FIG. 10.

The HindIII site is virtually inaccessible within the hsp82-ΔHSE1 promoter in both asynchronous and cell cycle-arrested cells. (A) Asynchronous SLY101 (WT) and BSY202 (ΔHSE1) cells were grown at 30°C in complete synthetic medium supplemented with 2% raffinose. Nuclei, isolated from spheroplasts and quantitated as previously described (17), were digested with either 12 or 40 U of HindIII per μg of DNA at 30°C for 60 min. Genomic DNA was purified, digested to completion with MspI, and subjected to linear PCR using the +26→−11 primer, and the amplified products were electrophoresed on an 8% sequencing gel. Bands were detected and quantitated using a PhosphorImager. Percent accessibility was calculated by dividing the total signal present in the daughter (D) fragment (resulting from HindIII cleavage) by that present in the sum of the parental (P) fragment plus daughter fragment. (B) As in panel A except that BSY202 cells were either maintained in log growth (async.) or arrested at the indicated phases in the cell cycle prior to isolation of nuclei. Samples were digested with 40 U of HindIII per μg of DNA and processed as above. See Fig. 2 for location of HindIII site with respect to mapped chromatin structure.