PMC

A gcn5Δ strain displays genetic interaction with DNA-bound kinetochore components. Yeast strains of genotypes W303, gcn5Δ (yPO4), cse4-1 (ySP1090), cse4-1, gcn5Δ (SVY102), mif2-3 (PDW370), mif2-3, gcn5Δ (SVY060), skp1-3(ySP422), skp1-3 gcn5Δ (SVY082), cep3-1 (PDW490), cep3-1 gcn5Δ (SVY069), ctf13-30 (ySP1107), ctf13-30, gcn5Δ (SVY081), ndc10-1 (ySP22), and ndc10-1 gcn5Δ (SVY083) (see Table ) were grown to log phase at 28°C. Fivefold serial dilutions were plated on YEPD and incubated at the indicated temperature for 48 h.

Defective nuclear migration and minichromosome loss in a gcn5Δ strain. Cells were grown at 28°C in YEPD, and nuclei were stained with propidium iodide. (A) Microscopic samples of large-budded doublets carrying two segregated nuclei, duplicated nuclei passing through the bud neck, and unsegregated nuclei in dividing cells. (B) Nuclear distribution in wild-type (WT) (W303) and gcn5Δ (yPO4) strains. (C) Percentage of centromeric plasmid loss (pDK243) measured in WT (W303) and gcn5Δ (yPO4) strains at either 28°C or 37°C after growth for 12 generations in nonselective medium. Error bars indicate standard deviations.

Deletion of Gcn5p induces G₂ delay and temperature sensitivity. (A) gcn5Δ strain accumulates in G₂/M. Flow cytometry analysis of wild-type (WT) (W303) and gcn5Δ (yPO4) strains is shown. (B) Fivefold serial dilutions of WT (W303), gcn5Δ (yPO4), and SAGA-deficient spt20Δ (SVY084) strains spotted on YEPD and grown at 25°C, 28°C, or 37°C for 48 h. (C) Analysis of cell morphology in WT (W303) and gcn5Δ (yPO4) strains grown at 28°C or 37°C. Percentages of G₁ cells (white bars), G₂/M doublets (gray bars), and G₂/M doublets with elongated buds (black bars) were determined (n = 450). Error bars indicate standard deviations.

Absence of Gcn5p causes sensitivity to microtubule-depolymerizing agents and spindle elongation defects. (A) Fivefold serial dilutions of wild-type (WT) (W303), gcn5Δ (yPO4), and spt20Δ (SVY084) strains were grown on YEPD supplemented with increasing benomyl concentrations (5, 10, and 15 μg/ml) and grown at 28°C for 60 h. (B) In vivo microscopy of spindle elongation visualized with GFP-tubulin (upper fluorescent panels). WT (W303) and gcn5Δ (yPO4) cells growing in SD medium were observed on microscope slides for 240 min. A selection of photographs collected every 3 min is shown. Nomarski pictures are given (bottom row) to show cell septation in a gcn5Δ strain.

Deletion of Gcn5p induces strong delay of G₂, spindle elongation, and chromatid separation. (A) FACS analysis of cells released in YEPD medium after α-factor blockage, collected every 10 min, and examined. WT, wild type. (B) After release, we calculated the percentages of budding cells (n = 250) (open circles), elongated anaphasic spindles (triangles), and separated centromeric GFP-tagged dots (closed circles) taken at 10-minute intervals. (C) Spindle (tubulin immunostaining) and nuclear (DAPI) images of WT (W303, left panel) and gcn5Δ (yPO4, right panel) G₂/M large-budded cells collected at 90 min and 130 min, respectively, showing normal (WT) or unaligned short (gcn5Δ) spindles. (D) Distribution of short unaligned spindles in WT and gcn5Δ strains.

Gcn5p physically interacts with the centromere and is required for the structure of variant centromeric nucleosome. (A) Schematic restriction map of the CEN3 region. The locations of DraI (black triangles) in the CDEII-CEN3 (II) region and EcoRI flanking sites (E) are indicated. The probe used for panel C is shown as gray bar. (B) Ethidium bromide staining of in vivo restriction of chromatin (wild type [WT] [W303] and gcn5Δ [yPO4] strains) at increasing DraI concentrations (0, 50, 100, and 150 U/ml), followed by EcoRI digestion after DNA extraction. (C) CEN3 accessibility to DraI cuts, showing the full-sized E/E 5.1-kb band and the E/D band of 2.2 kb corresponding to in vivo CDEII-CEN3 accessibility in WT and gcn5Δ strains. (D) Percentages of DraI cuts on the CDEII-CEN3 region and on a noncentromeric gene sequence (GLT1), used as an internal standard of digestion. Error bars indicate standard deviations. (E) ChIP of the inner kinetochore component Cep3p and 9Myc-Gcn5 in an epitope-tagged Gcn5-9Myc strain (SVY049). PCR amplification carried out with primers external to the core centromeric locus produced a 300-bp band spanning CEN3 and CEN16. As control, a telomeric sequence (TEL) was used in all ChIP samples.