FIG 6 .

Replicons containing the minimal replication origin and upstream enhancer sequences replicate extrachromosomally in keratinocytes for multiple passes. Gel-purified monomeric supercoils of the pCpGΔneo URR replicons were cotransfected together with the wild-type HPV 18 genome into primary keratinocytes and cultured for five passages. Each pass corresponded to approximately 4 population doublings, though this is an underestimate because of the low plating efficiency of primary keratinocytes. Total cellular DNA was extracted at each pass and analyzed by Southern blotting with a pCpGneo vector probe. The panel on the left contain samples digested with BstXI, which cleaves the replicon into two fragments. The samples in the panel on the right were digested with NdeI, which does not cut the replicons, allowing visualization of extrachromosomal supercoiled DNA. A representative image of two independent transfection experiments is shown, though subsets of the replicons were also analyzed in multiple other experiments with similar findings.