Further analysis of HSV-tk- subclones that showed no detectable change in the plasmid sequences by Southern blot analysis of genomic DNA digested with BamHI. Genomic DNAs from A3 and four of its HSV-tk- subclones, G1, G6, G8, and G38, that showed no change with BamHI, were digested with (A) BamHI and ClaI or (B) BgIII, and hybridization was performed with the pNCT-Δ probe. The presence of diffuse bands indicates that although the plasmid is still telomeric, the BgIII and ClaI sites between the HSV-tk gene and the telomeric repeat sequences are no longer present.