Figure 1 .
Northern analysis. (A) Verification of Δpre-2 and rescued strains. Total RNA was isolated from 6-day-old SCM plate cultures of mat A and mat a wild-type (WT) strains 74A and 74a; Δpre-2::hph knockout mutants 23a and 32a (Δpre-2), and Δpre-2::hph+, pre-2+ complemented strains 23-1 and 32-4 (COMP). Approximately 20 μg of total RNA was subjected to Northern analysis using the pre-2 ORF as a probe (see Materials and Methods). rRNA is the loading control. (B) pre-2 mRNA levels in various tissues. Strains were wild-type mat a (74a) or mat A (74A), as indicated. RNA samples representing vegetative tissues were isolated from 74a grown under the following conditions: freshly harvested conidia (0 hr), conidia germinated in liquid VM for 5, 14, and 24 hr, and on solid VM plates for 3 days. RNA representing unfertilized sexual tissues was prepared from 74A or 74a grown on solid SCM plates for 6 days. RNA for fertilized sexual tissues was from perithecia harvested 3 and 6 days after fertilization, respectively, from a cross of 74a with 74A. Northern blots were probed as in A. rRNA is used as a loading control. (C) Expression of pheromone receptor genes in mating-type mutants. Total RNA was prepared from 6-day-old SCM plate cultures of wild-type mat A strain 74A (WT A), wild-type mat a strain 74a (WT a), the mat Am44 and mat am1 sterile mutants, and the vegetative incompatibility mutant mat am33. Northern blots were probed using the pre-2 (A) or pre-1 ORF fragments (see Materials and Methods). rRNA is a loading control. (D) Pheromone receptor and pheromone precursor mRNA levels in strains lacking pre-1 and/or pre-2. Strains are wild-type (WT) 74A (WT mat A), 74a (WT mat a), 16A (Δpre-1, mat A), 16a (Δpre-1, mat a), 23A (Δpre-2, mat A), 23a (Δpre-2, mat a), P1P2A (Δpre-1 Δpre-2, mat A), and P1P2a (Δpre-, Δpre-2, mat a). Total RNA was isolated from 6-day-old SCM plate cultures. Northern blots were probed using pre-1, pre-2, ccg-4, and mfa-1 specific probes (see Materials and Methods). rRNA is the loading control.