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.

The adult sterile mutant, let-545, is likely to be a mutation in glh-1, a putative P-granule component.

Mike Gruidl (1), Sheldon McKay (2), Ann Rose (2) and Karen Bennett (1)

1. University of Missouri School of Medicine, Columbia, MO  USA          
2. University of British Columbia, Vancouver, British Columbia           

     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.