Oocyte meiotic spindles are bipolar but assemble without centrosomes. Three Caenorhabditis elegans genes that contribute are that for the calponin homology domain protein, aspm-1; the katanin mei-1; and the kinesin-12 family member klp-18. The results indicate that both microtubule severing and ASPM-1 promote pole assembly, whereas KLP-18 promotes bipolarity.
KLP-7/MCAK regulates kinetochore–microtubule attachment and spindle tension to promote the coalescence of early spindle pole foci, which produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly in C. elegans.
The adult Caenorhabditis elegans hermaphrodite gonad consists of two mirror-symmetric U-shaped arms, with germline nuclei located peripherally in the distal regions of each arm. The nuclei are housed within membrane cubicles that are open to the center, forming a syncytium with a shared cytoplasmic core called the rachis. As the distal germline nuclei progress through meiotic prophase, they move proximally and eventually cellularize as their compartments grow in size. The development and maintenance of this complex and dynamic germline membrane architecture are relatively unexplored, and we have used a forward genetic screen to identify 20 temperature-sensitive mutations in 19 essential genes that cause defects in the germline membrane architecture. 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.
Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.
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