The aim of the proposed research is to further our understanding of the mechanism of neoplastic cell transformation by polyoma virus at the molecular biological level. Advantage will be taken of our current knowledge of the genetics of the virus as it affects the transformation process, of DNA sequence data in the HR-T region of the viral genome the expression of which is known to be essential for transformation, and of RNA splicing events which occur in sequences encoded in this region of the viral DNA. We will pursue these aims by applying a specific in vitro site-directed mutagenesis procedure using synthetic single-stranded oligonucleotide DNA sequences. Sequences 10-12 nucleotides long and chosen to have a specific base mismatch with the viral DNA will be annealed to single-stranded circular viral DNA, and extended with DNA polymerase in vitro. These heteroduplexes will then be used to infect cells, and the resulting virus lysates will be used to obtain multiple single plaque isolates. Lysates grown from the latter will then be screened for the desired phenotype of T antigen analyses.
| Schaffhausen, B S; Liang, T J; Carmichael, G G et al. (1985) Residual transforming activity of PY1178T, a mutant lacking the principal in vitro tyrosine phosphorylation site, is not affected by removal of the secondary tyrosine phosphorylation site at residue 322. Virology 143:671-5 |
