Nucleic Acids Research, 1988, Vol. 16, No. 1 265-277
© 1988
Articles |
Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity
Molecular Biology Research and Development, Bethesda Research Laboratories, Life Technologies, Inc. 8717 Grovemont Circle, Gaithersburg, MD 20877, USA
Received August 17, 1987. Revised November 20, 1987. Accepted November 24, 1987.
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. coll, 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-HLV reverse transcriptase reside within separate structural domains.
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