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Identification of novel genes affecting body wall muscle in Caenorhabditis elegans Warner, Adam Dennis

Abstract

Muscular diseases affect many people worldwide. While we have learned much about the sarcomere, the basic building block of muscle cells, there are still numerous questions that remain to be answered. In fact, all of the proteins involved in the formation and maintenance of the sarcomere have yet to be catalogued and studied. We must learn more about proteins expressed in muscle and how they interact so that better treatments for myopathies can be produced. In this thesis, several novel sarcomeric proteins have been identified using Caenorhabditis elegans as a model organism. A list of genes expressed in muscle cells was compiled using available Serial Analysis of Gene Expression (SAGE) and microarray data. By eliminating or severely reducing the expression of each gene using RNA interference (RNAi), we were able to determine which genes were required for proper myofilament organization. Of 23 genes known to affect muscle, 16 were identified using this methodology. In total 119 genes were found to be necessary for proper myofilament organization, 103 of which are genes without a previously characterized role in muscle. In addition, a bioinformatics based screen that utilized tissue specific SAGE data in C. elegans yielded a number of potential candidate muscle affecting genes, and one, C28H8.6 was further studied. C28H8.6 consists of both an ' a ' and a ' b ' isoform, one of which, C28H8.6a has 4 LIM domains and bears striking sequence similarity to the focal adhesion protein paxillin. In animals homozygous for a mutation that knocks out both isoforms of C28H8.6, movement is uncoordinated and development is arrested at the first larval stage. We have demonstrated using isoform specific RNAi that knocking down expression of C28H8.6b has no observable effect on development, while C28H8.6a is required for proper larval growth and movement. Further study on this isoform verified that C28H8.6a co-localizes to dense bodies with a- actinin and is required for proper organization of myofilaments within the sarcomere.

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