Figure 9.

Crk1 controls the expression of prf1. (A) Crk1 is required for the trigging of prf1 expression after pheromone stimulation. The strains indicated at top were treated for 5 h with synthetic a2 pheromone dissolved in DMSO (+) or with the same volume of DMSO (-). RNA was isolated, and 10 μg of total RNA was loaded per lane. The blot was probed with mfa1 and with rRNA as loading control. (B) Heterologous expression of prf1 bypassed the requirement of Crk1 for pheromone gene expression. The strains indicated at top were grown in minimal medium with NH₄ or NO₃ as the nitrogen source. The prf1^nar allele is transcribed in the presence of NO₃ and is repressed in the presence of NH₄. RNA was isolated, and 10 μg of total RNA was loaded per lane. The blot was probed with mfa1 and with rRNA as loading control. (C) Constitutive expression of prf1 bypassed the requirement of Crk1 for infective filament formation. The strains indicated were spotted on charcoal-containing PD plates. (D) The UAS confers crk1-dependent expression to a reporter gene. (Top) Scheme of the prf1 promoter regulatory region. Prf1-binding sites (PRE, triangles) as well as the UAS region (filled arrow) are indicated. The reporter gene constructs indicated at right, consisting of a minimal promoter (Pmfa1) fused to the green fluorescent protein (sgfp) gene with and without three 85-bp fragments containing the UAS cloned in tandem. The bent arrows represent the transcription initiation points of the reporter gene. (Bottom) Results of fluorimetric measurements using strains harboring the constructs described above, carrying deletions in the genes indicated below. Relative fluorescence units (RFU) were measured and normalized to optical density.