Fig. 9. The expression of the mfa1 gene is regulated by the levels of Cru1 and Clb1. (A) Overexpression of cru1 induces mfa1 expression in nutrient-rich medium. The strains indicated on the top were incubated for 4 hours in YPD (noninducing conditions) or YPA (inducing conditions). RNA was isolated and 10 µg of RNA was loaded per lane. The blot was probed with mfa1, cru1 and rRNA. The cru1 mRNA appears as two different populations indicated by arrows: Pcru1 is the mRNA expressed from the native cru1 promoter and Pcrg1 is the mRNA expressed from the chimeric construction inserted in the ip locus. The different size is the result of the different transcription start point in both promoters. (B) Depletion of Clb1 bypassed the requirement of Cru1. The strains indicated on the top were grown in minimal medium with NO₃ (permissive conditions for the clb1^nar allele) until OD₆₀₀ of 0.2 and then were incubated for 4 hours in CMD (restrictive conditions for the clb1^nar allele). RNA was isolated and 10 µg of RNA was loaded per lane. The blot was probed with mfa1 and rRNA.