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Cloning, characterization and functional analysis of UBC-2, a gene encoding a ubiguitin-conjugating enzyme in the nematode Caenorhabditis elegans

University of British Columbia

ubc-2, a gene encoding a ubiquitin conjugating enzyme (E2) in Caenorhabditis elegans was cloned, and the expression and functions of this gene during the development of the nematode were investigated using molecular biological and genetic approaches. The ubc-2 gene was cloned by virtue of its high amino acid similarity to the yeast and Drosophila genes UBC4/5 and UbcDl, respectively. UBC4 and UBC5 encode ubiquitin conjugating enzymes which mediate selective degradation of short-lived and abnormal proteins in yeast, and are essential for viability under stressed conditions, ubc-2 encodes a protein sharing 85% amino acid identity with UBC4/5, and 95% with its Drosophila homolog UbcDl. Yeast complementation tests showed that UBC-2 rescues the growth deficiency of a yeast ubc4ubc5 mutant strain under normal and stressed conditions. These results suggest that this type of E2 is conserved among eukaryotes, and may play fundamental roles within cells. Northern blot analysis showed that ubc-2 is abundantly expressed in all developmental stages throughout the life cycle of C. elegans. Unlike yeast, the expression of this gene is not induced by heat shock or cadmium. ubc-2-lacZ fusions were constructed and transformed into the nematode to study the tissue specificity of ubc-2 expression in different life stages. The expression of the transgenes was observed from early embryogenesis (gastrulation) onward. Transgene expression was largely tissue general in embryonic and early-mid larval stages, but restricted to the nervous system in adults. UBC-2 protein was overexpressed in E. coli, and polyclonal antibodies were raised against the purified protein. In situ immunofluorescent staining showed that the endogenous UBC-2 protein is indeed specifically expressed in the adult nervous system. UBC-2 is the first ubiquitin-conjugating enzyme found to be tissue-specific in animals. Transformation of the nematode with constructs expressing antisense RNA against ubc-2 was lethal in early-mid larvae, suggesting that this gene is essential for early stage development. EMS-generated lethal mutations which map near ubc-2 were tested for rescue by transformation with a ubc-2 clone. One of the lethal mutations, let-70, was rescued. Both existing mutant alleles of let-70, SI 132 and S689, contain point mutations in ubc-2. SI 132 has a C to T substitution which leads to a His to Tyr change at residue 75. S689 contains a G to A transition at the splice donor site of the last intron of the gene. Both alleles exhibit developmental arrest in larvae, but the blockage in si 132 seems to be earlier (L1-L2 larval stage) than that in s689 (L3 stage), suggesting that the mutation in sll32 results in a more severe defect in the protein's function. These results clearly show that His75 and the C-terminal (amino acids 134-147) of UBC-2 are important for its functions. Since yeast UBC4 and UBC5 encode proteins with redundant functions, and knocking out either gene alone reveals no apparent cellular defect, it has been commonly assumed that multiple genes encoding functionally similar E2s would exist in multicellular organisms and that single gene mutations would therefore have little or no effect on organism viability. However the studies presented in this thesis suggest that in multicellular organisms, related E2 proteins may have become specialized for different life stages, different tissues or different environmental conditions, and thus essential for development and survival.

Thesis/Dissertation

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2009-03-19

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http://hdl.handle.net/2429/6227

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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