A novel ras-related gene family

Cell, Vol. 41, 31-40,

May 1985, Copyright

A Novel ras-Related

Pascal Madaule

and Richard

0092-8874/85/050031-10

0 1985 by MIT

$02.0010

Gene Family

Axel

Howard Hughes Medical Institute and Institute of Cancer Research College of Physicians and Surgeons Columbia University New York, New York 10032

Summary We have identified a new family of ras genes, the rho genes, which share several properties with the more classical ras gene family consisting of N-, K-, and H-ras. The rho genes, first isolated from a cDNA library from the abdominal ganglia of Aplysia, encode proteins that share 35% amino acid homology with H-ras. Evolutionarily conserved counterparts of rho have been detected in yeast, in Drosophila, in rat, and in man. Sequence analysis reveals over 85% homology between the human and Aplysia proteins. The ras and rho gene products share several common properties; both are 21,000 daltons, both reveal C-terminal sequences required for membrane attachment, and both show blocks of strong internal homology, suggesting that the two proteins may share common functions but may use these functions in different ways. Introduction The genome of all eukaryotes examined contains at least one member of a remarkably conserved family of ras genes (Shilo and Weinberg, 1981; DeFeo-Jones et al., 1983; Powers et al., 1984; Neuman-Silberberg et al., 1984). These genes were first identified as the transforming genes of both the Harvey and Kirsten sarcoma virus. Homologs of these viral sequences were subsequently identified in chromosomal DNA of several eukaryotes, suggesting that these viral oncogenes arose by transduction of cellular rat DNA during serial passage of leukemia viruses (for review, Ellis et al., 1982). Mutations in the cellular ras genes, which either alter the structure of the ras protein or increase the level of ras gene expression, have been demonstrated in several human tumor cell lines. Moreover, these mutant genes are capable of transforming fibroblasts to the tumor phenotype (Chang et al., 1982; Reddy et al., 1982; Shimizu et al., 1983; Stacey and Kung, 1984). The evolutionary constancy of the genes, taken together with the transforming potential of specific mutants, has led to the suggestion that the cellular ras genes must play an essential role in growth or development and, if altered, may lead to uncontrolled growth characteristic of transformed cells. Recent studies suggest that the ras gene product may function as a component of the complex of G proteins transducing information from the cell surface to regulate eukaryotic activities within the cell (Gilman, 1984). The mammalian ras gene products are associated with the inner leaflet of the cell membrane (Willingham et al., 1980;

Sefton et al., 1982) bind GTP (Scolnick et al., 1979), and exhibit a slow GTPase activity (Sweet et al., 1984). The ras genes of yeast also bind GTP (Tamanoi et al., 1984) and a deficiency of both ras genes is lethal (Kataoka et al., 1984; Tatchell et al., 1984). Genetic and biochemical analyses reveal a striking reduction in the activity of adenylate cyclase in these mutants, which can be restored by transformation with either the yeast or the human ras genes (Toda et al., 1985; Kataoka et al., 1985). Moreover, sequence comparisons between ras and the known members of the G protein complexes reveal significant homology between Go or transducin, two mammalian G proteins, and the N-terminal sequence of the ras proteins (Hurley et al., 1984a). Thus, the ras proteins share both structural and biochemical properties with the a-subunit of the G protein complex. In this study we have identified a new class of genes encoding proteins that share 35% homology with the classical ras proteins. This gene family, first identified in the marine snail Aplysia, shows striking evolutionary conservation among distant eukaryotes, including man. These observations suggest that the ras-like genes may be members of a super-family of genes comprised of individual families including those encoding the classical fas gene products, the G proteins, the Go and transducin, and a third set of ras-like proteins described in this study. Each of these proteins shares regions of structural homology, but also reveals regions of diversity, suggesting that they each share one set of biochemical functions, but may be divergent in others.

Results

and Discussion

A New ras Gene Is Expressed

in the Mollusc,

Aplysia

During the analysis of a cDNA library prepared from the Aplysia abdominal ganglian for clones homologous to mammalian peptide hormones, we inadvertently identified a new family of ras-related genes. We isolated a cDNA clone by low stringency screening that shared weak homology with the cDNA encoding the a subunit of human chorionic gonadotropin (hCG). The nucleotide sequence of this clone revealed a 5 amino acid homology with hCG embedded in an open reading frame of 157 nonhomologous amino acids. This short stretch of homology presumably afforded sufficient complementarity to be detected under our screening conditions but was not of sufficient length to suggest that this gene is related to hCG. We noted with interest, however, that the complementary strand also encoded an overlapping open reading frame of 192 amino acids. A computer-assisted search of the NIH protein database (the Protein Identification Resources of the National Institutes of Health) identified several regions of homology between the protein deduced from the sequence of this open reading frame and the sequences of several members of the ras gene family. Since the cDNA clone revealed open reading frames on

Cell

32

-10

* TTCGGCCAAGTGCCAACC

1 10 * * ATG GCA GCG ATA Met Ala Ala Ile

20 30 * * CGA AAG MG CTT GTT ATA GTC GGA GAT Arg Lys Lys Leu Val Ile Val Gly Asp

50 60 70 80 90 * * * * * TGT GGT AAA ACA TGT CTA CTT ATT GTC TTC AGC AAA GAC CAG TTC CCT GAA GTT Cys Gly Lys Thr Cys Leu Leu Ile Val Phe Ser Lys Asp Gln Phe Pro Glu Val 110 120 * * CCA ACA GTT TTT GAA AAT TAT Pro Thr Val Phe Glu Asn Tyr

