Cluster analysis of 150 genes with increased expression in the nrg1Δ nrg2Δ double mutant relative to that in the wild type. Clusters are indicated to the left. (Lanes 1 to 3) Expression of genes in glucose-grown nrg1Δ, nrg2Δ, and nrg1Δ nrg2Δ strains (MCY4710, MCY4706, and MCY4714) is compared to that of the wild type (WT, MCY4577). Genes with twofold-or-greater increases in expression in the double mutant relative to that of the wild type are shown; black bars mark 41 genes with expression increased by more than threefold. (Lanes 4 to 7) Glucose-grown WT cells (MCY4565) were shifted to 0.05% glucose for 15, 30, 45, and 60 min; expression is compared to that at time point zero. (Lanes 8 and 9) Expression of genes in glucose-grown reg1Δ (MCY4569) and reg1Δ gal83Δ (MCY4571) cells is compared to that in WT (MCY4565). (Lane 10) Expression in reg1Δ cells is compared to that in reg1Δ gal83Δ cells. Scale indicates increase (red) or decrease (green) (n-fold) relative to that of the control (black). See Table S1 in the supplemental material for data.

Northern blot analysis. RNA was prepared from glucose-grown cultures of WT, nrg1Δ, nrg2Δ, and nrg1Δ nrg2Δ strains (MCY4577, MCY4710, MCY4706, and MCY4714) and subjected to Northern blot analysis. Blots were hybridized with probes specific for each of the indicated RNAs and for the loading control, the small nucleolar RNA U3 encoded by SNR17A and SNR17B.

Cluster analysis of Nrg-repressed genes under various growth and stress conditions. Clusters are indicated to the left. Data for the 150 Nrg-repressed genes from Fig. were combined with data from DeRisi et al. (), Gasch et al. (), and Causton et al. (). Top rows show expression of NRG1 and NRG2. Triangles indicate a time course; in each case the zero time point is the reference. Expression during steady-state growth in ethanol (Eth), galactose (Gal), raffinose (Raf), glucose (Glu), mannose (Man), and sucrose (Suc) was compared to the pooled data for all carbon sources. Nitrogen depletion was achieved by a shift to limiting ammonium sulfate. Diamide, peroxide (H₂O₂), NaCl, and sorbitol were added to final concentrations of 1.5 mM, 0.4 mM, 1 M, and 1.5 M, respectively. Heat shock refers to a shift from 25°C to 37°C, and cold shock, vice versa. Heat transitions were shifts from various temperatures (17°C to 37°C) to 37°C for 20 min. Alkali and acid indicate media at pH 7.9 and at pH 4.0, respectively. The rightmost columns show expression in the msn2Δ msn4Δ mutant exposed to acid. Scale indicates change (n-fold) relative to the unstressed condition or time point zero. Bars mark genes containing C₄T, C₃TC, or (A/G)(A/G/C)C₃T, and the shade of blue corresponds to the number of elements, as indicated in the scale.

Regulation of stress response genes by Nrg1/Nrg2. Strains were (A and B) MCY5326, MCY5378, MCY5338, and MCY5385 (Σ1278b) and (C and D) MCY5444 and MCY5451 (W303). Cultures were grown to mid-log phase in YPD. (Panel A) Cells were collected by filtration and transferred to YPD containing 1 M NaCl. (Panels B to D) YPD containing 5 M NaCl was added to a final concentration of (panel B) 75 mM, (panel C) 0.5 M, or (panel D) 0.3 M NaCl. Samples were collected at the indicated times. RNAs were prepared, separated by denaturing agarose gel electrophoresis, and subjected to Northern blot analysis. Note that the 15-min sample for the nrg1Δ nrg2Δ mutant in panel C is underloaded, as judged by the loading control, the ACT1 (actin gene) RNA.

Assays of nrg1Δ nrg2Δ mutants for salt, NaAsO₂, H₂O₂, and cold tolerance. (A) WT and mutant strains (MCY4521, MCY4523, MCY4525, and MCY4549; S288C background) were grown overnight in YPD and diluted to an optical density at 600 nm of 0.3, and serial threefold dilutions were spotted on YPD and YPD plus 1 M NaCl. Plates were incubated for 5 days and photographed. (B) Strains MCY5326 and MCY5378 (Σ1278b background) were grown and spotted as described for panel A on YPD and YPD plus 150 mM NaCl. Plates were photographed after 2 days. (C) Strains MCY5326 and MCY5378 were grown to mid-log phase in YPD, and equal amounts of cells were plated on YPD. Sterile disks soaked with 5 μl of H₂O, 100 mM NaAsO₂, or 1.1 M H₂O₂ were placed on the plates. Plates were incubated for 2 days and photographed. (D) Wild-type and mutant strains (MCY5314 and MCY5414; S288C background) were grown to mid-log phase, and multiple aliquots (5 μl) were spotted onto sterile Whatman filters. Filters were placed in sterile tubes and incubated at −20°C for the indicated times. Cells were then resuspended in 1 ml of YPD, incubated for 15 min at 30°C, and plated onto YPD. Viability is shown relative to that at the initial time point prior to freezing. Data in top and bottom panels are from two independent experiments using different tubes and different freezers.