Worm Breeder's Gazette 13(5): 69 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
1. University of Missouri School of Medicine, Columbia, MO USA 2. University of British Columbia, Vancouver, British Columbia CANADA The glh-1 gene encodes a putative RNA helicase that is most similar to the Drosophila vasa, a component of the polar granules. By antibody localization, we have shown localization of the glh-1 protein to the P- cell lineage that produces all primordial germ cells (WM 93, p.149). With the help of Alan Coulson, the glh-1 gene has been physically mapped to LG I near dpy-14 on the cosmid T21G5. Sheldon McKay and Ann Rose have linked the physical map in this region to several lethal mutants generated in the Rose laboratory and have shown that T21G5 rescues the mutants let-394, let-534, let-545, dpy-14 and the strain polymorphism sP1 (WBG 12.5, p.87). The overlapping cosmid T14D10 rescues all of the above except let-545 and sP1. This result positions let-545 near the end of T21G5. Because the glh-1 cDNA hybridizes to T21G5 but not to T14D10, let-545, a mutant with an adult sterile phenotype, was considered a good candidate for containing a mutation in glh-1. Tim Schedl, Washington University, observed that the original let-545 strain contained an additional mutation not linked to the let-545 mutation that caused a tumorous germline phenotype. He crossed out this mutation and balanced let-545 with hT2. The phenotype of this new strain, when homozygous for let-545, is one of producing very few germ nuclei and no oocytes or sperm. We (M.G. and K.B.) have successfully repeated the rescue of let-545 by mating males heterozygous for let-545 (let-545 dpy-5/hT2) without the extra background mutation to a transgenic strain containing the Rol-6 plasmid and T21G5 (provided by Ann Rose, KR2384, lon2, hEx 24). After mating, the rolling wild type F1 animals were cloned and we found fertile dyp-5 animals in the F2 generation, indicating rescue of let-545. To further define the rescuing DNA region, we (M.G. and K.B.) used a 6.2 kb genomic glh-1 fragment and the same rescue strategy as used for the T21G5 cosmid, to produce four independent transgenic lines in the lon-2 (e678) background, three of which have successfully rescued the let-545 mutant. The rescued animals are very dumpy and lay fertilized eggs, some of which mature into dumpy sterile adults. The presence of the rescuing DNA was verified by PCR in all four original transgenic lines. Using antibodies generated against the glh-1 protein, we have also shown that the let-545 strain has reduced antibody staining in the few germ cells that are present in the gonad, while the rescued strains show strong staining similar to that seen for wild type animals (i.e. staining throughout the distal arm of the gonad, a cytoplasmically dispersed, even staining in oocytes and a punctate, perinuclear staining localized to the P cell lineage in developing embryos). The low levels of residual staining seen in the mutant let-545 animals could represent a maternally supplied glh-1 protein. Based on the rescue, as well as the antibody localization results, we predict that let-545 will prove to be a mutation in the glh-1 gene causing a germline proliferation defect. We also propose that the glh-1 protein is the first identified component of the C. elegans P granules.