Southern blot analysis of RFC gene copy number in parental WT cells and their PT523-resistant variants. High-molecular-weight gDNA (15 μg) was codigested with MfeI and XmaI (a 6-cutter; A) or MfeI and MspI (a 4-cutter; C), fractionated by electrophoresis on 0.8% agarose gels, and transferred to Zeta-Probe-GT nylon membranes. The blots were then hybridized with a [³²P]labeled 5′-genomic hRFC probe as detailed in “Materials and methods.” Following hybridization at 65°C, the membranes were washed under high stringency conditions and exposed to x-ray film. The intensity of the RFC band in panels A and C was estimated by scanning densitometry and normalization to an approximate 1.5-kb repetitive DNA band observed after ethidium bromide staining (B). The band obtained in panel A and the middle band observed in panel C (arrowhead) are both approximately 600 bp in length. The arrows shown on the right side in panel C denote the WT bands that were lost in the PT523-resistant cells.