Date of Defense
Date of Graduation
Human Performance and Health Education
UNC-82 is a kinase that is necessary for the correct development of body wall muscle in the invertebrate Caenorhabditis elegans. UNC-82 is a 1600 amino acid protein with homology to AMPK- related kinases. In vertebrates, UNC-82 orthologs may contribute to regulation of skeletal muscle metabolism. In invertebrates, UNC-82 is involved in the organization of myosin thick filaments in sarcomeres. To further explore the function of UNC-82, we looked at the effects of inactivating the catalytic domain of the kinase in C. e/egans. Site-directed mutagenesis was used to introduce a lysine to histidine substitution in template DNA that encodes UNC-82. The substitution inactivates the catalytic domain of the kinase, thus, eliminating the transfer of a phosphate to the substrate. The transgene was injected into wildtype C. e/egans gonads, and the organisms allowed to reproduce. The progeny were screened for transgenic Fl animals, and three independent stable transgenic lines were obtained. The muscle structure was examined using polarized light microscopy. The worms showed no apparent muscle defects, indicating that there were no dominant effects of the mutant UNC-82 protein on the action of the endogenous wild-type UNC-82 in organizing muscle. Fluorescence microscopy was used to determine the distribution of the mutant UNC-82 protein in the cell. The mutant protein showed disrupted patterning, including bright round accumulations within the muscle cell. The disrupted patterning and accumulations do not appear to affect locomotion. Toxicity that led to sterility and worm death was found in transgenic lysine to histidine worms. In the e1220 missense mutant background, worms had increased disrupted patterning and round accumulations. Control worms expressing a construct with the same promoter and YFP tag but wild-type kinase domain sequence had no muscle abnormalities or toxicity in wild type, and improved the muscle phenotype of the e1220 mutant. Therefore we conclude that the inactivated catalytic domain negatively affects muscle development and organismal viability.
Martinez Navarrete, Wenceslao, "Analyzing the Effects on Muscle in an Inactivated Catalytic Domain of UNC-82" (2019). Honors Theses. 3189.