T antigen and initiation of cell DNA synthesis in a temperature-sensitive mouse line transformed by an SV40tsA mutant and in heterokaryons of the transformed cells and chick erythrocytes

Abstract

The role of SV40 gene A product in initiation of cellular DNA synthesis was investigated, using a mouse kidney line [mKSA207] transformed by SV40tsA207. mKSA207 cells were temperature sensitive for growth, lost SV40 T antigen (Tag) when incubated in low serum at 40degreeC, and accumulated Tag in the cytoplasm when fed 10% serum and incubated at the nonpermissive temperature (39.7degreeC). Following serum addition, the percentage of mKSA207 cells synthesizing DNA was essentially the same at nonpermissive (39.7 degrees C) and permissive temperatures (33.5degreeC). The cells entered S phase asynchronously at both temperatures, but most cells entered S within 16 h, and before Tag accumulated. mKSA207 synchronized by a double thymidine block also synthesized DNA at 39.7degreesC and entered a second S phase. Tag-depleted or Tag-synchronized mKSA207, when fused with chick erythrocytes (CE), activated CE DNA synthesis. At nonpermissive temperatures (39.7degreesC), 40% of CE nuclei in heterokaryons with Tag-depleted mKSA207 displayed 3H-thymidine--labeled nuclei 28--40 h after fusion, when only 12% of CE nuclei were Tag+. The experiments indicate that SV40 gene A product probably does not have a direct role as initiator of cellular DNA synthesis.