130 * GTA GCA GAC ATT Val Ala Asp Ile

40 * GGT GCG Gly Ala 100 * TAC GTG Tyr Val

150 140 * * GAA GTT GAT GGC AAA CAG GTT Glu Val Asp Gly Lys Gln Val

160 * GAG CTA Glu Leu

170 180 * * GCT CTG TGG GAC ACA GCG GGA CAA Ala Leu Trp Asp Thr Ala Gly Gln

190 200 210 * * * GAG GAC TAT GAC AGA CTG AGG CCG CTG TCT Glu Asp Tyr Asp Arg Leu Arg Pro Leu Ser

220 * TAC CCT Tyr Pro

230 240 * * GAC ACA GAT GTC ATC CTC ATG TGT Asp Thr Asp Val Ile Leu Met Cys

250 260 270 * * * TTC TCT ATA GAC AGT CCA GAC AGT CTG GAG Phe Ser Ile Asp Ser Pro Asp Ser Leu Glu

280 * AAC ATA Asn Ile

290 300 310 320 330 * * * * * 1 CCG GAG AAG TGG ACG CCT GAG GTT CGT CAC TTT TGT CCA AAT GTT CCT ATA ATA Pro Glu Lys Trp Thr Pro Glu Val Arg His Phe Cys Pro Asn Val Pro Ile Ile

340 * CTT G'TG Leu Val

360 350 * * GGT AAC AAA AAG GAT CTT CGC AAC Gly Asn Lys Lys Asp Leu Arg Asn

380 390 370 * * * GAT GAA AGT ACC AAA CGT GAG CTC ATG AAA Asp Glu Ser Thr Lys Arg Glu Leu Met Lys

400 * ATG AAA Met Lys

410 420 * * CAG GAA CCA GTG AGA CCA GAG GAT Gln Glu Pro Val Arg Pro Glu Asp

430 440 450 * * * GGG CGC GCC ATG GCT GAG AAA ATC AAC GCC Gly Arg Ala Met Ala Glu Lys Ile Asn Ala

460 * bAC Tyr

470 480 * * CTT GAG TGC TCT GCT AAA ACC Leu Glu Cys Ser Ala Lys Thr

490 500 510 * * * AAG GAG GGC GTG AGG GAT GTG TTT GAG ACA Lys Glu Gly Val Arg Asp Val Phe Glu Thr

520 * GCT ACC Ala Thr

TAT Tyr

530 540 * * AGA GCT GCG CTG CAA GTG AAA AAG Arg Ala Ala Leu Gln Val Lys Lys

550 560 570 580 * * * * AAG AAG AAG GGT GGA TGT GTT GTA TTG TGA ATAAGTC Lys Lys Lys Gly Gly Cys Val Val Leu ---

590 600 610 620 630 640 650 * * * * * * * GCTGTTTTCTTCHATTCCCCACAACAGGGGCTGGTG~GGAGGACTGCATG~TTTTGTCTTTTG~GATATTCCTTT 670 680 690 700 * * * * TAAAATTATTTTTTTTAAAATACC~TTTTAAGATTAGTGC

710 *

720 *

660 *

730 740 * * AAAAAGACGGATGCTTCC

750 770 800 810 760 780 790 * * * * * * * CGACTGAAACACAAGAAAGGCCATATCAGAGTGGCATATGATCACAGT~ATATTTTGTGGCTTGTGTGATTGCATATAT Figure

1. Nucleotide

and Deduced

TCT Ser

Amino Acid Sequence

Aplysia rho cDNA was sequenced using strategies described by an arrow at position 186. The first five nucleotides reflect

of Aplysia

rho

In Experimental Procedures. a sequence of the synthetic

opposite strands, we first determined which of the two strands was transcribed: the strand encoding weak and statistically insignificant homology to hCG, or the strand encoding a ras homolog. To this end, the cDNA was cloned in both orientations in the RNA expression vector

820 *

The umque Hpa II site of the cDNA Eco RI linker used for cloning,

clone

IS indicated

pSP62 (Zinn et al., 1983), and highly radioactive RNA complementary to each of the strands was synthesized. These two RNAs were used to probe a Northern blot of poly(A)+ RNA from the Aplysia abdominal ganglion. Hybridization is observed to two discrete bands 4.5 kb and

A Novel ras-Related 33

Gene

Family

II-ras

Flgure 2. Comparison of the Amino Acid Sequences of Aplysla fbo and Human H-ras

Aplysia

*

H-raS Aplysia

*

* 110

I!-ZC&S Aplysia

****

As described In the text. the best alignment of the two sequences reveals 35% homology with three gaps. Conserved residues between Aplysia and H-ras are boxed. Residues conserved in these sequences as well as Dras2 and YP2 are indicated by asterisks. The numbering refers to that of the H-ras sequence.

