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Construction of promoter-detection plasmid in animal cells by using the metapyrocatechase gene

Research Project

Project/Area Number 03557013
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionYamaguchi University

Principal Investigator

NAKAZAWA Atsushi  Yamaguchi Univ.Sch.Med. Prof., 医学部, 教授 (90025594)

Co-Investigator(Kenkyū-buntansha) KUMAHARA Hiromi  Ube Industries Ube Inst.Director, 宇部研究所, 課長
NOMA Takafumi  Yamaguchi Univ.Sch.Med. Assist.Prof., 医学部, 講師 (40189428)
Project Period (FY) 1991 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1992: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1991: ¥4,400,000 (Direct Cost: ¥4,400,000)
KeywordsPromoter / Vector / Cultured cells / Catechol / C230 / ベペクター / プロモ-タ- / ベクタ- / カテュ-ル
Research Abstract

To test the ability of expression of the metapyrocatechase (C230) gene in the animal cells, we first constructed two plasmids. In one plasmid, the C230 gene was put under the control of the promoter and enhancer of SV40 early genes, while in the other, it was controlled by the promoter and enhancer of the human elongation factor gene. Neither of these plasmid showed production of active C230 in HeLa and CHO cells. Next, we made a gene fusion in which the N-terminal two residues of chicken adenylate kinase was ligated to the N-terminal truncated C230. The C230 gene of the above two plasmids was replaced by the gene fusion, and the plasmid products were checked for their expression in the animal cells. However, the results were negative. Since the transcription of these plasmids were thought to be active in the animal cells, failure in the expression of the C230 gene is probably due to a barrier in the process after the translation.
During the course of this study, we obtained a plasmid pKS230, in which the C230 gene is put under the control of the lac promoter. The restriction sites for HincII and EcoRV can be used as a cloning site for foreign DNA, since insertion to these sites make the plasmid inactive to produce the active C230. The inserted plasmid is easily detected by yellow color development after spraying a catechol solution. Therefore, this plasmid can be used a less expensive cloning vector comparing with the lac promoter vector that uses X-Gal as a detector.

Report

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

    (10 results)

All Other

All Publications (10 results)

  • [Publications] M.Shahjahan: "Cloning and characterization of the gene encoding bovine mitochondrial adenylate kinase isozyme 3" Gene. 107. 313-317 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] M.Gomada: "Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid" Molecular and General Genetics. 233. 419-426 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] A.NAKAZAWA: "Upstream regulatory sequence for transcriptional activation of catabolic genes on the TOL plasmid" Pseudomonas:Molecular Biology and Biotechnology (Eds.E.Galli,S.Siver and B Wiltholt) American Society for Microbiology,Washington,DC.353-357 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] A.NAKAZAWA: "A DNA-loop model for transcriptional activation of the xylene-metabolizing genes on Pseudomonas TOL plasmid" Molecular Structure and Life (Eds.Y.Ktogeku and Y.Nishimura) Japan Sci.Society Press,Tokyo. 251-260 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] M.Shahjahan et al.: "Cloning and characterization of the gene encoding bovine mitochondrial adenylate kinase isozyme 3" Gene. 107. 313-317 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] M.Gomada et al.: "Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida" Mol. Gen. Genet.233. 419-426 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] A.Nakazawa et al.: "Upstream regulatory sequence for transcriptional activation of catabolic genes on the TOL plasmid" Pseudomonas Molecular Biology and Biotechnology (Eds. E. Galli, S. Silver and B. Witholt) Amer. Soc. for Microbiol., Washington, DC.353-357

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] A.Nakazawa et al.: "A DNA-loop model for transcriptional activation of the xylene-metabolizing genes on Pseudomonas TOL plasmid" Molecular Structure and Life (Eds. Y. Kyogoku and Y. Nishimura) Japan Sci. Soc. Press, Tokyo. 251-260

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] M.Gomada: "Analysis of an upstream requlatory sequence required for activation of the requlatory gene xyls in xylene metabolism directed by the TOL plasmid" Molecular and General Genetics. 233. 419-426 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Shahjahan: "Cloning and characterization of the gene encoding bovine motochondrial ademylate kinase isozyme 3" Gene. 107. 313-317 (1992)

    • Related Report
      1991 Annual Research Report

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

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