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Basic Researches on Cell Fusion and Their Application to Cell Technology

Research Project

Project/Area Number 01102028
Research Category

Grant-in-Aid for Co-operative Research (A).

Allocation TypeSingle-year Grants
Research InstitutionOsaka University

Principal Investigator

OKADA Yoshio  Osaka Univ., Inst. Mol. Cell. Biol., Prof. Emeritus, 細胞工学センター, 名誉教授 (30029756)

Co-Investigator(Kenkyū-buntansha) ASANO Akira  Osaka Univ., Inst. Protein Research, Professor. Isi, 蛋白質研究所, 教授 (30029938)
OHNISHI S-I.  Kyoto Univ., Facul. Sci., Dept. Biophys., Professor, 理学部, 教授 (00025272)
UEDA Sigeharu  Osaka Univ., Inst. Microbial Des., Professor, 微生物病研究所, 教授 (90068453)
AKAMATSU Yuzuru  Natl. Inst. Health, Dept. Chem., Chairman, 部長 (00072900)
TANAKA Kiyoji  Osaka Univ., Inst. Mol. Cell. Biol., Professor, 細胞工学センター, 教授 (80144450)
米田 悦啓  大阪大学, 細胞工学センター, 助教授 (80191667)
岡田 泰伸  京都大学, 医学部, 講師 (10025661)
金 在萬 (金 在万)  京都薬科大学, 分子薬学研, 教授 (30029805)
Project Period (FY) 1989 – 1991
Project Status Completed (Fiscal Year 1991)
KeywordsCell Fusion / Membrane Fusion / HVJ (Sendai virus) / Endosome / Xeroderma pigmentosum / SSPE / Embryonic Stem Cell / Gene-targeting / カルシウム / DNA修復 / ES細胞 / 脂質合成変異株 / 筋芽細胞 / 核移植 / 核移行 / 核 / トランスポゾン
Research Abstract

On the mechanism of membrane fusion reaction, (1) Requirement of some species of lipid, such as phosphatidylserine, for fusion reaction was substantiated by using mutants defective for phospholipid biosynthesis. (2) Specific binding of cholesterol to N-terminal domain of F_1-subunit of F-glycoprotein was shown to be a prerequisite for fusion reaction between cell membranes and membrane of HVJ (Sendai virus). (3) A mutant with a defect in traffic between endosome and lysosome was isolated from mammalian cells which required cholesterol in LDL for their growth. This mutant seems to be very useful for analysis of fusion mechanism of endosomal membranes with lysosomal ones. (4) For electrofusion of mammalian cells, intracellular Ca_<2+> is required, but removal of extracellular Ca_<2+> did not hamper the reaction. Endogenous thiol-protease (s) which is different from calpain was shown to participate in the fusion reaction. (5) In case of HVJ-induced fusion of epithelial. cells, it was foun … More d that attack of the cells by the virus from apical side resulted in cell fusion at batholateral membranes. Thus, the sites of attack of the virus were far from the sites of membrane fusion, suggesting that some signal (s) for membrane fusion may be transmitted within the cells from the virus-attacked sites to the membrane fusion sites.
As an application of cell fusion for cell manipulation, (6) construction of model mouse for Xeroderma pigmentosum (XP), a defect of DNA repair mechanism, might be sited first. XPAC (XP group A complementing) gene was isolated by us several years ago. In this project, it was shown using site-directed mutagenesis that zinc finger motifs of the XPAC gene product are required for its DNA repair function, and that the protein contains nuclear localization signal in a basic domain. Disrupting XPAC gene of mouse ES (embryonic stem) cells by genetageting, and making chimera (s) containing the gene-disrupted cells by injecting them in normal mouse embryos, we have succeeded i. n obtaining a somatic cell chimera. Furthermore, we were also successful for disrupting both alleies of XPAC gene of ES cells. These cells showed defect in DNA repair after UV treatment. Thus, productions of model mouse with XPAC defect now become possible. (7) A method of production of germinal line chimeras from ES cells was established. Furthermore, a method of production of mouse derived from fusion of denucleated un-fertilized normal mouse eggs with gene-disrupted ES cells was almost established. (8) A curing method of SSPE (subacute sclerosing panencephalitis) using liposomes containing subunit A of diphteria toxin was established. Less

Report

(4 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • 1989 Annual Research Report
  • Research Products

    (37 results)

All Other

All Publications (37 results)