*

120 AART

;;;;DflMVviCB VPIILVGNKKDLRNDESTKRELMKMKQEPV **** **

H-rX Aplysia

130 140 VESRQAQDLARSYGIP[I~T,==\RQ~E~A[Y~L RPEDGRAMAEKINAYSYLECSAKTKEGVRDVFETA

Aplysia

T!Ft]AA

LMVMK

KhiK

150 *

* *

*

*

G G

3.3 kb in length only with the RNA complementary to the ras-like sequence (see Figure 3, below). The nucleotide sequence and deduced amino acid sequence encoded by this gene, which we denote rho, for fas homologous, is shown in Figure 1. The protein consists of 192 amino acids and has a predicted mass of 21,000 daltons. When the amino acid sequence of rho is compared with human H-ras using the Needleman and Wunsch algorithm (1970) the two sequences align with 35% homology and three gaps (Figure 2). The alignment score is 17 standard deviations above the average score for 100 random sequences of the same length and composition as rho. Comparison with other distantly ras-related sequences is also highly significant. YP2, a yeast gene sharing 34% homology with H-ras (Gallwitz et al., 1983), is 32% homologous to rho. Similarly, Dras2, a Drosophila gene sharing 50% homology with H-ras (NeumanSilberberg et al., 1984) is 29% homologous with rho. This strongly suggests that these genes share a common ancestor (Doolittle, 1981). Sequence comparisons among the members of the classical ras gene family reveal a conserved interposition of constant and variable regions, and it is useful to consider the homology between ras and rho in this context (Shimizu et al., 1983; Taparowsky et al., 1983). The three human proteins, H-, K-, and N-ras, are over 97% conserved from the NH, terminus to residue 120. Residue 121 demarcates the first variable region that extends to amino acid 132. A second highly conserved region extends from residues 133 to 165 followed by a highly variable C-terminal domain of 24 amino acids. This basic organization of constant and variable regions is also observed upon comparison of Drosophila ras 1 with human ras genes (Neuman-Silberberg et al., 1984) but breaks down when the yeast ras genes are included in the comparison. The two yeast ras genes maintain about 80% conservation through the first 80 amino acid residues. The remaining 100 amino acids show significant divergence, even in con-

stant regions (Powers et al., 1984; DeFeo-Jones et al., 1983). Overall, the Aplysia rho shares 35% homology with H-ras, but the homology is clustered, revealing regions of complete divergence and other regions of strong conservation. The region of maximal divergence between ras and rho extends from residues 121 to 140 (122 to 1.55 for rho), a region in which no homology is observed between rho and ras. This region, which coincides with the first variable domain of the classical ras gene family, encompasses a gap in the alignment because of the presence of a 14 amino acid block in rho not present in the ras sequence. The 12 C-terminal amino acids of rho correspond to the second variable domain of the classical ras gene family. This domain in rho is 12 amino acids shorter than that of ras and is homologous with ras at only two positions. Thus, rho diverges from ras most in regions in which the individual ras genes diverge. Thus, interposition of constant and variable domains is retained not only among members of a given ras gene family, but among the different ras gene families as well. Within the more conserved domains, strong homology is observed in four discrete regions. Between residues 5-17 (7-19 for rho), ras and rho share 67% homology. Further, two out of the four changes within this amino acid stretch are conservative. In this region rho retains a glytine at equivalent position 12, a residue that is altered in several transforming ras genes (Reddy et al., 1982; Seeburg et al., 1984). Residues 57 to 64 (59-66 for rho) show even stronger conservation, with a 7 out of 8 homology with one conservative change. Interestingly, these two regions of conservation include the specific residues of the cellular ras genes, which, if mutated, result in genes with transforming potential (Fasano et al., 1984). It has been suggested that the regions of ras between residues 5 and 17 (Wierenga and HOI, 1983; Sweet et al., 1984) and 57 and 64 (Shih et al., 1982a) may be involved in GTP binding or hydrolysis. The 20 N-terminal residues of H-ras, as

Cell 34

A

well as rho, share significant homology with a sequence present in the (I subunit of Go, a membrane GTP-binding protein. Even greater homology is observed between several FAD and NAD binding proteins with the N-terminal domain of rho:

B

C

D

rho NADH dehydrogenase (Young et al., 1961)

If the sequence conservation in these regions also results in the conservation of function, we may anticipate that rho will also reveal nucleotide binding and hydrolysis activities. In the comparison between ras and rho, a 7 out of 8 homology is also observed from residues 113-120 (114-121 for rho) and 11 of 18 homology with largely conservative changes is observed from residues 141-158 (156-173 for rho). No specific function has been attributed to these regions. However, residues 110-120 in both ras and rho share weak homology to the GTP binding protein of E. coli, EFTu (Leberman and Egner, 1984). When the distantly related Drosophila and yeast proteins, Dras2 and YP2, are compared with the H-ras and rho, conservation among the four sequences is clustered in two blocks from residues 57-62 and 114-120. Additional dispersed residues conserved among the four proteins are also observed in the second constant region (Figure 2). Two additional properties of the ras proteins have been conserved in rho. First, the C-terminal region, although highly divergent among different ras genes, often terminates with the sequence Cys-A-A-X, where A is an aliphatic amino acid, and X, the C-terminal amino acid (Powers et al., 1984). They subunit of transducin, a retinal G protein, terminates with this sequence as well (Hurley et al., 1984b). This characteristic is also retained in rho, which terminates with Cys-Val-Val-Leu. Ras proteins are synthesized as soluble precursors (Shih et al., 1982b) that migrate to the inner leaflet of the cell membrane where they acquire tightly bound lipids (Willingham et al., 1980; Sefton et al., 1982). Deletion of the six C-terminal amino acids in v-H-ras prevents lipid attachment and membrane binding, and abolishes its transforming potential (Willumsen et al., 1984). The C-terminal sequence of rho suggests it may also associate with the cytoplasmic face of the cell membrane through its C-terminal domain. Interestingly, just upstream of the putative binding site, rho contains a stretch of five lysines. A stretch of basic residues close to the C-termini has also been described for the Drosophila protein Dras2 (Neuman-Silberberg et al., 1984) and for human K-ras exon 48 (McGrath et al., 1983; Shimizu et al., 1983). These basic residues presumably facilitate or stabilize membrane binding by interaction with acidic lipids, One final property shared by the ras proteins is the striking conservation of size; all of the ras proteins, with the exception of those of yeast, retain a mass of 21,000 daltons. This size constraint suggests that these proteins may be integral components of a larger complex of proteins, each interacting with one another in such a way that a change

