let-60, a Gene That Specifies Cell Fates during C. elegans ...post.queensu.ca/~chinsang/a-worm-reader/han-and-sternberg-1990.pdf · during C. elegans Vulva1 Induction, Encodes a MS

Cell, Vol. 63, 921-931, November 30, 1990, Copyright 0 1990 by Cell Press

let-60, a Gene That Specifies Cell Fates during C. elegans Vulva1 Induction, Encodes a MS Protein Min Han and Paul W. Sternberg Howard Hughes Medical Institute Division of Biology, 156-29 California Institute of Technology Pasadena, California 91125

Summary

Genetic analysis previously suggested that the 181-60 gene controls the switch between vulva1 and hypodermal cell fates during C. elegans vulva1 induction. We have cloned the let-60 gene, and shown that it encodes a gene product ldentlcal in 84% of its first 164 amino acids to ras gene products from other ver- tebrate and invertebrate species. This conservation suggests that the let-60 product contains all the biochemical functions of fas proteins. Extrachromosom- al arrays of M-60 ras DNA cause cell-type misspecification (extra vulva1 fates) phenotypically opposite to that caused by let-60 ras loss-of-function mutations (no vulva1 fates), and suppress the vulvaless phenotype of mutatlons in two other genes necessary for vulva1 induction. Thus, the level and pattern of let-60 ras expression may be under strict regulation; increase in let-60 ras activity bypasses or reduces the need for upstream genes in the vulva1 induction pathway.

Introduction

During postembryonic development of the C. elegans hermaphrodite, each of six vulva1 precursor cells (VPCs) has the potential to generate either vulva1 cells or hypodermal cells. During vulva1 induction, three of the six VPCs are induced to generate vulva1 cells by a signal from the anchor cell in the gonad (Figure 1A). The other three VPCs generate nonspecialized hypodermal cells, the fate of all VPCs in the absence of inductive signal (Sulston and Horvitz, 1977; Sulston and White, 1980; Kimble, 1981; Sternberg and Horvitz, 1966).

We have taken a genetic approach to study the mechanism of VPC fate specification. Mutations that result in misspecification of VPC fates have defined genes necessary for the normal patterning process (Horvitz and Sul- ston, 1980; Greenwald et al., 1983; Ferguson and Horvitz, 1985, 1989; Ferguson et al., 1967; Sternberg and Horvitz, 1989; Han et al., 1990). Vulvaless (Vul) mutations cause fewer than three VPCs to generatevulval cells (Figure lB), often resulting in an egg-laying defect. Multivulva (Muv) mutations cause more than three VPCs to generate vulva1 cells, resulting in additional pseudovulvae (Figure 1C). Characterization of these mutations suggested that genes defined by these mutations act in a pathway that deter- mines whether a VPC adopts a vulva1 or a hypodermal fate in response to the inductive signal (Ferguson et al., 1987; Sternberg and Horvitz, 1989).

let-60 (let for lethal) is one of the key genes acting in the

vulva1 induction pathway (Han et al., 1990): loss of let-60 activity causes six VPCs to have the hypodermal fate, while closely linked mutations that elevate let-60 activity cause all VPCs to have vulva1 fates in the absence of inductive signal. Since genetic analysis led to the hypothe- sis that let-60 level controls VPC fates, we sought to clone the gene and determine its structure. In this paper, we demonstrate that let-60 encodes a protein very Similar in its overall structure to ras gene products in other organisms. Introduction of multiple copies of the let-60 gene into C. elegans by microinjection causes a dominant Muv phenotype and suppresses the Vul phenotype resulting from defects in two other genes, let-23 and lin-3, acting in the same pathway. These results provide clues about the nature of the mechanism underlying vulva1 induction, and about the role of ras in animal development.

Results

Summary of Known Genetic Properties of let-60 Our previous genetic analyses suggested that the activity level of let-60 controls the switch between vulva1 and hypodermal fates in response to the inductive signal (Han et al., 1990). Specifically, low let-60 activity results in hypodermal fate for all VPCs; high let-60 activity results in vulva1 fates for all VPCs. The genetics of let-60 can be briefly summarized as follows (Table 1). let-60 was initially defined by mutations isolated with a recessive larval lethal phenotype (Rogalski et al., 1982; Clark et al., 1986). These recessive lethal alleles most likely represent loss-of-function mutations, because tightly linked revertants of dominant alleles display the same recessive phenotype (Han et al., 1990). The function of let-60 in vulva1 development was defined by two observations. First, loss-of-function mutations result in a vulvaless phenotype once the recessive larval lethal phenotype is maternally rescued (Han et al., 1990). Second, dominant negative mutations cause a dominant vulvaless phenotype, and formally act as if the mutant protein competes with wild-type protein, leading to a reduction of gene function in heterozygotes. These dominant negative mutations were isolated as dominant suppressors of /in-75 Muv mutations (Han et al., 1990; S. Clark and R. Horvitz, personal communication). There is a class of mutations (collectively referred to as /in-34 mutations) that cause a mutant phenotype opposite to that of let-60 dominant negative mutations: all six VPCs adopt vulva1 fates, and hence the hermaphrodites are Muv (Ferguson and Horvitz, 1985; Han et al., 1990; G. Beitel, S. Clark, and R. Horvitz, personal communication; G. Jongeward and I? W. S., unpublished data). Based on genetic mapping, and on interactions between lin-34 and let-60 mutations, these h-34 Muv mutations are most likely gain-of-function (gf) mutations of let-60 (Han et al., 1990; G. Beitel and R. Horvitz, personal communication).

