Requirements for immortalization of primary mouse embryo fibroblasts probed with mutants bearing deletions in the 3' end of SV40 gene A

Abstract

The influence of specific contiguous stretches of amino acids predominantly in the carboxy terminal third of the SV40 large T antigen on the immortalization of cells in culture was investigated. Mutants that bear either small in-phase or frameshift deletions in the large T antigen coding sequence were transfected into primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF). The frequency of immortalization was determined as the number of colonies that developed from cells escaping senescence. The results indicated that the terminal 81 amino acids of large T antigen are not needed for efficient immortalization or tumorigenicity. In contrast removal of as few as three amino acids encoded in the vicinity of the Dde-1 site at 0.234 map units (m.u.) severely restricted immortalization, suggesting that this region of the coding sequence either structurally or functionally is essential to at least one parameter of the transformed cell phenotype. The T antigen produced by dlA2433 which bears a deletion of nine nucleotides at 0.234 m.u. fails to associate stably with the cellular protein p53. The results showed that the addition of long stretches of amino acids (96 or 97 residues) from the open reading frame at the 3' end of the early region inactivated immortalizing functions, although the addition of as many as 18 amino acids from other reading frames was not detrimental. The evidence presented also confirmed that wild-type levels of ATPase activity are not necessary for immortalization or tumorigenicity of B6/MEF. Finally, we show that one of the mutants that immortalized primary cells did not produce dense foci on a cell monolayer. This last result indicated that independent functions are required for these two parameters of the transformed cell phenotype.