No abstract

The viable polyoma mutants dl1013, dl1014, and dl1015 produced shortened middle and large T-antigens. In mouse 3T3 cells, dl1013 and dl1014 grew at normal rates, and dl1015 grew at a reduced rate. dl1015 behaved phenotypically as a double mutant, with deficiencies both in the stimulation of the host cell and the replication of viral DNA. Only the former defect could be complemented by the ts-a mutant, which produced a normal middle T-antigen and a temperature-sensitive large T-antigen. This result suggests that middle T-antigen is involved in the induction of cellular DNA synthesis. Of the three mutants, dl1015 alone failed to transform rat fibroblasts to growth in semisolid medium. This defect could not be complemented by the ts-a mutant. Determination of the base sequences of the mutant DNAs showed that dl1013 and dl1014 had overlapping deletions of 21 and 9 base pairs, respectively, and that the dl1015 deletion of 30 base pairs was contiguous to the other mutations on their 3' sides. Analyses of the mutant t-antigens showed that all three mutants produced shortened middle T-antigens, whereas only dl1015 large T-antigen was detectably reduced in size.

A variant mouse plasmacytoma (MPC)‐associated translocation chromosome has arisen by pericentric inversion and exchange of the distal segments of a Robertsonian 6;15 fusion chromosome in the CAK TEPC 1198 mouse plasmacytoma, as described earlier. In situ hybridization was performed on the normal and the inverted Rb chromosomes, using myc and kappa probes. On the normal Rb chromosome, myc was in the 15 D2/3 region, whereas kappa hybridized in the 6 C2 area, as expected. On the inverted Rb chromosome, myc remains on the centrometric side of the translocation breakpoint on the chromosome 15‐derived portion, whereas kappa has moved to the chromosome 6‐derived segment that joined the same breakpoint on the telomeric side. Taken together with our recent demonstration that the murine c‐myc locus is oriented ‘head up’ on chromosome 15, and with the results of Cory and co‐workers concerning the relationship between the kappa gene and the associated pvt‐1 region in the CAK TEPC 1198 tumor, the following conclusions can be drawn: (i) in the variant translocation of the CAK TEPC 1198 MPC, the breakage occurs 3′ of the c‐myc gene, as in the human Burkitt lymphoma‐associated variant translocations; (ii) the pvt‐1 gene on chromosome 15 is distal to the myc gene; (iii) the kappa light chain locus is oriented ‘head up’ on mouse chromosome 6 and faces pvt‐1 and, beyond it, c‐myc, in a head‐to‐tail configuration.

The movement of replication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction endonuclease HpaII which cleaves polyoma DNA into eight unique fragments. The terminus of in vitro DNA synthesis was identified by cleaving newly completed molecules with HpaII. The distribution of label in the restriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal terminus of replication. Analysis of replicative intermediates pulse-labeled in vitro further suggested that DNA synthesis in isolated nuclei is an ordered process similar to replication in intact cells. Replication forks moved with a constant rate from the origin towards the terminus of replication. The nonlinear course of the DNA synthesis reaction in the isolated nuclei seems to result from the random inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally synthesize a DNA chain about 1,000 nucleotides long. The results suggest that some replication forks might be initiated in vitro at the origin of replication.

Mouse 3T6 cells were infected with polyoma virus at high multiplicity, and survivors were isolated. Clones from single cells were then established and were found to be resistant to a second infection. However, in some clones viral functions could at least be partially expressed during reinfection, as judged from a stimulation of nuclear tumor antigen expression. One such clone was studied in detail. These cells were transformed and produced low amounts of virus (less than 1 PFU per cell per generation). The persistent infection did not seem to be a carrier-state phenomenon, since infectious-center assays showed that most cells produced virus. The resistance of the cells to reinfection can be explained by interference from viral DNA present in the cells, averaging about 1,500 "free" copies per cell. This DNA had the normal physical characteristics of polyoma DNA. However, it had a slightly larger size than authentic polyoma DNA. Mapping with restriction endonucleases showed that the addition to the DNA was about 5% of the wild-type genome and was located close to the origin of DNA replication. This DNA was infectious, although it had a 10-fold lower infectivity than wild-type polyoma DNA. Both virus and DNA from the polyoma-resistant cells had a small-plaque morphology, as opposed to the large-plaque morphology of the virus used for the initial selection of cells.