The relationship between let-60 and other genes presumably acting in the vulva1 induction pathway has been studied (Figure 1D; Han et al., 1990). Based on double

Cell 922

A wild type

IllU Hypcdemis Vulva HypodelTliS

B vulvaless (Vul) 0

C multivulva (Muv)

D

lin-15 vulva1 vulva1

inductive inductive signal --) h-23 ---) let-60 + h-1 signal --) h-23 ---) let-60 + lin-I

2 2 functions functions

\ \ hypodermal hypodermal functions functions

Figure 1. Specification of Vulva1 Precursor Cell Fates in Wild-Type and Mutant C. elegans hermaphrodites

(A) A simplified model for vulva1 induction. The six multipotent VPCs are located just ventral to the gonad. Their fates are specified by the anchor cell of the gonad (small oval). A signal from the anchor cell in- duces three VPCs to generate v&al, as opposed to hypodermal cell lineages. (For more detail, see Sternberg, 1988; Sternberg and Hor- vitz, 1989). (B) In vulvaless (Vul) mutant hermaphrodites, all six VPCs generate hypodermal cells even in presence of the anchor cell. (C) In multivulva mutant hermaphrodites, all six VPCs generate vulva1 cells even if the anchor cell is ablated (indicated by “X”). (D) The proposed functional relationship between let-60 and some other genes in the genetic pathway of VPC fate specification (Han et al., 1990). The arrows indicate positive regulation of one gene by another. “7 bars indicate negative regulation of one gene by another. Re- cent mosaic experiments by Herman and Hedgecock (1990) indicated that the /in-75 is likely to act in the hypodermis surrounding the VPCs to influence their fate. The sites of action of other genes in the pathway are not known, but many are expected to act within the VPCs in the response to the inductive signal. While /in-3 most likely acts upstream of let-60, its order of action with respect to /et-23 is not known, and thus has been left out of the diagram.

mutant phenotypes, let-60 acts downstream of the Muv gene h-15 but upstream of the Muv gene /in-l. The relationship between let-60 and let-23 is inferred from less direct evidence. Since the Vul phenotype of a let-23 mutation is suppressed by mutations resulting in elevation of let-60 activity, let-23 is likely to act upstream of let-60 (Han et al., 1990). In addition, the /in-3 gene is proposed to act before let-60 because Vul mutations in let-60 are epistatic to the h-75 multivulva mutations, which are epistatic to the /in-3 mutations (Ferguson et al., 1987; Han et al., 1990).

Table 1. Summary of let-60 and In-34 Mutations

Nature of Mutations Genotype Phenotypes

Loss-of-function (If) If/+ Wild type /f//f Larval lethal, vulvalessa

Dominant dn/+ Vulvaless negative (dn) dn/dn, dn//f Larval lethal, vulvalessa

Gain-of-function (gf) gf/gf Multivulva (>90%)b (h-34 Muv alleles) gf/+ , gf//f, gf/dn Weak multivulva (<20%)b

Data are according to Han et al. (1990). a When the recessive lethality is suppressed, the animals with these genotypes are vulvaless. b Percentage of animals in which additional VPCs are induced to generate vulva1 tissue.

Rescue of let-60 Mutations by DNAMediated Transformation Our strategy for cloning let-60 was to correlate the genetic and physical maps near let-60, and then to define the /et- 60 locus by DNA-mediated transformation and identify DNA lesions associated with let-60 mutations. The physical map in the region of linkage group IV containing let-60 was established by Coulson et al. (1986, 1988) and A. Coulson, J. Sulston, and R. Waterston (personal communication). let-60 maps between the dpy-20 gene and the left breakpoint of a deficiency (nDf27) (Figure 2; Clark et al., 1988; Han et al., 1990). dpy-20 has been localized to a cosmid on the C. elegans physical map by D. Clark and D. Baillie (personal communication). We localized the left breakpoint of nDf27 within two overlapping cosmids by identifying the restriction fragment length polymorphisms on Southern blots of genomic DNA from wild-type and nDf27 heterozygotes (data not shown; see Figure 2 for the map). Therefore, /et-60 was expected to lie within the cosmids covering the region between dpy-20 and the left breakpoint of nDf27, a region of approximately 70 kb.

To identify cosmids containing a functional let-60 gene, we transformed let-60 mutants with each of a set of overlapping cosmid DNAs that mapped between these two boundaries. A plasmid containing a dominant mutant gene of rol-6 (pRF4, a gift from J. Kramer) was used as a dominant transformation marker. Cosmid and marker DNA were coinjected into the gonad syncytium of C. elegans hermaphrodites. The recipient hermaphrodite strains carried either let-60&h) or /et-60(/f) mutations as heterozygotes (see Experimental Procedures). The Fl progeny of microinjected mothers were examined for animals with a Roller phenotype (caused by the coinjected marker DNA). The F2 progeny of these Fl transformants were then examined for stable (germline) transformants. Rescue of /et- 60 Let of Vul phenotypes were scored only among progeny of stable transformants. The results of these transformation rescue experiments are shown in Figure 2. These results suggest that let-60 lies within the overlapping region of three cosmids, ZK205, C3388, and ClOF4, because each rescues the recessive lethal phenotype of let-60.

A 10.5 kb Xhol-BamHI genomic subclone (pMH84) that

A C. elegans ras Gene Controls Vulva1 Induction 923

rescue of: causes Muv Figure 2. Rescue of let-60 Mutations by DNA-

dpy-20 let-60 Mediated Transformation with Cosmid DNA

C35H3 + - ClOF4 + +

C3388 + +

- ZK205 + -__ HD1

-- C50G2 F5863

+ + +

Overlapping cosmids lying between dpy-20 and the left breakpoint of deficiency nDf27 are indicated with their names. The location of dpy- 20 in cosmid C35H3 was previously determined by D. Clark and D. Baillie (personal communication). The approximate location of the left breakpoint of nDf27 was determined by

---i---T--- - -

identifying restriction fragment length polymorphisms on Southern blots as described in Ex- perimental Procedures. Each cosmid has been

lelt brenkpuint of nL$Z 7 dpy-20 tested for its ability to rescue the mutant phenotype of let-60 and dw20. The Muv phenotype

has been examined in animals carrying the injected DNA (display the Rol phenotype of the marker DNA) and have a genotype /e&6o(lf)/+ (which has no Muv phenotype by itself). All cosmids that rescue the let-60 lethal or Vul phenotypes display a low penetrance Muv phenotype (fewer than 20% of animals are Muv). These results suggest that let-60 lies within the overlap of the three cosmids that rescue let-60 defects and cause a Muv phenotype.

lies within the overlapping region of the three cosmids was then used in transformation rescue experiments, and was found to rescue the let-60 mutant phenotypes (Figure 3). Northern analysis using DNA probes within the Xhol-BamHI fragment detected two transcripts (Figure 4). To determine which of the two transcripts corresponds to let-60, further microinjection analyses were carried out with various subcloned DNA plasmids (shown in Figure 3). The results of these transformation experiments nar- rowed the let-60 rescuing activity to the region corresponding to transcript B.

