid cDNA Length
PTB1786 0.4Kbp pTB1810 1.4Kbp
PTB1819 0.6Kbp
Example 13 Determination of DNA synthesis inhibitory activity
Expression plasmids for animal cell were constructed by cleaving the plasmids pTB1786, pTB1810 or pTB1819 obtained in Example 12 with BamHI-NotI, and introducing into the Bglll site of pTB1698 each of the obtained cDNA portions by the method described in Example 8. Using these cDNA expression plasmids, DNA synthesis inhibitory activity was determined by the method described in Example 9. As shown in Tables 7 and 8, the cells incorporating the pTB1689-derιved cDNA underwent thymidine uptake inhibition in more than 2 experiments, as with the positive control.
[Table 7]
Ex. Clone Number of Labeled Labeling, DNA Synthesis Nuclei per Blue Index Inhibitory strained Cell (%) Rates (%)
1 pTB1698 60/88 38/72 60.5 0
PTB1699 12/25 11/22 49.0 19.0
PTB1786 9/17 10/23 48.2 20.3
PTB1810 23/52 31/62 47.1 22.1
2 pTB1698 74/156 33/89 42.3 0
PTB1699 36/143 28/126 23.7 44.0
PTB1786 24/73 36/116 32.0 24.5 pTB1819 18/58 24/82 30.2 28.7
3 pTB1698 48/132 35/118 33.0 0
PTB1699 24/72 29/105 30.0 9.0
PTB1786 35/127 38/135 27.9 15.6
PTB1810 28/89 23/93 26.4 20.0
PTB1819 22/79 21/80 27.0 18.0 [Tabl e 8]
Ex. Clone Number of Labeled Labeling DNA Synthesis
Nuclei per Blue Index Inhibitory strained Cell (%) Rates (%)
1 pTB1698 60/161 37.3 0
PTB1699 33/130 46/176 25.8 30.9
PTB1810 14/71 17/64 23.1 38.3
SEQUENCE LISTING
INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 25
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE Other nucleic acid (synthetic DNA) (iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
ACGCGTCCAG GATCCTGGTC GACGC 25
INFORMATION FOR SEQ ID NO:2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 23
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE Other nucleic acid (synthetic DNA) (iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGCCGCCCTT TAGTGAGGGT TAA 23
INFORMATION FOR SEQ ID NO:3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 26
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE Other nucleic acid (synthetic DNA) (iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CGCGTCCCTA TAGTGAGTCG TATTAC 26 INFORMATION FOR SEQ ID NO: k
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 27
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE Other nucleic acid (synthetic DNA) (iv) ANTI-SENSE Yes
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGCGGCCGCG TCGACCAGGA TCCTGGA 27
INFORMATION FOR SEQ ID NO:5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 20
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE Other nucleic acid (synthetic DNA) (iv) ANTI-SENSE Yes
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GATCTTAACC CTCACTAAAG 20
INFORMATION FOR SEQ ID NO:6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 25
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE Other nucleic acid (synthetic DNA) (iv) ANTI-SENSE Yes
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TCGAGTAATA CGACTCACTA TAGGG 25 INFORMATION FOR SEQ ID NO:7 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH : 637
(B) TYPE : Nucleic acid
(C) STRANDEDNESS : Single
(D) TOPOLOGY : Linear
(ii) MOLECULE TYPE : cDNA
(iv) ANTI-SENSE : No
(vi) ORIGINAL SOURCE :
(A) ORGANISM : Human
(F) TISSUE TYPE : Lung
(G) CELL TYPE : Fibroblast
(H) CELL LINE : MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 248..448
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCAGAT CCCCGGGGAG CTTCTGCCAG GGGTGGATGT ACTCCTGGAG GTGTTCCCTA 6
CCTGTTCGGT GGAGCAGGCC CAGTGGGTGC TGGCCAAAGC TCGGGGGGAC TTGGAAGAAG 12
CTGTGCAGAT GCTGGTAGAG GGAAAGGAAG AGGGCCTGCA GCCTGGGAGG GCCCCAACCA 18
GGACCTGCCC AGACGCCTCA GAGGCCCCCA AAAGGATGAG CTGAAGTCCT TCATCCTGCA 240
GAAGTACATG ATGGTGGATA GCGCAGAGGA TCAGAAGATT CACCGGCCCA TGGCTCCCAA 300
GGAGGCCCCC AAGAAGCTGA TCCGATACAT CGACAACCAG GTAGTGAGCA CCAAAGGGGA 360
GCGATTCAAA GATGTGCGGA ACCCTGAGGC CGAGGAGATG AAGGCCACAT ACATCAACCT 42
CAAGCCAGCC AGAAAGTACC GCTTCCATTG AGGCACTCGC CGGACTCTGC CCGAGCCTTC 48
TAGGCTCAGA TCCCAGAGGG ATGCAGGAGC CCTATACCCC TACACAGGGG CCCCCTAACT 540
CCTGTCCCCC TTCTCTACTC CTTTGCTCCA TAGTGTTAAC CTACTCTCGG AGCTGCCTCC 600
ATGGGCACAG TAAAGGTGGC CCAAGGAAGG TGAAAAA 637
INFORMATION FOR SEQ ID NO:8 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 874
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear
(ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE No (vi) ORIGINAL SOURCE (A) ORGANISM Human
(F) TISSUE TYPE Lung
(G) CELL TYPE Fibroblast (H) CELL LINE MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 279..752
