Recombinant DNA molecules comprising bovine papilloma virus type 1 DNA linked to plasmid DNA are maintained in a plasmidial state both in rodent fibroblasts and in bacterial cells.

Transformed cells obtained after transfecting FR3T3 rat fibroblasts with DNA of bovine papilloma virus type 1 ( BPV1 ) maintained only free copies of the viral genome. Transfection with BPV1 DNA inserted in a bacterial plasmid (pBR322 or pML2 ) did not produce transformants at a detectable rate, unless the viral sequences had been first excised from the plasmid. In contrast, transfer of the same plasmids by polyethylene glycol-induced fusion of bacterial protoplasts with FR3T3 rat or C127 mouse cells led to significant transformation frequencies. A total of eight cell lines were studied, three rat and five mouse transformants, obtained with various BPV1 - pML2 recombinants. In all cell lines, both BPV1 and plasmid sequences were maintained as non-integrated molecules, predominantly as oligomeric forms of the transforming DNA. In the three rat transformants and in two of the mouse lines, parts of the non-transforming viral region and some bacterial sequences were deleted. In the remaining three mouse lines, the monomeric repeat was a non-rearranged plasmid molecule which could be re-established as a plasmid in Escherichia coli after cleavage with "one-cut" restriction endonucleases and circularization of the molecule.     

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.