Immortalization of primary mouse embryo fibroblasts with SV40 virions, viral DNA, and a subgenomic DNA fragment in a quantitative assay.
In order to investigate the capacity of the N-terminal half of large T antigen to convert primary cells to continuous cell lines conditions were standardized for transforming primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF) with DNA. Using these conditions the transforming capability of SV40 virions, viral DNA, and a plasmid, pSV3T3-20-GV (C.E. Clayton, D. Murphey, M. Lovett, and P.W.J. Rigby, Nature (London), 299, 59-61, 1982) containing the 5' half of the SV40 early region, was determined in a quantitative immortalization assay. The plasmid pSV3T3-20-GV transformed B6/MEF at only 1/16 the frequency of a plasmid containing the entire early region. These results suggest that the 3' half of large T antigen which cannot be produced by this plasmid strongly influences the frequency of immortalization. It is not known whether this influence reflects the presence of a transformation domain in the carboxy terminus of large T antigen or, alternatively, results from an altered conformation or stability of the truncated polypeptide.
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis*; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000382:microbiology; D005796:Genes; D005814:Genes, Viral; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*; D014771:Virion / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins