INTRODUCTION

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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 cell-cell 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-of-function mutations in these genes show few or no abnormalities in vulval development (14, 18, 19, 30).

View larger version (46K): [in this window] [in a new window]   FIG. 1.   Vulval differentiation induced by cell-cell signaling. (A) Three-signal model for vulval cell fate specification (17, 31). A negative signal has been proposed to act from the surrounding epidermis to inhibit vulval differentiation of the six VPCs. An inductive signal from the anchor cell in the gonad induces the nearest three of six VPCs (P3.p to P8.p) to adopt the vulval cell fate, and the stronger signal received by P6.p due to its proximity to the anchor cell induces this cell into 1° vulval cell fate. A lateral signal acting between neighboring VPCs induces 2° cell fate in P5.p and P7.p and thus also prevents them from adopting the 1° cell fate. (B) In Vulvaless (Vul) mutants, more than three VPCs (often all six) adopt the nonvulval epidermal cell fate (3°), while Multivulva (Muv) mutations causes more than three VPCs (often all six) to adopt vulval cell fates. A block in the pathway transducing the inductive signal from the anchor cell causes the Vul phenotype, while constitutive activity of the pathway leads to the Muv phenotype. (C) Proposed sur-5 function (based on the work described in this paper) relative to some key factors in the main backbone of the pathway transducing the anchor cell (AC) signal.

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 three such suppressor mutations. Our genetic study of sur-5 indicates that it acts negatively on Ras, possibly by down regulating one of its activators. Our molecular analysis of the gene suggests that sur-5 encodes a novel and conserved protein.

	MATERIALS AND METHODS

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Strains and genetic methods. Methods for culturing, handling, and genetically manipulating C. elegans were previously described (3). All genetic experiments were done at 20°C except as otherwise noted. Methods for analyzing vulval defects under dissecting microscopes were performed as described previously (7, 9). Unless otherwise noted, the mutations used in this study are as described by Riddle et al. (23) and are as follows: lon-2(e678), unc-24(e138), dpy-6(e14), dpy-7(e88), unc-6(e78), unc-18(e81), let-60(n1046gf), lin-15(n765), lin-10(e1439), lin-1(e1275), dpy-20(e1282), dpy-20(e1362), him-5(e1490), mpk-1/sur-1(ku1), let-23(sy1), lin-45(sy96), lin-45(ku112) (30), let-65(s254), unc-22(s7), unc-119(ed3), let-60(sy94), let-60(sy93), let-60(sy101), let-60(sy100), let-60(n1531), let-60(n2031), and uDf1 (25).

Isolation and genetic characterization of suppressor mutants. Animals of the unc-24(e138) let-60(sy94 dn)/let-65(s254) unc-22(s7) genotype were mutagenized with 50 mM ethyl methanesulfonate (3), and the F₂ progeny were screened for egg-laying revertants. Candidates were picked and further characterized. The Vul percentage was determined by picking L4-stage animals and counting the number of Vul animals in the next 2 days. Two outcrosses were performed to eliminate false candidates and clean the genetic background. The first outcross was done by mating each candidate with dpy-20(e1282); him-5(e1490) males. F₁ Vul animals from the first outcross were picked out to produce F₂ animals. At this point, candidates for intragenic suppressors were identified as dominant suppressors of Vul but not the lethal phenotype. Non-Dpy non-Egl F₂ animals were allowed to propagate and were used for the second outcross. The second outcross was done by mating each surviving candidate from the first outcross to dpy-20(e1282)/dpy-20(e1362) unc-31(e169); him-5(e1490)/+ males. Non-Dpy F₁ Vul animals were picked to produce F₂ animals. Non-Dpy non-Egl F₂ animals were picked and allowed to propagate. These animals should have the genotype unc-24(e138) let-60(sy94 dn)/dpy(e1362) unc-31(e169); suppressor/suppressor. We screened 24,000 haploid genomes and found 3 potential intragenic revertants and 10 extragenic suppressors including three sur-5 alleles.

