The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies ofBPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.14.2 viral genome equivalent. It was concluded that maintenance oftransformation may be mediated by nonintegrated viral DNA.Cellular transformation induced by several DNA and RNA viruses has been studied in considerable detail. In most of these systems, cellular transformation is accompanied by integration of the viral sequences required for the maintenance of transformation. Plasmid viral DNA forms have been described in cells transformed by certain DNA viruses, but almost always in association with integrated viral DNA. The papillomaviruses represent a tumor virus system which for technical reasons has received relatively little attention. The papillomaviruses along with the polyomaviruses comprise the papovavirus family (Papovaviridae) (1). Despite the morphologic similarities of their virion and genomic structures, the papillomaviruses and the polyomaviruses are separate groups ofviruses, distinguished by biological, serological, and biochemical features (2-4).The papillomaviruses are widely distributed in nature and induce benign skin tumors (warts) in their natural hosts which include humans, cattle, rabbits, dogs, sheep, chaffinches, and horses. Most papillomaviruses are highly host specific, and this property, together with the lack of an adequate tissue culture system for their propagation, has hampered studies of their molecular biology and genetics. Recently, this liability has been partially circumvented by molecularly cloning the genomes of a number of different papillomaviruses in Escherichia coli by using the certified plasmid pBR322 as cloning vector (5, 6).Whereas the proliferative changes induced by most papillomaviruses are limited to epidermal cells, lesions induced by the bovine fibropapillomaviruses (BPV-1 and BPV-2) are exceptional in that they consist of both epidermal and mesenchymal proliferative components (7). These BPVs also differ in that they can induce ...

We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of mnethotrexate. Helper virus-free dihydrofolate reductasetransducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 x 106 per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.

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We describe a bovine papillomavirus hybrid plasmid containing the neomycin resistance gene from Tn5 inserted into a mammalian cell transcriptional unit. This plasmid is maintained as a stable extrachromosomal element (20 to 100 copies per diploid genome) in mouse cells selected either for the transformed phenotype or for resistance to the aminoglycoside G418. Cells selected for G418 resistance initially display a flat, nontransformed phenotype before exhibiting the gross morphological characteristics of transformation. The delay in the appearance of the transformed phenotype indicated that some intracellular event or series of events subsequent to the establishment of transcriptionally active bovine papillomavirus 1 hybrid plasmid is required for the manifestation of the transformed phenotype.