Figure 3.

Induction of 1.2-kb HAC1 mRNA correlates with the UPR. (A) The ERN⁺ (KMY1105) and ern1Δ (KMY1115) strains were transformed with a vector alone (V) or a Ern1p expression plasmid (a single-copy or multicopy plasmid indicated by Cen or 2 μm, respectively). Transformants were grown at 30°C in SC(−Ura, Leu) medium to midlogarithmic phase, and aliquots were incubated for 1 h in the presence (+) or absence (−) of tunicamycin (TM). Total RNAs were extracted and analyzed by Northern blot hybridization using DNA probes specific for HAC1, KAR2, or yeast actin ACT1. Positions of the 1.4-kb and 1.2-kb HAC1 mRNAs are indicated. In addition to these two mRNA species, tunicamycin treatment produced a faint band of 0.7 kb, that was not analyzed in this report. (B) The ERN⁺ and ern1Δ strains with the sec53 background in which the UPRE-CYC1-lacZ reporter gene had been integrated into the respective chromosome (KMY2105 and KMY2115, respectively) were grown at the permissive temperature of 23°C in SC(−Ura) medium to midlogarithmic phase, and aliquots were incubated for an additional hour at 23°C or at the seminonpermissive temperature of 30°C. Total RNAs were extracted and analyzed as in A. (C) The ERN⁺, SEC⁺ strain (KMY1105) was treated with tunicamycin at 30°C for the times indicated as in A, and RNA was similarly analyzed by Northern blot hybridization. Relative radioactivities of each band (HAC1, KAR2, and PDI1) were determined using BioImaging Analyzer BAS-2000 (Fuji Photo Film), corrected for ACT1 values, and plotted after normalization to the 0 time values.