"Onion skin" replication of integrated polyoma virus DNA and flanking sequences in polyoma-transformed rat cells: termination within a specific cellular DNA segment.

Abstract

Replication of integrated polyoma virus DNA and flanking cellular sequences was studied in an inducible line of polyoma-transformed rat cells, designated the LPT line, that contains a single viral integration site. Chromosomal DNA were purified from LPT cells treated with the virus-inducing agent mitomycin C and from untreated cells and were digested with restriction enzymes. The digests were analyzed by the Southern blotting technique. The virus DNA and a recombinant plasmid-containing flanking cell DNA were used as hybridization probes. The analysis showed that mitomycin C treatment caused a > 10-fold amplification of restriction fragments extending up to .apprx. 2.0 kilobase pairs into the cellular DNA flanking 1 end of the viral insertions, defined as the left joint. Fragments extending beyond this region were not amplified. Evidently, integrated polyoma virus DNA undergoes multiple rounds of replication in mitomycin C-treated LPT cells and the replication extends into the flanking sequences and is arrested within a 0.40-kilobase-pair cellular DNA segment located .apprx. 2.0 kilobase pairs beyond the left joint. This segment may include a terminator of a normal cellular replicon.