let-60 Is a C. elegans ras Gene We isolated let-60 cDNA clones from a library constructed by Barstead and Waterston (1989) by screening with probes covering the transcript B region (see Figure 3). Sixty one positives were found in a screen of about 1.2 x lo6 plaques. Characterization of nine clones indicated that all have inserts of approximately 1.4 kb (similar to the size of transcript B; see Figure 4). We sequenced one cDNA clone and approximately 4.5 kb of genomic DNA. The locations of exons and introns were inferred by comparison of cDNA and genomic sequences, and are illustrated in Figures 3 and 4. The cDNA sequence is shown

in Figure 5. The only open reading frame of significant length was translated conceptually into a protein of 184 amino acids, and compared with protein sequences in GenBank. The overall structure of let-60 is very similar to ras gene products of other organisms (Figure 8). For example, among the first 164 amino acids, 143 of them are identical between let-60 and the Drosophila rasl protein. This extremely conserved 164 amino acid region (89% of the protein) has been shown to contain the guanine nucleotide binding domains, intrinsic GTPase activity, and the so-called effector domain (residues 32-40), which has been suggested to be the site of interaction with downstream proteins (for reviews, see Barbacid, 1987; McCor- mick, 1989). The C-terminal sequence of /et-60 also fits the consensus Cys-A-A-X-COOH (A is any aliphatic amino acid and X is any amino acid), which has been proposed to be the site for farnesylation (Chen et al., 1985; Fujiyama and Tamanoi, 1986; Buss and Sefton, 1986).

To prove formally that the ras homolog we have cloned is indeed the genetically defined let-60 gene, we have searched for let-60 loss-of-function mutations within the ras gene coding region. Since no restriction fragment length polymorphisms associated with any of the let-60 alleles have been detected by Southern analysis of genomic

transcript A transcript B Figure 3. DNA-Mediated Transformation with 3’5’ DNA Subclones

XhoI H RV RVSAH 1 .a , 2.1 , 2.6 1.751.6

BaIlI 1.5 , 2.5

I I 1 .51 XhoII c R*

I ’ xba

Rescue MU-J

pMH64

pMH63

pHM1

p.MH72

pMH82

pMH81

p.MH65

pMH74

Bal +

XhOII +

C +

XhOII Xba +

RV

Bal/\ S

+ -

A partial restriction map of the Xhol-BamHI fragment, which lies within the overlapping region of the three let-60-rescuing cosmids in Figure 2, is shown. Abbreviations: H, Hindlll; C, Clal; RI, EcoRI; Bal, Ball; RV, EcoRV; S, Sall; A, Apal; and Barn, BamHI. DNA inserts of each plasmid are indicated by the thick lines below the map. The two transcripts displayed in Fig ure 4 are placed above the map according to the Northern results and restritiion analysis of cDNA clones (data not shown). The approximate positions of introns and exons corresponding to transcript B (let-60 cDNA) are also illustrated (se6 Figure 5 for precise positions of intron-exon boundaries in the cDNA).

Cell 924

M 1 2

4.4-

2.37- CA

Figure 4. Northern Analysis of Transcripts within the Xhol-BamHI DNA Fragment

Lane M: molecular weight markers (BRL). Lane 1: two transcripts were detected when the 2.6 kb EcoRV-EcoRV fragment was used as probe. These two transcripts are labeled A and B. Only these two bands were detected when a 13.5 kb BamHI-Barn-HI fragment (which contains the entire Xhol-BamHI fragment) was used as a probe (data not shown). Lane 2: only transcript B hybridized to the 0.6 kb Sall-Apal DNA fragment. Transcript B is also detected when the 0.75 kb EcoRV-Salt 0.5 kb Ball-EcoRV fragment, or 0.5 Kb Apal-Hindlll fragment was used as probe (data not shown).

DNAs (data not shown), all these mutations likely resulted from point mutations or other DNA alterations with no obvious size change. By using a chemical modification and cleavage method (see Experimental Procedures), we have detected mismatches between wild-type DNA and DNA carrying let-60 loss-of-function mutations within the ras gene coding sequence (Figure 7). Therefore, we conclude that the ras gene is the genetically defined let-60 gene. We now refer to this gene as let-60 ras.

Misspecification of VPCs Caused by Extrachromosomal let-60 ras DNA As shown in Figures 2 and 3, all DNA clones that suppress the loss-of-function mutant phenotypes of let-60 also cause a dominant, partially penetrant multivulva (Muv) phenotype. For example, 24% of wild-type hermaphrodites carrying approximately 30 copies of extrachromosomal let-60 ras DNA (pMH72) in an extrachromosomal array are Muv (see Figure 8 and Table 2). In contrast, cosmids or plasmids that do not suppress the let-60 ras mutant phenotypes do not cause a Muv phenotype. This Muv phenotype is unlikely to be caused by titration of a transcription factor by multiple copies of its binding sites, since overlapping DNA fragments that each contain a majority of the gene do not cause a multivulva phenotype (Figure 3). Based on what is known of /et-60 genetics, this Muv phenotype is expected to result from an increase in let-60 fas activity. Since the Muv phenotype has been observed consistently in independent transformed lines, it is unlikely that the Muv phenotype results from mutations generated within the ectopic DNA arrays through DNA recombination or rearrangement. The simplest explanation is that the Muv phenotype is caused by an increase in the cellular concentration of let-60 ras protein to an intolerable level, or by an abnormal spatial or temporal expression pattern of the


DTSAKTRMGVDEAFYTLVREIRKHRERRD

ETSAKTRQGVEDAFYTLVREIRQYRMKKLNSSDDGTQGCMGLPCVVM 189

(JKl , N-QKK'tfjQ;: ;;;

ETSAKTRQGVEDAFYTLVREIRQHKLRKLNPPDESGPGCMSCKC"

ETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNCRFKCKk$L

ETSAKQAINVEEAFYTLARLVRDEGGKYNKT (-130-j K S G S G G C C I I S 322

Figure 6. Comparison of the Protein Sequence of let-60 ras with Other Known ras Gene Products

Identical atTtin0 acids among more than three sequences are boxed. Abbreviations and references: Dros, ras-1, Drosophila ras-1 gene product (Neumann-Silberberg et al., 1984); Rat has (Ruta et al., 1986); Human N-ras (Taparowsky et al., 1983; Brown et al., 1984); Yeast RA.Q, RA.Q of Saccharomyces cerevisiae (Defeo-Jones et al., 1983; Powers et al., 1986). One hundred thirty amino acids near the C-terminus of the yeast ~,4&7 protein are omitted.

extrachromosomal let-60 ras genes. We therefore conclude that a gain-of-function mutant phenotype is generated by expression from the microinjected exogenous let- 60 ras DNA clones.

