Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses IX. Template Topography in the Early Region of Simian Virus 40

The DNAs of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses contain overlapping segments of the early region of wild-type SV40 DNA. The complementary DNA strands of these five viruses have been separated with synthetic polyribonucleotides in isopycnic cesium chloride gradients. The relative amounts of early and late SV40 template in the DNA of each virus were determined by RNA-DNA hybridization with late lytic SV40 RNA, which contains sequences complementary to both templates. From the distribution of early and late templates in the five overlapping SV40 segments, we conclude that either the entire early region of SV40 is symmetrically transcribed in vivo, or, more probably, that the early SV40 templates are not contiguous.     

Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix.

SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells. Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences. Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100%. The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low. Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column. Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact.     

SV40 DNA injected into Xenopus oocyte nuclei is transcribed by RNA polymerase B.

SV40 DNA I. injected into Xenopus oocyte nuclei is transcribed. The SV40-specific RNA molecules migrate on sucrose gradients as do viral RNAs formed in infected green monkey cells but a variable proportion of RNA sequences complementary to SV40 DNA is also found in the light region of the gradients. All SV40-specific RNA species seem to be synthesized by RNA polymerase B as their synthesis is completely sensitive to low concentrations (0.1 microgram/ml) of alpha-amanitin. Concomittantly, the formation of SV40-specific proteins (tumor antigens) is inhibited by injecting alpha-amanitin together with the SV40 DNA.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.