François Cuzin

The deoxyribonucleotide sequence containing the genetic information of a cell participates in two distinct chemical processes. In the first one, generally called replication, free deoxyribonucleotides are linearly assembled by specific base-pairing to form an identical sequence, or replica of the original structure. The second process, or transcription, allows the genetic material to perform its physiological functions consisting essentially in the production of specific proteins at a suitable rate. As a first step, transcription involves the production of messengers which carry to the protein-forming-centers the information necessary to specify the structure of the polypeptide chains. Messenger synthesis by DNA is a process probably closely similar to that of replication, with the differences that it involves ribo-, instead of deoxyribonucleotides and that, in all likelihood, only one of the DNA strands is used for copying into an RNA transcript.

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."