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High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects.

Lowry J, Yochem J, Chuang CH, Sugioka K, Connolly AA, Bowerman B - G3 (Bethesda) (2015)

Bottom Line: Using a combined genome-wide SNP mapping and whole genome sequencing strategy, we have identified the causal mutations in 10 of these mutants.Four of the genes we have identified are conserved, with orthologs known to be involved in membrane biology, and are required for proper development or maintenance of the adult germline membrane architecture.This work provides a starting point for further investigation of the mechanisms that control the dynamics of syncytial membrane architecture during adult oogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403.

No MeSH data available.


Related in: MedlinePlus

SNP mapping data for temperature-sensitive mutations with identified causal mutations. For each mutant, the frequency of homozygous parental alleles was plotted against chromosomal position in bins of either one megabase (gray bars) or half-megabase (red bars). Gene names on each plot are essential genes in which mis-sense mutations were detected. Underlined gene names are those for which deletion alleles exist for performing complementation tests. For complementation tests in which the mutations failed to complement each other, the gene names are in bold; for tests in which the mutations complemented, the gene names are in gray.
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fig2: SNP mapping data for temperature-sensitive mutations with identified causal mutations. For each mutant, the frequency of homozygous parental alleles was plotted against chromosomal position in bins of either one megabase (gray bars) or half-megabase (red bars). Gene names on each plot are essential genes in which mis-sense mutations were detected. Underlined gene names are those for which deletion alleles exist for performing complementation tests. For complementation tests in which the mutations failed to complement each other, the gene names are in bold; for tests in which the mutations complemented, the gene names are in gray.

Mentions: Because mutations in many essential genes required more generally for gene expression or metabolism might indirectly lead to both eggshell defects and larval arrest or sterility, we sought to identify the causal mutations in our set of 20 TS Eos/Ste mutants using a whole genome sequencing strategy before further examining the mutant phenotypes. Chemically mutagenized C. elegans strains have been shown previously to have hundreds of genomic mutations relative to the reference wild-type genome sequence, even after multiple rounds of backcrossing (Sarin et al. 2010). We therefore used an approach that provides both genome-wide SNP mapping and genome sequence data following a single outcross to a polymorphic Hawaiian strain called CB4856 (Doitsidou et al. 2010). In brief, we crossed CB4856 males to homozygous Eos/Ste hermaphrodites at the permissive temperature and then isolated 20–50 homozygous F2 animals scored for embryonic lethality after L4 up-shifts of F3 progeny to the restrictive temperature. We then pooled together for each mutant several individuals from the homozygous (ts/ts) F3 and F4 generations for genomic DNA isolation, and sequenced each pool using paired-end Illumina DNA sequencing (see Materials and Methods). This generated a pool of recombinant chromosomes with a shared lack of introduced CB4856 polymorphisms in the region linked to the causal mutation. This trait can be detected computationally using the CloudMap tool (Minevich et al. 2012), providing genetic mapping data. More specifically, the regions most tightly linked to the causal mutations often have the highest frequency of pure parental alleles (Figure 2). Previous work that applied this approach to viable mutants found that most causal mutations mapped to within 1 Mb of the highest peak in plots of pure parental allele frequency (Doitsidou et al. 2010, Minevich et al. 2012). We therefore used this approach to narrow the genomic regions of interest for these 20 TS Eos/Ste mutations.


High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects.

Lowry J, Yochem J, Chuang CH, Sugioka K, Connolly AA, Bowerman B - G3 (Bethesda) (2015)

SNP mapping data for temperature-sensitive mutations with identified causal mutations. For each mutant, the frequency of homozygous parental alleles was plotted against chromosomal position in bins of either one megabase (gray bars) or half-megabase (red bars). Gene names on each plot are essential genes in which mis-sense mutations were detected. Underlined gene names are those for which deletion alleles exist for performing complementation tests. For complementation tests in which the mutations failed to complement each other, the gene names are in bold; for tests in which the mutations complemented, the gene names are in gray.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4632044&req=5

fig2: SNP mapping data for temperature-sensitive mutations with identified causal mutations. For each mutant, the frequency of homozygous parental alleles was plotted against chromosomal position in bins of either one megabase (gray bars) or half-megabase (red bars). Gene names on each plot are essential genes in which mis-sense mutations were detected. Underlined gene names are those for which deletion alleles exist for performing complementation tests. For complementation tests in which the mutations failed to complement each other, the gene names are in bold; for tests in which the mutations complemented, the gene names are in gray.
Mentions: Because mutations in many essential genes required more generally for gene expression or metabolism might indirectly lead to both eggshell defects and larval arrest or sterility, we sought to identify the causal mutations in our set of 20 TS Eos/Ste mutants using a whole genome sequencing strategy before further examining the mutant phenotypes. Chemically mutagenized C. elegans strains have been shown previously to have hundreds of genomic mutations relative to the reference wild-type genome sequence, even after multiple rounds of backcrossing (Sarin et al. 2010). We therefore used an approach that provides both genome-wide SNP mapping and genome sequence data following a single outcross to a polymorphic Hawaiian strain called CB4856 (Doitsidou et al. 2010). In brief, we crossed CB4856 males to homozygous Eos/Ste hermaphrodites at the permissive temperature and then isolated 20–50 homozygous F2 animals scored for embryonic lethality after L4 up-shifts of F3 progeny to the restrictive temperature. We then pooled together for each mutant several individuals from the homozygous (ts/ts) F3 and F4 generations for genomic DNA isolation, and sequenced each pool using paired-end Illumina DNA sequencing (see Materials and Methods). This generated a pool of recombinant chromosomes with a shared lack of introduced CB4856 polymorphisms in the region linked to the causal mutation. This trait can be detected computationally using the CloudMap tool (Minevich et al. 2012), providing genetic mapping data. More specifically, the regions most tightly linked to the causal mutations often have the highest frequency of pure parental alleles (Figure 2). Previous work that applied this approach to viable mutants found that most causal mutations mapped to within 1 Mb of the highest peak in plots of pure parental allele frequency (Doitsidou et al. 2010, Minevich et al. 2012). We therefore used this approach to narrow the genomic regions of interest for these 20 TS Eos/Ste mutations.

Bottom Line: Using a combined genome-wide SNP mapping and whole genome sequencing strategy, we have identified the causal mutations in 10 of these mutants.Four of the genes we have identified are conserved, with orthologs known to be involved in membrane biology, and are required for proper development or maintenance of the adult germline membrane architecture.This work provides a starting point for further investigation of the mechanisms that control the dynamics of syncytial membrane architecture during adult oogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403.

No MeSH data available.


Related in: MedlinePlus