Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen

We have used two different, but complementary assays to characterize functi ons of SV40 T antigen that are necessary for its ability to immortalize rat embryo fibroblasts. In accordance with previous work, we found that severa l functions were required. These include activities that map to the p53 bin ding domain and the amino terminal 176 amino acids which contain the J doma in as well as the CR1 and CR2 domain required for binding and sequestering the RE family of pocket proteins. Moreover, we found that even though activ ities dependent only upon the amino terminus were sufficient for immortaliz ation they were unable to maintain it. This suggests that immortalization b y these amino terminal functions requires either additional events or immor talization of a subset of cells within the heterogeneous rat embryo fibrobl ast population, We further found that an activity dependent upon amino acid s 17-27 which remove a portion of the CR1 domain and the predicted alpha-1 helix of the J domain was not necessary to maintain growth but was required for direct immortalization suggesting that at least one of the functions r equired initially was not required to maintain the immortal state. This rep resents the first demonstration that some of the functions required for mai ntenance of the immortal state differ from those required for initiation of immortalization.