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGATCCCCGG CCCGGGAAAA ATCGCAGCCC TCAGAGAGTC GCTGGCTGAA GTATCTAGAA 60 AAGGACTCCC AAGAACTGGA GCTGGAAGGA ACAGGAGTGT GTTTCAGCAA ACAGCCTTCA 120 TCCAAAATGG AGGAGCCAGG CCCCCGCTTC AGTCAAGACC TGCCTAGAAA AAGGAAGTGG 180 AGCGGGAGCA CCGTCCAGCC TCCGTGCAGC CGTGGCGTGC AGGACTCGGG TGGCTCTGAG 240 GTCGCCTGGG GACCCCAGAA GGGACAGGCT GGCCTGACAT GGAAGGTGAA ACAAGCAGCA 300 GCCCCTGCCT TCAGGAGAAC TCTGCAGACT GCAGTGCCGG GGAGCTGAGG GGTCCTGGGA 360 AGGAGCTATG GAGTCCCATC CAGCAGGTTA CAGCCACATC CTCTAAATGG GCGCGATTTG 420 TCCTGCCACC TAGAAAAAGT TCACATGTGG ACAGTGAGCA GCCAAGGTCT CTTCAGAGGG 480 ACCCCAGGCC AGCTGGTCCA GCACAGGCTA AGCAAGGGAC CCCCAGAGCA CAGGCCTCAA 540 GAGAAGGCCT CAGCAGGCCC ACTGCCGCTG TCCAGCTTCC TCGGGCCACA CACCCCGTCA 600 CATCTGGGTC TGAGAGGCCT TGCGGGAAGA CCTCATGGGA CGCAAGGACT CCCTGGGCAG 660 AGGGTGGGCC CCTGGTCCTG GAGGCACAGA ATCCTCGACC CACACGACTA TGTGACCTCT 720 TTATAACTGG GGAAGACTTC GATGATGATG TGTGATCTGG GACTGGCAGG TTATTAATCG 780 AGATACACTT GTTAGGAGGG ACAGGGTTCC CCTAAGGCAC TTTTAAAGAT ACTCTGTAAG 840 AACCATTAAC AATAAACTTA CTGTCAATCA AAAA 874
INFORMATION FOR SEQ ID NO:9
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 640
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE No
(vi) ORIGINAL SOURCE : (A) ORGANISM Human
(F) TISSUE TYPE Lung
(G) CELL TYPE Fibroblast (H) CELL LINE MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 201. 377
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CTGGAAGGAC CTGCTGCTTT GCAGACCCAT GTATATATCC AGAAACCAAT CGGAACTCAG 6
GGTTACACTG ATTCCCTTTT GAGTATAATC TGTGCCATGA AGAAGGGGAT TTATTTGAGG 12
GAGGGACTTT TCTTCACCTG CACTCCTTTT ATTTTATTTT CCTATGTTTA GTTTTCTTTG 180
GAGTTGAACA GCTAGGCTGA ATGAGATTAA AGTTTTCCAA ACACACATGG CAGTATGGAG 240
GTTTTATGAA AAGTGATGGT GAAGAGTTGG GAGAGATGGA GGAAAAAAAA TGCAGTCAGA 300
AGTTTCAGAA CAAATACACA AAATCCTATG TTAGTTTGAA TCTTTATTTT TCTGGCACAC 360
TTTTAAAAGG GCTGTATTAA AATAGTGATT TTTTTTTTTT TTGCCTCAGG GAACCTCAGT 420
CAACAGGAAT ACCTCTGTTT CTAACCTAGA GAATAATATT GTGAAAATTG CTTTGTTAAT 480
TTTTTTTCCT CAGGAATAAT TTTCTCTTTT GGAAAGCACT TTCCCCGTCT CAGTAGAAAA 540
GTCTAGCAGT TGTAACTTCT TGTTTCTTAT TTGCTTTGGG GGAAATCAAA GAAAACAGAC 600
GGTGAAGGAA AGGGTGGGAA AAATTAAGTC TCATGAAAAA 640
INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 1560
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE No (vi) ORIGINAL SOURCE
(A) ORGANISM Human
(F) TISSUE TYPE Lung
(G) CELL TYPE Fibroblast (H) CELL LINE MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 296..1000 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGATCCCCGC GGACGAGGTG GCCGCGGCGG GGCAGCTGGG CCGCCAGCTT GGTGCCTCGG 60 GGACCGTCTC CCGCTGCTTT GGTCACCAGC CCCTGCCCGC CCGACCCGCT CCGTTCTCCG V20 GCCTGCGAGC CCTGCCGGCC GGACTTTGCG CCGCGTCCGG GCTGCTGCTG CGCTCGGGGC 180 CCCGCTCGGC GCCGGCGGTG ACCGGGAAGC CCGCGTTAAA GGGGCAACCG GGACCCTGGC 240 CCGGTATGGC TGAAGTCAGC ATCGACCAGT CCAAGCTGCC TGGAGTCAAG GAAGTATGCC 300 GAGATTTTGC TGTCCTGGAG GACCACACCC TGCTCACAGC CTGCAGGAAC AAGAGATTGA 360 GCATCATTTG GCATCGAACG TTCAGCGGAA CCGTTTGGTC CAGCATGATC TCCAGGTGGC 420 TAAGCAGCTC CAAGAGGAAG ATCTGAAAGC GCAGGCCCAG CTCCAGAAGC GTTACAAAGA 480 CCTTGAACAA CAAGACTGTG AAATTGCTCA GGAAATTCAG GAGAAGCTGG CTATTGAGGC 540 AGAGAGACGA CGCATTCAGG AGAAGAAGGA TGAGGACATA GCTCGCCTTT TGCAAGAAAA 600 GGAGTTACAG GAAGAGAAAA AGAGAAAGAA ACACTTTCCA GAGTTCCCTG CAACCCGTGC 660 TTATGCAGAT AGTTACTATT ATGAAGATGG AGGAATGAAG CCAAGAGTGA CGAAAGAAGC 720 TGTATCTACT CCATCACGAA TGGCCCACAG GGATCAGGAA TGGTATGATG CTGAAATTGC 780 CAGAAAACTG CAAGAAGAAG AACTTTTGGC TACCCAGGTG GACATGAGAG CCGCTCAAGT 840 AGCTCAAGAT GAAGAAATCG CTCGACTTCT AATGGCTGAA GAAAAGAAAG CTTACAAAAA 900 AGCCAAGGAG CGGGAGAAAT CATCTTTGGA CAAAAGAAAG CAAGACCCCG AGTGGAAGCC 960 AAAAACAGCT AAAGCAGCAA ATCAAAGTCA AAAGAGAGTA TGAACCTCAC CATTCTAAGA 1020 ATGAAAGGCC AGCACGGCCA CCACCACCTA TCATGACAGA TGGTGCAAGA TGCGGTACAC 1080 TCATTTTACA AACCAGCAGA GTTCCACACG GCATTTCTCA AAATCAGAGT CCTCTCATAA 1140 AGGTTTCATC ACAAACATTA AAAACCTAGG AATCTGCCTT GAAAATGGAC TCACTATAGC 1200 AAATATTACT GGGTGATACA GAATGAATTC TACACTTACT TTTTTTCTCC TGTGTTTGCA 1260 TGGCCTGGGA TTTACTCCTC AAGTGTCATT TCTGAACCAT AAGTAATTTT AATTCATTTC 1320 AAATGTTTTG GTTATTCATG ATCACTTGGG CAGTATAAGA AAATGTAGCT TCTGAATATT 1380 GGCCACCTCT ATGCTGCATA TACTTCTTGG GATATAGTAT CTAAGCCTTG TAAACTGCCA 1440 TTTGTTAGGT ATGGAGTTTG GTATCTAGGG AGTAGGCCTT ATTTAGCAAT TCAAATTTTA 1500 TGGAGATGAA TGATCAAAGT GAAACAATGT TTGGATGCAA CGCAGAATAA AAGAATATAA 1560
INFORMATION FOR SEQ ID NO: 11
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 67
(B) TYPE Amino acid (ii) MOLECULE TYPE Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Met Val Asp Ser Ala Glu Asp Gin Lys He His Arg Pro Met Ala
5 10 15
Pro Lys Glu Ala Pro Lys Lys Leu He Arg Tyr He Asp Asn Gin Val
20 25 30
Val Ser Thr Lys Gly Glu Arg Phe Lys Asp Val Arg Asn Pro Glu Ala
35 40 45
Glu Glu Met Lys Ala Thr Tyr He Asn Leu Lys Pro Ala Arg Lys Tyr
50 55 60
Arg Phe His
INFORMATION FOR SEQ ID NO: 12
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 158
(B) TYPE Amino acid (ii) MOLECULE TYPE Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Glu Gly Glu Thr Ser Ser Ser Pro Cys Leu Gin Glu Asn Ser Ala
