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Defense Mechanism and Adaptive Respense of Eukaryotic Cells against Active Oxygen

Research Project

Project/Area Number 03680225
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 分子遺伝学・分子生理学
Research InstitutionNational Institute of Radiological Sciences

Principal Investigator

INABA Hiroko  National Institute of Radiological Sciences, Division of Genetics, Senior Researcher, 遺伝研究部, 主任研究員 (70159953)

Co-Investigator(Kenkyū-buntansha) SHIMAZU Yoshie  National Institute of Radiological Sciences, Division of Chemical Pharmacology,, 薬理化学研究部, 主任研究員 (10162687)
MORIMYO Mitsuoki  National Institute of Radiological Sciences, Division of Genetics, Head, 遺伝研究部, 室長 (00166430)
Project Period (FY) 1991 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsAdaptive oxygen / Plumbagin / Mutant / S.pombe / CHO-K1 / DNA repair / Adaptive response / Gene cloning / CHO・KI / S.cerevisiae / CHO・Kl
Research Abstract

Active oxygen species are assumed to be an important cause of mutagenesis, tumorigenesis, diseases and aging. Although the defense mechanisms and responses of prokaryotic cells to toxic radicals have almost been reserved, those of eukaryotic cells remain unsolved. The mutants of yeast and mammalian cells sensitive to active oxygen generating agents, plumbagin(PG), were isolated and genetic analyzes including gene cloning were performed.
1) Isolation and Analysis of S.pombe mutants sensitive to active oxygen
S.pombe, G408 cells were mutagenized with EMS and about 400 mutants sensitive to PG were isolated. These mutants were classified into 3 groups ; (〕SY.encircled .〔)1 only sensitive to PG, (〕SY.encircled .〔)2 cross-sensitive to X-ray and/or UV, (〕SY.encircled .〔)3 cross-sensitive to oxygen. These results suggested that the defense mechanisms of yeast against active oxygen toxicity are attained by the elimination of active oxygen species and by DNA repair mechanisms. This is a good agree … More ment with the results of E.coli. Among 50 radiation-sensitive mutants, M12 was a new one, and the relevant gene radM12 was cloned. The gene was a new stress-response gene located on chromosome 1 of S.pombe consisted of 474bp encoding (157 amino acids) a new uniquitine-conjugating enzyme E2.
2) Analysis of adaptive mechanism of S.pombe against active oxygen
To clone directly stress-inducible genes, the fusion DNA library was made by ligation of cloning vector carrying reporter gene lacZ with fractionated S.pombe DNA.These DNA were transformed into a wild type S.pombe strain, and the colonies of fusion transformants exhibiting increased expression of lacZ by stress such as UV, X-ray, heat, and oxygen radicals were isolated. Plasmids were extracted from 71 candidates and 26 genes were identified from DNA sequence. Analyzes of genes are now in progress.
3) CHO-K1 cell mutants sensitive to active oxygen-generating agents
Nine mutants isolated from EMS-treated CHO-K1 cells with increased sensitivity to the lethal effect of PG, were classified into 5 groups A-E, by the sensitivity to PG and methyl viologen(MV). The strains of B and C groups were rather sensitive to both agents. Two mutants of group B (pa13, pb4) were sensitive to both drugs, and 2 mutants of group C(pa14, pa15) were sensitive to PG, extremely sensitive to MV.Genetic complementation analyzes of these 4 mutants were carried out using MV sensitivity. Group B was divided into 2, I and II.Pa14 and pa15 in group C belonged to the same complementation group III.These 4 mutants were also sensitive to mitomycin C (MMC) classified into 3 complementation groups as to MMC-sensitivity. As pa13 and pb4 were also sensitive to cis-diaminedichloroplatinum II, they may have a defect in the repair of DNA-crosslinks induced by these active oxygen generating agents. Less

