Rothenberg, Ellen and Baltimore, David (1977) Increased Length of DNA Made by Virions of Murine Leukemia Virus at Limiting Magnesium Ion Concentration. Journal of Virology, 21 (1). pp. 168-178. ISSN 0022-538X. http://resolver.caltech.edu/CaltechAUTHORS:ROTjvir77
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Conditions have been developed for reverse transcription by detergent-disrupted virions of Moloney murine leukemia virus which permit synthesis of molecules that appear to be complete transcripts of the 35S RNA subunits. At limiting Mg2+ concentration, DNA is synthesized in good yield, up to a maximum size of about 2.4 x 10^6 daltons. DNA larger than 2 x 10^6 daltons, taken from alkaline sucrose gradients, has no detectable self-complementarity and was protected from digestion by S1 nuclease to an extent of 90% by annealing to 70S RNA. All size classes ofDNA made in these reactions are primed with RNA, because all are initiated with a pApdA junction. To produce such long molecules, it is necessary to keep the concentration of Mg2+ in the reaction mixture below the total concentration of deoxyribonucleoside triphosphates. Under these conditions, degradation of the RNA template is minimized. The rate of DNA synthesis is also slowed by 30 to 50%, but products longer than 5,000 nucleotides, which are not found otherwise, are completed between 3 and 6 h of reaction.
|Additional Information:||Copyright © 1977, American Society for Microbiology. Reprinted with permission. Received for publication 23 June 1976 We are grateful to Daniel Donoghue and Phillip Sharp for their valuable advice and generous gifts of phage DNAs and restriction enzymes. William Haseltine kindly helped perform the electrophoresis on polyacrylamide gels. This research was supported by a contract from the Virus Cancer Program of the National Cancer Institute and grants CA-12174 and CA-14051 from the National Institutes of Health. E.R. was a predoctoral fellow of the National Science Foundation. D.B. is an American Cancer Society Professor of Microbiology.|
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