5 10 15
Asp Cys Ser Ala Gly Glu Leu Arg Gly Pro Gly Lys Glu Leu Trp Ser
20 25 30
Pro He Gin Gin Val Thr Ala Thr Ser Ser Lys Trp Ala Arg Phe Val
35 40 45
Leu Pro Pro Arg Lys Ser Ser His Val Asp Ser Glu Gin Pro Arg Ser
50 55 60
Leu Gin Arg Asp Pro Arg Pro Ala Gly Pro Ala Gin Ala Lys Gin Gly 65 70 75 80
Thr Pro Arg Ala Gin Ala Ser Arg Glu Gly Leu Ser Arg Pro Thr Ala
85 90 95
Ala Val Gin Leu Pro Arg Ala Thr His Pro Val Thr Ser Gly Ser Glu
100 105 no
Arg Pro Cys Gly Lys Thr Ser Trp Asp Ala Arg Thr Pro Trp Ala Glu
115 120 125
Gly Gly Pro Leu Val Leu Glu Ala Gin Asn Pro Arg Pro Thr Arg Leu
130 135 140
Cys Asp Leu Phe He Thr Gly Glu Asp Phe Asp Asp Asp Val 145 150 155 INFORMATION FOR SEQ ID NO: 13
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 59
(B) TYPE Amino acid (ii) MOLECULE TYPE Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Arg Leu Lys Phe Ser Lys His Thr Trp Gin Tyr Gly Gly Phe Met
5 10 15
Lys Ser Asp Gly Glu Glu Leu Gly Glu Met Glu Glu Lys Lys Cys Ser
20 25 30
Gin Lys Phe Gin Asn Lys Tyr Thr Lys Ser Tyr Val Ser Leu Asn Leu
35 40 45
Tyr Phe Ser Gly Thr Leu Leu Lys Gly Leu Tyr 50 55
INFORMATION FOR SEQ ID NO: 14
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 235
(B) TYPE Amino acid (ii) MOLECULE TYPE Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Pro Arg Phe Cys Cys Pro Gly Gly Pro His Pro Ala His Ser Leu
5 10 15
Gin Glu Gin Glu He Glu His His Leu Ala Ser Asn Val Gin Arg Asn
20 25 30
Arg Leu Val Gin His Asp Leu Gin Val Ala Lys Gin Leu Gin Glu Glu
35 40 45
Asp Leu Lys Ala Gin Ala Gin Leu Gin Lys Arg Tyr Lys Asp Leu Glu
50 55 60
Gin Gin Asp Cys Glu He Ala Gin Glu He Gin Glu Lys Leu Ala He
65 70 75 80
Glu Ala Glu Arg Arg Arg He Gin Glu Lys Lys Asp Glu Asp He Ala
85 90 95 Arg Leu Leu Gin Glu Lys Glu Leu Gin Glu Glu Lys Lys Arg Lys Lys
100 105 110
His Phe Pro Glu Phe Pro Ala Thr Arg Ala Tyr Ala Asp Ser Tyr Tyr
115 120 125
Tyr Glu Asp Gly Gly Met Lys Pro Arg Val Thr Lys Glu Ala Val Ser
130 135 140
Thr Pro Ser Arg Met Ala His Arg Asp Gin Glu Trp Tyr Asp Ala Glu 145 150 155 160
He Ala Arg Lys Leu Gin Glu Glu Glu Leu Leu Ala Thr Gin Val Asp
165 170 175
Met Arg Ala Ala Gin Val Ala Gin Asp Glu Glu He Ala Arg Leu Leu
180 185 190
Met Ala Glu Glu Lys Lys Ala Tyr Lys Lys Ala Lys Glu Arg Glu Lys
195 200 205
Ser Ser Leu Asp Lys Arg Lys Gin Asp Pro Glu Trp Lys Pro Lys Thr
210 215 220
Ala Lys Ala Ala Asn Gin Ser Gin Lys Arg Val 225 230 235
INFORMATION FOR SEQ ID NO: 15
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 230
(B) TYPE : Amino acid
(ii) MOLECULE TYPE : Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Val Lys Leu Phe He Gly Asn Leu Pro Arg Glu Ala Thr Glu Gin
5 10 15
Glu He Arg Ser Leu Phe Glu Gin Tyr Gly Lys Val Leu Glu Ser His
20 25 30
He He Lys Asn Tyr Arg Phe Val His He Glu Asp Lys Thr Ala Ala
35 40 45
Glu Asp Ala He Arg Asn Leu His His Tyr Lys Leu His Gly Val Asn
50 55 60
He Asn Val Glu Ala Ser Lys Asn Lys Ser Lys Thr Ser Thr Lys Leu 65 70 75 80 His Val Gly Asn He Ser Pro Thr Cys Thr Asn Lys Glu Leu Arg Ala
85 90 95
Lys Phe Glu Glu Tyr Gly Pro Val He Glu Cys Asp He Val Lys Asp
100 105 110
Tyr Ala Phe Val His Met Glu Arg Ala Glu Asp Ala Val Glu Ala He
115 120 125
Arg Gly Leu Asp Asn Thr Glu Phe Gin Gly Lys Arg Met His Val Gin
130 135 140
Leu Ser Thr Ser Arg Leu Arg Thr Ala Pro Gly Met Gly Asp Gin Ser 145 150 155 160
Gly Cys Tyr Arg Cys Gly Lys Glu Gly His Trp Ser Lys Glu Cys Pro
165 170 175
He Asp Arg Ser Gly Arg Val Ala Asp Leu Thr Glu Gin Tyr Met Ser
180 185 190
Asn Thr Glu Gin Cys Val Pro Leu His His Glu Leu Trp Gly Phe He
195 200 205
Val Leu Gin Gin Arg Val Arg Ser Ala Arg Cys Leu Leu Gin Ala Leu
210 215 220
Pro Cys Gly Ala Val Leu 225 230
INFORMATION FOR SEQ ID NO: 16 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 1585
(B) TYPE Nucleic acid
(C) STRANDEDNESS Sin le
(D) TOPOLOGY Linear (ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE No (vi) ORIGINAL SOURCE
(A) ORGANISM Human
(F) TISSUE TYPE Lung
(G) CELL TYPE Fibroblast (H) CELL LINE MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 51..740 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGATCCCCGG GGAGGAGGCC CTGCTGGTTT CTGTGCGGGC TCTTGTCAGG ATGGTGAAGC 6
TGTTCATCGG AAACCTGCCC CGGGAGGCTA CAGAGCAGGA GATTCGCTCA CTCTTCGAGC 12
AGTATGGGAA GGTGCTGGAA TCTCACATCA TTAAGAATTA CCGCTTTGTG CACATAGAAG 18
ACAAGACGGC AGCTGAGGAT GCCATACGCA ACCTGCACCA TTACAAGCTT CATGGGGTGA 24
ACATCAACGT GGAAGCCAGC AAGAATAAGA GCAAAACCTC AACAAAGTTG CATGTGGGCA 30
ACATCAGTCC CACCTGCACG AATAAGGAGC TTCGAGCCAA GTTTGAGGAG TATGGTCCGG 36
TCATCGAATG TGACATCGTG AAAGATTATG CCTTCGTACA CATGGAGCGG GCAGAGGATG 42
CAGTGGAGGC CATCAGGGGC CTTGATAACA CAGAGTTTCA AGGCAAACGA ATGCACGTGC 48
AGTTGTCCAC CAGCCGGCTT AGGACTGCGC CCGGGATGGG AGACCAGAGC GGCTGCTATC 54
GGTGCGGGAA AGAGGGGCAC TGGTCCAAAG AGTGTCCGAT AGATCGTTCA GGCCGCGTGG 60
