Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mutant revertants

Abstract

The growth properties of hamster cells transformed by wild-type SV-40, by early SV-40 temperature-sensitive mutants of the A complementation group and by spontaneous revertants of these mutants were studied. All of the tsA mutant-transformed cells were temperature sensitive in their ability to form clones in soft agar and on monolayers of normal cells except for CHLA-30L1, which was not temperature sensitive in the latter property. All cells transformed by stable revertants of well-characterized tsA mutants possessed certain growth properties in common with wild-type-transformed cells at both temperatures. Virus rescued from tsA transformants including CHLA30L1 was temperature sensitive for viral DNA replication, but that rescued from revertant and wild-type transformants was not thermolabile in this regard. T [tumor] antigen present in crude extracts of tsA-transformed cells including CHLA30L1, grown at 33 or 44.degree. C, was temperature sensitive by in vitro immunoassay, but that from wild-type-transformed cells was relatively stable. T antigen from revertant transformants was more stable than the tsA protein. Partially purified T antigen from revertant-transformed cells was nearly as stable as wild-type antigen in its ability to bind DNA after heating at 44.degree. C, but T antigen from tsA30 mutant-transformed cells was relatively thermolabile. T antigen is apparently a product of the SV-40 A gene. Significantly more T antigen was found in extracts of CHLA30L1 grown to high density at the nonpermissive temperature than in any other tsA-transformed cell similarly grown, which that the amount of T antigen synthesized in CHLA30L1 is large enough to allow partial expression of the transformed phenotype at the restrictive temperature. The increase in T antigen concentration may be secondary to .gtoreq. 1 genetic alterations that independently affect the transformed phenotype of these cells.