FIGURE 3:
Scc2 is required for REC8 gene expression. (A) Rec8 protein level in wild-type (HY1503C) and PCLB2-SCC2 (HY1586) cells. Yeast cells were induced to undergo synchronous meiosis, then subjected to immunoblot as for Figure 2A. Rec8'3HA was detected by an anti-HA antibody (16B12). (B) Smc3 protein level during meiosis. Note that the levels of Smc3 remain normal in the absence of Scc2 in meiosis. SMC3-3HA, HY1750C; PCLB2-SCC2 SMC3-3HA, HY1750. (C) Rec8 protein level in spo11Δ cells during meiosis. Note that, in the double mutant PCLB2-SCC2 spo11Δ (HY1975), only residual Rec8-3HA was detected. (D) Northern blot showing the levels of IME1, REC8, SCC2, and ACT1 transcripts during meiosis. Wild-type (NH144) and PCLB2-SCC2 (HY1279) cells were induced to undergo synchronous meiosis; aliquots were withdrawn at indicated time points. Total RNA was extracted and prepared for Northern blots. Gene-specific probes sequentially probed the same blots. The level of ACT1 served as a loading control. (E) Quantification of Northern blots. The ratio of the gene of interest to ACT1 is shown. Wild type, filled circles; PCLB2-SCC2, open circles. (F) RT-PCR assay of transcripts. Aliquots were withdrawn 6 h after induction of meiosis. Total mRNA was extracted and reverse transcribed to cDNA. A semiquantitative PCR was used to amplify target cDNA with gene-specific primers. (G) ChIP of Pol II from WT (NH144) and PCLB2-SCC2 (HY1279) cells during meiosis. Yeast cells were induced to undergo synchronous meiosis, and ChIP was performed with an anti-Pol II CTD antibody (8WT16). Semiquantitative PCR was used to determine Pol II enrichment at the REC8 locus. Two representative time points are shown.