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

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Abstract
In order to find out whether polyoma virus (Py) integration into chromosomes causes rearrangements in the cell DNA flanking the integration site, the flanking sequences in the inducible LPT line of Py-transformed rat cells and the corresponding sequences in normal rat fibroblasts were mapped and then the 2 maps were compared. To carry out this study a segment including Py DNA and flanking sequences were cloned in the bacteriophage vector .lambda.gtWES and the flanking cell DNA was subcloned in a bacterial plasmid. A Southern blot analysis of LPT and rat fibroblast DNA digested with various restriction enzymes was performed and the cloned flanking cell DNA and Py DNA were used as hybridization probes. Autoradiography of the LPT DNA blots revealed 2 sets of fragments. One set includes fragments containing both Py and cell DNA sequences; the 2nd set consists of fragments which contain no virus DNA sequences, and are identical to the fragments observed in the corresponding normal 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 2 types of cells. A comparison between the 2 maps revealed that a 3.0 kilobase cell DNA segment, which is located next to the unoccupied integration site in the normal rat chromosomes, was 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.