Mitotic Spindle Assembly and Function in C. elegans

University Of Oregon, Eugene OR

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Abstract

DESCRIPTION (provided by applicant): The goal of this competitive revision is to identify macromolecular interactions that influence microtubule stability and function in the nematode Caenorhabditis elegans, by finding conditional mutations in essential genes that are required for early embryonic cell division. Conditional (heat-sensitive) mutations remain the most valuable genetic tool available for the in vivo investigation of essential gene requirements, and C. elegans is unique as an animal model in which one can feasibly isolate large numbers of rare conditional mutations. This proposal seeks to substantially expand our identification of conditional mutations in essential C. elegans genes that influence microtubule stability and function. This will be done by (i) identifying and genetically characterizing a collection of early embryonic cell division defective conditional C. elegans mutants; (ii) using next generation whole genome DNA sequencing to rapidly map and identify the causal mutations in the mutant collection; and (iii) using DNA transformation to verify the identity of the causal mutations through rescue of conditional lethality with wild-type copies of the affected genes. Specific Aim 1 focuses on an examination of 2000 newly isolated temperature-sensitive, embryonic- lethal C. elegans mutants to identify those mutants with early embryonic cell division defects. These mutants will be examined using videomicroscopy of early embryogenesis to determine the nature and penetrance of the cell division defects. Genetic analysis will be done to determine if the mutations exhibit Mendelian inheritance and if the mutations are recessive or dominant. Specific Aim 2 focuses on the use of a polymorphic C. elegans strain and Illumina-based whole genome DNA sequencing to map the location of conditional mutations and to identify within whole genome sequences the likely causal mutations responsible for embryonic lethality. To our knowledge, we are the first laboratory to apply this high throughput and cost-effective approach to the mapping and positional cloning of large numbers of lethal C. elegans mutants. Specific Aim 3 seeks to confirm the identity of causal mutations by testing for rescue of viability after ballistic transformation of conditional mutants with wild-type copies of the implicated genes.