Figure 5.

The centromeric localization of Pol III genes is linked to mitotic chromosome condensation. (A) The temperature sensitivity phenotype of the condensin mutant, cut3-477, is suppressed by the sfc3-1 mutation. Logarithmically growing cells (OD ∼ 0.5) in YEA liquid medium at 27°C were serially diluted by 10-fold and spotted onto nonselective YEA plates that were incubated at indicated temperatures for 2–3 d. (B) The sfc3-1 mutation promotes the centromeric localization of Pol III genes. Wild-type (wt), cut3-477, cut3-477 sfc3-1, and sfc3-1 cells grown at 26°C were subsequently cultured at the restricted temperature (36°C) for 2 h and then subjected to FISH analysis. Most cells used for FISH analysis were in interphase. The Pol III gene locus was visualized using a FISH probe specific to the cosmid clone (c417), whose signal was merged with that of centromeres. The quantitative measurements of distances between the c417 locus and centromeres, and their plotting in the histogram, were carried out as described in Figure 2D. (C) The ϕ-shaped chromosomes phenotype of the cut3-477 mutant is suppressed by the sfc3-1 mutation. The indicated strains were cultured at 36°C for 2 h. Immunofluorescent images of cells stained for tubulin (red) were merged with DAPI signals (blue). The percentage of the ϕ-shaped chromosomes phenotype in each strain is shown on the right. More than 300 cells were counted for each strain. (D) The sfc3-1 mutation facilitates mitotic chromosome condensation. The indicated strains were cultured at 36°C for 2 h. The Pol III genes locus (c417) and non-Pol III gene locus (c162) visualized by FISH were merged with IF images of cells stained for tubulin, and distances between the two loci were measured in interphase cells (n > 100) and in mitotic cells with short spindles (n > 20). (E) The centromeric localization of the Pol III gene locus becomes prominent during prometaphase compared with interphase. For cell-cycle synchronization, exponentially growing cells were arrested in S phase by culturing in YEA medium containing 11 mM hydroxyurea (HU) at 30°C for 4 h, released by further culturing without HU for 1.5 h, and then subjected to IF-FISH experiments. Immunofluorescent images of cells stained for tubulin (cyan) were merged with FISH signals visualizing the c417 locus (green) and centromeres (red). During prometaphase, a few centromeric signals attached with short spindles were observed. Arrowheads indicate the positions at which foci derived from centromeres completely overlap with the c417 locus. Measurements of the distance between the c417 locus and centromeres in the respective images were indicated by linking the image with the histogram.