Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling of Saccharomyces cerevisiae to identify beneficial mutations in strain BY4741 that allow growth at increased pH.

ABSTRACT: The Low MP laboratory strain BY4741 was subjected to a stepwise selection procedure during 120-150 generations, enabling enrichment for beneficial mutations that allow growth at increasing pH. In order to identify the mutations responsible for the ability to grow at high pH, we used tiling arrays to genotype the Low MP ancestor and three evolved strains: two clones from two independent lines that had acquired the ability to grow at pH 8.5 and pH 8.6 (A8.5 and C8.6) and one clone from an intermediate stage of line C (C8.0). The identity and nature of the mutated alleles was further confirmed by direct DNA sequencing.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Ofer Lavi

PROVIDER: E-MEXP-2520 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function. Eight samples were analyzed: WT and the MCY5278 mutant strain, lacking both Msn2 and Msn4, in the presence of 20 mM KOH (pH 8) and in the presence of 20 mM KCl (non-induced conditions) for 10 and 30 min of stress. 2 biological replicates were analyzed for each condition, and dye-swapping was carried out for each comparison of samples. We compared the expression profiles of: 1) WT +KOH vs. WT +KCl after 10 min 2) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 10 min 3)WT +KOH vs. WT +KCl after 30 min 4) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 30 min Total number of chips analyzed: 16.

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Transcriptional profiling of Saccharomyces cerevisiae to identify beneficial mutations in strain BY4741 that allow growth at increased pH.

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