Figure 6

Localization of Spp1-GFP and Psf2-YFP after inactivation of Cdc23 using a degron mutant. A. Strain P1205 containing Spp1-GFP in the background of a degron cdc23tstd allele [25] was initially grown at 25°C in the absence of thiamine. 3 h before the temperature shift to 37°C, thiamine was added to repress cdc23 transcription, and 2 h after the shift, cells were analyzed using the chromatin binding assay (focus of Spp1-GFP shown (arrow), bar = 10 μm.) Quantitative analysis is shown in (D). B. Analysis of wild-type Psf2-YFP strain (P1411) in log phase after detergent extraction, showing retention of Psf2 in >50% binucleate (G1 or S phase) cells but not uninucleate (G2) cells. Quantitative analysis is shown in (E). C. Strain P1453, containing Psf2-YFP in the background of a degron cdc23tstd mutant, was treated as in (A) and analyzed by the chromatin binding assay. Cells shown had been shifted to 37°C for 2 h to inactivate Cdc23 before processing using the chromatin binding assay. Quantitative analysis is shown in (E). D. Analysis of data in experiments shown in (A); at least 200 cells were counted for each data point in each experiment. White bars show the percentage of total cells showing Spp1-GFP fluorescence coincident with chromatin (DAPI-staining region) after detergent extraction. Black bars show the percentage of cells showing foci of Spp1-GFP fluorescence in the absence of general nuclear fluorescence. 100% represents total number of cells counted for each data point. E. Analysis of Psf2-YFP in nuclei of wild-type cells, and in a cdc23tstd degron mutant at permissive and non-permissive temperatures, either fixed directly (-detergent), or after detergent extraction (+detergent). For the experiment with the cdc23tstd mutant, the percentages of all cells (i.e. binucleate and uninucleate) are shown.