Pleiotmpic Effects Caused by the Microinjected let-60 ras Gene let-60 ras also functions in several other aspects of C. elegans development. Animals carrying loss-of-function or dominant negative mutations are recessive lethal at an early larval stage and are defective in male spicule development (Han et al., 1990). We have observed that the microinjected let-60 ras gene can cause a dominant lethal phenotype: if the concentration of the microinjected let-60 plasmids exceeded 20 pglml, transformants were inviable (they die at various larval stages) and stable lines could not be established. In addition, we observed that homozygous let-60 loss-of-function (jr) mutant hermaphrodites containing injected let-60 ras DNA displayed a sterile phenotype. This sterile phenotype might be due to rescue of the lethal but not a sterile phenotype of /et-SO(F) by extrachromosomal DNA. It is also possible that the dominant effect of the extrachromosomai DNA generates the sterile phenotype. Unlike the phenotype caused by the microin- jetted wild-type let-60 ras gene, the penetrance of the Muv phenotype caused by /in-34 mutations (likely to be gain-of-

function mutations of let-60 ras) in homozygotes is above 900/o, with no obvious lethal or sterile phenotype (Fergu- son and Horvitz, 1985; Han et al., 1990; G. Beitel and Ft. Horvitz, personal communication; G. Jongeward and P W. S., unpublished data). These results indicate that in- jection of exogenous DNA causes gain-of-function mutant phenotypes for other let-60 ras functions, while h-34 mutations cause more specific defects in vulva1 development.

Exogenous let-60 DNA Suppresses the Vul Phenotype of /in-3 and 181.23 Since the presence of extrachromosomal arrays of let-60 ras DNA results in a gain-of-function mutant phenotype, we used these DNA arrays to study genetic interactions between let-60 ras and two other genes required for vulva1 induction, h-3 and let-23. The let-60 fas DNA arrays were genetically crossed into mutants defective in each of these two genes (see Experimental Procedures). Table 2 shows the results of the analyses of h-3 and let-23 mutants carrying an ectopic let-60 ras DNA array (named syExl), which causes a Muv phenotype in 24% of otherwise wiid- type animals. syEx7 suppresses the Vui phenotype of let- 23(sy97) from 100% Vul to 3% Vul, and that of lh3(n376) from 97% Vul to 12% Vul. These results suggest that an increase in let-60 ras activity bypasses or reduces the need for these two genes in specifying vulva1 cell types.

Cdl 926

Figure 7. Mapping of Point Mutations within the let-60 Coding Se- quences

DNA fragments containing both exons 2 and 3 were obtained by am- plifying genomic DNAs (3 r(g) from strains heterozygous for let-60 mutations by polymerase chain reaction. The chemical modification and cleavage method to detect the mismatch (point mutation) is described in Experimental Procedures. DNA samples were run on a 5.5% sequencing gel. Lane M, a sequencing ladder of an unrelated DNA sample run on the same gel; lane 1: DNA amplified from let-60@59/f)/+; lane 2: DNA amplified from let-6O@y107dnsy763/+/+; lane 3: DNA from /ef-6O(sr755ff,U+ ; lane 4: DNA from let-6O(syXdrtsy727~+. In each of the four lanes, a mismatch due to a point mutation in the let-60 mutants was detected by the appearance of a band running below the full- length fragment. The numbers on the left side of the Lane M indicate the sizes of DNA fragments at the corresponding positions.

Based on these results and previous genetic data, we be- lieve that let-60 ras acts downstream of h-3 and let-23 in the vulva1 induction pathway. Furthermore, syEx7 also causes a Muv phenotype in both the let-23 and h-3 mutants, indicating that the wild-type activity of let-23 and h-3 is not necessary to positively regulate let-60 fas to generate the Muv phenotype.

Like let-60 ras, let-23 is also required for larval growth (Ferguson and Horvitz, 1985; R. Aroian and F? W. S., un-

Figure 8. Exogenous let-6Ores Gene Copies Cause a Muv Phenotype

(A) dpy-20 hermaphrodite. The animals of this genotype are slightly dumpy but are wild type in vulva1 induction (arrow indicates the wild- type vulva. (B) Vulvaless hermaphrodite of genotype lef-6O(sy7OOdn) dpy-20/M- SO(sy7OOdn) dpy-20, which is viable only because it has been maternally rescued by a /in-34(gf) mutation (Han et al., 1990). Eggs have hatched inside the mother. (C) Multivulva hermaphrodite, which has the same genotype as the animal in (B) except that it carries an extrachromosomal let-60 ras DNA array (syfxl) (see Table 2). Extra vulva1 precursor cells generate pseu- dovulval tissue in the ventral hypodermis. This observation demon- strates that an increase in let-60 ras gene activity by the ectopic let-60 genes not only completely suppresses the Vul and Let phenotypes of let-6O(dn) but also displays a gain-of-function (Muv) phenotype. About 17% of the animals of this genotype display the Muv phenotype (Table

2); not all VPCs generate vulva1 tissues in those animals. Photomicro- graphs were taken under Nomarski optics (Zeiss Plan Neofluor 40x dry lens with Kodak 2415 film). All animals are positioned with ventral side toward the bottom. Scale bar is 20 urn.

published data). The let-23 mutation used in this study, sy97, causes more than 90% of the animals to die at an early larval stage (R. Aroian and P. W. S., unpublished data). This lethal phenotype is also suppressed nearly


