Figure 5.

Bal31 exonuclease analysis. Southern blot analysis of Bal31-digested genomic DNA shows that the telomeric ends of JM2860 (fig. 1A) are internal in the HACs. Zero (lanes 0), two (lanes 2), or four (lanes 4) units of Bal31 exonuclease were used, followed by digestion by XhoI. The 4.8-kb and 6.3-kb terminal XhoI fragments of I-SceI–cleaved JM2860 are detected, respectively, by Amp^R and repE probes (fig. 1B) in the control, in which the exonuclease is omitted (lanes 0). These fragments are degraded by Bal31, whereas the fragments detected by the probes in minichromosome cell lines are resistant (notwithstanding a band-intensity decrease expected in light of the extension of Bal31 degradation to internal regions). The 6.8-kb JM2860 fragment detected by the Amp^R probe probably results from incomplete digestion by I-SceI. Genomic DNA from the parental HT1080 cells was included as a negative control for the minichromosome probes used.