Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity

Nucleic Acids Res. 1988 Jan 11;16(1):265-77. doi: 10.1093/nar/16.1.265.

Abstract

Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide. Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity. It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity. We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E. coli, and purified the protein to near homogeneity. The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity. These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains.

MeSH terms

  • Chromosome Deletion
  • Cloning, Molecular*
  • DNA Restriction Enzymes
  • Genes
  • Genes, Viral
  • Molecular Weight
  • Moloney murine leukemia virus / enzymology
  • Moloney murine leukemia virus / genetics*
  • Plasmids
  • RNA-Directed DNA Polymerase / genetics
  • RNA-Directed DNA Polymerase / isolation & purification
  • RNA-Directed DNA Polymerase / metabolism*
  • Ribonucleases / metabolism*

Substances

  • RNA-Directed DNA Polymerase
  • Ribonucleases
  • DNA Restriction Enzymes