Report

(4 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • 1991 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] M.Morimyo, E.Hongo, H.Hama-Inaba and I.Machida: "Cloning and Characterization of the mvrC gene of Escherichia coli K-12 which Confers Resistance Against Methyl viologen Toxicity." Nucleic Acid Research. 20. 3159-3165 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Hama-Inaba, Y.Shimazu, E.Hongo and M.Morimyo: "Isolation and Characterization of CHO-cell Mutants Sensitive to Plumbagin, Active Oxygen Propagator." Biophysics and Synchrotron Radiation Abstracts. 269. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Y.Shimazu, H.Hama-Inaba and F.Sawada: "X-Ray-Induced Proteins in Cultured Human Melanoma Cells." Natl.Inst.Radiol.Sci.Ann.Rept.(NIRS-31). 25. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Morimyo, I.Machida, E.Hongo, T.Saeki, H.Hama-Inaba: "Cloning of a new DNA Repair Gene radM12 of Shizosaccharomyces pombe." Proc.16th Int.Conf.on Yeast Genetics and Molecular Biology. S275,5-29A.Wiley. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Morimyo, I.Machida, E.Hongo, T.Saeki, H.Hama-Inaba: "DNA Repair Gene radM12 of S.pombe Encoding a Novel Ubiquitin Conjugating Enzyme." Natl.Inst.Sci.Ann.Rept.(NIRS-32). 50-51 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Morimyo and K.Mita: "A Simple and Rapid Amplification Procedure for cDNA Cloned in Dephosphorylated Plasmid." Nucleic Acids Research. 21(7). 1679-1680 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] E.Hongo, M.Morimyo, H.Hama-Inaba, I.Machida: "Cloning of Stress Inducible Gene of S.pombe." J.Radiat.Res.34. 317 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] O.Ozawa, J.Ueda and Y.Shimazu: "DNA Single Strand Breakage by Copper (II) Complexes and Hydrogen Peroxide at Physiological Conditions." Biochemistry and Molecular Biology Int.31. 455-461 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Hama-Inaba, Y.Shimazu, M.Takusagawa, T.Nakajima, J.Ueda, T.Ozawa, O.Yukawa and M.Morimyo: "Characterization of CHO Cell Mutants Sensitive to Active Oxygen Propagators." Proc.VIth Int.Conf.Superoxide and Superoxide Dismutase, Elsevier Science Publishers. (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Y.Shimazu, H.Hama-Inaba, O.Yukawa and T.Ozawa: "Modification of Cytochrome C Method to Determine Superoxide Dismutase Activity in Living Organisms." Proc.VIth Int.Conf.Superoxide and Superoxide Dismutase, Elsevier Science Publishers. (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] I.Machida, M.Morimyo, T.Saeki: "Aberrant Transformation of Error-Prone Repair-Deficient, Mutants rad6-1 and rad18-2 of Saccharomyces cerevisiae by Integrating Plasmid Ylp5." J.Radiat.Res.(in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Morimyo,I.Machida,E.Hongo,T.Saeki,H.Hama-Inaba: "DNA Repair Gene radM12 of S.pombe Encoding a Novel Ubiquitin Conjugating Enzyme." Natl.Inst.Sci.Ann.Rept.32. 50-51 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Morimyo and K.Mita,: "A Simple and Rapid Amplification Procedure for cDNA Cloned in Dephosphorylated Plasmid" Nucleic Acids Research.21(7). 1679-1680 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] E.Hongo,M.Morimyo,H.Hama-Inaba,I.Machida: "Cloning of Stress Inducible Gene of S.pombe." J. Radial.Res.34. 317 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.Ozawa,J.Ueda and Y.Shimazu.: "DNA Single Stland Breakage by Copper(II) Complexes and Hydrogen peroxide at Physiological Conditions" Biochemistry and Molecular Biology Int.31. 455-461 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] H.Hama-Inaba,Y.Shimazu. M.Takusagawa,et al.: "Characterization of CHO CellMutants Sensitive to Active OxygenPropagators." Proc.VIth Int.Conf.Superoxide and Superoxide Dismulase, Elsevier Science Publishers. in press. (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] Y.Shimazu,H.Hama-Inaba,O.Yukawa ,and T.Ozawa,: "Modification of Cytochrome C Method to Determine Superoxide Dismutase Activity in Living Organisms." Proc.VIth Int.Conf.Superoxide and Superoxide Dismutase, Elsevier Science Publishers. in press. (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] I.Machida,M.Morimyo, T.Saeki.: "Aberrant Transformation of Error-Prone Repair-Deficient Mutants rad6-1 and rad18-2 of Saccharomyces cerevisiae by Integrating Plasmid Y1p5." J.Radiat. Res.in press. (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Mita,M.Morimyo,E.Hongo,: "One Step DNA Sequencing of Single-Stranded DNA with Reverse Dye-Primer." Nucleic Acids Res.in press. (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Morimyo,E.Hongo,H.Hama-Inaba,I.Machida: "Cloning and characterization of the mvrC gene of Escherichia coli K-12 which confers resistance against methyl viologen toxicity" Nucleic Acids Research. 20. 3159-3165 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Morimyo,I.Machida,E.Hongo,T.Saeki,H.Hama-Inaba,: "Cloning of a new DNA repair gene radM12 of Schizosaccharomyces pombe" Yeast. 8. S275- (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Morimyo,I.Machida,E.Hongo,T.Saeki,and H.Hama-Inaba: "Cloning and structure of DNA repair gene radM12 of Schizosaccharomyces pombe" Biophysics and Synchrotron Radiation Abstracts. 268- (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Hama-Inaba,H.,Shimazu,Y.,Hongo,E.,and Morimyo,M.: "Isolation and Characterization of CHO-cell Mutants Sensitive to Plumbagin,Active Oxygen Propagator" Biophysics and Synchrotron Radiation Abstracts. 269- (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Morimyo,I.Machida,and E.Hongo: "Defence Mechanisms of S.cervisiae Against Against Active Oxygen Toxity" Radiation Reseach Congress Abstracts. 1. 204 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Y.Shimazu,H.Hama-Inaba,and F.Sawada: "X-Ray-Induced protein Synthesis in Cultured Human Melanoma Cells" Radiation Reseach Congress Abstracts. 1. 221 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Estuko HONGO,Isamu MACHIDA,and Mitsuoki MORIMYO: "Mutants of S.cervisiae Sensitive to Active Oxygen Toxicity" J.Rad.Res.,. (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] I.MACHIDA,H.HAMA-INABA,E.HONGO,T.SAEKI and M.MORIMYO: "Mutants of Fission Yeast.S.pombe Sensitive to X-rays" J.Rad.Res.,. (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Hiroko HAMA-INABA,Yoshie SHIMAZU,Etsuko HONGO,and Mitsuoki MORIMYO: "Isolation and Characterization of plumbagin-Sensitive CHO Cell Mutants" J.Rad.Res.,. (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] M.Morimyo,E.Hongo,H.Hama-Inaba,and I.Machida: "Cloning and Characterization of the mvrC Gene of E.coli K-12 which confers Resistance against Methyl Viologen Toxity" Nucleic Acid Res.,.

    • Related Report
      1991 Annual Research Report
  • [Publications] Morimyo,M.,Machida,I.,Hongo,E.,Saeki,T.,and Hama-Inaba,H.: "RADIATION-SENSITIVE MUTANTS OF FISSION YEAST S.pombe" Japanese J.Genet.,. 66. 769 (1991)

    • Related Report
      1991 Annual Research Report

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Published: 1991-04-01   Modified: 2016-04-21  

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