Genetic mapping and complementation tests. We constructed genetic mapping strains for each chromosome except chromosome IV. The genotypes of the two-point mapping strains are dpy-20(e1282)IV; dpy-5(e61) unc-101(m1)I, dpy-20(e1282)IV; dpy-10(e128) unc-4(e120)II, dpy-20(e1282)IV; dpy-17(e164) unc-32(e189)III, dpy-20(e1282)IV; dpy-11(e224) unc-76(e911)V and dpy-20(e1282)IV; lon-2(e678)X. Standard complementation tests were performed for suppressors that map to the X chromosome (3).

Deficiency study. The deficiency uDf1 uncovers unc-6 and dpy-7. Thus, uDf1 uncovers sur-5. To determine the phenotype of sur-5(ku74)/deficiency, we constructed a strain of lon-2(e678) sur-5(ku74)/uDf1. lon-2(e678) sur-5(ku74); him-5(e1490) males were crossed with a single +/szT1[lon-2(e678)]I; uDf1/szT1X hermaphrodite. Each non-Lon F₁ progeny was picked individually onto a plate, and its phenotype was recorded. A total of 10% of both uDf1/+ and uDf1/lon-2(e678) sur-5(ku74) strains have a Egl phenotype that is independent of VPC induction, indicating that sur-5(ku74)/uDf1 is wild-type for VPC induction. The genotype of each F₁ progeny was determined by examining genetic markers in the F₂ generation.

Double-mutant constructions. We describe here how we constructed a lin-10(n1390); sur-5(ku74) double mutant as an example of our general procedure for constructing sur-5(ku74) double mutants. To construct the lin-10(n1390); sur-5(ku74) double-mutant animals, males of the genotype lon-2(e678) sur-5(ku74); him-5(e1490) were crossed with the lin-10(n1390) hermaphrodites. F₁ progeny were then picked to individual plates. Double-mutant candidates were then selected by picking F₂ progeny that are Lon and Vul, which indicated homozygous lin-10 and possible homozygous sur-5(ku74). Each candidate's progeny was tested for the presence of the sur-5(ku74) molecular lesion, a 295-bp deletion, by PCR. Candidates that are Lon and Vul and whose progeny showed only the ku74 molecular lesion on agarose gel were considered to be lin-10(n1390); sur-5(ku74) double mutants.

Microinjection transformation. All cosmids used in this study were obtained from A. Coulson and J. Sulston (Sanger Center, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom). DNA isolation, analysis, and subcloning were performed by standard molecular biology methods. Microinjection of cloned DNAs into the gonadal syncytia of C. elegans hermaphrodites was carried out as described previously (21). All the cosmids listed in Fig. 2 were initially injected as a pool of four overlapping cosmids at a concentration of ~12 ng/µl each. Positive cosmids were then injected individually at a concentration of ~60 ng/µl. The subclones were first injected as a pool of four nonoverlapping subclones at a concentration of ~15 ng/µl each. Subclones from positive pools were then injected individually at a concentration of ~15 ng/µl. A strain of let-60(sy94 dn)/dpy-20(e1362) unc-31(e169); lon-2(e678) sur-5(ku105) was used as the host strain for microinjection. This strain is <15% Vul. Rescue of the sur-5 mutant phenotype was indicated by a >40% Vul phenotype. Each injection was scored with at least four independent transgenic lines. The extent of rescue varied among different transgenic lines, ranging from 10 to 66% Vul phenotype. Plasmid pTG1 also rescues a strain of let-60(sy94 dn)/dpy-20(e1362) unc-31(e169); sur-5(ku74). Plasmid pTG1_1, which has an additional 1.8 kb 5' upstream DNA sequence added to the sur-5 5'-flanking sequence in pTG1, also rescues the strain let-60(sy94 dn)/dpy-20(e1362) unc-31(e169); sur-5(ku74). When pTG1_1 was injected at a concentration of ~45 and ~60 ng/µl into wild-type animals, no mutant phenotypes were observed. For all the above microinjections, pRF4, which contains the dominant rol-6 mutant gene, was used as the transformation marker injected at a concentration of ~100 ng/µl.

