Cell cycle dependent localization of Cdc25.
A. PCR amplification of Cdc25 and upstream sequence. B. Integration of pcdc25-GFP plasmid into the native cdc25 promoter by homologous recombination. C. Post-integration structure of cdc25-GFPint strain. D. Logarithmically growing cells expressing Cdc25-GFP from the chromosomal cdc25 promoter in YEA imaged by fluorescence microscopy. Bar represents 10 µm. E. Fluorescence images of Cdc25-GFP and Cdc25(9A)-GFP at mid-logarithmic phase in YEA at 30°C. Logarithmic phase 30°C cultures imaged using fluorescence microscopy. Bar represents 10 µm. F. Nuclear∶Cytoplasmic ratio of Cdc25-GFP and Cdc25(9A)-GFP fluorescence in logarithmically growing populations of cells. (n = 200. Each size category contains 20 to 40 cells. Error bars represent ±1 s.d. from the mean). G. Western blot comparing the relative Cdc25-GFP levels in the strains indicated. Cultures were grown to mid-log phase, harvested, lysed and analysed by SDS-PAGE and western blotting using mouse anti-GFP primary and anti-mouse HRP secondary antibodies.