The proposed project will examine whether the transcriptional transactivation function of the large T antigen contributes to cellular growth and transformation. The relationship between transactivation and growth will be studied in ts13 cells, cells which contain a temperature sensitive mutation in TAFII250. At the nonpermissive temperature, these cells arrest in G1 and are unable to transactivate specific cellular promoters. Large T rescues both the transactivational and cell cycle related defects in these cells, making ts13 cells an excellent system in which to study the relationship between the transactivational and growth promoting functions of large T.
The specific aims of this proposal are to map the domains of large T which contribute to the rescue of the transcriptional defects in ts13 cells and compare these to the domains which rescue the G1 phase arrest. The ability of large T to rescue the cyclin A promoter defect will be determined by transfecting ts13 cells with large T or various deletion mutants of large T and measuring the ability of these mutants to activate a cycle A promoter- luciferase reporter construct. The ability to rescue the G1 phase arrest will be measured by FACs analysis of large T antigen transfected cells. In addition, the mechanism of large T mediated rescue of the transcriptional defects in ts13 cells will be studied by determining whether large T is able to affect the composition of the basal transcriptional complexes in ts13 cells at the non-permissive temperature. The composition of these complexes will be determined by immunoprecipitation and Westerns.
| Buchmann, Ann M; Skaar, Jeffrey R; DeCaprio, James A (2004) Activation of a DNA damage checkpoint response in a TAF1-defective cell line. Mol Cell Biol 24:5332-9 |
| Chao, H H; Buchmann, A M; DeCaprio, J A (2000) Loss of p19(ARF) eliminates the requirement for the pRB-binding motif in simian virus 40 large T antigen-mediated transformation. Mol Cell Biol 20:7624-33 |
