Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication.
Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D005784:Gene Amplification; D005814:Genes, Viral; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics*; D051381:Rats; D011916:Rats, Inbred F344; D014779:Virus Replication
D004279:DNA, Viral; D004262:DNA Restriction Enzymes