PMID:20729566

Citation	Timmermann, B, Jarolim, S, Russmayer, H, Kerick, M, Michel, S, Krüger, A, Bluemlein, K, Laun, P, Grillari, J, Lehrach, H, Breitenbach, M and Ralser, M (2010) A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging. Aging (Albany NY) 2:475-86
Abstract	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.
Links	PubMed PMC2954039
Keywords	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
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