FIG. 9.

Southern blot analysis demonstrating increasing methylation of telomeric plasmid sequences in clones A211 and A405 upon passaging in culture. Genomic DNAs isolated from A211 and A405 cultures, grown for various numbers of passages after removal of G418 selection, were digested with SacI, followed by digestion with either HpaII, which does not cut DNA methylated at the restriction site, or MspI, which is insensitive to DNA methylation. Hybridization was performed with the pNPT plasmid lacking telomeric repeat sequences. Digestion with SacI generated plasmid-containing fragments of 3 and 4 kb with clone A211 and of 1.9 and 8 kb with clone A405 (arrows). These large fragments disappeared if the DNA was cut with either MspI or HpaII. The presence of these bands in DNA digested with HpaII denotes its failure to cut due to extensive methylation of its recognition sites. In contrast, the presence of small plasmid-containing fragments (bracket) demonstrates extensive digestion of the same DNA preparations with MspI.