Nucleotide Sequence of the Two Rat Cellular rash Genes

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1 MOLECULAR AND CELLULAR BIOLOGY, May 1986, p Vol. 6, No /86/5176-5$2./ Copyright 1986, American Society for Microbiology Nucleotide Sequence of the Two Rat Cellular ras Genes MARTIN RUTA,l* RONALD WOLFORD,2 RAVI DAR,3 DEBORA DEFEO-JONES,4 RONALD W. ELLIS,4t AND EDWARD M. SCOLNICK4 Laboratory of Cellular and Molecular Biology,' Laboratory of Experimental Carcinogenesis,2 and Laboratory of Tumor Virology,3 National Cancer Institute, Bethesda, Maryland 2892; and Merck Sharpe & Dohme, West Point, Pennsylvania Received 14 October 1985/Accepted 2 January 1986 We present the nucleotide sequence of the coding region of the rat c-ras11 gene and a partial sequence analysis of the rat c-ras-2 gene. By comparing these sequences with the arvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the arvey virus differs from the cellular c-ras-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-ras-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NI 3T3 cells, and differs in sequence from both c-ras-1 and v-ras at several base pair positions. The arvey murine sarcoma virus (-MuSV) is an acutely transforming retrovirus that was derived during passaging of a Moloney murine leukemia virus in rats (12). Rat cellular sequences transduced by -MuSV (v-ras) encodes a 21,-dalton protein that is required for the induction and maintenance of transformation (4, 11, 21, 26). The rat cellular c-ras gene from which the viral v-ras gene was presumably derived is part of a multigene family that is highly conserved in evolution, having been detected in Saccaromyces cerevisiae (5, 16) as well as humans (7, 18, 24). There are at least three members of the ras gene family in higher eucaryotes: the cellular c-ras, c-rask, and c-rasn genes (2, 15, 22). Each of these genes encodes similar but distinct p21 proteins that are thought to play a role in the normal cell cycle (2, 14, 15, 18, 22, 24). Of particular interest is the finding that ras genes are activated in human tumors. There are two cellular c-ras genes present in the normal rat genome, the c-ras_1 and c-ras-2 genes (4). These genes differ in their structural properties. eteroduplex comparison with v-ras suggests that the c-ras_1 gene contains at least four exons, whereas the c-ras-2 gene is apparently colinear with the viral ras gene (4). In addition, the c-ras-1 and c-ras-2 genes apparently differ in their biological properties. When ligated to a viral promoter, the c-ras_1 gene, like viral ras, transforms NI 3T3 cells in transfection assays (3, 4). Transformed cells transfected with the activated c-ras_1 gene synthesize high levels of a p21 protein that has similar properties to the viral ras p21. Both the cellular and viral p21 proteins are localized to the inner surface at the plasma membrane and can bind GTP. owever, viral ras p21 differs from c-ras p21 in being a phosphoprotein at position 59 and in its impaired ability to hydrolyze GTP (9, 19, 2, 23). In contrast, the c-ras-2 gene is not activated by ligation to a viral long terminal repeat. To identify the similarities and differences between the v-ras gene and the two cellular rat ras genes, we determined the complete nucleotide sequence of the c-ras-1- coding region and a partial sequence analysis of the c-ras_ 2-coding region and compared these sequences with the known v-ras sequence (6). Our results indicate that v-ras * Corresponding author. t Author to whom reprint requests should be sent. p21 differs from the c-ras_l p21 protein at only two amino acids, those at positions 12 and 59. Alterations at both of these positions are thought to activate the c-ras gene and alter the biochemical properties of p21. In contrast, the c-ras-2 gene differs from the v-ras gene at numerous positions and is likely to be a nonfunctional