The germ cells in many metazoan organisms contain distinctive granules, which may serve as determinants of germ-cell fate. We want to elucidate the role of P granules, the germ-line granules of C. elegans, by studying a gene,
pgl-1, which likely encodes a component of P granules.
pgl-1 was identified in genetic screens:
pgl-1 mutant worms lack some P-granule epitopes (those recognized by the MAbs K76 and OIC1D4), and show a temperature-sensitive maternal-effect sterile phenotype (
pgl-1/pgl-1 hermaphrodites from heterozygous mothers are themselves fertile but produce all sterile progeny at 26oC). The temperature-sensitive period for
pgl-1 mutants is during larval development: when
pgl-1 hermaphrodites are shifted from permissive to restrictive temperature before the L4 stage, they develop into sterile adults; when shifted during or after the L4 stage, they develop into fertile adults but produce all sterile progeny. We mapped the
pgl-1 gene to the
lin-45-
deb-1 interval of LGIV by 3-factor crossing. Further mapping of the
pgl-1 gene relative to Bristol/Bergerac RFLPs narrowed the
pgl-1-containing region down to several cosmids. One of the cosmids, B0318, and a 20 kb restriction fragment of this cosmid rescued both the P-granule staining defect and the ts Mes phenotype of
pgl-1 mutants. The 20 kb rescuing fragment hybridizes to two transcripts (2.7kb and 1.3kb) on Northerns. Both transcripts are highly enriched in the germ line. We isolated cDNA clones corresponding to the two transcripts from R. Barstead's cDNA library. Injection into N2 worms of in vitro synthesized antisense (and sense) RNAs from one of the cDNA clones generated sterile progeny, at restrictive temperature, that resemble
pgl-1 mutant worms (displaying the same germ line defects and an absence of P-granule staining by K76 and OIC1D4!), which made it an excellent candidate for the
pgl-1 transcript. Injection of sense and antisense RNAs to the other transcript did not result in any sterile progeny. The 2.7kb
pgl-1 transcript encodes a protein of 730 amino acids. Putative PGL-1 is a novel protein with a glycine-rich stretch of 60 amino acids which includes ten RGG (arg-gly-gly) repeats (RGG box) at its carboxy terminus. RGG boxes are seen in many RNA-binding proteins, including fibrillarins, nucleolins, hnRNP proteins, and RNA helicases like Drosophila Vasa. PGL-1 may be an RNA-binding component of P granules that is required for specification and/or maintainance of germ-line fate in C. elegans.