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Purification of low-abundance mRNAs by polysome immunoprecipitation and its use for cDNA cloning

Research Project

Project/Area Number 59870009
Research Category

Grant-in-Aid for Developmental Scientific Research

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

Principal Investigator

MORI Masataka  千葉大学, 医, 助教授 (40009650)

Project Period (FY) 1984 – 1985
Project Status Completed (Fiscal Year 1985)
Budget Amount *help
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1985: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1984: ¥4,400,000 (Direct Cost: ¥4,400,000)
KeywordsFree and membrane-bound polysomes / polysome immunoprecipitation / cDNA clonong / ornithine transcarbamylase / 3-oxoacyl-CoA thiolase / 3-ケトアシル-CoAチオラーゼ
Research Abstract

cDNA cloning of low abundance mRNA species is one of the most critical steps in recombinant DNA studies. Polysome immunoprecipitation is one of the most powerful means to purify low-abundance mRNAs and the purified mRNAs should facilitate cDNA cloning. We developed a simple and rapid procedure for isolation of polysomes from rat liver and applied the procedure for cDNA cloning.
1. A procedure was developed for preparation of free and membrane-bound polysomes from rat liver. The procedure involves separation of the two classes of polysomes by differential centrifugation of liver homogenate and magnesium precipitation of both classes of polysomes. The polysomes were essentially undegraded and highly active in cell-free synthesis.
2. A polysome class synthesizing ornithine transcarbamylase was purified about 50-fold from the free polysomes by immunoprecipitation. A cDNA clone with a 1600base insert was isolated from the enriched mRNA and the entire primary structure of the transcarbamylase precursor was determined.
3. 3-Oxoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase mRNAs were purified 25-40-fold and cDNA clones for the two enzymes were isolated. The longest clones containing 1400- and 1200-base insert, respectively, are expected to carry more than 80% of the thiolase and dehydrogenase mRNAs.
4. The mRNA for carbamyl phosphate synthetase (subunit molecular weight, 160,000) could not be effectively enriched by the polysome immunoprecipitation procedure. Thus, the present procedure may not be suitable for purification of very large mRNAs.

Report

(1 results)
  • 1985 Final Research Report Summary
  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] J.Biol.Chem.259-10. (1984)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] Proc.Natl.Acad.Sci.USA. 81-23. (1984)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] J.Biochem.97-5. (1985)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] Eur.J.Biochem.(1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] J. Biol. Chem.259-10. (1984)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] Proc. Natl. Acad. Sci. USA. 81-23. (1984)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] J. Biochem.97-5. (1985)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1985 Final Research Report Summary
  • [Publications] Eur. J. Biochem.(1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1985 Final Research Report Summary

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Published: 1987-03-31   Modified: 2016-04-21  

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