CAGACTTGAC CGAGCAATAT ATGAGCAATA CGGAGCAGTG CGTACCCTTA CACCATGAGC 66
TATGGGGATT CATTGTATTA CAACAACGCG TACGGAGCGC TCGATGCCTA CTACAAGCGC 72
TGCCGTGCGG CGCGGTTCTA TGAGGCAGTG GCAGCTGCAG CCTCCGTGTA TAATTACGCA 78
GAGCAGACCC TGTCCCAGCT GCCACAAGTC CAGAATACAG CCATGGCCAG TCACCTCACC 84
TCCACCTCTC TCGATCCCTA CGATAGACAC CTGTTGCCGA CCTCAGGAGC TGCTGCCACA 90
CTGCGTGCTC GGACGAGCCG CTGCTGCTGT TACTGCAGCT TCCACTTCAT ATACGGGCGG 96
GATCGGAGCC CCCTCGTCCC TACAGCCCCA GTCCCCACTG TTGGAGAGGG CTACGGTTAC 102
GGGCATGAGA GTGAGTTGTC CCAAGCTTCA GCAGCCGCGC GGAATTCTCT GTACGACATG 108
GCCCGGTATG AGCGGGAGCA GTATGCCGAT CGGGCGCGGT ACTCAGCCTT TTAAAGCTTG 114
AGGTGGGATG TGTGTGGGCT GAAATTCCGA GCTGCGGTTG TGCATGAGAA TCACCCTTCG 120
TGGTACCCCA TCTTCGGGAC GTTCTCGGCT CTGTGCGTTC AGTCCCTCAG GAACCGTGGA 126
CCTTAATTTA CCTTGCTAAG TTCAGACCTT CTCTTCCTTT CCTTTCCTTT CCTCTCCTGC 132
CCATTTTCCT GTTCTTCTGT CCTCCAATAC TTCTGTAGCT ACCCATTCAT GTTCTCTTCT 138
CCCAGAAGGC CTCATTGTGT GCAGAAACTG TGGTGGGGGC TGTGCTGTCT CCTCCCTGCC 144
TCCTGCCTCT GCGGCTGTTG GATTTGGGAA TGACCTTGGT GAGAGTCTCA CTGCTCCAGG 150
GTCTCTTTTT GGTCCAAAGG CTAGACCTAT AGAGTTGGAT CACTCTTTTT CTTTCCGGTG 156
AGATAAATGG TTTTTCAACT TAAAA 158
INFORMATION FOR SEQ ID NO: 17
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 2500
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear
(ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE : No (vi) ORIGINAL SOURCE :
(A) ORGANISM : Human
(F) TISSUE TYPE : Lung
(G) CELL TYPE : Fibroblast (H) CELL LINE : MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 1062..1736
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GGATCCCCGG GAACAGCGGC TCCGGGCCGC GCCGTCGCTG CTGCTGCTGC TGCTGTGGCT 60
GCTCGCGGTT CCCGGCGCTA ACGGGCCCCG GGTCGGCGCT CTATTCGCCT TCCGACCCGC 120
TGACGCTGCT GCAGGCGGAC ACGGTGCGCG GCGGGTGCTG GGCTCCCGCA GCGCCTGGGC 180
CGTGGAGTTC TTCCCCTCCT GGTGCGGCCA CTGCATCGCC TTCGCCCCGA CGTGGAAGGC 240
GCTGGCCGAA GACGTCAAAG CCTGGAGGCC GGCCCTGTAT CTCGCCGCCC TGGACTGTGC 300
TGAGGAGACC AACAGTGCAG TCTGCAGAGA CTTCAACATC CCTGGCTTCC CGACTGTGAG 360
GTTCTTCAAG GCCTTTACCA AGAACGGCTC GGGAGCAGTA TTTCCAGTGG CTGGTGCTGA 420
CGTGCAGACG CTGCGGGAGA GGCTCATTGA CGCCCTGGAG TCCATCATGA CACGTGGCCC 480
CCAGCCTGTC CCCCACTGGA GCCTGCCAAG CTGGAGGAGA TTGATGGATT CTTTGCGAGA 540
AATAACGAAG AGTACCTGGC TCTGATCTTT GAAAAGGGAG GCTCCTACCT GGGTAGAGAG 600
GTGGCTCTGG ACCTGTCCCA GCACAAAGGC GTGGCGGTGC GCAGGGTGCT GAACACAGAG 660
GCCCAATGTG GTGAGAAAGT TTGGTGTCAC CGACTTCCCC TCTTGCTACC TGCTGTTCCG 720
GAATGGCTCT GTCTCCCGAG TCCCCGTGCT CATGGAATCC AGGTCCTTCT ATACCGCTTA 780
CCTGCAGAGA CTCTCTGGGC TCACCAGGGA GGCTGCCCAG ACCACAGTTG CACCAACCAC 840
TGCTAACAAG ATAGCTCCCA CTGTTTGGAA ATTGGCAGAT CGCTCCAAGA TCTACATGGC 900
TGACCTGGAA TCTGCACTGC ACTACATCTG CGGATAGAAG TGGGCAGGTT CCCGGTCCTG 960
GAAGGGCAGC GCCTGGTGGC CCTGAAAAAG TTTGTGGCAG TGCTGGCCAA GTATTTCCCT 1020
GGCCGGCCCT TAGTCCAGAA CTTCCTGCAC TCCGTGAATG AATGGCTCAG AGGCAGAAGA 1080
GAAATAAAAT TCCCTACAGT TTCTTTAAAA CTGCCCTGGA CGACAGGAAA GAGGGTGCCG 1140
TTCTTGCCAA GAAGGTGAAC TGGATTGGCT GCCAGGGGAG TGAGCCGCAT TTCCGGGGCT 1200
TTCCCTGCTC CCTGGGCCTC CTCTTCCACT TCTTGACTGT GCAGGCAGCT CGGCAAAATG 1260
TAGACCACTC ACAGAACACC AAGGCCAAGG AGGTCCTCCC AGCCATCCGA GGCTACGTGC 1320
ACTACTTCTT CGGCTGCCGA GACTGCGCTA GCCACTTCGA GCAGATGGCT GCTGCCTCCA 1380
TGCACCGGGT GGGGAGTCCC AACGCCGCTG TCCTCTGGCT CTGGTCTAGC CACAACAGGG 1440
TCAATGCTCG CTTGCAGGTG CCCCCAGCGA GGACCCCCAG TTCCCCAAGG TGCAGTGGCC 1500
ACCCCGTGAA CTTTGTTCTG CCTGCCACAA TGAACGCCTG GATGTGCCCG TGTGGGACGT 1560 GGAAGCCACC CTCAACTTCC TCAAGGCCCA CTTCTCCCCA AGCAACATCA TCCTGGACTT 1620 CCCTCAGCTG GGTCAGCTGC CCGGAGGGAT GTGCAGAATG TGGCAGCCGC CCCAGAGCTG 1680 GCGATGGGAG CCCTGGAGCT GGGAAGCCGG AATTCAACTC TGGACCCTGG GAAGCCTGAG 1740 ATGATGAAGT CCCCCACAAA CACCACCCCA CATGTGCCGG CTGAGGGACC TGAGCTTATT 1800 TGAAGTCCTG CCTCATTCTC ACTGGAGCCT CAGTCTCTCC TGCTTGGTCT TGGCCCTCAA 1860 CTGGGGCAAG TGAAGCCAGA GGAGGGTCCC CCAGCTGGGT GGGCTGGAAT GGAACTCCTC 1920 ACTAGCTGCT GGCTCCGCCC ACCCTGCTCC CTTCCGGACA ATGAAGAAGC CTTTGCACCC 1980 TGGGAGGAAG GACCACCCCG GGCCCTCTAT GCCTGGCCAG CCTCCAGCTC CTCAGACCTC 2040 CTGGGTGGGG TTTGGCTTCA GGGTGGGGTT TGGAAGCTTC TGGAAGTCGT GCTGGTCTCC 2100 CAGGTGAGGC AAGCCATGGT TGCTGGGCTG TAGGGTGAGG TGGCTTCCTT GGTGGGACCT 2160 GACGAGTTGG TGGCATGGGA AGGATGTGGG TCTCTAGTGC CTTGCCCTGG CTTAGCGGCA 2220 GGAGAAGATG GCGGCTTTCA CTTCCCCCCA ATTGAGCTCT GCTCCCTCTG AGCCTGGGTC 2280 TTTTGTCCTT TTTTATTTTG GTCTCCAAGA TGAATGCTCA TCTTTGGAGG GTCCCAGGTA 2340 GAAGCTAGGG AGGGGAGTGT CTTCTCTCTC CCAGGTTTCA CCTTCCAGTG TGCAGAAGTT 2400 AGAAGGGTCT GGCGGGGGCA GTGCCTTACA CATGCTTGAT TCCCACGCTA CCCCCTGCCT 2460 TGGGAGGTGT GTGGAATAAA TTATTTTTGT TAAGGCAAAA 2500
INFORMATION FOR SEQ ID NO: 18
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 1246
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear
(ii) MOLECULE TYPE cDNA
(iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GGATCCCCGG GCCCGGGAGA AGCAGAGCTC AGAGGAAGAA GAAAAGGAAA CAAGAGGGGT 60