2kb-

Figure

3. Northern

Blot Analysis

of rho RNA in Aplysia

Tissues

Poly(A)’ RNA was electrophoresed on a 1% formaldehyde agarose gel and was hybridrzed to highly radioactive Aplysia rho antisense RNA synthesized in vitro using the SP6 transcription system. Lanes A, C, and D contain 7 rg of poly(A)’ RNA from hepatopancreas, buccal mass, and ovotestis, respectively. Lane S contains 2 rg of poly(A)’ RNA from both the abdominal and ring ganglia. Lane S is exposed for 48 hr, while the remaining lanes are exposed for only 16 hr. Analogous experiments with sense-strand probes show no signal on parallel Northern blots.

in size in one protein while still maintaining

could not be readily accommodated, the function of the larger complex.

Expression of the Aplysia rho Gene We have examined a number of Aplysia tissues for the expression of rho RNA by Northern blot analysis (Figure 3). Two major bands 4.5 kb and 3.3 kb in length are observed in hepatopancreas, ganglion, and ovotestis poly(A)+ RNA. A single band, 4.5 kb, is observed in the buccal muscle. Thus neurons, secretory epithelial cells, and muscle appear to express this gene. At present we can offer no molecular explanation for the existence of two discrete mRNA species, and we do not know whether these two RNAs are the product of the transcription of two genes or whether they derive from alternative processing of a single transcript. A minimum estimate for the relative frequency of rho RNA in ganglion RNA populations can be obtained from the frequency of rho positive clones in an abdominal ganglion cDNA library. We observe a frequency of one in 30,000. This is obviously a minimum estimate since the smallest rho mRNA is 3.3 kb long, and the coding se-

A Novel 35

ras-Related

A

Gene

Family

B

C

D

kb

A

Human ‘I

-

234

,

56789101112’1

Rat DIYIC

kb

23

-(l-*-o*

~clr~.

*‘y,

II

m

-.

-

4.4

-20 -09 -

2.3 1.3 I. I 0.6 0.3

2.3 -2.0 B

Human ‘I

Figure

4. Southern

Blot Analysis

of rho Genes

in Drfferent

2 3 4

5 6

Rat 7 8 9

IO II 12’ I I 2

3’

DI YI C

kb

0

Organisms

-4.4

Cellular DNA is digested with Eco RI separated on a .@/a agarose gel and probed with the 1 kb rho cDNA insert from Aplysra. (A) 4 rg yeast DNA; (B) 10 pg Drosophila DNA; (C) 20 pg human DNA; (D) 10 rg Aplysia DNA. Hybridization was performed under conditions of moderate stringency at 42% in 30% formamide as descrrbed in the Experrmental Procedures.

I

-m* e

-2.3 -2.0 - 1.3 I.1 -0.9 -0.6 -0.3

quence only occupies 600 nucleotides. Since the probes we have used in the screen consist largely of coding sequences, the bulk of the somewhat shorter rho clones could not have been detected. It is of interest that both nervous ganglion and buccal muscle consist largely of nondividing cells, thus rho gene expression may not be restricted to proliferating cells.

Aplysia rho is a Member Evolutionarily Conserved

of a New Family of ras-like Genes

Given the remarkable conservation of the classical ras genes among distant species, we asked whether we could detect homologs of rho in organisms other than Aplysia. This question was initially addressed by performing Southern blotting on Eco RI-digested DNA obtained from Aplysia, Drosophila, yeast, and man (Figure 4). Under conditions of moderate stringency, a series of intense bands is observed with Aplysia, yeast, and Drosophila DNA. A weaker array of bands is observed with human DNA, presumably because of the larger complexity of the human genome. The molecular weight of the Eco RI digestion products of Drosophila and yeast DNA differ from those observed when classical ras is used as a probe in low stringency hybridizations (Shilo and Weinberg, 1981; Powers et al., 1984). If this blotting experiment is performed at high stringency, no hybridization is observed with heterologous DNA. The lowest molecular weight band detected with Aplysia DNA disappears under these annealing conditions, suggesting the presence of a small gene family in Aplysia. These data further suggest that the hybridizing sequences represent evolutionarily conserved homologs of rho. This conclusion is supported by the analysis of human cDNA clones described below.

