Fig. 2. Overproduction of Plo1p and Spo12p do not cause release of Clp1p. cdc25-22 clp1-GFP cells carrying either a plasmid expressing plo1⁺ or spo12⁺ from the thiamine-repressible nmt1 promoter were grown at 25°C in the absence of thiamine for 12 hours. The cells were then shifted to 36°C for an additional 4 hours in the absence of thiamine to inactivate the Cdc25-22 mutant protein and arrest cells in G2 phase. The cells were then fixed, stained with DAPI, and representative DAPI, GFP and merged images are shown. Note that the nmt1 promoter does not become active until 12 hours after removal of thiamine.