Stabilization of the large T protein in temperature-independent (type A) FR 3T3 rat cells transformed with the simian virus 40 tsA30 mutant.
The stabilities of in vivo [35S]methionine-labeled large T and small t proteins, synthesized in temperature-sensitive (type N) and temperature-insensitive (type A) FR 3T3 rat cells transformed by an early temperature-sensitive mutant of simian virus 40 (SV40), tsA30, were analyzed at the permissive and restrictive temperatures. The two polypeptides, detected in greatly reduced amounts in cells of the N type at the restrictive temperature, were also unstable at the permissive temperature. However, both were made in similar amounts and were apparently stable in cells of the A type, irrespective of the temperature. The structures of the viral RNAs present at the permissive temperature were analyzed for transformants representative of each type, and containing a single integration of viral DNA. The two cell lines synthesized transcripts identical to the large T and small t mRNAs identified in SV40-infected monkey cells. Similar amounts of viral RNA were found in A and N transformants in active growth at the permissive and restrictive temperatures, which argued against a control at a transcriptional level. Assay of a defined function of the protein, namely, the binding of nucleotide detected by affinity labeling with periodate-oxidized [alpha-32P]ATP, clearly showed that the large T proteins from both types of transformants exhibited, at least for that particular biochemical function, the same in vitro temperature sensitivity. In transformants of the A type only could a reduced binding activity be detected in extracts from cells grown at the restrictive temperature. Thus, the temperature-independent behavior of the A transformants may result from an in vivo partial stabilization of the newly synthesized large T protein, probably through interaction with a cellular component(s).
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D002460:Cell Line; D002472:Cell Transformation, Viral; D004262:DNA Restriction Enzymes; D005814:Genes, Viral; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D012367:RNA, Viral; D014764:Viral Proteins; D004262:DNA Restriction Enzymes