Integration of Polyoma Virus DNA into Chromosomal DNA in Transformed Rat Cells Causes Deletion of Flanking Cell Sequences

In order to find out whether polyoma virus (Py) integration i to chromosomes causes rearrangements in the cell DNA flanking the integration site, we have mapped the flanking sequences in the inducible LPT line of Py-transformed rat cells and the corresponding sequences in normal rat fibroblasts, and then compared the two maps. To carry out this study we have cloned a segment including Py DNA and flanking sequences in the bacteriophage v ctor 2gtWES and subcloned the flanking cell DNA in a bacterial plasmid. We performed a Southern blot analysis of LPT and rat fibroblast DNA digested with various restriction enzymes and used the cloned flanking cell DNA and Py DNA as hybridization probes. Autoradiography of the LPT DNA blots revealed two sets of fragments. One set includes fragments containing both Py and cell DNA sequences; the second set consists of fragments which contain no virus DNA sequences, and are identical tothe fragments observed in the corresponding ormal rat DNA digests. These data indicate that LPT cells are heterozygous with respect to the Py inserts. The same data were used to map the flanking sequences in the two types of cells. A comparison between the two maps revealed that 3.0 kb cell DNA segment, which is located next to the unoccupied integration site in the normal rat chromosomes, has been deleted from the LPT chromosome which carries Py DNA, but not from the LPT chromosome which does not carry the virus DNA. The implications for papovavirus integration are discussed