Distinctive effects of erh1Δ and mmi1Δ on pho1 expression. (A) Northern analysis. RNAs isolated from WT, erh1Δ, and mmi1Δ cells were resolved by formaldehyde-agarose gel electrophoresis and stained with ethidium bromide to visualize 28S and 18S ribosomal RNAs (bottom panel). The RNAs in the gel were transferred to membrane and hybridized to the pho1 probe. Annealed probe was visualized by autoradiography. The positions and sizes (in kilobases) of RNA markers are indicated on the left. The pho1 mRNA and prt–pho1 readthrough transcript are indicated on the right. The three RNA samples were analyzed in parallel on the same gel, probed on the same membrane, and visualized in a single exposure; intervening lanes of the stained gel and blot were cropped out during figure preparation. (B) Primer extension analysis. Total RNA from WT and mmi1Δ cells was analyzed by reverse transcription primer extension using a mixture of radiolabeled primers complementary to the pho1 and act1 mRNAs. The reaction products were resolved by denaturing PAGE and visualized by autoradiography. The positions and sizes (nt) of DNA markers are indicated on the left. (C) mmi1+ (WT) and mmi1Δ cells deleted for pho1 and bearing the prt–pho1 reporter plasmid (shown in Fig. 3A) were assayed for acid phosphatase activity as described in Figure 3D.
