Origin of replication in episomal bovine papilloma virus type 1 DNA isolated from transformed cells

Abstract

The origin of replication of bovine papilloma virus type 1 (BPV-1) has been determined by isolating replicative intermediates (RI) of BPV-transformed hamster embryo fibroblasts (HEF-BPV). These RI were treated with single cut restriction enzymes to determine the start-position (origin) of the extending replication eyes using electron microscopic techniques. 'Cairns'-type RI molecules were shown to contain one replication eye in monomeric as well as in dimeric molecules. The position of this eye was localized at 6940 +/- 5% bp in the physical map. In a second set of experiments BPV-1 DNA fragments cloned in pBR322 were tested for transient episomal replication. Transfected cells were harvested after increasing periods of time and screened for replication with isoschizomeric restriction enzymes to differentiate between input and replicated DNA. The part of the BPV genome harboring the replication origin spans the BPV ClaI-C restriction fragment corresponding to the non-coding region of the BPV genome and coincides with the DNase I-hypersensitive control region in the chromatin, isolated from transformed cells.