UBC Theses and Dissertations
Characterization and developmental expression patterns of the ubiquitin-conjugating enzyme UBC-2 in the nematode Caenorhabditis elegans Stevens, Tracy Alison
The role of the essential ubiquitin-conjugating enzyme, UBC-2, during development in the nematode Caenorhabditis elegans was examined. The mutant strain, let-70, which corresponded to ubc-2, was L3 larval lethal, and indicated that UBC-2 was required post-embryonically. Additionally, a maternal contribution of UBC-2 was necessary for embryonic development. Detailed analysis of let-70(s1132) and let-70(s689) mutant phenotypes revealed defects in muscle cell positioning, attachment and sarcomere assembly. Intestinal cell attachment and maturation were also affected. A small number of let-70(s689) mutants developed into sterile adults with gonadal defects. In these mutants, the somatic gonad and vulva did not develop properly, only a small number of germ cells were produced, and oocytes and spermatocytes failed to mature. UBC-2 protein was undetectable in let-70(s1132) and let-70(s689) mutants. The let-70(s689) mutation altered the splice donor site of the fourth intron. In affected animals, the fourth intron was not removed from the ubc-2 pre-mRNA, and the transcript was subject to smg-mediated mRNA surveillance. The mutant UBC-2 protein produced in let-70(s1132) appeared to be highly unstable. The let-x(s2293) strain was identified in a non-complementation screen for additional alleles of let-70 and was a large deletion that spanned several essential genes, including ubc-2. The UBC-2 expression pattern during development was determined by immunofluorescence staining and examination of transgenic animals carrying a UBC-2::GFP fusion construct. UBC-2 expression was largely tissue general in embryonic stages. Post-embryonically, UBC-2 remained tissue general, although a large amount of protein became concentrated in the nucleoli of a number of cells, including intestinal, body wall muscle, hypodermal, somatic gonad, germline, pharyngeal muscle, pharyngeal-intestinal valve and several neurons. In addition, UBC-2 localized to the dense bodies or M-lines of body wall muscle, and a few neuronal cell processes. In let-70 mutants, this expression pattern was lost. Somatic and germline expression of an extrachromosomal array that contained ubc-2 could rescue let-70(s689), but not let-70(s1132) mutants. A cold-sensitive allele of ubc-2 was constructed which was also capable of rescuing let-70(s689) mutants in a temperature-dependent manner. ubc-2 forms part of a polycistronic unit with two other genes, apg-7 and Y5F2A.4. Both ubc-2 and Y5F2A.4 are exclusively SL1-spliced while apg-7 is SL1- and SL2-spliced. Comparison of the ubc-2 polypeptide sequence between C. elegans and C. briggsae revealed 100% identity; however, C. briggsae UBC-2 was unable to rescue let-70 mutants. A high degree of synteny exists between the genomic sequences surrounding the C. elegans and C. briggsae ubc-2 genes, with the exception of sequences immediately surrounding the ubc-2 operon.
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