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Comments on April M Orsborn et al. (2003) International Worm Meeting "Loss of Glycine Repeats and Zinc Fingers, or Helicase Motifs: Each Results in Non-functional GLH-1 Proteins." (0)
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April M Orsborn, Nicole Meyer, Susan Strome, & Karen L Bennett (2003). Loss of Glycine Repeats and Zinc Fingers, or Helicase Motifs: Each Results in Non-functional GLH-1 Proteins presented in International Worm Meeting. Unpublished information; cite only with author permission.
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.