Fig. 8. Increased expression of flp 1⁺ arrests cells in G₂. A single copy of the flp 1⁺ gene was integrated at the leu 1 locus of wild-type cells. Expression of flp 1⁺ was induced by incubation in medium without thiamine at 25°C. Samples were removed at intervals, processed for FACS analysis, and fixed and stained with Calcofluor and DAPI. (A) Uninduced control cells (top), cells 20 hours after induction (bottom). (B) FACS analysis of uninduced (top) and induced (bottom) cells. The position of the G₁ peak was established by incubating cells in 12 mM hydroxyurea (HU) for 3 hours. -N are cells grown in medium lacking a nitrogen source. (C) leu 1-32 cells were transformed to leucine prototrophy with a REP3 plasmid expressing flp 1p(C286S). Expression was induced for 24 hours at 25°C, cells were fixed, and stained with DAPI and Calcofluor. (D) leu 1-32 cells were transformed to leucine prototrophy with a pREP3-flp 1⁺. Expression was induced (-T) and samples were removed at the indicated times thereafter. Protein extracts were prepared and western blots probed for the indicated antigens. For cdc25p, the two arrows indicate the position of the upper (hyper-phosphorylated) and lower bands. A portion of culture resuspended in medium containing thiamine served as a control. (E) leu 1-32 cdc25-HA₃ was transformed with either pREP3 or pREP3-flp 1⁺ and expression was induced for 20 hours at 25°C. Protein samples were prepared and a western blot was probed with 12CA5. The samples were run in adjacent lanes, on the same gel.