Eric Lambie et al. (2003) International Worm Meeting "Identification of genes that interact with gon-2"

Rachel West, Eric Lambie, Diane Church, Murline Gelin, Wenhua Wu

[International Worm Meeting, 2003]

gon-2 encodes a TRP-family cation channel whose function is required for the post-embryonic divisions of the gonadal precursors (1). In order to better understand how gon-2 is regulated and how it regulates the cell cycle, we have screened for extragenic suppressors of the hypomorphic allele, gon-2(q388). In this screen, we identified four loci, gem-1, 2, 3 and 4 (gon-2 extragenic modifier). We have determined that gem-4 encodes a member of the copine family. Copines were originally identified based on their ability to bind to phosphatidylserine-containing membranes in a Ca2+-dependent manner (2). The suppressor alleles of gem-4 all appear to be loss-of-function mutations. This suggests that gem-4(+) antagonizes gon-2(+). gem-4 mutations do not rescue gonad development when gon-2 activity is eliminated by gon-2[RNAi] in a gon-2(q388) background. Therefore, gem-4 acts either upstream of gon-2 or in a parallel, non-essential, negative regulatory pathway. As an independent method for identifying antagonists of gon-2 activity, we have begun screening the Fraser et al. (3) RNAi feeding library for clones that can suppress gon-2(q388). Unexpectedly, many of clones that suppress gon-2(q388) encode components of the protein synthesis machinery. The significance of this is not clear; however, we are currently testing whether these clones are able to suppress gon-2(0) and whether cycloheximide treatment can also suppress gon-2(q388). A progress report on the characterization of the various suppressor loci will be presented at the meeting.1. West et al., 2001. Gene 266:103; 2. Creutz et al., 1998. JBC 273:1393; 3. Faser et al., 2000. Nature 408:325.

David Wynne et al. (2001) International C. elegans Meeting "Approaches to Studying the Function of APH-1"

Valerie Hale, Katie Scangos, Caroline Goutte, Murline Gelin, David Wynne

[International C. elegans Meeting, 2001]

The aph-1 gene is involved in Notch-mediated signaling events in the C. elegans embryo. Mutations in aph-1 were isolated in the Priess lab by virtue of their embryonic lethal phenotype which showed a striking resemblance to the phenotype caused by mutations in the glp-1 gene. Further analysis of the aph-1 mutant phenotype demonstrated that the aph-1 gene product was necessary for GLP-1-mediated events at both the 4-cell and 12-cell stages of embryogenesis, thus identifying a new component of the GLP-1 signaling pathway in the early embryo. We have cloned the aph-1 gene and found that it encodes a novel protein with unknown function. Several lines of experimentation are underway to determine what is the specific function of the APH-1 protein. One approach we have taken is to build an aph-1::GFP fusion construct to look at the cellular and subcellular localization of the APH-1 protein in embryos. We have established transgenic lines with these constructs and shown that the fusion protein can rescue the aph-1 phenotype, albeit with low penetrance. Given that the fusion has some wild type activity, we are now analyzing the fluorescence pattern obtained from these constructs. A second approach we have taken is to search for genes that interact with aph-1. We have used a leaky allele of aph-1 to isolate extragenic suppressor mutations. Six independent mutations were found to suppress the aph-1 embryonic lethal phenotype. Two of these are dominant mutations in two different genes, and the other four mutations are recessive and identify two additional genes. Mapping data and further characterization of these four genes will be presented.