Restriction endonuclease mapping of unintegrated proviral DNA of Kirsten murine sarcoma virus.

Linear unintegrated Kirsten murine sarcoma virus DNA synthesized after acute infection of NIH/3T3 cells or by detergent-disrupted virions, was studied by restriction enzyme cleavage and agarose gel electrophoresis. Labelled DNA (cDNA) synthesized by reverse transcription of a virion RNA template was used to detect viral DNA sequences by filter hybridization. The sites of cleavage for eleven enzymes were located on the genome. Hybridization studies using a cDNA probe specific for the 3' end of the genome defined the orientation of the physical map with respect to virion RNA and identified terminally redundant sequences on the genome. Further evidence for terminal redundancy was obtained by restriction mapping of linear and circular viral DNA, where the duplication was shown to be 0.34 x 10(6) (500 base pairs) in length and in a tandem orientation. Circular viral DNA was found predominantly in the nucleus of acutely infected cells and it existed in two major size classes. The larger size class represented an exact circularization of linear DNA (with no other sequence permutation) whilst in the smaller DNA species one of the terminally redundant sequences was absent.