FIG. 4.

Identification of a minimal p5 (p5D12) element able to replicate in a cell-free assay. (A) In vitro replication assay of the p5-containing plasmids. Plasmids were digested with AflIII and XmnI and incubated with cell extracts from adenovirus type 5-infected HeLa cells (75 μg) and 300 ng of either Rep68 (+) or β-galactosidase (−) protein in the presence of [α-³²P]dCTP. The reaction products were visualized by autoradiography of the membrane. The p5 element is contained in the lower DNA band. C, control pSP72 plasmid with no p5 element. (B) Quantification of the in vitro replication assays. The level of Rep-dependent replication of each individual p5-containing fragment was expressed as ³²P incorporation calculated as described in Materials and Methods. The mean and standard deviation were calculated from three independent experiments.