A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging

• Published in: Aging (Albany, NY.) Vol. 2; no. 8; pp. 475 - 486
• Main Authors: Timmermann, Bernd, Jarolim, Stefanie, Russmayer, Hannes, Kerick, Martin, Michel, Steve, Krüger, Antje, Bluemlein, Katharina, Laun, Peter, Grillari, Johannes, Lehrach, Hans, Breitenbach, Michael, Ralser, Markus
• Format: Journal Article
• Language: English
• Published: United States Impact Journals LLC 13.08.2010
• Subjects: Alleles; Animals; Base Sequence; Benzene Derivatives - toxicity; Cell Proliferation; Ethyl Methanesulfonate - toxicity; Gene Expression; Genome, Fungal; Haploinsufficiency; Mutagenesis; Mutagens - toxicity; Oxidants - toxicity; Oxidative Stress - genetics; Peroxidases - genetics; Phenotype; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - genetics; Sequence Analysis; Sequence Analysis, DNA
• ISSN: 1945-4589; 1945-4589
• DOI: 10.18632/aging.100187

The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.