Cloning of DNA Fragment Coding for <i>Tetrahymena</i> Ribosomal RNA with<i> Escherichia coli</i> Plasmid pBR 322

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  • HIGASHlNAKAGAWA Toru
    Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health
  • SAKAKI Yoshiyuki
    Department of Biochemistry, Kyushu University, School of Medicine
  • IIO Masako
    Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health
  • SAIGA Hidetoshi
    Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health
  • MITA Takashi
    Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health

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Other Title
  • TetrahymenaリボソームDNAの大腸菌プラスミドpBR322によるクローニング

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Description

The DNA coding for ribosomal 17s and 25s RNA of Tetrahymena pyriformis GL was digested with restriction endonuclease Hind Ⅲ, and the largest fragment (fragment 1; 2.5x106 dalton) was inserted into the Hind Ⅲ site of plasmid pBR 322. Out of 11 bacterial clones which were resistant to ampicillin and sensitive to tetracycline, 2 clones, termend pTpr 2 and pTpr 4, were positive in a colony hybridization assay with 32P-labelled rRNA as a probe. Plasmids were purified from these clones through CsCl-Ethidium bromide centrifugation, and analyzed with several restriction endonucleases. Upon Hind Ⅲ digestion both plasmids gave two bands with similar intensity on agarose gel electrophoresis which comigrate with fragment 1 and linear molecule of pBR 322. Digestion with Bam HI, and further with Pst I demonstrated that the fragment 1 was inversely oriented between pTpr 2 and pTpr 4. Multimers of pTpr 2 and pTpr 4 were revealed by electronmicroscopic observation of the plasmids, in which the monomers were tandemly repeated as evidenced by EcoRI digestion pattern. R-Ioop formed between pTpr 2 and rRNA confirmed that the genes for both rRNAs were inserted in the expected manner.

Journal

  • Journal of UOEH

    Journal of UOEH 2 (4), 449-462, 1980

    University of Occupational and Environmental Health, Japan

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