Random-Choice Replication of Extrachromosomal Bovine Papillomavirus (BPV) Molecules in Heterogeneous,

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ED13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromo-somal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis ofBPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPVDNA and ofBPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachro-mosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism. Bovine papillomavirus type 1 (BPV-1) normally infects and transforms fibroblists and epithelial cells, causing warts in cattle, its natural host (18), but it can also replicate in an