We have previously described several series of biochemical transformation experiments in which small defined portions of herpes simplex virus (HSV-1 and HSV-2) DNA encompassing the thymidine kinase (TK) gene were introduced into Ltk cells by the calcium transfection procedure. The presence of authentic virus TK enzyme in several subcloned cell lines derived from these experiments was confirmed by either the specific incorporation of [I]iododeoxycytidine into their nuclei or the inhibition of cell growth by the antiviral drug arabinosyl thymine. A panel of 24 independent Ltk cell lines receiving either isolated virus DNA fragments or cleaved plasmid DNAs was examined by blot hybridization for both the presence and copy number of virus TK DNA sequences. Most cell lines contained a single virus DNA fragment covalently joined to host (or carrier Ltk) mouse DNA sequences, but several contained multiple copies of the TK gene. Examination of the structural arrangement of the virus DNA in two early passage multicopy cell lines indicated that the TK gene had integrated into Ltk cell DNA and then subsequently both viral and flanking cellular sequences were amplified to create up to 20 tandem duplications. In one case, mapping of the adjacent cellular sequences has revealed that the total repeat unit is greater that 23 kilobases (kb) in size. On subsequent passaging, even in HAT medium, the amplified repeat units were not stable and fell to only three to four copies per haploid cell genome. These cell lines should prove useful for additional studies to examine the expression of co-selected non-TK virus sequences and the influence of adjacent cellular DNA sequences on transcription and retransfection of the resident TK gene.


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