Table 2. Suppression of Vul Mutant Phenotypes by Extrachromosomal let-60 fas DNA

Extrachromosomalb Genotypea let-60 ras % VUIC % MuvC N

0 0 >200 Wild type + 0 0 290

let-6o(sy700 dn) - 1006 Od >200 /et-So(sy700 dn) + 0 17 156

let-23(sy97) - 100 0 >200 le&23(sy97j + 3 23 112

/in-3fn37.6) - 97 0 257 /in-3(n378) + 12 9 196

a let-6OfsylOOdn) is linked to dpy-20 as a marker and let-23(sy97) is linked to uric-4. b Obtained by microinjection with DNA mix of pMH72 (15 @ml) and pRF4 (rol-6 mutant gene as a marker, 65 vg/ml). pMH72 contains the entire let-60 ras gene (see Figure 3). Construction of the strains with the extrachromosomal DNA array is described in Experimental Procedures. c Percentage of vulvaless (Vul) and percentage of multivulva (Muv) hermaphrodites. The Vul phenotype is indicated by an Egl (egg-laying defective) phenotype (Ferguson et al., 1965). Control experiments also showed that the dominant Roller phenotype of the roC6 mutant DNA has no effect on vulva1 development in wild-type, Vul, and Muv strains used in this study. d let-6o(sylOCdn) homozygotes are progeny of /at-6O(syVlOdn)4i~34(gfj heterozygous mothers (maternal h-34 product recues lethality of /et-SOfsylOOdn) homozygotes; Han et al., 1990).

completely by the extrachromosomal let-60 ras DNA, based OF deviations from expected ratios of progeny segregating from a let-23 heterozygote carrying the let-60 ras array (see Experimental Procedures). This observation suggests that let-60 ras and /et-23 act together in a commonly used developmental control pathway.

Discussion

/ef-60 ras Plays a Key Role in VPC Fate Specification We have shown that let-60, whose activity controls the specification of the fate of VPCs, encodes a protein very similar to ras gene products in other organisms. We have also shown that an increase in let-60 ras gene activity caused by extrachromosomal let-60 ras DNA results in a dominant Muv phenotype, and suppresses the Vul phenotype of two other genes necessary for vulva1 induction. Our results suggest that the let-60 ras gene product functions similarly to other known ras gene products. Figure 9 illustrates a simple model to explain the function of wild- type and mutant let-60 ras proteins in VPC fate specification. We propose that the let-60 fas protein binds to the inner membrane of VPCs and specifies whether they have a vulva1 of hypodermal fate. The let-60 ras protein might be activated in response to the inductive signal through a receptor and other upstream proteins. Loss of let-60 ras function blocks the signal transduction pathway, while in- creased let-60 ras function causes a constitutive response. The site of the let-60 ras action is not known, so

A

Inductive signal

membrane

c

Inductive signal

membrane

B I tive x hypodermal fate

hypodermal fate vulval fates

Figure 9. A Model for let-60 ras Function in Wild-Type and Mutant Animals

We propose that let-60 ras binds to the inner membrane of each VPC and acts in response to an inductive signal. (A) In a wild-type hermaphrodite, reception of the inductive signal acti- vates the res protein, presumably through a receptor complex (indicated by the rectangle crossing the membrane). The activated ras protein promotes the vulva1 cell fate in the VPC. (B) If a VPC does not receive the inductive signal due to a large dis- tance between the distal VPC and the anchor cell (see Figure l), or if the anchor cell has been ablated with a laser microbeam, the 11)s protein is not activated and the VPC adopts a hypodermal cell fate. (C) In the absence of let-60 ras function by loss-of-function or dominant negative mutants, the ras protein cannot be activated by the signal. Therefore, the VPCs always adopt the hypodermal fates. (D) In the presence of hyperactive let-60 ras, resulting from multiple copies of the let-60 ras gene in an extrachromosomal array or from lin- 34(gf) mutations, the vulva1 fate is always promoted in the VPCs, even in the absence of the signal. This model illustrates the simplest explanation of our genetic and molecular results. However, the site of the let-60 ras action is not known, so it is possible that let-60 ras acts in other tissues to indirectly control the fates of VPCs. Whether the inductive signal from the anchor cell directly interacts with receptors on the VPCs is also not known. If the signal acts indirectly through the hypodermal tissue (Herman and Hedgecock. 1990), let-60 ras might act in those cells as well.

it is possible that let-60 ras acts in other tissues to indirectly control the fates of VPCs. However, our model provides the simplest explanation of our genetic and molecular results.

Function and Regulation of M-60 ras Activity The high degree of similarity between the sequences of the C. elegans let-60 ras protein and other known ras proteins reveals the biochemical identity of the let-60 protein. All the biochemical functions of extensively studied ras genes (for review, see Barbacid, 1967) are expected to ap- ply to let-60 ras. For example, amino acids l-164, containing the guanine nucleotide binding activity, are extremely conserved between let-60 ras and other ras gene products (Figure 6), and thus the activity of let-60 ras likely depends on the exchange between the GDP-bound and GTP- bound states. Other structures consented between let-60

Cell 928

ras and known ras genes include the “effector” domain, which has been proposed to interact with a downstream protein in the signal transduction pathway; the C-terminal Site for posttranslational modification, which is required for the attachment of ras proteins to the plasma membrane; and the amino acids at which alterations lead to dominant oncogenic transformation (for reviews, see Bar- bacid, 1987; McCormick, 1989; Hall, 1990). These known biochemical properties of ras proteins allow us to link our genetic studies to specific biochemical functions.

Our genetic analyses suggested that the level of /et-60 ras activity controls the switch between vulva1 and hypodermal cell types during vulva1 induction (Han et al., 1990). Such a role for a C. elegans ras gene is consistent with the proposed function of ras in signal transduction pathways in mammalian cells (for review see Barbacid, 1987). During vulva1 induction, the activity of the let-60 ras protein might be regulated in two different ways. First, the let-60 ras protein could be activated by an intercellular signal acting through a receptor and other upstream proteins, presumably by converting ras from its GDP-bound state to a GTP-bound state. Second, let-60 ras activity might also be controlled by regulation of its spatial and temporal expression, which could be at the level of transcription, translation, and/or posttranslational modification. If the let-60 ras protein is regulated primarily by increasing its GTP-binding activity, the protein might be present consti- tutively, but in a nonactive (GDP-bound) state until induction.

