FIG. 3.

Remodeling of TALS chromatin assessed by changes in topology in unsynchronized and arrested yeast cells. (A) Cells (YJ0α) harboring TALS and having GAL4 under control of LexA-ER-VP16 were grown in raffinose medium in the presence or absence of hormone, either unsynchronized or arrested as indicated, and DNA was isolated for analysis of TALS topoisomer distributions. Hormone induction was for 3 h at 100 nM β-estradiol. The band near the top is nicked circular DNA, and the lower bands represent topoisomers differing in linking number from adjacent bands by one; under the conditions used, faster-migrating species are more positively supercoiled. Values shown for ΔLk indicate the differences between the calculated centers of the Gaussian distributions in the lanes indicated. (B) Topoisomer distributions of TALS from cells (FY24) harboring TALS and a multicopy plasmid bearing the GAL4 gene grown in glucose medium in the presence of nocodazole (10 μg/ml) for 3 h or in its absence, as indicated. Cells were spun down and taken up in galactose medium with or without nocodazole and incubated for the additional intervals indicated. The uppermost band corresponds to nicked circular TALS, and faster-migrating topoisomers are more positively supercoiled. The linking number changes between samples grown in glucose and galactose are indicated at the bottom. o/n, overnight.