Figure 7.

Pheromone Response in Δpcl12 Cells.

(A) Formation of conjugation tubes in wild-type and Δpcl12 cells after 6 h of a2 pheromone addition. Note that conjugation tubes in Δpcl12 cells were shorter and showed a dramatic vacuolization. Bars = 15 μm.

(B) Quantitative analysis of conjugative tube formation.

(C) pcl12 expression is enhanced after pheromone treatment. FB1 cells were treated with a2 pheromone or DMSO as a solvent control, and after 6 h, RNA was extracted and submitted to RNA gel blot analysis using pcl12 and 18s RNA probes.

(D) pcl12 is induced by the MAPK cascade. UMN4 cells carrying fuz7^DD, a constitutive allele of the Fuz7 MAPK kinase, under the control of Pcrg1 promoter were grown to an OD₆₀₀ of 0.2 in noninducing conditions (CMD) and then shifted to noninducing conditions (CMD) and inducing conditions (CMA) for 6 h.

(E) Pcl12 is required for sustained MAPK-induced cell cycle arrest. Cells carrying fuz7DD under Pcrg1 control and grown for 6 h in arabinose produce structures resembling conjugation tubes (left column, top). Prolonged incubation (24 h, left column, middle and bottom images) resulted in a single-celled filament that is cell cycle arrested and leaves septa behind. However, in pcl12 defective cells (right column), there is a clear delay in the production of conjugation tubes (top image), and prolonged incubation resulted in a filament that is not cell cycle arrested (see the multiple nuclei in DAPI image, bottom). Bars = 15 μm.