UDP- N -acetylglucosamine:alpha-6-D-mannose
beta-1,6- N -acetylglucosaminyltransferase V (GlcNAc-TV) is a Golgi enzyme that catalyzes the addition of
beta-1,6-GlcNAc in the production of complex N -glycans. GlcNAc-TV is of particular interest because its activity is directly involved with cancer transformation and metastasis in mice and is a prognostic indicator of breast cancer survival in humans. Mice deficient in GlcNAc-TV via deletion in the Mgat5 locus experience dramatic suppression of metastasis. Analysis of the molecular mechanism of Mgat5 in mice is confounded by the pleiotropic nature of N -glycosylation. Perturbation of Mgat5 affects all
beta-6-GlcNAc substituted glycoproteins so that identification of specific molecules that control specific systems is difficult. The C. elegans genome contains an ortholog,
gly-2 , which can rescue CHO cells mutant at the Mgat5 locus. Strain XA766
gly-2(
qa703)I qaEx743[C55B7.3 C55B7.10
gly-2(+),
rol-6(d)] was constructed and used in a pilot scale pre-complementation screen of 650 haploid genomes to identify loci that are synthetic lethal in a
gly-2 null background. Two alleles, not integrants, both of which are strongly dependent on the qaEx743 array were isolated. The allele
nh4 is lethal in a
gly-2 null background unless pre-complemented by the qaEx743 array. The second allele,
nh3 , exhibits a temperature-sensitive dauer constitutive phenotype that only exits dauer at restrictive temperature in the presence of the array. Both
nh3 and
nh4 are linked to
gly-2 on LGI. One plausible explanation for the array dependence is that
nh3 and
nh4 are alleles of loci underneath the qaEx743 array. Formally then,
nh3 or
nh4 could interact to create a synthetic lethal phenotype with
gly-2 ,
rol-6 or be due to a single site mutation in C55B7.3 or C55B7.10. We are currently mapping the exact locations of
nh3 and
nh4 . In the case that
nh3 and
nh4 are
gly-2 dependent, they should reconstruct with
gly-20 , the gene immediately upstream of
gly-2 in the glycosylation pathway. We have separated
nh3 from the
gly-2 locus and are currently reconstructing it with
gly-20 .