SV40 large T Antigen (T Ag) can serve as both a potent transforming oncoprotein and transcriptional activator. Several domains of T Ag have been identified .that contribute to the transforming and transactivation functions and, in some cases, these domains appear to overlap. However, it is not known whether the ability of T Ag to activate transcription from a wide variety of viral and cellular promoters contributes to cellular transformation. The dual effects of T Ag on the temperature sensitive Syrian hamster cell line, ts 13, may provide the t6ols to address this question. This cell line undergoes a GI arrest at the restrictive temperature due to a mutation in TAFII250, an essential component of the TFIID RNA polymerase Il complex. Transcription from a limited number of promoters including cyclin A is significantly reduced in tsl3 cells at the restrictive temperature. Expression of wild type TAF1I250 or SV40 large T Ag can override the cell cycle block and rescue the cyclin A promoter defects at the restrictive temperature. We propose to perform a genetic and biochemical analysis of the T Ag domains that are required to neutralize the cell cycle and transcriptional defects in tsl3 cells. We will examine whether the ability of T Ag to rescue the cyclin A promoter defect in tsl3 cells correlates with its ability to bind to certain TBP-Associated-Factors (TAFs) contained in the TFIID complex. Finally, we will examine the correlation between T Ag's ability to rescue the cyclin A promoter defect in tsl3 cells with its ability to override a GI/S arrest in these cells at the restrictive temperature. The goal here would be determine whether the specific domains of T Ag required for transactivation and rescue of the cyclin A promoter and for growth promotion in the tsl3 cells at the restrictive temperature were also required for cellular transformation. To the extent that this is so, the information extracted from these experiments could speak to the overall T Ag transforming mechanism.
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