Figure 5. Southern Aplysia rho

Blot Analysrs

of Cloned

DNAs

Homologous

to

I phage DNAs were cut with Eco RI, were fractionated on 1.2% agarose gels, and were analyzed using the moderate stringency hybridization procedures described in Experimental Procedures. Human and rat lanes represent 15 independently isolated cDNA clones, 200-500 ng of phage DNA per lane; lane Dl contains 150 ng of a Drosophrla melanogaster clone; lane Yi contains 25 ng of a Saccharomyces cerevisiae clone; lane C contains 2 ng of Aplysra rho cDNA, cut with Hpa II. (A) Hybridization was performed with nicktranslated 300 bp Hpa II fragment encodrng the N-termrnal half of Aplysia rho. (B) Parallel blot probed wrth a 700 bp Hpa II fragment encoding the C-terminal half of Aplysra rho.

Isolation and Characterization from Rat and Humans

of rho, cDNAs

To demonstrate that the Aplysia rho genes are conserved through evolution, we isolated cross hybridizing clones from cDNA libraries from distantly related organisms and determined the extent of conservation by nucleotide sequencing. Aplysia rho cDNA was used as a probe to obtain homologous cDNAs from both human peripheral T cell and adult rat brain cDNA libraries constructed in the A cloning vector gtl0. Cross-hybridizing clones were obtained from both the T cell and brain cDNA libraries at a frequency of one in twelve thousand. These libraries were screened in duplicate with either rho cDNA or a mixture of N- and K- ras probes. Clones that hybridize with rho cDNA do not hybridize with the classical ras probes, suggesting that they reflect transcripts of conserved homologs of Aplysia rho. In addition, we have isolated crosshybridizing genomic clones from a library of chromosomal DNA from both Drosophila and yeast. Preliminary analyses of these clones by restriction

Cell 36

10 20 30 40 * * * * * TTCGGC GAG TTC CCC GAG GTG TAC GTG CCC ACC GTC TTC GAG AAC TAT Glu Phe Pro Glu Val Tyr Val Pro Thr Val Phe Glu Asn Tyr 1

60 70 * * GAG GTG GAC GGC AAG.CAG Glu Val Asp Gly Lys Gln

80 90 100 * * * GTG GAG CTG GCG CTG TGG GAC ACG GCG GGC CAG GAG Val Glu Leu Ala Leu Trp Asp Thr Ala Gly Gln Glu

130 140 120 150 * * * * GAC CGC CTG CGG CCG CTC TCC TAC CCG GAC ACC GAC GTC ATT Asp Arg Leu Arg Pro Leu Ser Tyr Pro Asp Thr Asp Val Ile 180 190 209 * * * GAC AGC CCG GAC TCG CTG GAG AAC ATC Asp Ser Pro Asp Ser Leu Glu Asn Ile 240 * TGT CCC AAT Cys Pro Asn

250 * GTG CCC ATC ATC CTG Val Pro Ile Ile Leu

300 310 * * GTC CGC ACA GAG CTG GCC CGC ATG Val Arg Thr Glu Leu Ala Arg Met

ATG Met

50 * GTG GCC GA<: ~XTT Val Ala Asp Ile

TGC TTC Cys Phe

170 * TCG GTG Ser Val

230 210 220 * * * CCC GAG AAG TGG GTC CCC GAG GTG AAG CAC TTC Pro Glu Lys Trp Val Pro Glu Val Lys His Phe

260 270 280 * * * GTG GCC AAC AAA AAA GAC CTG CGC AGC GAC Val Ala Asn Lys Lys Asp Leu Arg Ser Asp 320 330 340 * * * AAG CAG GAA CCC GTG CGC ACG GAT Lys Gln Glu Pro Val Arg Thr Asp

360 370 380 * * * GCC GTG CGC ATC CAA GCC TAC GAC TAC Ala Val Arg Ile Gln Ala Tyr Asp Tyr

420 430 * * GTG CGC GAG GTC TTC GAG ACG GCC Val Arg Glu Val Phe Glu Thr Ala

160 * CTC ATG Leu Met

110 * GAC TAC Asp Tyr

290 * GAG CAT Glu His

350 * GAC GGC CGC GCC Asp Gly Arg Ala

390 400 * * CTC GAG TGC TCT GCC AAG ACC AAG Leu Glu Cys Ser Ala Lys Thr Lys

410 * GAA GGC Glu Gly

440 450 460 * * * ACG CGC GCC GCG CTG CAG AAG CGC TAC GGC Thr Arg Ala Ala Leu Gln Lys Arg Tyr Gly

470 * TCC CAG Ser Gln

480 490 500 510 520 530 * * * * * * AAC GGC TGC ATC AAC TGC TGC AAG GTG CTA TGA GGGCCGCGCCGTCGCGCCTGCCCCTGCCGGC Asn Gly Cys Ile Asn Cys Cys Lys Val Leu --Figure

6. Nucleotide

and Deduced

Ammo Acid

Sequence

of a Human

rho cDNA

The human cDNA clone homologous to Aplysia rho, clone 6, was sequenced by the chain-terminator technique usrng the strategy presented in Experimental Procedures. The first five nucleotides represent the sequence of the synthetic Eco RI lanker used for cloning. The sequence begrns at presumed amino acid residue 29.

