title: A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging.
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.
authors: Next Generation Sequencing Group, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany.
authors: Bernd
authors: B
authors: Timmermann
authors: Stefanie
authors: S
authors: Jarolim
authors: Hannes
authors: H
authors: Russmayer
authors: Martin
authors: M
authors: Kerick
authors: Steve
authors: S
authors: Michel
authors: Antje
authors: A
authors: Krüger
authors: Katharina
authors: K
authors: Bluemlein
authors: Peter
authors: P
authors: Laun
authors: Johannes
authors: J
authors: Grillari
authors: Hans
authors: H
authors: Lehrach
authors: Michael
authors: M
authors: Breitenbach
authors: Markus
authors: M
authors: Ralser
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001555:Benzene Derivatives / Q000633:toxicity; D049109:Cell Proliferation; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D015870:Gene Expression; D016681:Genome, Fungal; D057895:Haploinsufficiency; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000235:genetics*; D010544:Peroxidases / Q000235:genetics*; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA
chemical_list: D001555:Benzene Derivatives; D009153:Mutagens; D016877:Oxidants; D029701:Saccharomyces cerevisiae Proteins; D005020:Ethyl Methanesulfonate; D010544:Peroxidases; C496253:Tsa1 protein, S cerevisiae; C007164:cumene hydroperoxide