Amino acids affect stress response genes in a Rim15-dependent way.

<p>(A) Nutrient response assay of DBY746 (WT) and of the isogenic derivatives involved in stress response gene transcription. Auxo means the addition of the organic compounds necessary to compensate auxotrophies. (B) DBY746 bearing a wild type (WT) or a null (rim15Δ) <i>RIM15</i> allele were grown until day 3, then switched into fresh synthetic medium containing only ‘auxo’ amino acids or ‘all’ amino acids (for a detailed description of auxo mixture see the legend to the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004113#pgen-1004113-g001" target="_blank">figure 1A</a>); after 4 hours RNA was extracted and retro transcribed as reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004113#s4" target="_blank">materials and methods</a> section; quantitative RT-PCR was performed using <i>SOD2</i> specific primers and gene expression levels were normalized using <i>ACT1</i> as an internal control. The bar chart shows <i>SOD2</i> expression level after SDC incubation with respect to <i>SOD2</i> expression after incubation only with L, H, W as amino acids source. The data represent the mean ± standard errors of three different experiments. (C) Beta galactosidase assay of PDS and STRE reporters. Post-diauxic yeast cell (about 10⁸ cells at day 2 in liquid culture) were exposed to nutrients (4 hrs with the indicated mixtures). Cells were then disrupted and beta galactosidase activity was measured. The activity obtained with medium containing only the amino acids necessary to compensate strain auxotrophies (auxo) was referred to as 100% activity. Statistical significance was evaluated by 2-tailed T-test for groups with unequal variants. Data shown are mean and standard deviation of three independent samples assayed. * , p<0.5; **, p<0.05; ***, p<0.01.