We are interested in studying the function of the ubiquitin system during C. elegans development. Ubiquitin is a small, highly conserved protein found in all eukaryotic cells. The conjugation of ubiquitin to substrate proteins occurs through a multi-step enzymatic process and generally targets the substrate proteins for degradation. A gene encoding the ubiquitin conjugating enzyme,
ubc-2, has been previously cloned and characterized in our lab (Zhen et al., 1993; Zhen et al., 1996).
ubc-2 belongs to a highly conserved gene family which includes yeast UBC4 and UBC5.
ubc-2 can functionally substitute for UBC4 and UBC5 in yeast
ubc4ubc5 double mutants. Northern analysis has shown that
ubc-2 is abundantly expressed at all life stages during C. elegans development. Transgenic worms containing
ubc-2-lacZ fusions show a tissue general pattern of
ubc-2 expression during embryonic and larval stages which becomes restricted to the nervous system in adults. This expression pattern has been confirmed by in situ immunoflourescence. To further examine the role of
ubc-2 in development, three
ubc-2 mutants were isolated by complementation analysis:
let-70(
s689),
let-70(
s1132), and
let-70(
s2293). Both
let-70(
s689) and
let-70(
s1132) are L3-L4 lethals. Sequencing analysis revealed that
let-70(
s689) has a G to A transition in the splice donor site of the last intron (Zhen et al., 1996). The
let-70(
s1132) mutation is a C to T change which results in the substitution of histidine 75 for tyrosine. His75 is a highly conserved residue among all the ubiquitin-conjugating enzymes sequenced to date. The
let-70(
s2293) strain is embryonic lethal. When
let-70(
s2293) is placed over either
let-70(
s689) or
let-70(
s1132), the resulting mutant progeny are L3-L4 lethals, suggesting that
let-70(
s2293) is a null allele. PCR analysis of the mutant gene from arrested embryos also suggests that
let-70(
s2293) is a null allele. The pattern of
ubc-2 expression during development in the mutants is currently being examined by in situ immunoflourescence. Preliminary analysis of
let-70(
s689) mutant embryos indicates that
ubc-2 is expressed in all cells throughout embryonic development. The expression pattern of
ubc-2 in the other
let-70 mutants is underway. References: Zhen M, Heinlein R, Jones D, Jentsch S and Candido EPM. 1993. The
ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation. Mol Cell Biol. 13:1371-1377. Zhen M, Schein JE, Baillie DL and Candido EPM. 1996. An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in the nematode Caenorhabditis elegans. EMBO J. 15:3229-3237.