TCAGAAGAGG CGAGGAGGGA GCACAGTACC CAAAGATGGG CCAGTGAGAC CTCAGAACGC 120
TGAAGAAGAA AAAAGAGGCT TAGACCTGCG TGTGTCGGGG TACCTGAATC TGGCTGCTGA 180
CTTGGCACAC AACTTCACTG ATGGTCTGGC CATTGGGGCT TCCTTTCGAG GGGGCCGGGG 240
ACTAGGGATC CTGACCACAA TGACTGTCCT GCTACATGAA GTGCCCCACG AGGTCGGAGA 300
CTTTGCCATC TTGGTCCAGT CTGGCTGCAG CAAAAAGCAG GCGATGCGTC TGCAACTACT 360
GACAGCAGTA GGGGCACTGG CAGGCACAGC CTGTGCCCTT CTCACTGAAG GAGGAGCAGT 420
GGGCAGTGAA ATTGCAGGTG GTGCAGGTCC TGACTGGCTC CTACCATTTA CTGCAGGTGG 480
CATTATCCTA CGTAACAAAT AGTGTGTGTG TTCCCCGAGC TGCTGAGGGA GGCATCACCA 540
TTGCAATCAC TTCTGGAGGG TGCTGGGGCT GCTGGGGGGA ATTATCATGA TGGTGCTGAT 600 TCCCCACCTT GAGTGAGGGG TGGATAAACT ACCCCTCCCC AAACCTCTAC CCCTAACTCC 660
AGGTCAGGGG TGCGTAGAGG TTGGGGGCCC TGGCCAGGGA CATCTGCCAA AGGAAGGAAC 720
TGTAGCCTGG GAGAATGGTT ACTTTGGCAT TAGGGCCTTC AAGGGCTGGC AGTCTTACAG 780
AGGCTGGAGC GGTGAGAATG AGAGGCCAGA GGGACCATAG TGTTGGGCAC TGTCTGACCA 840
TGTTGCATTT GGAAGGCTAA ATGGGGCCAT GAAGAAGGCT GGAAGGGACA GGGGGTGATG 900
GCAGCCTACC TGGTGTCCCC TACCCCACCT GTTCTCGGAG AACCAAGTTG CTACACAGGA 960
AGTTCTCCAA GGTCCAGTTT CCTTTCTCCC ACCAGTTGGT GGAGGCTTCA GGGAAGACCA 1020
GAGTCCTGGA CAGAGAGGGT AACAGGAGGA GTCGGGGATA AACATCAAAC ATCAATCGTG 1080
TGTCCTGATT TGGGAGTGAT TGGGGGGATG GGGTGGGAGA GGGTTAGTTG GTATTCTCAT 1140
GGCCTGATTT TTTTTGTTTC TATTCCTTTT ATATCACTGT GTTTGAATCG AGGGGGAGGG 1200
GTGGTAACCG GAAATAAAGA CCTCCGATCT TCCGCCCCAC CAAAAA 1246
INFORMATION FOR SEQ ID NO: 19
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 661
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear
(ii) MOLECULE TYPE cDNA
(iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GGATCCCCGG GCTCCTGTCC ATGAACTGGG CCACGTGGCC GACATTCTGC TGTACGTGGT 60
GATCCCTACC CGACGCTCCA CCGCCGAGGC CTTCCAGATC GTGCTGTCCC ACCTGCTGGG 120
TGATGCTGGG AGCCCCTACC TCATTGGCCT GATCTCTGAC CGCCTGCGCC GGAACTGGCC 180
CCCCTCCTTC TTGTCCGAGT TCCGGGCTCT GCAGTTCTCG CTCATGCTCT GCGCGTTTGT 240
TGGGGCACTG GCGCGCGCAG CCTTCCTGGG CACCGCCATC TTCATTGAGG CCGACCGCCG 300
GCGGGACAGC TGCACGTGCA GGGCCTGCTG CACGAAGCAG GGTCCACAGA CGACCGGATT 360
GTGGTCCGCA GCGGGGCCGC TCCACCCGCG TGCCCGTGGG CAGGGTGCTC ATCTGAGAGG 420
CTGCCGCTCA CCTACCAGCC TGACATCTCC ACAGCTGCCC TGGCCCACCC ACAAGGGGCC 480
TGGCCTAACC CCTTGGCCTG GCCCAGCTTC CAGAGGGACC CTGGGCCGTG TGCCAGCTCC 540
CAGACACTAC ATGGGTAGCT CAGGGGAGGA GGTGGGGGTC CAGGAGGGGG ATCCCTCTCC 600
ACAGGGGCAG CCCCAAGGGC TCGGTGCTAT TTGTAACGGA ATAAAATTTG TAGCCAGAAA 660
A 661
INFORMATION FOR SEQ ID NO:20
(i) SEQUENCE CHARACTERISTICS :
oo- (A) LENGTH : 1951
(B) TYPE : Nucleic acid
(C) STRANDEDNESS : Sin le
(D) TOPOLOGY : Linear
(ii) MOLECULE TYPE : cDNA
(iv) ANTI-SENSE : No
(vi) ORIGINAL SOURCE
(A) ORGANISM Human
(F) TISSUE TYPE Lung
(G) CELL TYPE Fibroblast
(H) CELL LINE MRC-5
(ix) FETURE :
(A) NAME KEY : CDS
(B) LOCATION : 55..1488
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GTCGCGGCCG CTGTTCCTGG GACGTCCGGT TGACCGCGGT CTGCTGCAGA GACCATGTCT 6
GCCGACGGGG CAGAGGCTGA TGGCAGCACC CAGGTGACAG TGGAAGAACC GGTACAGCGG 12
CCCAGTGTGG TGGACCGTGT GGCCAGCATG CCTCTGATCA GCTCCACCTG CGACATGGTG 18
TCCGCAGCCT ATGCCTCCAC CAAGGAGAGC TACCCGCACG TCAAGACTGT CTGCGACGCC 24
GCAGAGAAGG GAGTGAGGAC CCTCACGGCG GCTGCTGTCA GCGGGGCTCA GCCGATCCTC 30
TCCAAGCTGG AGCCCCAGAT TGCATCAGCC AGCGAATACG CCCACAGGGG GCTGGACAAG 36
TTGGAGGAGA ACCTCCCCAT CCTGCAGCAG CCCACGGAGA AGTGCTGGCG GACACCAAGG 42
AGCTTGTGTC GTCTAAGGTG TCGGGGGCCC AAGAGTTGGT GTCTAGCGCC AAGGACACGT 48
TGGCCACCCA ATTGTCGGAG GCGGTGGACC GACCCGCGGT GCTGTGCAGA GCGGCGTGGA 54
CAAGACAAAG TCCGTAGTGA CCGGCGGCGT CCAATCAGTC ATGGGCTCCC GCTTGGGCCA 60
GATGGTGCTG AGTGGGGTCG ACACGGTGCT GGGGAATTCG GAGCCGTGCG CGCACAACCA 66
CCTGCCCTTA CGGATGCCGA ACTGGCCCGC ATCGCCACAT CCCTGGATGG CTTCGACGTC 720
GCGTCCGTGC AGCAGCAGCG GCAGGAACAG AGCTACTTCG TACGTCTGGG CTCCCTGTCG 78
GAGAGGCTGC GGCAGCACGC CTATGAGCAC TCGCTGGGCA AGCTTCGAGC CACCAAGCAG 840
AGGGCACAGG AGGCTCTGCT GCACGTGTCG CAGGCCCTAA GCCTGATGGA AACTGTCAAG 900
CAAGGCGTTG ATCAGAAGCT GGTGGAAGGC CAGGAGAAGC TGCACCAGAT GTGGCTCAGC 960
TGGAACCAGA AGCAGCTCCA GGGCCCCGAG AAGGAGCCGC CCAAGCCAGA GCAGGTCGAG 1020
TCCCGCGCGC TCACCATGTT CCGGGACATT GCCCAGCAAC TGCAGGCCAC CTGTACCTCC 1080
CTGGGGTCCA GCATTCAGGG CCTCCCCACC AATGTGAAGG ACCAGGTGCA GCAGGCCCGC 1140
CGCCAGGTGG AGGACCTCCA GGCCACGTTT TCCAGCATCC ACTCCTTCCA GGACCTGTCC 120
AGCAGCATTC TGGCCCAGAG CCGTGAGCGT GTCGCCCGCG CCCGCGAGGC CCTGGACACA 126 TGGTGGGATA TGTGGCCCAG CACACACCTG TCACGTGGCT CGTGGGACCC TTTGCCCCTG 1320
GAATCACTGA GAAAGCCCCG GAGGAGAAGA AGTAGGGGGA GAGGAGAGGA CTCAGCGGGC 1380
CCCGTCTCTA TAATGCAGCT GTGCTCTGGA GTCCTCAACC CGGGGCTCAT TTCAAACTTA 1440
TTTTCTAGCC ACTCCTCCCA GCTCTTCTGT GCTGTCCACT TGGGAAGCTA AGGCTCTCAA 1500
AACGGGCATC ACCCAGTTGA CCCATCTCTC AGCCTCTCTG AGCTTGGAAG AAGCCTGTTC 1560
TGAGCCTCAC CCTATCAGTC AGTAGAGAGA GATGTCCAGA AAAAATATCT TTCAGGAAAG 1620
TTCTCCCCGG CAGAATTTTT TTTCCTTGTT AGATATCAGG GATATAGGCC GGGTGCGGTG 1680
