FIGURE 7:
Feedback control of cohesin on REC8 promoter. (A) Rec8 activates its own promoter. Yeast cells were induced to undergo meiosis, and samples were prepared for Northern blot as for Figure 3D. PREC8-GFP::LEU2 (HY2157); PREC8-GFP::LEU2 rec8Δ (HY2229). (B) Quantification of Northern blots. Wild type, filled circles; rec8Δ, open circles. (C) GPF protein level by immunoblot. Cells were induced to undergo synchronous meiosis as for A, and protein extracts were prepared for immunoblot. (D) GFP production in live cells. Aliquots were withdrawn 6 h after induction of meiosis. GFP intensity was measured by fluorescence microscopy as for Figure 6D. PCLB2-SCC3, HY2285. (E) Ectopic production of Rec8 in Scc2-depleted cells. To induce PCUP1-REC8 (HY2122) expression, 100 μM CuSO4 was added to the culture medium after induction of meiosis. (F) ChIP of Rec8 at centromere III. Chromosome III SGD coordinates are shown on the x-axis. Ratio of immunoprecipitation to input is shown on the y-axis. Rec8'3HA, filled circles; PCUP1-Rec8'3HA, PCLB2-SCC2, open circles. (G) Rec8 localization on meiotic chromosomes. Yeast cells were induced for synchronous meiosis and nuclear spreads were prepared for immunofluorescence as for Figure 1A. Bar, 2 μm.