  • [Publications] O.Kuge,Yuzuru Akamatsu,M.Nishijima: "Abortive infection with Sindbis virus of a Chinese hamster overy cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis" Biochim.Biophy.Acta. 986. 61-69 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] J.Kim,T.Adachi,Y.Yoneda,Y.Okada: "Fusion of Mardin-Darby canine kidny cells by HVJ(Sendai virus):Absence of direct association of virus particles with the site of membrane fusion" E.J.Cell Biol. 51. 128-134 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Kato,M.Nakanishi,Y.Kaneda,T.Ushida,Y.Okada: "Expression of hepetitis B virus surface antigen in adult rat liver.Cointroduction of DNA and nuclear protein by a simplified liposome method" J.Biol.Chem.266. 3361-3364 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] N.Miura,I.Miyamoto,H.Asahina,I.Satokata,K.Tanaka,Y.Okada: "Identification and characterization of XPAC protein,the gene product of human XPAC (Xeroderma pigmentosum group A complementing)gene" J.Biol.Chem.266. 19786-19789 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] I.Miyamoto,N.Mirua,H.Niwa,J.Miyazaki,K.Tanaka: "Mutational analysis of the structure and function of the XPAC protein:Identification of essential domains for nuclear localization and DNA excission repair" J.Biol.Chem.267. (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y.Kato and Y.Tsunoda: "Synchronous division of mouse two cell embryos with nocodazole in vitro" J.Reprod,Fert.(1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T.Ohono-Shosaku,and Ya.Okada: "Role of protease in electrofusion of mammalian cells,In"Electroporation and Electrofusion in Cell Biology",(E.Neuman,A.E Sowers,& C.A.Jordan,eds.)" Plenum Publ.,New York, 9 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Asano,and A.Asano: "A molecular study of the fusion reaction between viral and cell membranes;Advancesin Membrane Fluidity,Vol.6,"Membrane Interactions of HIV,"Aloia,R.C.,Curtain,C.C.,& Gordon,L.M.(eds)" Wiley-Liss,Inc.,New York,N.Y., 275-282 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] O. Kuge, Yuzuru Akamatsu, & M. Nishijima: "Abortive infection with Sindbis virus of a Chinese hamster overy cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis." Biochim. Biophys. Acta. 986. 61-69 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] J. Kim, T. Adachi, Y. Yoneda, & Y. Okada: "Fusion of Mardin-Darby canine kidny cells by HVJ (Sendai virus) : Absence of direct association of virus particles with the site of membrane fusion." E. J. Cell Biol.51. 128-134 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Kato, M. Nakanishi, Y. kaneda, T. Uchida, & Y. Okada: "Expression of hepetitis B virus surface antigen in adult rat liver. Cointroduction of DNA and nuclear protein by a simplified liposome method." J. Biol. Chem.266. 3361-3364 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] N. Miura, I. Miyamoto, H. Asahina, I. Satokata, K. Tanaka, &Y. Okada: "Identification and characterization of XPAC protein, the gene product of human XPAC (Xeroderma pigmentosum group A complementing) gene." J. Biol. Chem.266. 19786-19789 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] I. Miyamoto, N. Mirua, H. Niwa, J. Miyazaki, & K. Tanaka: "Mutational analysis of the structure and function of the XPAC protein : Identification of essential domains for nuclear localization and DNA excission repair." J. Biol. Chem.267. (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y. kato and Y. Tsunoda: "Synchronous division of mouse two cell embryos with nocodazole in vitro." J. Reprod. Fert.(1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Ohono-Shosaku, and Ya. Okada: "Role of protease in electrofusion of mammalian cells." Electroporation and Electrofusion. Plenum Publ.9 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Asano, and A. Asano: "A molecular study of the fusion reaction between viral and cell membranes." Advances in Membrane Fluidity. 6. 275-282 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] O.Kuge,M.Nishijima,Yuzuru Akamatsu: "A cloned gene encoding phosphatidylserine decarboxylase complements the phosphatidylserine biosynthetic defect of a Chinese hamster ovary cell mutant" J.Biol.Chem.266. 6370-6376 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] O.Kuge,M.Nishijima,Yuzuru Akamatsu: "A Chinese hamster cDNA encoding a protein essential for phosphatidylserine synthetase I activity" J.Biol.Chem.266. 24184-24189 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Y.ーS.Bae,M.Chiba,M.Ohira,H.Ikeda: "A shuttle vector for analysis of illegimate recombination in mammalian cells:Effects of DNA topoisomerase inhibitors on deletion frequency" Gene. 10. 