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Selective isolation of human promoter sequences that carry a bent DNA structure and analyzes of their structures and function

Research Project

Project/Area Number 07680751
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Molecular biology
Research InstitutionKONAN UNIVERSITY

Principal Investigator

OHYAMA Takashi  Konan University Department of Biology (Faculty of Science) Associate Professor, 理学部, 助教授 (60268513)

Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
Keywordsbent DNA / promoter / human genome / unusual DNA / higher order structure / chromatin / ベントDNA (bent DNA)
Research Abstract

Information about the localization of bent DNA around promoters and about the conformational characteristics of these curvatures is likely to help explain the role of DNA curvature in eukaryotic transcription. We developed a method that begins the first step in that process, namely selective isolation of curved eukaryotic promoters.
Bent DNA fragments in a digest of human genomic DNA was separated from "normal" fragments by using a biotinylated oligonucleotide and streptavidin-coated magnetic particles. The oligonucleotide was designed to hybridize specifically to bent DNA fragments. The sequence of the oligonucleotide was 5'-(T_4N_6)_3T_4-3'(N=A,G,or C). The magnetic isolation of bent DNA fragments were successful. It took only about five hours to accomplish the isolation. In addition, about 80% of the isolated fragments were bent DNAs. Taking it into consideraton that the conventional isolation method, which employs 2D gel electrophoresis, occupies about two days, our method seemed much superior to the conventional method.
Then, we tried to introduce curved fragments into a promoter trap vector pATO or into the vector pGL2-basic for luciferase assay. However, these fragments could not be introduced efficiently. It seemed that the bent DNAs captured by the above method possessed the conformations which were not favored by pATO and pGL2-basic. Therefore, we prepared bent DNA segments by the conventional method. The bent fragments thus obtained could be introduced into pGL2-basic successfully. Starting from about 700 clones, we obtained 15 bent DNA fragments that showed promoter activities and determined the sequences of two clones. Now, we are analyzing the transcription initiation site and the curved center of each clone.

Report

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

    (13 results)

All Other

All Publications (13 results)

  • [Publications] Masaru Miyamo: "Cloning of human promoter sequences that carry a curved DNA structure" Nucleic Acids Symposium Series. No.35. 265-266 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takashi Ohyama: "Possible mechanistic roles of roles bent DNA in transcriptional" Viva Origino. vol.24. 199-210 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shigenobu Tone: "Cloning of DNA fragments derived from 30kbp DNA occuring in early phase of apoptosis" Nucleic Acids Symposuium Series. No.35. 215-216 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takashi Ohyama: "Easy screening of recombinant plasmids" Technical Tips Online. http:11www.elsevier.coml locate/tto T40073. (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Masaru Miyano, Shigenobu Tone, Yuzo Kadokawa, and Takasi Ohyama: "Cloning of human promoter sequences that carry a curved DNA structure." Nucl.Acids Symp.Ser.35. 265-266 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takashi Ohyama: "Possible mechanistic roles of bent DNA in transcriptional regulation." Viva Origino. 24. 199-210 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Shigenobu Tone, Keiko Kurihara, Takashi Ohyama, and Kunio Shinohara: "Cloning of DNA fragments derived from 30kbp DNA occuring in early phase of apoptosis." Nucl.Acids Symp.Ser.35. 215-216 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takashi Ohyama, Hideki Tagashira, and Hiromi Tsujibayashi: "Easy screening of recombinant plasmids." Technical Tips Online http : //www.elsevier.com/locate/tto T40073. (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Masaru Miyano: "Cloning of human promoter sequences that carry a curved DNA structure" Nucleic Acids Symposium Series. No.35. 265-266 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Takashi Ohyama: "Possible mechanistic roles of bent DNA in transcriptional regulation" Viva Origino. vol.24. 199-210 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Shigenobu Tone: "Cloning of DNA fragments derived from 30 kbp DNA occuring in early phase of apoptosis" Nucleic Acids Symposium Series. No.35. (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Shigenobu Tone: "Cloning of DNA fragments derived from 30 kbp DNA occuring in early phase of apoptosis" Nucleic Acids Symposium Series. No.35. (1996)215-216

    • Related Report
      1996 Annual Research Report
  • [Publications] Takashi Ohyama: "Easy screening of recombinant plasmids" Technical Tips Online. http : //www.elsevier.com/locate/tto T40073. (1997)

    • Related Report
      1996 Annual Research Report

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

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