Figure 6.

The mitotic phenotypes of H2A-E57A (TBS-II mutant) and H4-L97A (TBS-III mutant) cells. (A) H2A-E57A and H4-L97A cells show perturbation of cell-cycle progression when released from mitotic arrest with nocodazole (+Noc). Samples were harvested at the times indicated and analysed by flow cytometry as described in Materials and methods. (B) The spindle assembly checkpoint in H2A-E57A and H4-L97A cells is functional. A deletion mutant of Mad2, a spindle assembly checkpoint protein, was used as a positive control (see Figure 2F). Samples were taken at the times indicated and analysed by flow cytometry as described in Materials and methods. (C) Immunoblotting of Pds1-3HA. Cells were subjected to immunoblotting as described in Figure 2H. (D) The rate of mono-polar attachment in H2A-E57A and H4-L97A cells. Kinetochore-microtubule attachment was evaluated by the behaviour of GFP-labelled CEN5 in metaphase-arrested cells. (E) The protein level of Sgo1 is reduced in nocodazole-treated H2A-E57A and H4-L97A cells. (F) The localization of Sgo1 is impaired in H2A-E57A cells and moderately impaired in H4-L97A cells. Cells were subjected to ChIP as described in Figure 4A–D. (G) Htz1 is a multi-copy suppressor of TBZ/benomyl sensitivity in H4-L97A cells but not in H2A-E57A or -I112A cells. The sensitivity of the indicated histone point mutants to 25 μg/ml TBZ and 10 μg/ml benomyl was determined using three-fold serial dilutions of cells. Each strain was transformed with a YEplac195 multi-copy vector (URA3 marker, 2 μ) containing the HTZ1 gene. The plates were incubated for 3 days at 25 °C. (H) The localization of Htz1 is severely impaired in H4-L97A cells, but not in H2A-I112A or -E57A cells. Cells were subjected to ChIP as described in Figure 4A–D. Input DNA and DNA co-immunoprecipitated with the anti-FLAG antibody (IP) were amplified with primer sets targeting the left or the right side of CEN3 nucleotide sequences.