Intragenic transcription from the ASP3 ORF is detected in both ammonium-containing medium and under nitrogen starvation. (A) The wild-type strain was pre-grown in ammonium-containing medium. Cells were then washed with phosphate buffer and transferred to a 3% glucose solution for nitrogen starvation for 2 h. Northern analysis of the two different transcripts of ASP3 was conducted using an ASP3 probe. The blot was subsequently probed against ACT1 as a loading control. The sizes of transcripts are labeled at right. (B,C) Intragenic transcription is independent of GAT1 and GLN3. (B) The wild-type, gat1Δ, gln3Δ, and gat1Δ gln3Δ mutant strains were treated as described in A, and then analyzed by Northern blotting. (C) Strains described in B were pre-grown in ammonium-containing medium, treated with rapamycin, and then analyzed by Northern blotting. In B and C, ASP3 and ncASP3 signals were normalized to ACT1 signals. Values are displayed below the blots.