GCTCACACCT GTAATCCCAG CACTTTGGGA GGCTGAGGCG GGCGGACACC TGAGGTCAGG 1740
TGTTCGAGAC CAGCCAGGCC AACATGGTGA AACCCCGTCT CTACTAAAAA TACAAAAAAA 1800
AATGAGCCGC GCATGGTAGC AGGTGTCTGT TATCCCAGTT AGGAGGCTGA GGGAAGAGAA 1860
TCTCTTGAAC CTGAGAGGCG GAGGTTGCAG TGAGCCAAGA TCGCGCCTTG CACTCCAGCC 1920
TGGGGAACAA GAGTGAGACT TAGTCTCAAA A 1951
INFORMATION FOR SEQ ID NO:21
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 513
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear
(ii) MOLECULE TYPE cDNA
(iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21 :
GGATCCCCGG GGCGCAGGAC AGCCTCTTCC AGCTATGGAA GAAGAAGCGC GGGGTGCTCA 60
CCTCCGACCG CCTGAGCCTG TTCCCCGCGA GCCCCCGGGC GCGCCCCAAG GAGCTGCGCT 120
TCCACTCCAT CCTAAGGTGG ACTGCGTGGG GGACGGCAAG TACGTGTACT CACCATCGTC 180
ACCACCGACC ACAAGGAGAT CGACTTCCGC TGCGGGCGAG AGCTGCTGGA ACCGGCCATC 240
GGCGGCGCTC ATCGATTTCC AGAACCGCCG CGCCCTGCAG GACTTTCGCA GCCGCCAGAA 300
CGCACCGCAC CCGCCGCACC CGCCGAGGAC GCCGTGGCTG CCGCGGCCGC CGACCCTCCG 360
AGCCCTCGGA GCCCTCCAGG CCATCCCCGC AGCCCAAACC CCGCACGCCA TGAGCCCGCC 420
GCGGGCCATA CGCTGGACGA GTCGGACCGA GGCTAGGACA TGGCCCGCGC TCTCCAGCCC 480
TGCAGCAGAA GAACTTCCCG TGCGCGCGGA TCC 513
INFORMATION FOR SEQ ID NO:22
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 225
(B) TYPE : Amino acid (ii) MOLECULE TYPE : Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Ala Gin Arg Gin Lys Arg Asn Lys He Pro Tyr Ser Phe Phe Lys
5 10 15
Thr Ala Leu Asp Asp Arg Lys Glu Gly Ala Val Leu Ala Lys Lys Val
20 25 30
Asn Trp He Gly Cys Gin Gly Ser Glu Pro His Phe Arg Gly Phe Pro
35 40 45
Cys Ser Leu Gly Leu Leu Phe His Phe Leu Thr Val Gin Ala Ala Arg
50 55 60
Gin Asn Val Asp His Ser Gin Asn Thr Lys Ala Lys Glu Val Leu Pro 65 70 75 80
Ala He Arg Gly Tyr Val His Tyr Phe Phe Gly Cys Arg Asp Cys Ala
85 90 95
Ser His Phe Glu Gin Met Ala Ala Ala Ser Met His Arg Val Gly Ser
100 105 110
Pro Asn Ala Ala Val Leu Trp Leu Trp Ser Ser His Asn Arg Val Asn
115 120 125
Ala Arg Leu Gin Val Pro Pro Ala Arg Thr Pro Ser Ser Pro Arg Cys
130 135 140
Ser Gly His Pro Val Asn Phe Val Leu Pro Ala Thr Met Asn Ala Trp 145 150 155 160
Met Cys Pro Cys Gly Thr Trp Lys Pro Pro Ser Thr Ser Ser Arg Pro
165 170 175
Thr Ser Pro Gin Ala Thr Ser Ser Trp Thr Ser Leu Ser Trp Val Ser
180 185 190
Cys Pro Glu Gly Cys Ala Glu Cys Gly Ser Arg Pro Arg Ala Gly Asp
195 200 205
Gly Ser Pro Gly Ala Gly Lys Pro Glu Phe Asn Ser Gly Pro Trp Glu
210 215 220
Ala 225
INFORMATION FOR SEQ ID NO: 23
(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 240 (B) TYPE : Amino acid (ii) MOLECULE TYPE : Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Ser Ala Asp Gly Ala Glu Ala Asp Gly Ser Thr Gin Val Thr Val
5 10 15
Glu Glu Pro Val Gin Arg Pro Ser Val Val Asp Arg Val Ala Ser Met
20 25 30
Pro Leu He Ser Ser Thr Cys Asp Met Val Ser Ala Ala Tyr Ala Ser
35 40 45
Thr Lys Glu Ser Tyr Pro His Val Lys Thr Val Cys Asp Ala Ala Glu
50 55 60
Lys Gly Val Arg Thr Leu Thr Ala Ala Ala Val Ser Gly Ala Gin Pro 65 70 75 80
He Leu Ser Lys Leu Glu Pro Gin He Ala Ser Ala Ser Glu Tyr Ala
85 90 95
His Arg Gly Leu Asp Lys Leu Glu Glu Asn Leu Pro He Leu Gin Gin
100 105 110
Pro Thr Glu Lys Cys Trp Arg Thr Pro Arg Ser Leu Cys Arg Leu Arg
115 120 125
Cys Arg Gly Pro Lys Ser Trp Cys Leu Ala Pro Arg Thr Arg Trp Pro
130 135 140
Pro Asn Cys Arg Arg Arg Trp Thr Asp Pro Arg Cys Cys Ala Glu Arg 145 150 155 160
Arg Gly Gin Asp Lys Val Arg Ser Asp Arg Arg Arg Pro He Ser His
165 170 175
Gly Leu Pro Leu Gly Pro Asp Gly Ala Glu Trp Gly Arg His Gly Ala
180 185 190
Gly Glu Phe Gly Ala Val Arg Ala Gin Pro Pro Ala Leu Thr Asp Ala
195 200 205
Glu Leu Ala Arg He Ala Thr Ser Leu Asp Gly Phe Asp Val Ala Ser
210 215 220
Val Gin Gin Gin Arg Gin Glu Gin Ser Tyr Phe Val Arg Leu Gly Ser 225 230 235 240
INFORMATION FOR SEQ ID NO:24
(i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 448
(B) TYPE : Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY : Linear
(ii) MOLECULE TYPE : cDNA
(iv) ANTI-SENSE : No
(vi) ORIGINAL SOURCE :
(A) ORGANISM : Human
(F) TISSUE TYPE : Lung
(G) CELL TYPE : Fibroblast
(H) CELL LINE : MRC-5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GGATCCCCGG GACCAAGAAC TTATCGGAAG TGTGCCTCTG TGTCTCCTTC CTCGGGGTAA 6
GGAGGGGACA GTGCTTCCCA AGTTCCAGCT GCAAGTCCAA CTTAACCAAC TTTCCTTCAA 12
AGTCAGTTAC TGCCAATTTT CTGAAAAAAG CATGTTCCAT ATACTAAGTC TCTCTTCTCA 18
CGGTAGGAAA TAATACAGCC AAGATATGCA GCATCCTTCT CATTGATGTA GAAAATTCTG 24
AAAATTCTGC GATAGACCAG AAAAATCCTG GCAGCTTTTC TCCAGGCATC TGGGTCACTA 30
AAAACTGATT TTCTAAAATT ATTGGATTTG TATTTTGTTA TTAAGGGGGG AAATGTGATT 36
TGTGCCTGAT CTTTCATCTG TGATTCTAAT AAGAGCTTTG TCTTCAGAGA AACTAAAAAT 42
AAAAGGCATT GACTTAAACA GCTGAAAA 44
INFORMATION FOR SEQ ID NO:25 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 1352
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GGATCCCCGG GCTGCCGCGG CTCCCCGAGC TGTTCGAAAC TGGTAGACAG TTACTGGACG 6
