Caenorhabditis elegans SUR-5, a Novel but Conserved Protein, Negatively Regulates LET-60 Ras Activity during Vulval Induction

Abstract

The let-60 ras gene acts in a signal transduction pathway to control vulval differentiation in Caenorhabditis elegans. By screening suppressors of a dominant negative let-60 ras allele, we isolated three loss-of-function mutations in the sur-5 gene which appear to act as negative regulators of let-60 ras during vulval induction. sur-5 mutations do not cause an obvious mutant phenotype of their own, and they appear to specifically suppress only one of the two groups of let-60 ras dominant negative mutations, suggesting that the gene may be involved in a specific aspect of Ras activation. Consistent with its negative function, overexpressing sur-5 from an extragenic array partially suppresses the Multivulva phenotype of an activated let-60 ras mutation and causes synergistic phenotypes with a lin-45 raf mutation. We have cloned sur-5 and shown that it encodes a novel protein. We have also identified a potential mammalian SUR-5 homolog that is about 35% identical to the worm protein. SUR-5 also has some sequence similarity to acetyl coenzyme A synthetases and is predicted to contain ATP/GTP and AMP binding sites. Our results suggest that sur-5 gene function may be conserved through evolution.The Ras-mediated signal transduction pathway plays important roles in specifying cell fates in a number of developmental events in the nematode Caenorhabditis elegans including vulval cell differentiation (17, 31), male spicule cell differentiation (4), germ nucleus exit from pachytene (5), sex-myoblast migration (32), and excretory duct cell differentiation (38). Vulval differentiation has been the main system used in a number of laboratories to identify new components of this pathway and study its regulation. Vulval differentiation in C. elegans hermaphrodites is controlled by the combination of several cellcell signaling events ( Fig. 1A) (17,31). In particular, an inductive signal from the anchor cell induces three of the six vulval precursor cells (VPCs, P3.p to P.8p) to differentiate into vulval cells (Fig. 1A). The let-60 ras gene acts in a conserved signal transduction cascade to transduce the anchor cell signal encoded by the lin-3 gene (Fig. 1C). Previous genetic screens in several laboratories have identified many components that are either key factors acting in the main backbone of the signaling cascade (Fig. 1C) or factors that regulate the activity of these key players in the pathway (17, 31). The functions of several genes as regulators of the signaling pathway (e.g., unc-101, sli-1, and ksr-1) have been identified only by the genetic suppressor phenotypes and the synergistic effects of their mutations in other mutant backgrounds, since mutants with loss-offunction mutations in these genes show few or no abnormalities in vulval development (14,18,19,30).To identify negative factors that down regulate the let-60 ras activity, we screened for mutations that suppress the Vulvaless phenotype caused by a let-60 ras dominant negative (dn) mutation, let-60(K16N). In this paper, we describe the gene sur-5, defined by thr...

Targeted gene alteration in Caenorhabditis elegans by gene conversion

“…We cloned this fragment into vector pCR4-TOPO (Invitrogen), and the resultant plasmid was partially digested with MfeI to remove B1.5 kb of sequence and then religated. This plasmid was introduced as a transgene into strains VJ136 and VJ151 (dpy-18 (e364) mut-7 (pk204) III; frm-3 (cxP4618) X), along with the marker plasmids pRF4 and pTG96 (a sur-5-GFP fusion 22 ), by injection 2 . The transgenic VJ136 strain generated in this way was crossed into the mutator backgrounds TW404 (dpy-5 (e61) mut-2 (r459); a gift from Q. Boese and J. Collins) and VJ151.…”

“…22), which confer, respectively, rolling or green fluorescent protein (GFP) phenotypes. We tested tkr-1 in the mut-2 mutator background and frm-3 in both the mut-2 and the mut-7 backgrounds.…”

Demonstration of thein vivointeraction of key cell death regulators by structure-based design of second-site suppressors

“…At 20 and 25°C, the death-inducing activity of EGL-1(D64G) and EGL-1(D64A) was reduced significantly, further confirming the temperaturesensitive nature of these mutations. In contrast, wild-type EGL-1 *The hsp constructs (at 50 g/ml each) were injected into ced-1(e1735); egl-1(n1084 n3082) unc-76(e911) animals with pTG96 (at 20 g/ml), which expresses GFP in many somatic cells in most of the developmental stages (35), and p76-16B (at 50 g/ml), which rescues unc-76 uncordinated phenotype (36). The transgenes were then crossed into ced-1(e1735); ced-9(n1950) mutant background.…”

“…EGL-1(D64G) and EGL-1(D64A) induce stronger cell killing in ced-9(n1950) mutants than EGL-1. A 10-kb egl-1 wild-type genomic fragment (hatched box) containing 3.8 kb upstream of the egl-1 start codon and 5.7 kb downstream of the egl-1 stop codon (34) or the corresponding egl-1 genomic fragment carrying either the D64G (gray box) or D64A (empty box) mutation was introduced at 40 g/ml into ced-1(e1735); egl-1(n1084 n3082) unc-76(e911) or ced-1(e1735); ced-9(n1950) animals with pTG96 (20 g/ml) (35) and p76 -16B (50 g/ml) (36). The transgenic animals were cultured at three temperatures (15, 20, or 25°C), and the cell-killing activity of the EGL-1 proteins was assessed by counting the number of cell corpses in the head region of 3-fold or later-stage transgenic embryos.…”

Strongyloides stercoralis: Cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3′ UTR

“…By contrast, gfp-based reporter constructs incorporating regulatory elements from C. elegans are not expressed in the firstthrough third-stage larval progeny of microinjected free-living female S. stercoralis but only in dysregulated fashion in degenerating embryos (Li, et al, 2006). We have previously detected DNA of the C. elegans-derived plasmid construct pTG96_2 (Gu, et al, 1998) in transgenic S. stercoralis up to the F5 generation (Li, et al, 2006), but we have not yet succeeded in deriving a stable transgene expressing line; that is, one that transmits an expressed transgene to its progeny at a constant frequency through successive generations. Grant et al (2006) have achieved heritable transformation of P. trichosuri with β-gal-based reporter constructs, and have succeeded in maintaining transformant lines through multiple in vitro and in vivo passages, providing proof of principal for our studies in S. stercoralis.…”