Generating UBC-1 transgenes for expression analysis in C. elegans

CREVELING 117

Within the ubiquitin proteasome pathway, the E2 ubiquitin-conjugating enzymes interact with E3 ubiquitin ligase to covalently attach a chain of ubiquitin to a lysine residue on the substrate which allows for proteasomal degradation. UBC-1 is an E2 ubiquitin-conjugating enzyme in C. elegans which, when mutated, display poor health, low brood size, uncoordinated movement, and synaptic defects. The yeast homolog of UBC-1 is involved in regulating DNA Repair pathways and histone H2B, but UBC-1 substrates in C. elegans have not been defined. This project seeks to characterize the subcellular expression pattern of UBC-1 by creating two ubc-1::GFP transgenes that can be analyzed in live animals using epifluorescence microscopy. The first transgene was created under the control of a pan-neuronal promoter, while the second transgene used a GABAergic neuron-specific promoter from the unc-25 gene. The promoter of the unc-25 gene was excised from an existing plasmid construct with restriction enzymes PstI and BglII and religated into a plasmid containing the ubc-1::GFP. After the ligation mix was transformed into bacteria, the recombinant plasmid DNA was purified from transformed cells and analyzed by gel electrophoresis. Gel electrophoresis confirmed that the transgenic product was the predicted punc-25::gfp::ubc-1 with predicted band sizes. A similar strategy was used to create a transgene under the pan-neuronal promoter. Both transgenes are now being used in microinjections into C. elegans with a cotransformation marker to generate strains that stably transmit the transgenes. When completed, the transgenic strains will be examined by conventional and confocal fluorescence microscopy to detail the UBC-1 expression pattern.