To study germline development in Caenorhabditis elegans, we are focusing on a family of four proteins, the germline helicases (GLHs). These proteins are homologous to the Drosophila protein VASA, but the GLHs differ in that they contain multiple FGG repeats and retroviral-like CCHC zinc fingers (with the exception of GLH-3, which does not contain FGG repeats). The GLH proteins are components of P granules, non-membranous aggregates of protein and RNA that segregate with the germline throughout development and are essential for fertility. Through the collaboration of our two laboratories and the efforts of the C. elegans Knockout Consortium, three
glh-1 mutant strains have been generated; the set includes two deletion mutations,
glh-1(
ok439) and
glh-1(
gk100), and the transposon insertion allele
glh-1::Tc1
(bn103). All three strains are maternal effect steriles at 25-26oC. Surprisingly the two
glh-1 deletion strains each produce GLH-1 proteins. By western analysis each strain produces a GLH-1 protein that is smaller than wild type. The
glh-1(
ok439) strain is predicted to produce a protein lacking 235 amino acids from the C-terminus, removing three of the eight conserved RNA helicase motifs. By comparison, the internal deletion in
glh-1(
gk100) results in the loss of 183 amino acids, eliminating three of the four CCHC zinc fingers and four of the nineteen FGG repeats. Because of the indistinguishable sterility phenotypes of
glh-1(
ok439) and
glh-1(
gk100), both deletions eliminate motifs critical for GLH-1 function. Immunocytochemistry with the deletion alleles to determine whether these smaller GLH proteins still localize to P granules is ongoing. We are also generating transgenic worms expressing GLH-1 (and
glh-1 mutations) fused to green fluorescent protein (GFP) using the gene gun method and the PIE-1 expression vector of G. Seydoux. In addition, we plan to create a vector driven by the endogenous
glh-1 promoter.