FIG. 8.

An esa1 mutation affects FACT binding and ARG3 expression. (A) FACT binding to the ARG3 open reading frame is reduced in an esa1 mutant. Strains DY150 (wild type), DY10398 (rco1), and DY7558 (esa1-Δ414) were grown at 25°C in rich medium and transferred to minimal medium containing SM to starve cells for amino acids, and samples were taken for ChIP before and 10 min after induction. ChIP assays were performed with cross-linked extracts, and a mock precipitation without antibody was performed with extracts from wild-type cells. PCRs measured binding to the ARG3 promoter (−295 to −53) (white), the ARG3 5′ open reading frame (−34 to +169) (light gray), the ARG3 middle open reading frame (+214 to +415) (dark gray), and the ARG3 3′ open reading frame (+707 to +926) (black). The results give the ratio of the ChIP signals at the ARG3 region to the control interval, and error bars show the standard deviation of the ChIP PCRs performed in triplicate. (B) An esa1 mutation reduces ARG3 expression. Strains DY150 (wild type [WT]), DY10398 (rco1), DY7558 (esa1-Δ414), and DY11116 (esa1-Δ414 rco1) were starved for amino acids as in panel A. RNA was isolated, and mRNA levels were determined by RT-PCR for ARG3 and ACT1 (internal control). “0” and “10” indicate time after the shift to starvation conditions. The results are given as the ratio of ARG3 to the ACT1 internal control, with the error bars showing the standard deviations of the triplicate PCRs.