Allelic Ratios and the Mutational Landscape Reveal Biologically Significant Heterozygous SNVs

Chu, Jeffrey Shih-Chieh; Johnsen, Robert; Chua, Shu-Yi; Tu, Domena; Dennison, Mark; Marra, Marco A.; Jones, Steven J.M.; Baillie, David L.; Rose, Ann M.

doi:10.1534/genetics.111.137208

Cited by 12 publications

(20 citation statements)

References 32 publications

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“…For the remaining 76 genomes, we filtered out the background mutations and found on average 44 SNVs that show >40% allelic ratio in the sDp2 region. Most of the SNVs are G > A or C > T changes as expected and previously observed after EMS treatment [30, 38]. We also found an average of 7 small indels of 1–2 bps.…”

Section: Resultssupporting

confidence: 88%

High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans

et al. 2014

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BackgroundEssential genes are critical for the development of all organisms and are associated with many human diseases. These genes have been a difficult category to study prior to the availability of balanced lethal strains. Despite the power of targeted mutagenesis, there are limitations in identifying mutations in essential genes. In this paper, we describe the identification of coding regions for essential genes mutated using forward genetic screens in Caenorhabditis elegans. The lethal mutations described here were isolated and maintained by a wild-type allele on a rescuing duplication.ResultsWe applied whole genome sequencing to identify the causative molecular lesion resulting in lethality in existing C. elegans mutant strains. These strains are balanced and can be easily maintained for subsequent characterization. Our method can be effectively used to analyze mutations in a large number of essential genes. We describe here the identification of 64 essential genes in a region of chromosome I covered by the duplication sDp2. Of these, 42 are nonsense mutations, six are splice signal mutations, one deletion, and 15 are non-synonymous mutations. Many of the essential genes in this region function in cell cycle, transcriptional regulation, and RNA processing.ConclusionsThe essential genes identified here are represented by mutant strains, many of which have more than one mutant allele. The genetic resource can be utilized to further our understanding of essential gene function and will be applicable to the study of C. elegans development, conserved cellular function, and ultimately lead to improved human health.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-361) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 88%

High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans

et al. 2014

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“…To remedy this, one might use balancer chromosomes to maintain lethal mutations (Chu et al 2012). Indeed, some balanced strains fortuitously appear within the collection with a higher proportion of nonsense alleles.…”

Section: Discussionmentioning

confidence: 99%

The million mutation project: A new approach to genetics in Caenorhabditis elegans

et al. 2013

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We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

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“…Genomic DNA was prepared as described and sequenced using Illumina Solexa GAII (BC Cancer Agency Genomic Sciences Centre). Sequences were aligned to C .…”

Section: Methodsmentioning

confidence: 99%

“…Genomic DNA was prepared as described 28 and sequenced using Illumina Solexa GAII (BC Cancer Agency Genomic Sciences Centre). Sequences were aligned to C. elegans genome version WS200 using Burrows-Wheeler Aligner (BWA) 29 under default settings.…”

Section: Whole Genome Sequencingmentioning

confidence: 99%

Degenerin channel activation causes caspase-mediated protein degradation and mitochondrial dysfunction in adultC. elegansmuscle

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Shephard

Chu

et al. 2015

Journal of Cachexia, Sarcopenia and Muscle

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BackgroundDeclines in skeletal muscle structure and function are found in various clinical populations, but the intramuscular proteolytic pathways that govern declines in these individuals remain relatively poorly understood. The nematode Caenorhabditis elegans has been developed into a model for identifying and understanding these pathways. Recently, it was reported that UNC‐105/degenerin channel activation produced muscle protein degradation via an unknown mechanism.MethodsGeneration of transgenic and double mutant C. elegans, RNAi, and drug treatments were utilized to assess molecular events governing protein degradation. Western blots were used to measure protein content. Cationic dyes and adenosine triphosphate (ATP) production assays were utilized to measure mitochondrial function.Results unc‐105 gain‐of‐function mutants display aberrant muscle protein degradation and a movement defect; both are reduced in intragenic revertants and in let‐2 mutants that gate the hyperactive UNC‐105 channel. Degradation is not suppressed by interventions suppressing proteasome‐mediated, autophagy‐mediated, or calpain‐mediated degradation nor by suppressors of degenerin‐induced neurodegeneration. Protein degradation, but not the movement defect, is decreased by treatment with caspase inhibitors or RNAi against ced‐3 or ced‐4. Adult unc‐105 muscles display a time‐dependent fragmentation of the mitochondrial reticulum that is associated with impaired mitochondrial membrane potential and that correlates with decreased rates of maximal ATP production. Reduced levels of CED‐4, which is sufficient to activate CED‐3 in vitro, are observed in unc‐105 mitochondrial isolations.ConclusionsConstitutive cationic influx into muscle appears to cause caspase degradation of cytosolic proteins as the result of mitochondrial dysfunction, which may be relevant to ageing and sarcopenia.

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Allelic Ratios and the Mutational Landscape Reveal Biologically Significant Heterozygous SNVs

Cited by 12 publications

References 32 publications

High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans

High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans

The million mutation project: A new approach to genetics in Caenorhabditis elegans

Degenerin channel activation causes caspase-mediated protein degradation and mitochondrial dysfunction in adultC. elegansmuscle

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