FIG. 2.

Mapping of the p53 domains necessary for suppression of papillomavirus amplificational replication. (A) Schematic representation of p53 mutants. Numbers indicate positions on the amino acid sequence. (B) Southern blot analysis of the transient replication of BPV-1 origin plasmid pNeoBgl40 in the presence of different p53 mutants in the CHO4.15 cell line. Episomal DNA was extracted from cells at 48, 72, and 96 h after transfection and digested with restriction endonucleases PstI and DpnI. Filters were probed with radiolabeled HPV-11 URR containing plasmid p7072-99; 100 ng of pNeoBgl40 together with 250 ng of p53 expression plasmid was transfected into the cells. Lanes 1 to 8 correspond to the transfections with p53 mutants in the same order as depicted in panel A. Carrier, mock-transfected cells; BPV1 ori, control with no added p53. (C) Relative inhibition of replication of the BPV-1 replication origin by different p53 mutants. The replication signals of three independent experiments (72 h posttransfection) were quantified with a PhosphorImager and signals from the cells transfected with origin plasmid only were used as a control to normalize the results. (D) Southern blot analysis of transient replication of the BPV-1 origin plasmid pUCAlu in the presence of additional N-terminal p53 deletion mutants in the CHO4.15 cell line. Episomal DNA was extracted from cells at 72 and 96 h after transfection and digested with restriction endonucleases PstI and DpnI. Filters were probed with radiolabeled pUCAlu plasmid. Lanes 1, 7, 9, 10, and 11 correspond to transfections with p53 mutants as depicted in panel A.