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ANALYSIS OF MOLECULAR STRUCTURE AND REACTION MECHANISM OF RESTRICTION ENDONUCLEASE FROM ACIDOPHILIC BACTERIA

Research Project

Project/Area Number 07680688
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionOKAYAMA UNIVERSITY

Principal Investigator

INAGAKI Kenji  FACULTY OF AGRICULTURE,OKAYAMA UNIVERSITY ASSOCIATE PROFESSOR, 農学部, 助教授 (80184711)

Co-Investigator(Kenkyū-buntansha) TANAKA Hidehiko  FACULTY OF AGRICULTURE,OKAYAMA UNIVERSITY PROFESSOR, 農学部, 教授 (90065912)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsACIDOPHILIC BACTERIA / RESTRICTION ENDONUCLEASE / DNA METHYLASE / GENE CLONING / RESTRICTION-MODIFICAITON SYSTEM / 分子構造 / 結晶化
Research Abstract

1) Cloning and sequencing of the retriction-modification system gene from acidophilic bacteria.
The Afa22MI restriction-modification system from Acidocella facilis 22M recognizes the nucleotide sequence CGATCG.The M.Afa22MI gene was cloned into E.coli XL-1 Blue MRF^1 and the nucleotide sequence was determined. The homologous regions observed in 5-methyl cytosine methyltransferases (C5-MTase) were identified in the deduced amino acid sequence of M.Afa22MI.These conserved regions and the carboxyl terminal sequence of the M.Afa22MI showed high similarity with those of M.XorII which recognized same nucleotide sequence. The ORF located upstream of MTase gene showed high homology with the very short patch repair endonuclease of XorII and other vsr gene found in C5-MTase-endonuclease system.
2) Purification and characterization of DNA methylases.
Two DNA methyltransferases, M.Afa22MI and M.Afa16RI,were purified and characterized from same species Acidocella facilis 22M and 16R,respectively.
3) Determination of methylation specificity of sequence-specific DNA methylase using MALDI-TOF MS.
Time-dependent phosphodiesterase digestion of methylated oligonucleotide coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has enabled a sensitive and straightforward method for determing the methylation specificity of type II DNA methyltransferase. The mass deference between the peaks corresponded to the individual nucleotide released by the enzymatic cleavage, and the methylation site can be explicitly identified in reference to the know sequence of the substrate DNA.
4) Crystallization of type II restriction endonuclease.
We prepared large amounts of the homogeneous R.Aor 13HI,and crystallized it with an oligonucleotide containing it's recognition site (5'-TCCGGA-3') by hanging drop vapor diffusion method. A small crystal was obtained by precipitation with polyethylene glycol 4000 or 6000,100 mM HEPES (pH8.0), 100mM NaCl.

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Iichiro Ohhira: "Antimicrobial Activity against MRSA in the Culture broth of Enterococcus faecalis TH10" Jpn.J.Dairy Food Sci.45. 93-96 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hiroyuki Inoue: "Molecular Characterization of the mde Operon Involved in L-Methionine Catabolism of Pseudomonas putida." J.Bacteriol.179(in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Seow Teck Keong: "Alanine Racemase from an Acidophilic Bacterium,Acidiphilium organovorum." Sci.Rep.Fac.Agric.Okayama Univ.86. 13-19 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takashi Tamura: "Protocols for Selenium Biochemistry." Biryo-Eiyoso Kenkyu. 13. 1-6 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 稲垣賢二: "好酸性細菌の機能開発と利用に関する研究" 日本農芸化学会誌. 71・1. 1-8 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] 谷本保英: "好熱性好酸性細菌の検索・単離および諸性質の検討" 岡山大学農学部学術報告. 85. 15-21 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hiroyuki Inoue, Kinji Inagaki, Shin-ichi Eriguchi, Takashi Tamura, Nobuyoshi Esaki, Kenji Soda, and Hidehiko Tanaka: "Molecular Characterization of the mde Operon Involved in L-Methionine Catabolism of Pseudomonas putida." J.Bacteriol.(in press.).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Seow Tech Keong, Kenji Inagaki, Takashi Tamura, and Hidehiko Tanaka: "Alanine Racemase from an Acidophilic Bacterium, Acidiphilium organovorum." Sci.Rep.Fac.Agric.Okayama Univ.86. 13-19 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Kenji Inagaki: "Studies on Microbial Function of Acidophilic Bateria and Their Application." Nippon Nogeikagaku Kaishi. 71. 1-8 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Ichiro Ohhira, Takashi Tamura, Nozomi Fujii, Inagaki Hidehiko Tanaka: "Antimicrobial Activity against Methicillin-Resistant Staphylcoccus aureus in the Culture Broth of Enterococcus faecalis TH10." Jpn.J.Dairy Food Sci.45. 93-96 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takashi Tamura, Kenji Inagaki, Hidehiko Tanaka, Joe N.Davis, and Thressa C.Stadtman: "Protocols for Selenium Biochemistry." Biryo-Eiyouso Kenkyu. 13. 1-6 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Yasuhide Tanimoto, Kenji Inagaki, and Hidehiko Tanaka: "Screening, Isolation and Characterization of Thermophilic and Acidophilic Bacteria." Sci.Rep.Fac.Agric.Okayama Univ.85. 15-21 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Iichiro Ohhira: "Antimicrobial Activity Against MRSA in the Culture broth of Enterococcus faecalis TH10" Jpn.J.Dairy Food Sci.45. 93-96 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Hiroyuki Inoue: "Molecular Characterization of the mde Operon Involved in L-Methionine Catabolism of Pseudomonas putida." J.Bacteriol.179. in press. (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Seow Teck Keong: "Alanine Racemase from an Acidophilic Bacterium,Acidiphilium organovorum." Sci.Rep.Fac.Agric.Okayama Univ.86. 13-19 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Takashi Tamura: "Pritocols for Selenium Biochemistry." Biryo-Eiyoso Kenkyu. 13. 1-6 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 稲垣賢二: "好酸性細菌の機能開発と利用に関する研究" 日本農芸化学会誌. 71・1. 1-8 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Noriaki Kishimoto: "Distribution of bacteriochlorophyll a among aerobic and acidic bacteria" FEMS Microbiology Ecology. 16. 291-296 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Hiroyuki Inoue: "Structural analysis of the L-Methionine γ-Lyase Gene" Journal of Biochemistry. 117. 1120-1125 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 谷本保英: "好熱性好酸性細菌の検索・単離および諸性質の検討" 岡山大学農学部学術報告. 85. 15-21 (1996)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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