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.
Highlights
The free radical theory of aging implies that oxidative stress, and the generation of free radicals, are causally involved in the process of aging [1,2,3]
This theory is supported by many observations, including that yeast mother cells retain oxidatively damaged macromolecules, whereas the daughter cells are formed from a juvenile set of proteins [4], or inherit functional enzymes whereas the damaged species are retained with www.impactaging.com
Mitochondrial ROS production correlates with aging but is not sufficient to alter lifespan [9]. All these observations are further complicated by the fact that mutants which are long-living under one environment/condition do not necessarily show this phenotype under other circumstances, i.e. yeast mutants with prolonged survival at 4°C are not enriched for mutants that are long-living at a higher temperatures [10]
Summary
The free radical theory of aging implies that oxidative stress, and the generation of free radicals, are causally involved in the process of aging [1,2,3] This theory is supported by many observations, including that yeast mother cells retain oxidatively damaged macromolecules, whereas the daughter cells are formed from a juvenile set of proteins [4], or inherit functional enzymes whereas the damaged species are retained with www.impactaging.com. Mitochondrial ROS production correlates with aging but is not sufficient to alter lifespan [9] All these observations are further complicated by the fact that mutants which are long-living under one environment/condition do not necessarily show this phenotype under other circumstances, i.e. yeast mutants with prolonged survival at 4°C are not enriched for mutants that are long-living at a higher temperatures [10]. Oxidative stress and free radicals are important players, their exact role during aging and the complex interplay of the involved genetic and biochemical components has yet to be clarified
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