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

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 DNAs 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 more than 10-fold amplification of restriction fragments extending up to about 2.0 kilobase pairs into the cellular DNA flanking one end of the viral insertions, defined as the left joint. Fragments extending beyond this region were not amplified. These results showed that (i) integrated polyoma virus DNA undergoes multiple rounds of replication in mitomycin C-treated LPT cells and (ii) the replication extends into the flanking sequences and is arrested within a 0.40-kilobase-pair cellular DNA segment located about 2.0 kilobase pairs beyond the left joint. This segment may include a terminator of a normal cellular replicon.