Northern analysis, cDNA isolation, and DNA sequencing. pTG1 was used as a template for making radioactive probes for Northern blots. Low levels of sur-5 transcript were detected in poly(A)⁺ early embryonic RNAs and total RNAs. pTG1 was used to screen approximately 10⁶ plaques from a gt11 early embryonic cDNA library (a gift from P. Okkemma and A. Fire). Four clones were identified. All four clones lack one or two nucleotides that caused deletion of the first amino acid, methionine, based on the sequence data and on a prediction by the C. elegans genome project.

Plasmid construction. The rescuing plasmid, pTG1, has a 8-kb BamHI fragment with the overlapping region of cosmids K03A1 and R12E12 cloned into pBluescript. Plasmid pTG1_1 was constructed by first inserting an 1.8-kb NsiI-BamHI fragment upstream of sur-5 into the PstI-BamHI site of the pUC19 vector. The 8-kb BamHI fragment of pTG1 was then inserted into the BamHI site of the resulting plasmid.

Mammalian sur-5 homology search. We use the BLAST computer program to search for homologs. Only in the EST database did we find mammalian sequences that are similar to SUR-5. They consist of mouse and human cDNA sequences. Based on these sequences, it is likely that all the EST sequences are incomplete cDNAs made from the same transcript from each organism. We then cloned additional human and mouse sequences by PCR by screening about 3 × 10⁶ plaques of a mouse thymus and a human brain cDNA library (Stratagene), using lambda reverse or forward primers as the 5'-end primers and specific EST sequences as the downstream 3' primer. We finally cloned an additional 1.4-kb 5' human cDNA fragment by PCR from a psport I human brain cDNA library, using psport forward primer as the 5'-end primer and specific sequences as the downstream 3' primer.

	RESULTS

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Isolation of suppressors of dominant negative let-60 ras. To identify negative regulators in the let-60 ras pathway, we screened for suppressors of a let-60 dominant negative allele, let-60(K16N dn) (genetic allele name, sy94 [12]). Homozygous let-60(K16N dn) mutations are completely lethal, while heterozygous let-60(K16N dn)/+ worms display a moderately reduced LET-60 protein activity as indicated by the partial defect in vulval development (about 30% VPC induction and 97% egg-laying defective [Egl]) (Table 1) (11). This allele was chosen for the screen because (i) it does not poison the let-60(+) Ras activity completely in the heterozygous mutant, which may allow us to isolate a wide variety of mutants with mutations in different let-60 negative regulators; and (ii) it has a relatively tight Egl phenotype (97%), which would facilitate the screening for egg-laying competent (non-Egl) revertants.

Genetic interactions between sur-5 and mutations in other genes in the signaling pathway. To determine where sur-5 acts in the let-60 Ras pathway, we constructed double mutants between mutants containing sur-5(ku74) and other loss-of-function mutations of the let-60 ras pathway and then examined the vulval phenotype of these double mutants (Table 2).

