Previous studies have demonstrated that EGL-8 (PLC) mediates only part of the effects of the C. elegans EGL-30 (Gq) pathway (Lackner et al., 1999; Miller et al., 1999; Bastiani et al., 2003). Aldicarb resistance screens, which easily identify mutants with reduced EGL-30 or EGL-8 function, have thus far failed to identify the missing component(s). It thus seems likely that knocking out the second effector branch does not confer aldicarb resistance, in all likelihood because the EGL-8 (PLC) branch is able to compensate and generate near normal levels of release. If this is the case, and there are only two branches, then knocking out both branches should give a phenotype like the
egl-30 null (paralyzed, larval arrested, but rescueable with phorbol esters). To identify the missing branch, we are performing an
egl-8 enhancer screen, in which we look for animals that are dependent on having the
egl-8 null mutation to give a larval arrest phenotype. To do this, we have produced a strain in which the
egl-8 null mutation
md1971 is rescued with an extrachomosomal transgenic array that contains a full-length
egl-8(+) cDNA driven by the pan-neural
rab-3 promoter. The array is marked with
rab-3::GFP and exhibits ~70% transmittance. To carry out each round of screening, we will plate 2000 synchronous adult grandprogeny of EMS mutagenized animals in separate wells of 24 well plates containing solid media. These 2000 F2s will have been randomly derived from ~500 F1s, such that ~4 F2s are plated for every F1 in the starting population. Each F2 will be allowed to lay ~40 50 eggs before being picked off. We will screen the plates after these progeny reach adulthood and clone animals from wells whose only non-green animals are larval arrested or otherwise synthetically more severe than
egl-8 null single mutants, and whose
egl-8(+) rescued siblings are wild type or exhibit only mild sluggishness. We plan to repeat this screening procedure until at least 20,000 F2s have been screened in this manner. We will map the new mutations entirely with respect to SNPs and entirely in the transgenically rescued
egl-8 background using our standard SNP mapping technology (Schade et al., 2005). This poster will highlight our progress in the screens and describe the mapping strategy. Handouts describing our SNP mapping protocol will be available.