Induction of cell DNA replication in G1-specific ts mutants by microinjection of SV40 DNA

ts13 and tsAF8 are temperature-sensitive mutants of BHK cells that become arrested in G1 at the non-permissive temperatures of 39.6-40.6 °C. When these mutants are made quiescent by serum deprivation and are subsequently stimulated by serum, they enter S phase at the permissive but not at the non-permissive temperature. The purpose of these experiments was to determine whether Simian Virus 40 (SV40) could induce cellular DNA synthesis in these mutants at the non-permissive temperatures. Because both cell lines are resistant to SV40 infection we cloned an SV40 DNA fragment (from residue 1705 counter-clockwise to residue 2455) inclusive of the A gene into the plasmid pBR322, and we microinjected the recombinant plasmid DNA in both ts13 and tsAF8 cells. Cells microinjected with the cloned SV40 DNA fragment entered DNA synthesis at both permissive and non-permissive temperatures. The newly synthesized DNA in the microinjected cells was shown to be cellular in origin. Stimulation of cell DNA replication was inhibited when antibodies against T-antigen were microinjected together with the recombinant plasmid DNA. In addition SV40-specific mRNA microinjected into ts13 cells also stimulated cellular DNA synthesis. We conclude that the product(s) of the SV40 A gene can induce cell DNA synthesis in ts13 and tsAF8 at either permissive or non-permissive temperatures, thus dispensing with at least two cellular functions that are required by serum-stimulated cells for progression through G1.