Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli

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Abstract

It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2–4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied.

This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.

References (34)

  • T. Terao et al.

    J. Biol. Chem.

    (1972)
  • S.K. Niyogi et al.

    J. Biol. Chem.

    (1965)
  • S.K. Niyogi

    J. Mol. Biol.

    (1972)
  • C.F. Fox et al.

    J. Biol. Chem.

    (1964)
  • S. Cory et al.

    J. Mol. Biol.

    (1975)
  • S. Cory et al.

    Biochim. Biophys. Acta

    (1976)
  • E. Southern

    J. Mol. Biol.

    (1975)
  • S.B. Zimmerman et al.

    Anal. Biochem.

    (1966)
  • S.K. Niyogi et al.

    J. Biol. Chem.

    (1965)
  • C.F. Kreijl et al.

    Biochem. Biophys. Res. Comm.

    (1973)
  • R. Williamson et al.

    FEBS Lett.

    (1969)
  • E. Blackburn

    J. Mol. Biol.

    (1975)
  • M. Szekely et al.

    J. Mol. Biol.

    (1969)
  • J.G. Gall et al.

    Methods Enzymol.

    (1971)
  • J. Taylor et al.

    Biochim. Biophys. Acta

    (1976)
  • J.N. Vournakis et al.

    Biochem. Biophys. Res. Comm.

    (1976)
  • W. Gilbert et al.
  • Cited by (0)

    Present address: Basel Institute of Immunology, 487 Grenzacherstrasse, Postfach 4005, Basel 5, Switzerland.

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