Genetic interactions between sur-5(ku74) and let-60 dn mutations. To determine whether suppression by sur-5(ku74) of the dominant Vul phenotype caused by let-60(K16N dn) is allele specific, we tested whether sur-5(ku74) can suppress other let-60 dn mutations (1, 11, 12). We found that sur-5(ku74) was able to suppress four let-60 dn alleles, sy94 (K16N), sy101(G10R), n1531(G15D), and n2301(G15S), but failed to suppress two other let-60 dn alleles, sy93(E119N) and sy100(S89F) (Table 3). We have also constructed and tested double mutants between let-60(S89F) and the other two sur-5 alleles and found that these sur-5 alleles also fails to suppress the dominant Vul phenotype caused by let-60(S89F) (data not shown). All of the let-60(dn) alleles that can be suppressed by sur-5(lf) (called group I alleles) altered the residues within the first conserved loop of the Ras protein that is involved in GTP/GDP binding (35). One of the sur-5(lf) nonsuppressible alleles (group II alleles), E119N, mutates residue 119 on loop 8, which is also involved in nucleotide binding, while the other group II allele S89F mutates residue 89, which might be involved in interacting with other factors (35). It is interesting that the group II allele let-60(S89F) is the weakest dominant negative allele, based on the percentage of vulval differentiation, while the other group II allele let-60(E93N) is the strongest dominant negative allele (Table 3) (11). Therefore, it is unlikely that the difference in sur-5(lf) suppression between the two groups is due to the strength of the dominant negative effect of the let-60 ras(dn) alleles. The results in Table 3 may suggest that the dominant negative effects of these two groups of ras(dn) mutations are due to two different mechanisms (see Discussion).

Phenotypes due to overexpression of sur-5. If sur-5 is a negative regulator of let-60 ras, it is possible that elevating sur-5 activity will suppress the Muv phenotype caused by an activated let-60 ras mutation. Since an extrachromosomal array can contain hundreds of copies of injected plasmids (21), we tested if we could observe an effect of overexpressing sur-5 transgenes (see below for cloning the sur-5 gene). The data in Table 4 indicates that an extrachromosomal array carrying a sur-5(+) plasmid partially suppresses the Muv phenotype of let-60(G13E gf) from 89 to 34%. This result is consistent with the notion that sur-5 plays a negative role in the let-60 ras pathway.

The sur-5 gene encodes a novel gene product. We genetically mapped sur-5 to a small chromosome region between two cloned genes, unc-6 and dpy-7 (0.37 map unit) (Fig. 2). sur-5 is estimated to reside between 0.14 and 0.16 map unit away from dpy-7 (see Materials and Methods). We used DNA-mediated microinjection methods to identify cosmids that span the region containing the sur-5 gene, with the dominant rol-6 mutant DNA as a marker (21). The host strain has the genotype let-60(K16N dn)/dpy-20(e1362) unc-31(e169); lon-2(e678) sur-5(ku105). We assayed DNA-mediated rescue by scoring transformants for reversion of the suppressor phenotype of sur-5(ku74) on the let-60 dominant Vul phenotype. We determined that each of two overlapping cosmids, K03A1 and R12E12, is capable of rescuing the sur-5(ku74) suppression phenotype (data not shown). After further subcloning and injection, we determined that an 8-kb BamHI restriction fragment (pTG1) within the overlapping region of cosmids K03A1 and R12E12 rescues the sur-5 mutant phenotype. Using this DNA fragment as a probe to screen a cDNA library (a gift from P. Okkema and A. Fire), we isolated four positive clones from about 10⁶ plaques. All four cDNA clones had the same sur-5 gene sequence. The sur-5 cDNA sequence was predicted to encode a protein that has 700 amino acids (Fig. 3). Northern analysis with the sur-5 genomic DNA as a probe detected a single transcript of approximately 2.2 kb which is present in the embryos and larvae (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Positional cloning of the sur-5 locus. (A) Genetic map position of sur-5 on the X chromosome. (B) Identification of DNA clones containing the sur-5 gene. A collection of cosmid clones near the sur-5 gene were used in microinjection transformation tests. A partial restriction map within the overlapping region of the two positive cosmids is depicted. B, BamHI; K, KpnI. (C) Exon and intron structure of the sur-5 gene. A total of 12 exons (open boxes) and 11 introns (lines) are shown proportional to their actual size. The genomic sequence of sur-5 was determined by the C. elegans genome project. The intron/exon boundaries were predicted by Genefinder software and confirmed by sequencing a cDNA clone.