AAGTAGAAGT GGTGACTGAA CCCGCCGGTT CCCGGATAGT CCAGGAGAAG GTGTTCAAGG 12
GCTTGGACCT CCTTGAGAAG GCTGCCGAAA TGTTATCGCA GCTCGACTTG TTCAGCCGAA 18
ATGAAGATTT GGAAGAGATT GCTTCCACCG ACCTGAAGTA CCTTTTGGTG CCAGCGTTTC 24
AAGGAGCCCT CACCATGAAA CAAGTCAACC CCAGCAAGCG TCTAGATCAT TTGCAGCGGG 30 CTCGAGAACA CTTTATAAAC TACTTAACTC AGTGCCATTG CTATCATGTG GCAGAGTTTG 360
GGCTATCCCA AACCATGAAC AACTCTGCTG AAAATCACAC TGCCAATTCC TCCATGGCTT 420
ATCCTAGTCT CGTTGCTATG GCATCTCAAA GACAGGCTAA AATACAGAGA TACAAGCAGA 480
AGAAGGAGTT GGAGCATAGG TTGTCTGCAA TGAAATCTGC TGTGGAAAGT GGTCAAGCAG 540
ATGATGAGCG TGTTCGTGAA TATTATCTTC TTCACCTTCA GAGGTGGATT GATATCAGCT 600
TAGAAGAGAT TGAGAGCATT GACCAGGAAA TAAAGATCCT GAGAGAAAGA GACTCTTCAA 660
GAGAGGCATC AACTTCTAAC TCATCTCGCC AGGAGAGGCC TCCAGTGAAA CCCTTCATTC 720
TCACTCGGAA CATGGCTCAA GCCAAAGTAT TTGGAACTGG TTATCCAAGT CTGCCAACTA 780
TGACGGTGAG TGACTGGTAT GAGCAACATC GGAAATATGG AGCATTACCG GATCAGGGAA 840
TAGCCAAGGC AGCACCAGAG GAATTCAGAA AAGCAGCTCA GCAACAGGAA GAACAAGAAG 900
AAAAGGAGGA AGAGGATGAT GAACAAACAC TCCACAGAGC CCGGGAGTGG GATGACTGGA 960
AGGACACCCA TCCTAGGGGC TATGGGAACC GACAGAACAT GGGCTGATCT TCCCACAACA 1020
CCACAGGACT GCAGGGTGCA CAACTCCCCT GCCAAGGAAA ACCATGCAGT CCTCCCCTCC 1080
CTGGTCTCCT GCTTCAGCTC TGTACAACGA GGGCAAAGAT GCTAAATCTT GCTTTGCATT 1140
CAGTAAAGTG TCAAGTGATT AAGTGTGTAT TTGTACCCTA GATGATATGA ACCAGCAGTC 1200
TTGTTTTGGC ATCATCCTCA TCATGTTGTA TTCCAGCTTC TTAAGTGGAA GGAAAAGAGT 1260
GCTGAGAAAT GGCTCTGTAT AATCTATGGC TATCCCGAAT TCTCTGAAAA AATAATAAAA 1320
GTCCCCTCTA TTATATGAGC CTGTACAGAA AA 1352
INFORMATION FOR SEQ ID NO:26
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH 632
(B) TYPE Nucleic acid
(C) STRANDEDNESS Single
(D) TOPOLOGY Linear (ii) MOLECULE TYPE cDNA (iv) ANTI-SENSE No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GGATCCCCCC CGGGCCGATT TTCTCCTGCT GCTGTGGCCC GGACATGGCG ACTCCCGGCC 60
CTGTGATTCC GGAGGTCCCC TTTGAACCAT CGAAGCCTCC AGTCATTGAG GGGCTGAGCC 120
CCACTGTTTA CAGGAATCCA GAGAGTTTCA AGGAAAAGTT CGTTCGCAAG ACCCGCGAGA 180
ACCCGGTGGT ACCCATAGGT TGCCTGGCCA CGGTGGGCGN CCTCANCTAC GGTCTCTACT 240
CCTTCCACCG GGGGAACAGC CAGCGCTCTC AGCTCATGAT GCGCACCCGG ATCGCCGCCC 300
AGGGTTTCAC GGTCGCAGCC ATCTTGCTGG GTCTGGCTGT CACTGCTATG AAGTCTCGAC 360
CCTAAGCCCA GGGTCTGGCC TTGAAAGCTC CGCAGAAATG ATTCCAAAAC CCAGGGAGCA 420
ACCACTGGCC CTAACCGTGG GACTTACTCC CTCCTCTCCT TTGAGAGGCC CATGTGTCGC 480 TGGGGAGGAA GTGACCCTTT GTGTAACTGT AACCGAAAGT TTTTTCAAAA ATCCTAGATG 54 CTGTTGTTTG AATGTTACAT ACTTCTATTT GTGCCACATC TCCCCTCCAC TCCCCTGCTT 60 AATAAACTCT AAAAATCCAC TTGTATTTAA AA 63
INFORMATION FOR SEQ ID NO:27
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 285
(B) TYPE : Amino acid
(ii) MOLECULE TYPE : Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Met Leu Ser Gin Leu Asp Leu Phe Ser Arg Asn Glu Asp Leu Glu Glu
5 10 15
He Ala Ser Thr Asp Leu Lys Tyr Leu Leu Val Pro Ala Phe Gin Gly 20 25 30
Ala Leu Thr Met Lys Gin Val Asn Pro Ser Lys Arg Leu Asp His Leu 35 40 45
Gin Arg Ala Arg Glu His Phe He Asn Tyr Leu Thr Gin Cys His Cys 50 55 60
Tyr His Val Ala Glu Phe Gly Leu Ser Gin Thr Met Asn Asn Ser Ala 65 70 75 80
Glu Asn His Thr Ala Asn Ser Ser Met Ala Tyr Pro Ser Leu Val Ala 85 90 95
Met Ala Ser Gin Arg Gin Ala Lys He Gin Arg Tyr Lys Gin Lys Lys 100 105 110
Glu Leu Glu His Arg Leu Ser Ala Met Lys Ser Ala Val Glu Ser Gly 115 120 125 Gin Ala Asp Asp Glu Arg Val Arg Glu Tyr Tyr Leu Leu His Leu Gin 130 135 140
Arg Trp He Asp He Ser Leu Glu Glu He Glu Ser He Asp Gin Glu 145 150 155 160
He Lys He Leu Arg Glu Arg Asp Ser Ser Arg Glu Ala Ser Thr Ser 165 170 175
Asn Ser Ser Arg Gin Glu Arg Pro Pro Val Lys Pro Phe He Leu Thr 180 185 190
Arg Asn Met Ala Gin Ala Lys Val Phe Gly Thr Gly Tyr Pro Ser Leu 195 200 205
Pro Thr Met Thr Val Ser Asp Trp Tyr Glu Gin His Arg Lys Tyr Gly 210 215 220
Ala Leu Pro Asp Gin Gly He Ala Lys Ala Ala Pro Glu Glu Phe Arg 225 230 235 240
Lys Ala Ala Gin Gin Gin Glu Glu Gin Glu Glu Lys Glu Glu Glu Asp 245 250 255
Asp Glu Gin Thr Leu His Arg Ala Arg Glu Trp Asp Asp Trp Lys Asp 260 265 270
Thr His Pro Arg Gly Tyr Gly Asn Arg Gin Asn Met Gly 275 280 285

Claims

CLAIMS What is claimed is:
1. A method of screening for a DNA encoding a eukaryotic cell growth inhibiting factor comprising :
(a) introducing a human DNA operably linked to an inducible promoter into a eukaryotic host cell ;
(b) testing said host cell for the presence of said DNA by measuring host cell growth rate under conditions in which the promoter is induced and not induced ; and
(c) determmg the differential growth of these two groups at selected times whereby a host cell showing at least about 25% growth rate inhibition under the inducible condition as compared with the cell growth rate under the non-mducible condition is identified as containing the DNA encoding a eukaryotic cell growth inhibiting factor.
