title: Expression of the large T protein of polyoma virus promotes the establishment in culture of "normal" rodent fibroblast cell lines.
abstract: Transfer into mouse and rat embryo fibroblasts in primary culture of cloned polyoma virus genes encoding only the large T protein led to the establishment of flat colonies in sparse subcultures at a frequency equal to that of transformation by wild-type virus. Cell lines could be derived from such colonies and maintained in culture for large numbers of generations without entering crisis. They exhibited a normal phenotype, by the criteria of growth on plastic to a low saturation density and of anchorage dependency. However, they required a lower serum concentration for growth than spontaneously established 3T3 cells. Similar results were obtained after transfer of recombinant DNA molecules encoding only the amino-terminal 40% of the large T protein, suggesting that this "immortalization" function corresponds to the activity of an amino-terminal domain of the protein. Immunoprecipitation analysis of T antigens in cell lines established after transfer of the full-size and of the truncated large T genes demonstrated the expression of the full-size large T protein and of a Mr 40,000 antigen expressed from the amino-terminal part of the gene, respectively. After transfer of a "large T only" plasmid that carries a tsa mutation, cell lines were established at 33 degrees C with the same efficiency as with the wild-type large T gene, but their growth was arrested after a shift to 40 degrees C, with a progressive loss in cell viability. This result indicates a continuous requirement for a large T function in the maintenance of "immortality."
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000502:physiology; D051379:Mice; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics*; D051381:Rats; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004274:DNA, Recombinant; D014764:Viral Proteins; D004262:DNA Restriction Enzymes