Our results suggest that C. elegans has a strict requirement for either the level of let-60 ras or its temporal and spatial expression pattern. The presence of multiple COP- ies of the wild-type let-60 ras gene in an extrachromosomal array does not consistently rescue loss-of-function mutations of this gene and often causes gain-of-function mutant phenotypes (larval lethal and multivulva). The expression level and pattern of extrachromosomal let-60 ras genes could be significantly different from the endoge- nous let-60 gene. High level expression of the ras prOtO-

oncogene has also been previously shown to cause certain manifestations of malignant phenotypes in mammalian cells (Chang et al., 1982; Pulciani et al., 1985; McKay et al., 1986; Quaife et al., 1987). Furthermore, the above two modes of regulation of let-60 activity could be closely related. For example, there could be a factor that normally represses let-60 ras activity (e.g., by blocking ras-GTP binding) until induction; a high cellular level of let-60 ras could titrate out this negative factor so that the let-60 ras is active without induction. A GAP protein, which stimulates the intrinsic GTPase activity of ras proteins, could be such a negative regulator of let-60 ras.

ras-Mediated Signal Transduction Pathways Searches for genes acting upstream and downstream of ras proteins have been carried out extensively in other organisms. For example, in mammalian cells, tyrosine kinase proto-oncogenes have been suggested to act upstream of the ras proteins, because the ras gene products

al., 1986; Noda et al., 1983). Recently, studies on GAP Proteins have drawn a further connection between ras and UPStream growth factor receptors. GAP is phosphorylated on tyrosine in response to PDGF, EGF, and the oncogene products v-src or v-fps (Molloy et al., 1989; Ellis et al., 1990; Kaplan et al., 1990). GAP interacts with ras-GTP to Catalyze the conversion of ras-GTP to ras-GDP (pahey and McCormick, 1987; Gibbs et al., 1988), and can inhibit morphological transformation by the normal H-ras gene, but not by the gain-of-function mutant ras gene (Zhang et al., 1990). These results suggest that GAP might act as a negative regulator of ras, and that GAP might mediate the regulation of ras by growth factor receptors. Genetic ap- proaches have been taken to identify the biological functions of the ras proteins in the yeast Saccharomyces cerevisiae. The studies in yeast have revealed the relationship between ras and other proteins (e.g., Toda et al., 1985; Powers et al., 1986; Robinson et al., 1987; Broek et al., 1987; Garrett and Broach, 1989; Tanaka et al., 1989; Hughes et al., 1990; Fedor-Chaiken et al., 1990).

A similar genetic approach to study ras function can now be taken in C. elegans. Since the C. elegans let-60 ras protein is closely related to mammalian ras proteins (see Figure 6), genetic and molecular analysis of let-60 ras in C. elegans provides strong in vivo evidence for a developmental control function of the ras gene family in animals. The extensive genetics of C. elegans vulva1 induction now provides an opportunity to identify gene products that act upstream and downstream of the ras protein in a signal transduction pathway. For example, based on double mutant phenotypes, we proposed that let-60 acts downstream of the h-3, let-23, and lin-15 genes, but upstream of /in-7 to specify the VPC fates in response to the anchor cell signal (Figure 1D). Characterization of these and other genes that act in vulva1 induction might allow us to identify genes encoding known or novel products that interact with let-60 ras, and thereby relate the genetic pathway to a ras-mediated biochemical regulatory pathway.

Experimental Procedures

Strains and Genetic Methods Methods for culturing, handling, and genetic manipulation of C. elegans have been described by Brenner (1974). All genetic experiments were performed at 20%. Vul, Muv, Egl. Let, Dpy, Uric, and Rol refer to vulvaless, multivulva, egg-laying defective, lethal, dumpy, uncoordi- nated movement, and roller phenotypes respectively. The standard wild-type strain N2 is that of Brenner (1974). The alleles of various mutants used in this study are listed below.

LGII linked-double mutations: let-23(sy97) one-4(e720) (Ft. Aroian and f? W. S., unpublished data); mnC7 [dpy-70@7282) uric-52@444)](11j (Herman, 1978).

LGIV single mutations: dw-20(67362) (Brenner, 1974); /in-3(n378) (Ferguson and Horvitz, 1985); /in-34(sy73Cgf) (Han et al., 1990); nDf27 (Ellis and Horvitz. 1988).

LGIV linked-double or multiple alleles: let-SO(s59) uric-22(s7) uric-37 (e769) (Rogalski et al., 1982); let-60@7724/f) uric-22(s7) uric-37(e769) and /et-SO(s77551f) uric-22(s7) uric-37(e769) (Clark et al., 1988); let- 6O(sy7OOdn) dpy-2O(e7282), uric-24(e738) let-dO(syXdn ~~727). let- 8O(sy707dn ~~763) dpy-2O(e7282) and lh34(n7046gf) uric-22(s7) (Han et al., 1990). let-(m435)nTl(lV; V) (obtained from D. Riddle’s laboratory).

LGV: him-5(e7490) and him-5(e7467) (Hodgkin et al., 1979)

are required for serum stimulation of prolif&ation and for Cosmld Clones and C. elegans Physical Maps transformation by tyrosine kinase oncogenes (Smith et All cosmid clones shown in Figure 3 and the physical map overlapping


cosmids were obtained from A. Coulson and J. Sulston (MRC Labora- tory of Molecular Biology, Cambridge, England).

Localization on the Phyeical Map of the Left Breakpoint of Deflclency nDf27 Genomic DNA was prepared from a C. elegans strain heterozygous for deficiency nDf27 and from strains without the deficiency. The DNA samples were digested with restriction enzymes (EcoRI, EcoRV, Hindlll, Bglll, and Clal plus Xhol) and run on a standard 1% agarose gel. Southern hybridization analysis was performed with cosmid clones as probes. Restriction fragment length polymorphisms were identified when two cosmids, F56B3 and C5OG2, were used as probes to detect shifts in migration of restriction fragments (shifts were ob- SeNed with all the above enzymes except Hindlll). Therefore, the left breakpoint of nDf27was determined to lie within 6 kb of the DNA region covered by both cosmids.

