Discovery of Mutations in<i>Saccharomyces cerevisiae</i>by Pooled Linkage Analysis and Whole-Genome Sequencing

Birkeland, Shanda R.; Jin, Natsuko; Ozdemir, Alev Cagla; Lyons, Robert H.; Weisman, Lois S.; Wilson, Thomas E.

doi:10.1534/genetics.110.123232

Cited by 74 publications

(88 citation statements)

References 40 publications

(39 reference statements)

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“…There are a number of other possible antimutagenesis mechanisms involving multiple pathways and candidate genes (Herr et al 2011b). Whole-genome sequencing should greatly facilitate identification of the chromosomal eex alleles (Birkeland et al 2010;Wenger et al 2010;Dunham 2012). These eex alleles may reveal novel pathways of mutation suppression and help clarify the division of labor between Pols e and d at the replication fork.…”

Section: Perspectives and Conclusionmentioning

confidence: 99%

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

2013

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DNA polymerases (Pols) e and d perform the bulk of yeast leading-and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10-to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol d proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol e proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2D, mlh1D, msh3D msh6D, or pms1D mlh3D) but not with partial MMR loss (msh3D, msh6D, pms1D, or mlh3D), indicating that high levels of unrepaired Pol e errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2D eex mutants encoded second-site changes in Pol e that reduced the pol2-4 mutator phenotype between 3-and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol e and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading-and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol e gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity.O RGANISMS must accurately duplicate their genomes to avoid loss of long-term fitness. Consequently, cells employ high-fidelity DNA polymerases (Pols) equipped with proofreading exonucleases to replicate their DNA (reviewed in McCulloch and Kunkel 2008;Reha-Krantz 2010). Mismatch repair (MMR) further ensures the integrity of genetic information by targeting mismatches for excision from newly replicated DNA (reviewed in Kolodner and Marsischky 1999;Iyer et al. 2006;Hsieh and Yamane 2008). These polymerase error-correcting mechanisms, together with DNA damage repair (Friedberg et al. 2006), maintain the genome with less than one mutation per 10 9 nucleotides per cell division (Drake et al. 1998). Defects in proofreading or MMR result in mutator phenotypes characterized by increased rates of spontaneous mutation (Kolodner and Marsischky 1999;Iyer et al. 2006;Hsieh and Yamane 2008;McCulloch and Kunkel 2008; RehaKrantz 2010).Mutator phenotypes can be an important source of genetic diversity, which facilitates adaptation to environmental change. In bacterial and yeast populations, unstable environments favor mutator strains that readily acquire adaptive mutations (Chao and Cox 1983;Mao et al. 1997;Sniegowski et al. 1997...

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Section: Perspectives and Conclusionmentioning

confidence: 99%

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

2013

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“…Once (or if) complementation is successful, the mutated allele of the candidate gene still needs to be sequenced by traditional Sanger sequencing. In contrast, methods based on microarrays (5) or high-throughput DNA sequencing (3,4,11,44) make it possible to quickly generate high-density SNP maps that allow direct identification of specific point mutations or indels by sequence analysis. Moreover, this approach does not rely on a specific set of reference strains.…”

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confidence: 99%

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1 , To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

