Isolated simian virus 40 (SV40) and polyoma nucleoprotein complexes contain endonuclease that, under in vitro conditions, converts part (up to 30%) of the covalently closed superhelical DNA to full-length linear rods. The positions of the cleavage sites within the genomes of SV40 and polyoma were determined by digestion with various single-cut restriction endonucleases and subsequent agarose gel electrophoresis of the cleavage products. Both SV40 and polyoma covalently closed superhelical DNA were cleaved open at their respective origins of DNA replication (±75 base pairs). The full-length linear DNA rods whose ends map adjacent to the origin of DNA replication could also be isolated by sodium dodecyl sulfate/phenol extraction both from SV40-infected permissive cells and from purified SV40 virions. These data reveal the resence of a unique structure of the papovavirus chromatin cose to the initiation site of DNA replication.Isolated papovavirus chromatin has been used as a simple model for the study of the fine structure of chromosomes (1-5). A close similarity between the structural organizations of viral and eukaryotic chromatins was revealed with the main feature being that beaded globular structures termed "nucleosomes" (6), which consist of cellular histones (5, 7) are aligned along the DNA filaments on a small number of distinct alternative positions (4).Viral chromatin also has been successfully used to determine the in vitro factors and conditions that are required for DNA replication. The availability of soluble nucleoprotein complexes derived from the nuclei of simian virus 40 (SV40)-infected cells (8) has fostered such studies (8,9). A number of enzymes such as a and y DNA polymerases (10,11) and RNA polymerase (12) are associated with papovavirus nucleoprotein complexes. As will be shown in this report, there is, in addition, an endonuclease activity present that introduces one double-strand break into the viral DNA. In the case of both SV40 and polyoma DNA, the position that is susceptible to the endonuclease activity within the chromatin was shown to be located at the origin of replication. This finding has revealed a specific structure of the papovavirus chromatin in a biologically important region of the genome. MATERIALS AND METHODSVirus and Cells. SV40 strain Rh 911 was grown on CV-1 cells as described (13). Propagation of polyoma virus and infection of 3T6 cells were carried out as described (14). For isolation of nucleoprotein complexes the cells were infected at a multiplicity of 10 plaque-forming units per cell.Preparation of Nucleoprotein Complexes. Nuclei were isolated from SV40-infected CV-1 cells (107 cells in a 15-cm petri dish, 38 hr after infection) and from polyoma-infected 3T6 cells (2 X 107 cells in a 15-cm petri dish, 26 hr after infection) as described (14). For preparation of nuclear extracts as initially described by Su and De Pamphilis (8) the nuclei were suspended in 2 ml of modified hypotonic extraction buffer (10 mM Hepes, pH 8.0/1 mM MgCl2/0.5 mM CaCl2/1 mM dithio...

No abstract

No abstract

No abstract

Tumorigenicity and immunogenicity of two adenovirus 12 (Ad 12)‐transformed cell lines was studied in syngeneic LIS rats. One cell line (RT2), transformed with Ad 12 virions, contains most of the Ad 12 genome and transcribes its E1, E3 and E4 regions. The other line (RFC1) was transformed with isolated EcoRI‐C fragment of Ad 12 DNA. Both cell lines are equally tumorigenic in newborn rats; in weanling and mature animals the virion‐transformed cells are more tumorigenic as determined by the number of cells required to induce tumors in 50% of animals (TPD50) and by the proportion of “takes” after transplantation of tumor fragments. Serological analysis indicates that antibodies reactive with Ad 12 T‐antigen (product of the E1 region of Ad 12) appear later than those reactive to other Ad 12 antigen(s) present in RT2 cells. The “early” RT2 antibodies are non‐reactive with RFC1 cells. Sera of RT2 tumorbearing rats become reactive with RFC1, cells and isolated Ad 12 T‐antigen after a period of 1–4 weeks. The sera of RFC1 tumor‐bearing rats become reactive with transformed cells after a similar period. The RFC1 sera and the “late” RT2 sera are cytotoxic to both RFC1 and RT2 cells. None of the immunoreactive sera of tumor‐bearing rats are active in antibody‐dependent cellular cytotoxicity (ADCC). The spleen cells of rats bearing RFC1, tumors were cytotoxic to both RFC1 and RT2 cells. The spleen cells of animals with RT2 cell tumors, however, did not exert any cytotoxicity against either cell line. When the spleen cells of tumor‐bearing rats were further educated by incubation with transformed cells in vitro, significant cytotoxicity was observed. The spleen cells of rats bearing RFC1 tumors were equally stimulated in vitro with RFC1 and RT2 cells. The cells of rats bearing RT2 tumors were cytolytic after in vitro exposure to RFC1, cells; much less cytotoxicity was observed with cells restimulated in vitro with RT2 cells. The RFC1 and “late” RT2 sera effectively blocked the cytolysis of both RFC1 and RT2 cells. The “early” RT2 antibodies blocked the cellular cytotoxicity against RT2 cells but not RFC1 cells. Interestingly, the blocking effect was observed even with the effector cells primed in vivo and educated in vitro with only RFC1 cells. These results demonstrate that the E1 gene block of Ad 12 codes for a membrane‐associated antigen active in both humoral and cellular immunity.