Rolling-circle replication of a high-copy BPV-1 plasmid.
We investigated the replicating form of a bovine papillomavirus type 1 (BPV-1) deletion mutant by direct electron-microscopic analysis of low molecular weight cellular DNA fractions. The detection of viral plasmid DNA replication intermediates was facilitated by the isolation of a spontaneously transformed mouse cell subclone containing an unusually high viral genome copy number (approx. 1000 per cell), and by employing a slight modification of the Hirt fractionation procedure to reduce the level of contaminating linear chromosomal DNA fragments. We observed exclusively rolling-circle-type viral DNA replication intermediates, at a frequency of detection of approximately one replication intermediate per 200 monomeric circular viral DNA molecules. The demonstration of rolling-circles with longer-than-genome-length tails indicated that this high-copy viral plasmid was not subject to a strict once-per-cell-cycle mode of DNA replication. Our observations provide further evidence in favour of an alternative replication mode of the BPV-1 genome, and may help to explain earlier conflicting findings concerning the mechanism of stable BPV-1 plasmid copy-number-control.
Dasgupta
S
S
Department of Medical Genetics, Biomedical Centre, Uppsala, Sweden.
Zabielski
J
J
Simonsson
M
M
Burnett
S
S
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics* / Q000648:ultrastructure; D002460:Cell Line; D006224:Cricetinae; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics*
D004279:DNA, Viral