View larger version (85K): [in this window] [in a new window]   FIG. 3.   Amino acid sequence alignment of SUR-5 and its potential mammalian homologs. SUR-5 is shown aligned with a potential human homolog (HsSUR-5) and a C-terminal partial mouse sequence (MuSUR-5). Boxed letters indicate amino acid identity (black) or similarity (gray) between at least two organisms. Gaps are represented by dots. Potential ATP or GTP binding motifs and AMP binding motifs are indicated by bars below the sequences. The deleted region (amino acids 76 to 145) in the ku74 allele is indicated by the open bar above the letters. The point mutation in the ku105 allele is also indicated.

Isolation of a potential human SUR-5 homolog. Using BLAST software, we identified partial DNA sequences of potential mouse and human sur-5 homologs in the EST database. We then used PCR screening of mouse thymus and human brain cDNA libraries to clone additional cDNA sequence 5' to the EST fragments. The total cloned cDNA length (>3.2 kb including >1.2 kb of the 3' untranslated region of the EST clones) is similar to the size of a single transcript displayed in a Northern blot, suggesting that the cDNA clone is either full-length or close to full-length. The predicted amino acid sequence of the human clone is close to the size of the C. elegans protein. The predicted human protein is about 38% identical to the worm protein and contains all the key features mentioned above (Fig. 3). There is about 90% amino acid identity between the potential human SUR-5 homologs and the partial mouse sequence. This result suggests that the sur-5 gene may be conserved from C. elegans to humans. Northern analysis also shows that the human gene was expressed ubiquitously with higher abundance in brain and testis (data not shown). We did not identify a yeast gene that has a high degree of similarity in overall structure to the sur-5 gene.

sur-5 is strongly expressed in most of the cells in C. elegans. To visualize sur-5 expression in vivo, we constructed several SUR-5-GFP fusion protein constructs. One construct, pTG96, includes a 3.68-kb fragment of the 5'-flanking sequence and the full-length sur-5 genomic sequence fused at its C terminus to GFP containing a potent, artificial nuclear localization signal (NLS) sequence. When transgenic animals carrying pTG96 on an extrachromosomal array (kuEx75) were examined, the fusion protein, as judged by fluorescence of GFP, was observed tightly localized to the nuclei of most cells. SUR-5 appeared to be expressed in the VPCs, consistent with a function in regulating Ras activity during vulval induction (Fig. 4). However, we have no data demonstrating that SUR-5 functions in the VPCs.

View larger version (117K): [in this window] [in a new window]   FIG. 4.   SUR-5-GFP expression in vulval cells in a transgenic animal carrying a SUR-5-GFP construct (N2; kuEx75) examined under a compound microscope with Nomarski optics. (A) Nomarski image of the mid-body. The three VPCs (P5.p to P7.p) have just completed the second round of cell division. (B) The same animal examined under a fluorescent light source. The SUR-5 and GFP proteins are expressed mostly in the nuclei, and all cells derived from VPCs express the fusion protein.

	DISCUSSION

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Multiple negative factors influence the let-60 ras signaling pathway. We have screened for suppressors of a dominant negative ras mutation to identify negative regulators of the Ras-mediated signal transduction pathway. We have isolated 10 extragenic suppressors that may define six genes involved in the Ras-mediated signaling process. Since the number of worms screened was relatively small (~24,000 haploid genome) and since it is possible that only rare mutations in some essential or multifunctional genes were recovered in the suppressor screen, the screen is probably not close to saturation. None of the 10 mutations we isolated displayed any mutant phenotypes except suppression of the Vulvaless phenotype caused by the let-60(K16N dn) mutation. If these mutations are null or severe-loss-of-function alleles, this result suggests that negative regulators defined by these mutations play a limited regulatory role or that there is functional redundancy among two or more negative regulators. In the case of the sur-5 gene, our genetic and molecular data do not exclude the possibility that the mutations isolated are only partial-loss-of-function mutations.