digestion and Southern blotting suggest that the human cDNA clones reflect the transcription of not one gene but of a small family of rho genes in the human genome. Aplysia rho cDNA was cut at a single Hpa II site at codon 96, generating N-terminaland C-terminal-specific fragments. Under conditions of moderate stringency, all but one of the 12 human cDNA clones hybridized with the fragment encoding the N-terminal segment of the protein (Figure 5). In contrast, only human clones 6 and 9 hybridize strongly with the C-terminal probe. These observations suggest that we have identified at least three classes of human cDNA clones as follows: clone 6, which hybridizes strongly with both N- and C-terminal probes; clone 9, which hybridizes only with the C-terminal probe; and a remaining set of 10 clones, which hybridize strongly with

N-terminal probes and weakly with C-terminal probes (Figure 5). The variation in intensity observed upon hybridization of C-terminal probes to the human cDNAs suggested that these classes may differ by virtue of the presence of variable C-terminal domains. In subsequent experiments all 12 human cDNA clones were hybridized under high stringency with cDNA derived from representatives of the following three putative classes: human clones 1, 6, and 9. Under conditions of high stringency clones 6 and 9 hybridize only to themselves. The remaining 10 human cDNAs all hybridize strongly to human clone 1. These experiments strongly suggest that three distinct rho mRNAs are expressed in human T cells, which are likely to derive from three independent genes. Transcripts characterized by the hu-

A Novel raMelated

Gene

Family

37

1

10

20

Figure 7. Comparison of the Amino Acid quences of Aplys~a and Human rho

Aplysia Human 6 40

Aplys1?3 HL,J?Xl5

50

110 120 FCPNVPIILVGNKKDLRNDESTKRELNKMKQEPVR FCPNVPIILVANKKDLRSDEIiVRTELARMKObPVR 150

Fplysia

Hm6

130

140

q i

160

170

190

~Iii:::;:~:::CKVL

man clone 1 cDNA are most abundant. This conclusion is supported by preliminary sequence analysis of the clones described below. A similar analysis of cDNA clones obtained from rat indicate that in this species as well there are at least two classes of cDNA, one most homologous to human clone 1 and a second homologous to the human cDNA clone 9. Finally, it should be noted that we have isolated both yeast and Drosophila genomic clones (Figure 5) that contain DNA inserts that hybridize at moderate stringency with Aplysia rho cDNA. In the Drosophila clone, Eco RI fragments are generated that hybridize either to N- or C-terminal probes, while in yeast, hybridization is only observed with the N-terminal probe. These observations with isolated genomic clones confirm our previous data that rho, like classical ras, may be highly conserved from yeast to human.

Sequence

The human cDNA (clone 6) begins at residue 29, allowing comparison over the 168 C-terminal amino acids. Homologous residues are boxed.

PEDGRAMAEKINAYSYLCCSAKTKEGVRDVFETAT TDDGRAIlAVRIQAYDYLECSAKTKEGVREVFETRT j IoIIi 180

Aplysia Humm6

70

PTVFENYVADIEVDGKQVELALWDTAGQEDYDRLR PTVFENYVADIEVDGKQVELALUDTAGQEDYD~

H-“Fimn

Aplysia

60

Se-

Analysis

of Human

rho cDNAs

Human clone 6, which hybridizes equally intensely with N- and C-terminal probes (Figure 5) was initially chosen for sequence analysis. The nucleotide sequence and deduced amino acid sequence of human clone 6 is shown in Figure 6, and a comparison of the amino acid sequences of human and Aplysia rho is shown in Figure 7. This cDNA clone begins at residue 29. From residue 30 to residue 85 there is complete identity between the human and Aplysia sequences, and only four differences are observed between residues 85 and 122. The human sequence, like Aplysia, contains a 14 amino acid insertion beginning at residue 122, which is not present in the classical ras genes. From residues 122 to 155, we observe a region of marked divergence between human and Aplysia rho that coincides with the first variable region described for the classical ras genes. From residues 156 to 180, which correspond to the second constant domain of the classical ras family, the two sequences are almost identical, with a single conservative amino acid change. Maximal divergence is observed in the 16 C-terminal amino acids. The highly basic character of the Aplysia rho and several ras C termini is not evident in human rho. This protein terminates with the sequence Cys-Lys-Val-Leu, a se-

quence more similar to the C terminus, Cys-Lys-Met-Leu, of the Drosophila gene Drasl (Neuman-Silberberg et al., 1984). Interestingly, the regions of maximal variance among the different members of the classical ras gene family also coincide with the variable regions among members of the rho family. We have obtained a preliminary sequence extending from the 5’end of human cDNA clone 1. The 50 N-terminal amino acids are identical with those of Aplysia rho. The sequence we have obtained thus far for clone 1 overlaps that of clone 6 over a short region of 66 nucleotides or 22 amino acids. The amino acid sequence in this overlapping region diverges at a single position, and several third position changes are observed in the nucleotide sequence. These observations clearly demonstrate the presence of at least two genes encoding the human rho cDNAs we have isolated. In humans, the rho genes comprise a small gene family that shares greater than 85% homology with the rho genes of the mollusc Aplysia.