285-289 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] N.Miura,I.Miyamoto,H.Asahina,I.Satokata,K.Tanaka,Y.Okada: "Identification and characterization of XPAC protein,the gene product of human XPAC (Xeroderma pigmentosum group A complementing) gene" J.Biol.Chem.266. 19786-19789 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] I.Miyamoto,N.Mirua,H.Niwa,J.Miyazaki,K.Tanaka: "Mutational analysis of the structure and function of the XPAC protein:Identification of essential domains for nuclear localization and DNA excission repair" J.Biol.Chem.267. (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Y.Kato and Y.Tsunoda: "Synchronous division of mouse two cell embryos with nocodazole in vitro" J.Reprod.Fert.(1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Asano,and A.Asano(Aloia,R.C.,Curtain,C.C.,&Gordon,L.M.(eds)): "A molecular study of the fusion reaction between viral and cell membranes;Advancesin Membrane Fluidity,Vol.6,“Membrane Interactions of HIV,"" WileyーLiss,Inc.,New York,N.Y, 275-282 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Harada,M.Nishijima,Yuzuru Akamatsu: "A temperatureーsensitive mammalian cell mutant with thermorablle serine pimitoyi transferase for the sphingolipid biosynthesis" J.Biol.Chem.265. 22137-22142 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Y.Sako,S.Sato,S.Ohnishi: "Subpopulations of endosomes generated at sequential stages in the endocytic pathway of asialoganglyosideーcontaining ferrite ligands in rat liver" J.Biochem.107. 846-853 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] H.Taira,L.Ranin,Kentaro Iwasaki: "Construction of expression plasmids for the fusion protein of Sendai virus,and their expression in E.coli cells and eucaryotic cells" FEBS Letters. 262. 39-41 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Tanaka,N.Miura,I.Satokata,I.Miyamoto,M.Yoshida,Y.Okada et al.: "Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and cotaining a zincーfinger domain" Nature. 348. 73-76 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] I.Satokata,K.Tanaka,N.Miura,I.Miyamoto,Y.Satoh,S.Kondo,Y.Okada: "Characterization of a splicing mutation in group A xeroderma pigmentosum" Proc.Natl.Acad.Sci.U.S.A.87. 9908-9912 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] N.ImamotoーSonobe,Y.Matsuoka,T.Semba,Y.Okada,T.Uchida,Y.Yoneda: "A protein recognized by antibodies to AspーAspーAspーGluーAsp shows specific binding activity to heterologous nuclear transport" J.Biol.Chem.265. 16504-16508 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Jeman Kim,Toshiyuki Adachi,Yoshio Yoneda,Yoshio Okada: "Fusion of Mardin-Dorby canine kidney cells by HVJ(Sendai virus):Absence of direct association of virus particles with the site of membrane fusion" European Journal of Cell Biology. 51. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kiyoji Tanaka,I.Satokata,Z.Ogita,T.Uchida,Yoshio Okada: "Molecular cloning of a mouse DNA repair gene that complements the defect of group A xeroderma pigmentosum" Proc.Natl.Acad.Sci.USA. 86. 5512-5516 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Y.Sako,S.B.Sato and S.Ohnishi: "Two Subpopulations of endosomes generated on sequential.stages in endocytic pathway of asialoganglioside-containing ligands in rat liver" J.Biochem.(1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Osamu Kuge,Yuzuru Akamatsu,Masahiro Nishijima: "Abortive infection with Sindbis virus of a Chinese hamster overy cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis" Biochim.Biophys.Acta. 986. 61-69 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Hideharu Taira,Razar Ranin,Kentaro Iwasaki: "Construction of expression plasmids for the fusion protein of Sendai virus,and their expression in E.coli cells and eukaryotic cells" FEBS Letters. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] 中村公章、吉中一朗、角田幸雄: "核移植実験系におけるHVJ不活性化法が操作卵子の融合及び発生率に及ぼす影響" 家畜繁殖学会誌. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] T,Ohono-Shosakyu,& Y.Okada: "Role of proteases in electrofusion of mammalian cells.In“Electroporation and Electrofusion in Cell Biology",ED.by E.Neumwnn,A.E.Sowers & C.A.Jordan" plenum Publ.,New York, 9 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Akira Asano: "Gunma Synposia on Endocrinology,Vol.26" Center for Academic Publications,Japan, 10 (1989)

    • Related Report
      1989 Annual Research Report

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Published: 1993-03-16   Modified: 2016-04-21  

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