Roughly 10% of C. elegans genes give a phenotype when individually reduced in function using RNAi. Presumably, many genes are redundant for function or important only in special circumstances. We are undertaking a high-throughput screen to identify additional genes from the remaining 90% of the genome that function during early embryogenesis. Our screen identifies new gene functions based on synthetic interactions with known temperature-sensitive mutants. Our goal is to identify genes that display few requirements when reduced in function on their own, but become important in sensitized conditions. We are currently applying this procedure to better understand centrosome function.The centrosome is the organizing center of the microtubule cytoskeleton and critical for cell division and cell polarity. Identifying, at a genetic and molecular level, the regulators and components of centrosomes is an important step in understanding cell division. Our screen involves using sensitized genetic backgrounds generated by exploiting temperature-sensitive (ts), embryonic-lethal mutations in genes known to be required for centrosome function. By using low or high ranges of temperature-lethality curves, we are identifying genes that enhance or suppress known ts alleles of
dhc-1 (a cytoplasmic dynein heavy chain) and
dnc-1 (dynactin),
spd-5 and
spd-2 (centrosomal scaffolds), and
zyg-1 (a kinase required for centrosome duplication). We hope to identify positive regulators of centrosome function as synthetic lethal genes and negative regulators as suppressor genes. Our screen takes advantage of the Ahringer labs genome-wide RNAi feeding library, which enables us to rapidly survey the genome for loci important in centrosome function. Currently, we are examining the reproducibility of interactors identified in primary screens that tested over 16,000 loci for each of the five conditional mutants. Thus far we have confirmed in secondary screens over 250 enhancers and 212 suppressors that consistently and specifically interact with our strains (including 36 enhancers for
spd-5 and 61 suppressors for
dhc-1). These are now being ranked with respect to the penetrance of embryonic lethality. Genes confirmed thus far are intriguing. For example, subunits of the dynein motor complex specifically suppress the lethality of our
dhc-1 allele, suggesting that light chains of the motor may mediate negative regulation, as well as cargo attachment. Also, a kinase-like protein that strongly and specifically suppresses the lethality of
spd-5 has a human ortholog, Madm, which localizes to centrosomes in mammalian cells and physically associates with the Mlf1 oncoprotein.