De novo synthesis of the nucleotide sugar UDP-N-acetylglucosamine (UDP-GlcNAc) requires the enzyme glucosamine 6-phosphate acetyl transferase (GNA1/ PAT1 (yeast), EMeg32 (mouse)) of the hexosamine synthesis pathway. UDP-GlcNAc is an essential substrate in chitin synthesis, in N- and O-linked glycosylation of membrane and secreted proteins (by Golgi-resident transferases), in O-linked cytosolic protein glycosylation and in GPI protein anchoring. In yeast, deletion of GNA1 is lethal, causing cells to have defects at multiple steps in the cell cycle, resulting in some cells with multiple or no nuclei. Mouse embryos homozygous for a null mutation in EMeg32 (GNA1) die at E7.5 and in vitro differentiated null ES cells show multiple changes, including defects in proliferation and adhesiveness, resistance to apoptotic stimuli, decreased actin depolymerization increased PIP 2 levels and increased PKB activation. To try to elucidate the role played by UDP-GlcNAc in development and the cell cycle, we have begun studies of GNA1 in C. elegans . RNA interference has been done using the two predicted worm homologues of GNA1 (B0024.12, T23G11.2). Interference with B0024.12 had no discernible effect on phenotype. However, interference with T23G11.2 resulted in F1 progeny that were sterile. We have isolated a mutant of T23G11.2 in which all four predicted exons have been deleted. Worms that are heterozygous for this mutation are viable and produce viable progeny. However, mirroring the RNAi results, homozygous mutants are viable but sterile, laying masses of eggs that do not hatch. We have rescued the aberrant phenotype using a line carrying the cosmid T23G11 in an extrachromosomal array. The mutant does not appear to be Spe, since it was not rescued by mating with wild-type males. Mating with
fem-1 is being done to determine if the mutant produces fertile sperm and UDP-GlcNAc in the mutant is being quantified by HPLC. In addition, we are characterising the phenotype of the embryos in the uterus by staining with DAPI, anti-tubulin and calcofluor (for chitin).