2. The method as claimed in claim 1, wherein a host cell showing at least about 50% growth rate inhibition is identified as containing the DNA.
3. The method as claimed in claim 1, wherein a host cell that has at least about 75% growth rate inhibition is identified as containing the DNA.
4. The method as claimed in claim 1, further comprising the step of isolating the DNA encoding the eukaryotic cell growth inhibiting factor from the host identified as containing the DNA.
5. The method as claimed in claim 1 , the inducible promoter is, PH05 promoter, nmtl promoter or hsp promote .
6. The method as claimed m claim 1, the selected times is about 12 to 144 hours after culture.
7. A method of screening for a DNA encoding a eukaryotic cell growth inhibiting factor, which comprises introducing a human DNA to be tested into a eukaryotic cell host so as to be controlled by an inducible promoter and selecting the host cell which does not grow under the inducible condition of said promoter but grows under the non-mducible condition.
8. The method as claimed in claim 1 or 7, wherein said eukaryotic cell host is a yeast.
9. The method as claimed in claim 8, wherein said yeast is a fission yeast.
10. The method as claimed in claim 9, wherein said fission yeast is a Shizosaccharomyces pombe.
11. An isolated DNA encoding a eukaryotic cell growth inhibiting factor, which is screened by the method as claimed in claim 1.
12. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 11.
13. The DNA as claimed in claim 12, wherein said DNA comprises a nucleotide sequence at least from the 248th to the 448th residues of the nucleotide sequence represented by SEQ ID NO. 7.
14. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 12.
15. The DNA as claimed in claim 14, wherein said DNA comprises a nucleotide sequence at least from the 279th to the 752nd residues of the nucleotide sequence represented by SEQ ID NO. 8.
16. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 13.
17. The DNA as claimed in claim 16, wherein said DNA comprises a nucleotide sequence at least from the 201st to the 377th residues of the nucleotide sequence represented by SEQ ID NO. 9.
18. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the am o acid sequence of SEQ ID NO. 14.
19. The DNA as claimed in claim 18, wherein said DNA comprises a nucleotide sequence at least from the 296th to the 1000th residues of the nucleotide sequence represented by SEQ ID NO. 10.
20. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 15.
21. The DNA as claimed in claim 20, wherein said DNA comprises a nucleotide sequence at least from the 51st to the 740th residues of the nucleotide sequence represented by SEQ ID NO. 16.
22. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 22.
23. The DNA as claimed in claim 22, wherein said DNA comprises a nucleotide sequence at least from the 1062nd to the 1736th residues of the nucleotide sequence represented by SEQ ID NO. 17.
24. A DNA which codes for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 23.
25. The DNA as claimed in claim 24, wherein said DNA comprises a nucleotide sequence at least from the 55th to the 1488th residues of the nucleotide sequence represented by SEQ ID NO. 20.
26. A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 18.
27. A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 19.
28. A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 21.
29. A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 27.
30. The DNA as claimed in claim 29, wherein said DNA comprises a nucleotide sequence at least from the 150th to the 1004th residues of the nucleotide sequence represented by SEQ ID NO. 25.
31. A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 24.
32. A DNA coding for a eukaryotic cell growth inhibiting factor which comprises the nucleotide sequence of SEQ ID NO. 26.
33. A vector comprising any one of DNAs as claimed in claim 11 to 32.
34. A transformant harboring the vector as claimed in claim 33.
35. A eukaryotic cell growth inhibiting factor which is coded by the DNA obtained by the method as claimed in claim 1.
36. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 11.
37. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 12.
38. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 13.
39. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 14.
40. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 15.
41. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 22.
42. A eukaryotic cell growth inhibiting factor which comprises the ammo acid sequence of SEQ ID NO. 23.
43. A eukaryotic cell growth inhibiting factor which comprises the amino acid sequence of SEQ ID NO. 27.
44. A eukaryotic cell growth inhibiting factor which is encoded by the DNA as claimed in claim 26.
45. A eukaryotic cell growth inhibiting factor which is encoded by the DNA as claimed in claim 27.
46. A eukaryotic cell growth inhibiting factor which is encoded by the DNA as claimed in claim 28.
47. A eukaryotic cell growth inhibiting factor which is encoded by the DNA as claimed in claim 31.
48. A eukaryotic cell growth inhibiting factor which is encoded by the DNA as claimed in claim 32.
49. A method for preparing the eukaryotic cell growth inhibiting factor as claimed in claim 35 which comprises cultivating a transformant containing a DNA encoding said factor under conditions suitable for expression of the said factor and recovering said factor .
50. A pharmaceutical composition which comprises an effective amount of any one of eukaryotic cell growth inhibiting factors as claimed in claim 35.
51. Use of the eukaryotic cell growth inhibiting factor as claimed in claim 35 for preparing an anticancer agent or infection remedy.
52. A method for treating for a patient suffering from cancer or infection which comprises administering to said said patient an effective effective amount of the eukaryotic cell growth inhibiting factor as claimed in claim 35 in the form of a pharmaceutical composition containing said factor as the effective component.
53. A method for inhibiting nucleic acid synthesis in target cell comprising containing said cell with an effective amount of a eukaryotic cell growth inhibiting factor encoded by the DNA of claim 11.
PCT/JP1995/002488 1994-12-09 1995-12-05 Dna encoding a cell growth inhibiting factor and its product WO1996017933A2 (en)

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JP30660294 1994-12-09
JP6/306602 1994-12-09
JP5771695 1995-03-16
JP7/057716 1995-03-16
JP7/136252 1995-06-02
JP13625295 1995-06-02

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WO1998017798A1 (en) * 1996-10-22 1998-04-30 Amgen Inc. Placental-derived prostate growth factors
WO2005025624A3 (en) * 2003-09-15 2005-05-26 Cenix Bioscience Gmbh The use of eukaryotic genes affecting cell cycle control or cell cycle progression for diagnosis and treatment of proliferattive diseases
US8140148B2 (en) 1998-01-20 2012-03-20 Boston Scientific Scimed Ltd. Readable probe array for in vivo use
CN110249048A (en) * 2016-09-15 2019-09-17 太阳基因组学公司 For extracting the universal method of nucleic acid molecules from one of sample or the diverse populations of a plurality of types of microorganisms

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Cited By (10)

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Publication number Priority date Publication date Assignee Title
WO1998017798A1 (en) * 1996-10-22 1998-04-30 Amgen Inc. Placental-derived prostate growth factors
US5914251A (en) * 1996-10-22 1999-06-22 Amgen Inc. Nucleic acid molecules encoding placental-derived growth factors
US6025204A (en) * 1996-10-22 2000-02-15 Amgen Inc. Diagnostic method for detection of placental-derived prostate growth factors
US6075008A (en) * 1996-10-22 2000-06-13 Amgen Inc. Method for promoting cell proliferation and growth
US6197939B1 (en) 1996-10-22 2001-03-06 Amgen Inc. Placental-derived prostate growth factors
US8140148B2 (en) 1998-01-20 2012-03-20 Boston Scientific Scimed Ltd. Readable probe array for in vivo use
WO2005025624A3 (en) * 2003-09-15 2005-05-26 Cenix Bioscience Gmbh The use of eukaryotic genes affecting cell cycle control or cell cycle progression for diagnosis and treatment of proliferattive diseases
US7648827B2 (en) 2003-09-15 2010-01-19 Cenix Bioscience Gmbh Use of eukaryotic genes affecting cell cycle control or cell cycle progression for diagnosis and treatment of proliferative diseases
AU2004271725B2 (en) * 2003-09-15 2010-04-29 Cenix Bioscience Gmbh The use of eukaryotic genes affecting cell cycle control or cell cycle progression for diagnosis and treatment of proliferative diseases
CN110249048A (en) * 2016-09-15 2019-09-17 太阳基因组学公司 For extracting the universal method of nucleic acid molecules from one of sample or the diverse populations of a plurality of types of microorganisms

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