Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.
Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.
D000818:Animals; D010582:Bacteriophage lambda / Q000032:analysis; D001483:Base Sequence; D002460:Cell Line; D004274:DNA, Recombinant / Q000032:analysis*; D004278:DNA, Superhelical / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D005347:Fibroblasts; D051379:Mice; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000032:analysis; D009053:Sarcoma Viruses, Murine / Q000032:analysis* / Q000235:genetics; D014162:Transfection
D004274:DNA, Recombinant; D004278:DNA, Superhelical; D004279:DNA, Viral; D009711:Nucleotides