High-Throughput Cloning of Temperature-Sensitive Mutants Affecting Germline Membrane Dynamics in Caenorhabditis elegans
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At the core of Genetics as a field of study is the ability to understand how changes at the genetic level cause changes at the level of the organism. As a part of carrying out these types of investigations, genetic mutations are induced at random in the DNA of the organism of interest; however, for any meaningful associations between genotype and phenotype to be made, it is necessary to identify the causal molecular lesion. Massively parallel sequencing (MPS)-based methods have been developed to alleviate the difficulty in making this identification. In this dissertation, I describe the use of this technology to identify the causal mutation in mutant strains of the nematode Caenorhabditis elegans. The mutants themselves are of particular interest due to the nature of the mutations induced, the genes affected by these mutations, and the biological processes they affect. These mutants are temperature-sensitive, and embryonic lethality occurs when late stage larvae are moved to the restrictive temperature (26°C); specifically, the embryos are eggshell defective and either swell or lyse when dissected into water. When these same mutants are moved to 26°C as early larvae, they grow into sterile adults, the sterility being due to germline development defects. Based on commongenetic requirements for eggshell formation and germline development, we expected to find mutants with defects in membrane trafficking, an expectation borne out by the results. Using MPS-based cloning techniques, I have identified the causal mutation in a subset of these mutants and have characterized the germline phenotypes observed in these mutants. These novel phenotypes provide insight into the development and maintenance of germline membrane architecture and dynamics. This dissertation includes unpublished co-authored material.