Bennett 9604552 The basic mystery of development is how a fertilized egg can correctly become a complex adult organism. The Bennett laboratory is interested in how a particular cell lineage, the germline, is established. In the 1 6-cell embryo of the soil nematode Caenorhabditis elegans, one founder cell, P4, will become the entire adult germline. Surrounding the nucleus of the P4 primordial germ cell are non-membrane-bound particles called P granules. Germ granules similar to the P granules have been described in the primordial germ cells of many animals. It is important for understanding the overall role of germ granules in animal development to consider how germ granules function in Caenorhabditis and humans. The Bennett laboratory has recently reported on two P-granule components, the germline RNA kelicases, GLH-1 and GLH-2. By now focusing on the function of GLH-1 and GLH-2, this project will begin to discriminate their roles in the P granules and in germline development. Transposon-tagged glh-1 and glh-2 mutant strains will be identified and will be phenotypically analyzed. In addition, the RNA helicase activity of the GLH proteins will be confirmed by standard biochemical assays. The predicted GLH proteins contain several motifs not found in other RNA helicases, motifs likely to be critical to GLH function. These include multiple retroviral-like CCHC zinc fingers. Similar zinc fingers, best studied in HIV, bind RNA with selectivity. It is proposed that the various GLH zinc fingers add RNA binding specificity. In addition, N-terminal glycine-rich repeats, found in both GLH-1 and GLH-2, may function in P granule aggregation. These hypotheses will be tested by in vitro analyses. If RNA specificity can be demonstrated for in vitro-produced GLH proteins, the RNAs bound can begin to be identified. Detailed studies of the function and regulation of glh genes and the identification of the RNAs they bind may reveal that GLHs are critical germline determinants.
