Figure 6.

The MBF complex restricts CENP-A transcription to G1 phase. (A) Quantitative RT-PCR analysis of cnp1 RNA levels in cells with a WT or ∆MCB1 cnp1 promoter in combination with mutation of MBF genes. Cells carrying an extra copy of cnp1 tagged with GFP under control of the WT or ∆MCB1 cnp1 promoter were crossed with mutants for nrm1, yox1, or res2 genes. Graph shows RNA fold changes for the cnp1-gfp transgene, endogenous cnp1 and cdc18 genes for the indicated genotypes. Fold change was calculated normalizing to levels in WT cells with the cnp1-gfp under the WT cnp1 promoter. (B) cnp1 and hht1 mRNA levels measured by quantitative RT-PCR in synchronized cells with a WT or MCB1 box mutant promoter on cnp1. Dotted line indicates the time window when most of the cells are undergoing S phase, measured by the septation index. (C) TBZ sensitivity assays for Cnp1 promoter mutants. Serial dilutions of indicated strain cultures were plated onto YES medium supplemented with 15 μg/ml TBZ or control YES medium without TBZ. Pictures of colony growth were taken 72 or 96 hr postplating. (D) Overexpression of Cnp1-GFP causes chromosome segregation defects in MCB1 mutant cells. Cells were fixed and stained with DAPI staining. The percentage of mitotic cells showing bridged, and lagging chromosomes was quantified as in Figure 2C. MBF, MluI box-binding factors; MCB, MluI cell cycle box; TBZ, thiabendazole; WT, wild-type; YES, yeast extract with supplements.