Increased length of DNA made by virions of murine leukemia virus at limiting magnesium ion concentration

Abstract

Conditions were 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 .apprx. 2.4 .times. 106 daltons. DNA > 2 .times. 106 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 of DNA 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 slowed by 30-50%, but products > 5000 nucleotides, which are not found otherwise, are completed between 3-6 h of reaction.