FIGURE 4:

The specificity of Scc2 to REC8 promoter activation. (A) Ectopic expression of MCD1 in meiosis. The MCD1 open reading frame was inserted at the REC8 locus and was under the control of the endogenous REC8 promoter and its 5′ upstream sequence. Yeast cells were induced to undergo synchronous meiosis, and samples are prepared for immunoblot as for Figure 2A. An Mcd1-specific antibody was used to determine the Mcd1 protein level. Strains used: HY2500 and HY2502. (B) Depletion of Scc2 in vegetative cells. A DEGRON-SCC2 (HY1869) construct is under the control of the MET1 promoter. Yeast cells were arrested at G1 with α-factor; the culture was split into two and released to 25°C and 37°C simultaneously. Protein extracts were prepared for immunoblots. Note that the Mcd1 protein levels remained normal in Scc2-depleted vegetative cells. The level of β-tubulin served as a loading control. (C) A heterologous reporter assay of the REC8 promoter. The GFP open reading frame was inserted at the REC8 locus as in A. Black bars show cohesin enrichment on the chromosome as determined by ChIP on chip. The scale of the y-axis is log₂ ratio of immunoprecipitation to input. SGD coordinates of chromosome XVI are shown at the bottom. Asterisks mark the long-terminal repeat (LTR) sequence as putative peaks because of its repetitive nature. The first CAR that is 5′ upstream of the REC8 promoter is depicted as a black triangle (∼3 kb away). (D) Meiotic expression of P_REC8-GFP::REC8 (HY2203 and HY2107). Yeast cells were induced to undergo synchronous meiosis, and aliquots were withdrawn at indicated time points. Samples were prepared for northern blots as for Figure 3D and probed with GFP- and ACT1-specific probes. (E) GFP protein level by immunoblot. Yeast cells were induced to undergo synchronous meiosis as in C. The GFP protein was detected by an anti-GFP antibody. This antibody recognizes a major GFP band and a minor one with a lower molecular weight.