A functional simian virus 40 origin of replication is required for the generation of a super T antigen with a molecular weight of 100,000 in transformed mouse cells

Abstract

We used two recombinant plasmids, one containing wild-type simian virus 40 DNA (pSVR1) and the other containing a simian virus 40 genome with a defective origin of replication (pSVR1-origin-minus) to transfect NIH3T3 cells. Quantitation of T-antigen synthesis by indirect immunofluorescence at 48 h after transfection with either DNA revealed the same percentage of T-positive nuclei. The transformation frequencies observed were also similar with both plasmids. Immunoprecipitation of [35S]methionine-labeled cell extracts showed the expected 94,000-dalton (94K) T and 17K t antigens in all clones examined. In pSVR1-generated transformants, a 100K super T antigen was also detected. Transformants isolated from pSVR1-origin-minus transfection, however, never expressed this 100K super T antigen, and some of these clones originally also showed greatly reduced levels of 94K T antigen. However, after growth in culture for several generations, the levels of 94K T antigen synthesis in these underproducer clones were dramatically increased. A direct correlation between the amounts of T antigen synthesized and the ability to grow independently of anchorage was observed. The mechanism which brings about increasing levels of T-antigen synthesis in some of the clones is not clear, but it appears not to be due to changes in either the copy number or the methylation pattern of the integrated simian virus 40 DNA.