EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans

AbstractBackground: Cell-fate determination requires that cells choose between alternative developmental pathways. For example, germ cells in the nematode worm Caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. Germ-line mitosis depends on a somatic signal that is mediated by a Notch-type signaling pathway. The ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for oogenesis. Here, we provide more insight into the role of ego-1 in germ-line development.Results: We have determined the ego-1 gene structure and the molecular basis of ego-1 alleles. Putative ego-1 null mutants had multiple, previously unreported defects in germ-line development. The ego-1 transcript was found predominantly in the germ line. The predicted EGO-1 protein was found to be related to the tomato RNA-directed RNA polymerase (RdRP) and to Neurospora crassa QDE-1, two proteins implicated in post-transcriptional gene silencing (PTGS). For a number of germ-line-expressed genes, ego-1 mutants were resistant to a form of PTGS called RNA interference.Conclusions: The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery

A Genomic Screen for Yeast Vacuolar Membrane ATPase Mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, ∼4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Δ mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Δ, vps15Δ, vps45Δ, and vps16Δ, caused both Vma− growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Δ mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma− growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma− growth phenotype