The Kinetochore Is an Enhancer of Pericentric Cohesin Binding (original) (raw)
Figure 5
Centromere Excision
(A) CEN1 on CHRI was replaced with a CEN3-URA3 cassette flanked by head-to-tail-oriented site-specific recombination target sites (red arrows) for the R recombinase from Zygosaccharomyces rouxii, as described in Materials and Methods. This strain (1824-23B) contained the R recombinase under the control of a galactose-inducible promoter. Genomic DNA samples, taken prior to the addition of galactose to the culture medium (0) and at 0.5-h intervals for 4.5 h after the galactose addition, were digested to completion with PvuII (black arrows) and analyzed by Southern blot analysis using a 1.25-kb probe corresponding to CHRI SGD coordinates 151823 to 153080.
(B) The percentage of centromere excision was determined for the timecourse shown in (A). Briefly, a phosphorimage of the Southern blot and ImageQuant software were used to determine the pixel intensities of the unexcised and excised bands (top and bottom bands, respectively). The percent excision was then calculated as the pixel intensity present in the excised band divided by the total pixel intensities of both bands at each timepoint.
(C) A Southern blot analysis of centromere excision from CHRIII. The endogenous CEN3 on CHRIII was replaced by R-recombinase target-site-flanked CEN3 in strain 1829-15B, as described in Materials and Methods. The efficiency of centromere excision from CHRIII was determined by Southern blot analysis in two independent experiments using genomic DNA samples digested with SnaBI and a probe corresponding to CHRIII SGD coordinates 113799-114336. Lanes 1 and 3 represent uninduced controls, and lanes 2 and 4 represent the extent of centromere excision after 2 h of recombinase induction. The percent excision was determined as in (B).