FIG. 3.
Northern blot hybridization for detection of transcripts in the orf5-orf4-amfR region (A) and S1 nuclease mapping for determination of the transcriptional start point in the presence and absence of A-factor (B). S. griseus HH1 (A-factor negative) and the wild-type strain IFO 13350 (A-factor positive) were grown for the indicated periods. Total RNA was isolated from mycelium grown for the indicated period and subjected to Northern hybridization and S1 mapping. (A) When the amfR sequence (see Fig. 1) was used as the 32P probe, a single transcript of 3.5 kb was seen in the mycelium from the wild-type strain but not from strain HH1. When the orf5 sequence (see Fig. 1) was used, two transcripts of 3.5 and 2.2 kb were detected in the mycelium from the wild-type strain but not from strain HH1. (B) The amounts of transcripts directed by PORF5 were examined by S1 nuclease mapping with mRNAs isolated from the wild-type and HH1 strains. The mRNAs were prepared from the wild-type strain grown for 24 h (lane 1) and 48 h (lane 3) and from strain HH1 grown for 24 h (lane 2) and 48 h (lane 4). Protected fragments were analyzed in parallel with sequencing ladders (lanes: left, T plus C; right, A plus G).