Characterization of a mutant polyoma that expresses in F9 embryonal carcinoma cells: morphology, tumorigenicity, and restriction enzyme analysis

A mutant polyoma virus (lT340), which replicates in F9 embryonal carcinoma (EC) cells and contains 2500 base pairs (bp) of additional DNA located in the early noncoding region of the genome, was analyzed to determine the DNA origin of the mutant insertion. Two fragments, representing repeated units of the 2500-bp insert, were isolated from TT340, labeled, and hybridized to the parental wild-type viral DNA. A BglI 500-bp unit, of which there are approximately live copies within the 2500-bp insert, contains sequences homologous to regions on the early and late side of the viral origin of replication. A HpaII 400-bp repeated fragment shows homology to sequences on the early side with little hybridization to the late side. Removal of the 2500-bp insert results in the loss of infectivity on F9 EC cells but not on 3T6 or mouse embryo fibroblasts. Insertion of the BglI SOO-bp repeat element into wildtype DNA at the BglI site allows replication of the constructed virus in F9 cells. The mutant virions were tumorigenic in newborn Syrian hamsters and the morphology of the virus was that of wild-type as assayed by electron microscopy. Q