Discussion We have identified a new family of ras-related genes, the rho genes, which share several properties with the more classical ras gene family. The rho genes, first detected in the mollusc, Aplysia, encode a protein that shares 85% homology with proteins encoded by the rho genes present in man. Furthermore, homologous counterparts can be detected in yeast, Drosophila, and rat. A yeast gene, YP2, and a gene expressed in human melanoma cells (Gallwitz et al., 1983; Padua et al., 1984), also share partial homology with the classical ras genes, but these genes are likely to differ from the rho family, and their conserved counterparts in other species have not yet been identified. These observations suggest that the ras genes may comprise a family of genes significantly larger than previously expected, a super-family of genes. Since both the rho genes and YP2 were identified in the course of analyzing cloned DNA purely by chance, it is likely that several other members exist within the genome of all eukaryotes. The ras and rho gene families reveal an interesting pattern of parallel evolution. Aplysia and human rho share a

Cell 38

35% amino acid homology with the ras proteins, indicating that the two gene families have arisen from a common ancestral gene. Divergence to generate the two gene families must have occurred early in evolutionary time since the ras and rho genes are present within the genome of primitive eukaryotes. Following this early divergence, the individual families show remarkable sequence conservation. Aplysia rho, for example, shows 85% amino acid homology with a rho protein of man. Comparison of the sequence of both ras and rho through evolution reveals that the minimal divergence observed within the individual families is restricted to two short variable stretches interposed between two highly conserved or constant domains Thus, the ras and rho genes appear to have evolved from a common ancestor that diverged early in evolution to generate two gene families that show striking conservation through subsequent evolution. ras and rho share the following properties: both are 21,000 daltons, both reveal C-terminal sequences required for attachment to the inner leaflet of the cytoplasmic membrane, and both show blocks of strong internal homology, suggesting that the two proteins share common functions (Powers et al., 1984; Willumsen et al., 1984). Interestingly, two regions of maximal conservation between rho and ras, residues lo-17 and 57-65, have been implicated in GTP binding and hydrolysis. Moreover, mutations at specific residues within these regions result in ras proteins with transforming potential (Fasano et al., 1984). Thus, ras and rho reveal common properties that suggest that they may share common functions, but they also have regions of complete divergence, suggesting that members of the two families may use common functions in very different ways. What may these shared and unique functions be? The classical ras proteins exhibit several biochemical and structural features of the a subunit of the complex of G proteins. The G protein complex is coupled both to surface receptors and to internal effector molecules, such as adenylate cyclase (Gilman, 1984). The specific association of ligand with receptor results in the binding of GTP to G, and this activated complex may then interact with effector molecules, either stimulating or inhibiting their activity. This activity is terminated by the hydrolysis of bound GTI? Evidence that yeast ras may act as a G protein to activate adenylate cyclase has emerged from an analysis of cyclase activity in mutants deficient in ras gene function and in transformants with restored function. Further, the 20 N-terminal amino acids of ras and rho show homology with sequences present in Go, the a subunit of a bovine brain G protein complex, as well as with bovine transdutin, a G protein of the retina (Hurley et al., 1984a). This region also shows homology with sequences in several dinucleotide binding proteins (Wierenga and HOI, 1983). We may speculate that the individual members of the super-family of ras-like genes may encode different components of the G protein complex that serves as intermediary proteins capable of transducing information from the cell surface to effector proteins within the cell. The conserved domains among the super-family of ras-like proteins may function to bind and hydrolyze GTP or to interact

with the more conserved subunits of the G protein complex. The variable domains may therefore serve to link the ras proteins to several different receptors or effector proteins. This may afford flexibility in function such that a single cell may respond identically to different ligands or, alternatively, two different cells may respond differently to identical ligands. Experimental

