Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.
AUTOR(ES)
Hager, G L
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=353507Documentos Relacionados
- Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.
- Molecular Cloning of the Harvey Sarcoma Virus Circular DNA Intermediates II. Further Structural Analyses
- Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones.
- Molecular Cloning of B- and N-Tropic Endogenous BALB/c Murine Leukemia Virus Circular DNA Intermediates: Isolation and Characterization of Infectious Recombinant Clones
- Molecular cloning and characterization of avian sarcoma virus circular DNA molecules.