2011

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With the advent of high-throughput DNA sequencing, it is now straightforward and inexpensive to generate high-density small nucleotide polymorphism (SNP) maps. Here we combined high-throughput sequencing with bulk segregant analysis to expedite mutation mapping. The general map location of a mutation can be identified by a single backcross to a strain enriched in SNPs compared to a standard wild-type strain. Bulk segregant analysis simultaneously increases the likelihood of determining the precise nature of the mutation. We present here a high-density SNP map between Neurospora crassa Mauriceville-1-c (FGSC2225) and OR74A (FGSC2489), the strains most typically used by Neurospora researchers to carry out mapping crosses. We further have demonstrated the utility of the Mauriceville sequence and our approach by mapping the mutation responsible for the only existing temperature-sensitive (ts) cell cycle mutation in Neurospora, nuclear division cycle-1 (ndc-1). The single T-to-C point mutation maps to the gene encoding ornithine decarboxylase (ODC), spe-1 (NCU01271), and changes a Phe to a Ser residue within a highly conserved motif next to the catalytic site of the enzyme. By growth on spermidine and complementation with a wild-type spe-1 gene, we showed that the defect in spe-1 is responsible for the ts ndc-1 mutation. Based on our results, we propose changing ndc-1 to spe-1 ndc , which reflects that this mutation results in an ODC with a specific nuclear division defect.Single nucleotide polymorphism (SNP) maps between organisms with different genetic backgrounds are useful for identifying point mutations that result in an observable phenotype. By mating a mutant with a strain of a different genetic background and then selecting progeny with and without a phenotype, one is able to map the underlying mutation(s) by bulk segregant analysis (29). After undergoing recombination, each progeny exhibiting a specific phenotype will have the same genetic background as the mutant parent in the vicinity of the mutation but a mixture of DNA inherited from either parent in regions of the genome that are unlinked to the mutation. By analyzing DNA from a mixture of phenotypically mutant progeny from the cross, it is possible to define the genetic region containing the mutation and even to identify the mutation itself. This is what is meant by "bulk segregant analysis."Most recently, molecular mapping strategies based on restriction fragment length polymorphisms (RFLP) (28), cleaved amplified polymorphic sequences (CAPS) (20, 22), or microarray-based restriction-site-associated DNA mapping (RAD mapping) (1, 23) have been used to map mutations in Neurospora crassa. These methods can be time-consuming and laborious and therefore expensive. In addition, once the mutation is mapped to a specific region of the genome, additional primer pairs for CAPS mapping need to be ordered and additional SNPs identified to more precisely map the mutation. Typically, researchers will then attempt complementation of phenotypes by candidate genes. On...

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“…Although 1 to 2 SDs might be subtle for ENU screens, a single point mutation affecting a trait by 1 SD is considered quite strong for a QTL. Use of new sequencing technologies has already been applied to the cloning of mutants in Drosophila, Caenorhabditis elegans, Arabidopsis, and yeast (56)(57)(58)(59)(60). Recently Bruce Beutler and colleagues (61) used bulk segregation mapping along with next-generation sequencing to identify ENU mutation with coat color and circling phenotypes in mice.…”

Section: Discussionmentioning

confidence: 99%

Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants

Kumar

Kim

Joseph

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Forward genetic screens have been highly successful in revealing roles of genes and pathways in complex biological events. Traditionally these screens have focused on isolating mutants with the greatest phenotypic deviance, with the hopes of discovering genes that are central to the biological event being investigated. Behavioral screens in mice typically use simple activity-based assays as endophenotypes for more complex emotional states of the animal. They generally set the selection threshold for a putative mutant at 3 SDs (z score of 3) from the average behavior of normal animals to minimize false-positive results. Behavioral screens using a high threshold for detection have generally had limited success, with high false-positive rates and subtle phenotypic differences that have made mapping and cloning difficult. In addition, targeted reverse genetic approaches have shown that when genes central to behaviors such as open field behavior, psychostimulant response, and learning and memory tasks are mutated, they produce subtle phenotypes that differ from wild-type animals by 1 to 2 SDs (z scores of 1 to 2). We have conducted a second-generation (G2) dominant Nethyl-N-nitrosourea (ENU) screen especially designed to detect subtle behavioral mutants for open field activity and psychostimulant response behaviors. We successfully detect mutant lines with only 1 to 2 SD shifts in mean response compared with wild-type control animals and present a robust statistical and methodological framework for conducting such forward genetic screens. Using this methodology we have screened 229 ENU mutant lines and have identified 15 heritable mutant lines. We conclude that for screens in mice that use activity-based endophenotypic measurements for complex behavioral states, this G2 screening approach yields better results.

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Discovery of Mutations inSaccharomyces cerevisiaeby Pooled Linkage Analysis and Whole-Genome Sequencing

Cited by 74 publications

References 40 publications

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

Bulk Segregant Analysis Followed by High-Throughput Sequencing Reveals the Neurospora Cell Cycle Gene, ndc-1 , To Be Allelic with the Gene for Ornithine Decarboxylase, spe-1

Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants

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