Procedures

cDNA and Genomic Libraries cDNA libraries ware constructed in the 1 cloning vector gtl0 (Thanh Huynh, Stanford University, personal communication) using procedures described by Littman et al. (1985). An Aplysia abdomrnal ganglion cDNA library was generously provided by Michael Palazzolo, the T cell library by Dan Littman, and the rat brain cDNA library by David Anderson. The Drosophila melanogaster genomic library constructed in the I clonrng vector, Charon 4A, was a grft of Tom Mamatrs (Maniatrs et al., 1978) and the yeast (Saccharomyces cerevisiae) genomic library was a gift of John Woolford (Woolford and Rosbash, 1981). DNA Probes lnrtial screenrng of the Aplysia cDNA library was performed wrth a 600 bp Hind Ill insert encompassing the codmg region of the a subunit of human chorionic gonadotropin derived from the plasmid a-HCGlpBR322 (Fiddes and Goodman, 1979). We thank John Fiddes for the gift of this plasmid. The N-ras DNA probes were synthesized from a 450 bp Sal INco I fragment of the plasmid p6al (Taparowsky et al., 1983). The K-ras probe was comprised of 1 kb Eco RI insert of the plasmid HiHi 3 (Ellis et al., 1981) and includes the viral K-ras sequences. These plasmids were generously provided by Mitchell Goldfarb, Michael Wigler, and Edward Scolnick. The inserts were labeled with 3*P by nick translatron to specific activities of 5-10 times lo* dpmlpg (Weinstock et al., 1978). Northern Blot Analysis RNA was extracted from various frozen tissues of Aplysia californica using the guanidine thiocyanate homogenization procedure, followed by ultracentrifugation through a CsCl cushion (Chirgwin et al., 1979). Polyadenylated RNA selected on an oligo(dT) cellulose column was denatured, was run on a formaldehyde-agarose gel, and was transferred onto nitrocellulose as described (Scheller et al., 1982). A singlestranded RNA probe was synthesized according to Zinn et al. (1983) from pSP62 plasmids containing the 1 kb Aplysia rho cDNA insert in both orientations at the Eco RI cloning site. Prior to in vitro transcrrption, the plasmids were linearized using the unique Pvu I restriction site of the vector. Blots were analyzed with strand-specific probes according to Zinn et al. (1983). Moderate Stringency Southern Blot Analysis Eco RI-digested DNA was electrophoresed on agarose gels and was transferred onto nitrocellulose filters as described (Roberts and Axel, 1982). These filters were prehybridized for 8 hr at 42OC in 30% formamide, 5 x SSC (1 x SSC = 150 mM NaCl and 15 mM Na citrate), 5 mM EDTA, 0.1% SDS, 0.1% Ficoll, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 12 mM Na, HPO,, 9 mM NaH,PO,, 0.06% (w/v) Na, P,O,.lO H,O (pH 7.0) and 100 pglml of sonicated and denatured salmon sperm DNA. Hybridization was performed for 20 hr at 42% in the above buffer with salmon sperm DNA at 50 pglml, 0.04% bovine serum albumin, Ficoll, and polyvinylpyrrolidone. The rho cDNA probe labeled by nick translation was denatured and was used at a concentration of 2 nglml. Following hybridization, filters were washed in 2 x SSC, 0.1% SDS, 2.5 mM EDTA, 12 mM Na, HPO,, 9 mM NaH,PO,, 0.06% (w/v) Na,P,O, .lO H,O (pH 7.0). The filters were washed several times at room temperature and then at 55% for 2 hr. We thank Michael Young, Laura Sarokin, Dan Littman, and James Jackson for providing DNA samples. Screening of Libraries The Aplysia cDNA library constructed in the A vector gtl0 was plated on E. coli CGOO/HFL and was transferred onto nitrocellulose according to Maniatis et al. (1982). The filters were prehybndized at 45% for 4 hr in SET 4 x (SET 1 x = 150 mM NaCI, 2 mM EDTA, 30 mM Tris-HCI

A Novel 39

ras-Related

Gene

Family

Tumorigenic transformation of mammalian human gene homologous to the oncogene virus. Nature 297, 479-483.

cells induced by a normal of Harvey murine sarcoma

Chirgwin, J., Przybyla, A., McDonald, R., and Rutter, W. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Humon6

ECORI 5”

Hl”fl t .

XII01 t

Nod t .

/-

ECORI t 3’ a

Figure

8. Sequencing

Strategy

for Aplysia

and for Human

rho cDNAs

Circles at the origin of arrows indicate sequences determined using the chemical degradation procedure after 3’OH end-labeling (Maxam and Gilbert, 1980). Filled circles at the origrn of arrows indicate sequences determined using the chain-terminator procedure (Sanger et al., 1977). (pH 8) at 25%) 0.1% SDS, 0.1% Ficoll, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 10 kg/ml of sonicated, denatured salmon sperm DNA. The filters were hybridized at 45% for 20 hr in the above buffer minus SDS and salmon sperm DNA. Nick-translated hCG cDNA probe was added at a concentration of 1 nglml. The filters were then washed at 47’C in 1.3 x SET and 0.1% SDS, until no background was detectable by a Geiger counter and were then exposed at -70°C with intensifying screens for 3 days. Aplysiarho cDNA was used as a probe to isolate homologous clones from other species. Genomic libraries from Drosophila and yeast were plated on E. coli LE 392. Four replrca nitrocellulose filters were obtained from each plate. Two filters were hybridized with nick translated Aplysia rho cDNA insert and one filter was hybridized wrth a mixture of nick-translated inserts representative of human N-ras and viral K-ras. Hybridization was performed as described above for screening the Aplysia library except the temperature was adjusted to 50%. Filters were washed usrng the conditions described above for moderate stringency Southern blot. A fourth filter was hybridized at high stringency with nick-translated pBR322 to exclude contaminating clones. Sequence Analysis The Aplysia rho cDNA was sequenced using both chemical procedures of Maxam and Gilbert (1980) and the chain-terminator technique of Sanger et al. (1977) using Ml3 derivative vectors (Vieira and Messing, 1982). Human cDNA clones were sequenced entirely by the chain-terminator method. Strategies for sequencing are summarrzed in Figure 8. Other Techniques Preparation of A phage DNA, plasmid DNA, subclonings, and high stringency screening of libraries were performed according to Maniatis et al. (1982). High stringency Southern blot analysis was performed as described by Roberts and Axe1 (1982).

We wish to thank Drs. Russell Doolittle and Mrchael Wigler for helpful discussions in the analysis of this data. We also thank John Fisher for salvaging this clone by suggesting a homology search with the strand complementary to the HCG homology. We are grateful to Philip Bourne for his help with the program. We also thank Drs. Mitch Goldfarb and Steve Goff for their suggestions and careful reading of this manuscript and Phyllis Kisloff for preparing the manuscript. This work was supported by grants from the Howard Hughes Medical Institute (R. A. and P M.), the Public Health Servrce (R. A ), and by the French Government (P M.). 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 wrth 18 U.S.C. Sectron 1734 solely to indrcate this fact. January

25, 1985

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