An analysis of transformed clones obtained by coinfections with hr-t and ts-a mutants of polyoma virus.

Clones of stably transformed rat cells from the F-111 (Fisher rat embryo) and NRK (normal rat kidney) cell lines have been obtained by complementation using pairs of nontransforming polyoma mutants of the hr-t and ts-a classes. A total of 78 clones were isolated and studied, 21 from the NRK line, and 57 from the F-111 line. These "complementation transformed clones" were then examined for the presence and expression of each of the parental mutant viruses. The expression of viral T (tumor) antigens was analyzed by immunoprecipitation. Every one of 54 clones examined express the 22K (small) and 56K (middle) T antigens which are the products of the hr-t viral gene. This indicates a requirement for the presence of a wild-type hr-t allele contributed by the ts-a mutant parent. Ten clones lack detectable large T antigen, while four show thermolabile large T antigen. These results support the conclusion that middle and small but not large T antigen(s) are essential for the maintenance of the transformed phenotype. Most of the 57 F-111 clones are virogenic when fused to mouse cells under permissive conditions and yield both mutant types. Surprisingly, wild-type recombinants have been recovered from 40 of the F-111 clones. Three clones show evidence of retention of only the ts-a mutant genome. Virus cannot be rescued from the majority of the 21 NRK clones. The few clones which are virogenic, however, yield both hr-t and ts-a mutants. Integration patterns of several clones analyzed by Southern blotting confirm the expectations based on viral T antigen(s) and virus rescue analyses. Hr-t mutant genomes, though not required for the maintenance of the transformed state, are frequently retained in complementation transformed clones. Tandem integration of the two parental mutants is clearly demonstrated in one clone and implicated in the other eight out of nine virogenic F-111 complementation transformed clones examined. This observation provides the basis of a model for the generation of wild-type recombinants following fusion of F-111 clones.