DNA-Medlated Ttansformatlon of /et-60 Mutants Microinjection of cloned DNAs into the gonad syncytia of C. elegans hermaphrodites was carried out according to the method developed by C. Mello, V. Ambros, J. Kramer, and D. Stinchcomb (unpublished data). In this method, only the distal arms of the hermaphrodite gonad are targets of microinjection. A plasmid containing a dominant mutant gene of m/-6 (pRF4) is used as a dominant marker. Since multiple copies of pRF4 are required to generate a dominant roller (Rol) phenotype, transformants generally contain a large array of extrachromosomal DNA (over 100 copies of the m/-6 gene; C. Mello and V. Ambros, personal communication; M. H. and P. W. S., data not shown). In our experiments, the concentration of pRF4 was 50-65 @ml; the concentra- lion of DNA to be tested for its ability to rescue let-60 mutants was l-50 rig/ml.

Three strains were constructed and used as host strains for the micminjected DNA: (1) /et-6O(syloOdn) dpy2O(e7282ylh34(nlO46gf) uric-22; (2) /et-60@1724/f) unc-22Mpy-2O(el362); and (3) /et-SO(sSS/f) unc-22@7ww20(e7362) uric-31. For all three strains, only one-fourth of the progeny of a hetemzygous mother are expected to be let-60. homozygotes. After microinjection of parents with cloned DNA, only Fl heterozygous Rol animals were selected. Typically about 10% of these Fl transformants are germline transformants (stable transformant lines). We screened for let-60 rescue at the F2 or F3 generation, In the case of strain 1, Dpy (dumpy) progeny of stable Rol parents were screened; usually more than 30 Dpy animals were picked to examine if any of them gave viable progeny. These Dpy animals are homozygous for /et-6O(syPNdn) dw20, and are viable for one generation because of the maternal effect of h34(nlO46gf) in the parent strain (Han et al., 1990). However, only Dpy animals containing extrachmmosomat let-60 DNA are expected to give viable progeny; those Dpy animals containing no extrachromosomal let-60 DNA are Egl (egg-laying defective) and segregate only dead larvae (Han et al., 1990). We also observed that dpy-20 mutations suppress the Rol phenotype of the m/-G mutation such that the stable transformants of genotype let-60 dpy-20 homozygotes were Dpy but nonRol. In addition, because let-80 is less than 0.01 map units from dpy-20, (Han et al., 1990) (or as we now know, about 5 kb), recombination between these two is extremely rare.

For strains 2 and 3, homozygous Uric-22 animals were screened from animals of stable heterozygous parents. Recombinants were dis- tinguished from let-60 transformants because Uric-22 recombinants segregated Dpy-20 progeny: recombinants between uric-22 and let-60 will pick up the dpy-20 mutation and be of the genotype let-60 unc- 22Aiw20 uric-22. Strain 2 was used for microinjecting all the cosmids and plasmids listed in Figures 2 and 3A; strain 1 was used for microin- jetting all the cosmids and plasmids except cosmid C35H3; and strain 3 was used for micminjecting cosmids C35B8,ZK205, and C35H3. In all the transformation experiments with let-68.rescuing clones (see Figures 2 and 3), stable M-60 transformant lines could always be established from strain 1. Rescue of /et-60(/f) in strains 2 and 3 was generally difficult. In many cases, stable transformant hetemzygotes segregated only sterile let-60 homozygous animals. However, several stable lines were established from strain 2 (with C3388,ZK205, and pMH82) and strain 3 (with C33B8 and pMH82). They were established either by direct microinjection, or by genetic crosses to transfer the ectopic DNA arrays from rescued /ef+Sqsylm) ~2tlfet-so(svl0&fn) dty20 animals to a strain of genotype /et-So(n) unc-22/let-6O(/f) uric-22. The

Muv phenotype was examined among transformants from strains 2 and 3. For some let-60 rescuing plasmids, the Muv phenotype was also examined in /et-6o(sy7OOdn) homozygotes, and otherwise wild-type animals (see Table 2). Quantitation of the Muv phenotype was described previously (Han et al., 1990). All let-60 transformants have a small brood size (lo-50 progeny) compared with about 300 progeny in wild-type animals.

Suppmsslon of the Vul Phenotypes of Other Genes The Vul mutations /h3(n378) and let-23(Sy97) were used in the sup pression study. The ectopic DNA arraysyEx7 used for the suppression study was obtained by microinjecting a DNA sample containing 65 uglml pRF4 and 15 ug/ml pMH72. The fin-3; syEx7 strain was constructed by crossing /&?@378); him-5(eI490) males with /et-6o(sylOOdn) dpy-20/7et-6o(sy7OOdn) dw20; syEx7 hermaphrodites. Fl Roller hermaphrodites were picked; they had the genotype let-So(sy7OOdn) dpy- 2O//in-3; syEx7 (/in-3 is linked to let-60 and is about 0.5 map units away; Clark et al., 1988; R. Hill and P W. S., unpublished data). F2 Rollers were individually picked, and /in-3/lin-3; syEx1 animals were recog- nized by examining their progeny (which contained no Dpy animals; nonRol animals, which had lost syfxl, are mostly Egl).

let-23(sy97); syEx7 was constructed by crossing let-23(sy97) unc- 4(e120jImnC7 males (mnC7 is a chromosome balancer [Herman, 19781) with /et-6O(sylOOdn) dpy-2OAet-6O@ylOOdn) dpy-20; syEx7 hermaphrodites. Roller progeny were followed for a few generations until animals were obtained that displayed both the Rol and Uric phenotypes, and did not segregate Dpy progeny. These animals have the genotype let-23fsy97) uric-Met23(sy97) uric-4; syExl. This genotype was confirmed by the observation that nearly 100% nonRol animals (which had lost syEx7) were Egl and segregated mostly dead larvae. let-23(sy97) has a strong larval lethal phenotype; about 90% of the homozygotes die at an early larval stage (R. Aroian and P W. S., unpublished data). To determine whether syEx7 also suppresses the lethal phenotype of let-23(sy97), N2 males were crossed with let-23(sy97) uric-4/&23(sy97)unc-4; syEx7 hermaphrodites. Fl Roller nonUnc hermaphrodites (let-23(sy97) uric-4/+ +; syEx7) were selected and their Roller progeny were examined. If the lethality of sy97 was completely suppressed, the number of Uric and Roller animals would be one- fourth of the number of total Rol animals; if the lethality of sy97 was not suppressed, the number of Uric and Roller animals was expected to be lo-fold lower. It was found that among 95 F2 Rollers, 23 were Uric and Rot indicating that the lethal phenotype of /e&23&97) was also significantly suppressed by the presence of syEx7.