Role of sur-5 in regulating ras activity. Although sur-5(ku74) is an effective suppressor of several dominant negative let-60 ras mutations, it does not suppress mutations in sem-5 or let-23 receptor tyrosine kinase, both of which act upstream of let-60 ras. Since sem-5(n2019) and let-23(sy1) homozygous mutants do not display a more severe Vul phenotype than that of let-60(K16N dn)/+ animals, loss of sur-5 appears to elevate vulval induction more in the let-60 ras (dn)/+ background than in the lowered let-60(+) background.

Indication of multiple Ras activators. The observation that sur-5 mutations fail to suppress the Vul phenotype caused by one of the two groups of let-60 dn mutations suggests there are at least two mechanisms which account for the dominant effect of ras(dn) mutations and that sur-5 mutations affect only one of these mechanisms. Since all let-60 ras(dn) mutations are completely suppressed by the let-60(G13E gf) mutation in trans-heterozygotes (1, 11, 12), both mechanisms are likely to involve toxic effects on upstream activators rather than downstream effectors. One such activator would probably be a GNEF, since previous studies showed that increased expression of the Saccharomyces cerevisiae CDC25 (a GNEF) gene can suppress the toxic effect caused by either mammalian H-Ras (G15E dn) or yeast RAS2(G22A dn) (22). Residue 22 of S. cerevisiae RAS2 corresponds to residue 15 of H-Ras. We could thus imagine that the sur-5(lf)-suppressible let-60 ras(dn) mutations (group I), which are all located on loop 1 of Ras, behave similarly to the G15A mutations of H-Ras. The sur-5(lf) nonsuppressible LET-60 Ras(dn) proteins (S89F and E119N) may be toxic to a different activator. The possibility of having multiple positive regulators was raised previously. Overexpression of GNEF can suppress only one of the two types of Ras dominant negative mutants in Schizosaccharomyces pombe (15). Although alternative explanations are possible, this work suggests that there could be more than one upstream Ras activator in S. pombe. There is as yet no direct evidence that C. elegans has multiple Ras activators.

sur-5 overexpression phenotypes. Consistent with the role of sur-5 as a negative regulator of Ras-mediated vulval signaling, multiple copies of sur-5 in an extrachromosomal array partially suppress the Muv phenotype caused by the let-60(G13E gf) mutation. This result is still consistent with the potential role of sur-5 in interacting with a Ras activator, since vulval induction in the let-60(G13E gf) mutant is known to be influenced to a certain degree by gene activities upstream of Ras (30, 36). The level of suppression of the Muv phenotype of the let-60(gf) allele by the sur-5 transgenes (89 to 38% Muv) is significant, considering that the sur-5(+) transgenes do not cause any phenotype in wild-type worms. It is possible that vulval induction is more sensitive to elevated sur-5 activity in the let-60 ras(G13E gf) mutant than in the let-60 ras(+) strains due to the sur-5 function on a specific regulatory aspect of Ras. Mutations in the ksr-1 gene were also shown to have a stronger effect on the let-60 ras(gf) gene than on the let-60 ras(+) gene (30), suggesting that the function of ksr-1 is closely related to Ras activity.

The sur-5 gene product may be conserved during evolution. We show in this paper that sur-5 encodes a novel protein, and we have identified candidate mammalian homologs of sur-5. The human protein sequence is about 38% identical to the SUR-5 sequence (Fig. 3). There is about 90% identity between the human sequence and the partial mouse sequence. Like C. elegans sur-5, which is expressed in most of the cell lineages, the potential human sur-5 homolog is also expressed ubiquitously in many tissues. Further studies of these mammalian genes may determine whether they are also involved in negative regulation of Ras functions. We have not identified an obvious sur-5 homolog in S. cerevisiae.