Genomlc DNA and cDNA Manipulations and Sequencing Northern analyses were carried out to map the transcripts to the genomic DNA fragments. Both transcripts A and B (Figure 3) were detected when the Hindlll fragment (6.6 kb), the EcoRV fragment (2.6kb), or a BamHl fragment (13.5 kb, which contains the entire 10 kb Xhol-BamHI fragment) was used as probe. Only transcript A was detected with the Hindlll-EcoRV fragment (1.9 kb, 0.8 kb to the left of the Xhol site) used as probe. Only transcript B was detected when the Apal-Hindlll (0.5 kb) fragment, the Sall-Apal fragment (0.6 kb), the EcoRV-Sall fragment (0.75 kb), or the Ball-EcoRV fragment (0.5 kb) was used as probe.

cDNA clones were obtained from a ZAP library kindly provided by Dr. R. Barstead and R. Waterston (Barstead and Waterston, 1989). Af- ter screening approximately 1.2 million plaques with genomic DNA probes overlapping the let-60 gene region (DNA fragments from the EcoRl site to BamHl site in Figure 48) we identified 61 positive plaques. Nine candidates were purified and characterized further. All nine clones have the same internal restriction sites and are approximately 1.4 kb in size. Restriction enzyme analyses of the cDNA inserts were carried out with the cDNA inserts directly amplified from the i clones by polymerase chain reaction with primers hybridizing to the tinker region of the vector. For sequencing, plasmids containing the inserts were obtained by in vivo excision of the AZAP phages. DNA subclones in Bluescript (SK+) were constructed from the cDNA clones, Double-strand DNA sequencing was performed by dideoxy chain ter- mination methods with Sequenase. We have sequenced through all the restriction sites within the cDNA clone.

We have also sequenced about 4.5 kb of genomic DNA covering the entire cDNA region (from the EcoRl site to the region close to the Xbal

Cell 930

site in Figure 48) (data not shown). The introns and exons were determined by comparing the genomic and cDNA sequences,

The cDNA sequence was translated into amino acids, which were compared to the existing protein sequences in GenBank with the TFASTA program.

Standard molecular biology methods including DNA enzymatic reactions, DNA and RNA purification and DNA cloning techniques. Southern analysis, Northern analysis, plaque hybridization, and DNA sequencing were performed essentially as described by Sambrook et al. (1989) and Ausubel et al. (1989).

Mapping Point Mutations wlthln the let-80 ras Codlng Region Chemical modification of mismatched cytosines (with hydroxylamine) and cleavage of DNA at the modified nucleotides were carried out based on the procedure described by Cotton et al. (1988). This method will detect all but A:T to TA mutations. We followed a protocol communi- cated by Ft. Barstead with minor modifications of our. own. Genomic DNA from animals heterozygous for let-60 alleles were used as reaction substrates. Each let-60 mutation in Figure 7 was placed in trans to a balancer chromosome (/ef(m345)nT1). Genomic DNA fragments were amplified by PCR with oligonucleotides complementary to flank- ing noncoding sequence (about 100 bp away from the intron-exon boundaries). The two primers used for the samples in Figure 7 are 5’-CCTGCTCGTGTCGTGTGAGGAGT-3’and 5’-GACTGAATTTGTACCT- TAATTGTAC-3’. About 3 ug of purified genomic DNA was used in each PCR reaction so that any initial PCR mistake would be diluted. The mutant and wild-type let-60 DNA mixes were labeled at the 5’ ends with szP by T4 kinase. Mismatches of the nucleotides were obtained by denaturation and renaturation of the mixture of DNAs of two genotypes. In addition, we ran the DNAsamples through G-25 spin columns rather than perform DNA precipitations at each step. After chemical modification and cleavage of the mismatched base, DNA samples were run on a 5.5% sequencing gel. A mismatch due to a point mutation in let-60 was detected by the appearance of a single band running below the full-length fragment. The polymorphism detected by this method is reproducible: in more than 20 reactions performed to detect mismatches within the first three exons of the gene, no random, false positive bands have been detected.

Acknowledgments

We thank Hiro Mori and Phoebe Tzou for their help during this study. We thank Alan Coulson and John Sulston for providing us with the cosmid maps in the let-60 region, and for their efforts in helping us to re- solve the physical map near the let-60 DNA region. Our thanks also to R. Barstead and R. Waterston for the cDNA library. J. Kramer for pRF4, the C. elegans Genetic Center (University of Missouri) for useful strains, and J. Mendel, R. Hill, G. Jongeward, R. Aroian, and other members of our laboratory for strains and other reagents. We thank C. Mello, V. Ambros, J. Kramer,.and D. Stinchcomb for the transformation procedure, D. Clark and D. Baillie for their communication of unpublished dpy-20 cloning results, R. Barstead and R. Waterston for the protocol to map point mutations, and G. Beitel, S. Clark, and R. Horvitz for communication of their unpublished genetic results on/in-34/let-60. We also thank W. Boorstein, A. Golden, Y. Hajdu, W. Katz, H. Lipshitz, H. Mori, S. Parkhurst, and B. Wold for comments on the manuscript. M. H. is a Genentech Fellow of the Life Science Research Foundation. P W. S. is an investigator of the Howard Hughes Medical Institute. This research has been supported by a grant to t? W. S. from the USPHS (HD23890).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 5, 1990; revised October 3, 1990.

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GenBank Accession Number

The accession number for the sequence reported in this paper is M5535.

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let-60, a Gene That Specifies Cell Fates during C. elegans ...post.queensu.ca/~chinsang/a-worm-reader/han-and-sternberg-1990.pdf · during C. elegans Vulva1 Induction, Encodes a MS