pseudogene. MATERIALS AND METODS DNA sequencing of ras. The molecular cloning of c-ras-1 and c-ras-2 has been described previously (4). The pbr322 clone LXB3 contains a 2.3-kilobase-pair insert of rat cellular sequences spanning the coding region of c-ras-1. LXB3 was derived from the previously described clone -MuSV-sarcII (4) by blunt ending the EcoRI and XbaI sites and adding BamI linkers. The pbr322 clone GAB contains 1.5 kilobase pairs of the c-ras-2 gene and was derived from the clone -MuSV-sarc I (4) by blunt ending the 1.5-kilobase-pair AccI fragment and adding BamI linkers. Nucleotide sequencing was performed by the chemical modification method of Maxam and Gilbert (13). Sequence data were analyzed by the computer program of Wilbur and Lipman (28). Activation of c-ras-2. Molecular constructs between a portion of v-ras and c-ras-2 were prepared by previously described methods (4). These constructs were analyzed for their transforming ability on NI 3T3 cells by a CaPO4 coprecipitation procedure (27). 176 RESULTS Sequence analysis of c-ras-l. To identify the similarities and differences between v-ras and the rat cellular c-ras-1 and c-ras-2 genes, we determined the nucleotide sequence of the c-ras_1 gene and a partial sequence of the c-ras-2 gene and compared these sequences with the known sequence of v-ras. In Fig. 1 is presented the DNA sequence of the coding region of the c-ras_1 gene, as well as the sequencing strategy that was employed. By comnparing the c-ras_l gene sequence with the viral ras and human c-ras_1 gene sequences (1, 6, 17), we identified the p21 protein-coding sequences. The protein-encoding sequence began with the ATG codon at nucleotides 161 to 163 and was followed by an open reading frame corresponding to the remainder of the

2 VOL. 6, 1986 RAT CELLULAR ras GENE NUCLEOTIDE SEQUENCE 177 A Pvu,, inc Pt Bam i ind III Mst I Pat I Xma I / pa I Pvu II Pst I Bam i Sa I lac I l Sac I D ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ GGATCCGGAATTCGGCCACTGGCTTGCTTGCCTACTCAGTGCTGGAGAGAGCCCTTGGGTTTTCATCTATTAGCAGTCTCCAATGGCTAGGGCTGGCTAGTGTATTCTCATTGGCA EXON Met Thr GIu Tyr Lys Leu Yal Val Val Giy Al a Fiy Gly Val Gly Lys Ser Ala Leu Thr GTGGGGCAGGAGCTCCTGGMGGCAACCCCTGTAGMGCG ATG ACA GAA TAC MG CTT GTG GTG GTG GGC GCT w GGC GTG GGA MG AMT 6CC CTG ACC Ile Gln Leu Ile Gln Asn is Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu 325 ATC CAG CTG ATC CAG AAC CAT m GTG GAC GAG TAT GAT CCC ACT ATA GAG GTGAGCTCTGACTACCTGCCAGAGGTCGGCTCTGGCAGTGGTCATGGGTTGA GTCCCAAACAACTAGGTCTTGAAGTTGGTATGGGCCTGATTCCTACCTGATCCTGATCCATCAGGGTATGAGAGGTGCAAGGGTAGGCGGATTCTCTGTCTAAGAGGTAGGACCCTTA EXON Asp Ser Tyr Arg Lys GIn Val Val Ile Asp Giy GIu Thr Cys Leu Leu Asp Ile Leu Asp Thr ii Gly GIn Glu GIu AGCTGTGTTCTTGCAG GAC TCC TAC CGG AAA CAG GTA GTC ATT GAT GGC -GAG ACG TGT TTA CTG GAC ATC TTA GAC ACA E GGT CM GM GAG 632 Tyr Ser Ala Met Arg Asp GIn Tyr Met Arg Thr Giy GIu Giy Phe Leu Cys Val Phe Ala IlIe Asn Asn Thr Lys Ser Phe GIu Asp Ile TAT AGT 6CC ATG CGG GAC CAG TAC ATG CGC ACA GGG GAG GGC TTC CTC TGT GTA m 6CC ATC AAC AAC ACC MG TCC m GM GAC ATC is Gin Tyr Ar 749 CAT CAM TAC AM GTGACCACCTATGGCTAGCCCGTGGCCCATGGCATATGTGAGGAAGGTTCTGTGTGCACACTGAGGCTTTATGTCTTTTiTTGMTGTCATGGACACGTCAMGCCT EXON 3 85 g Giu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val Leu Val Gly Asn 6CAGTCT6CTA6CTGGCTCATATACAACCCCATCCCCCTCCAG 6 GAG CAG ATC MG CGG GTG AM GAT TCA GAT SAT GTG CCA ATG GTG CTG GTG GGC AC 91 Lys Cys Asp Leu Ala Gly Arg Thr Val Glu Ser Arg Gln Ala Gln Asp Leu Ala Arg Ser Tyr Giy Ile Pro Tyr Ile Glu Thr Ser Ala MG TGT GAC CTG GCT 6CT C6C ACT GTT GAG TCT CGG CAG GCC CAG SAC CTT GCT CGC A6C TAT GGC ATC CCC TAC ATT GM ACA TCA GCC Lys Thr Arg Gln 157 AM ACC CCC CAG GTGTGA6CTTGTTCCCCTCTCCACMGCTAGTCAGGUATTCACCATACCCCACCCCAGCCGGAGCGAGCTCATC6CCCTCTCCCTCMCACAG6CAGCCGCTCT 1178 GGCTCTGGCTCCAGCTCTGGACCCTCTGGGACCCCCCCGGGACCCATGTGACCCATGTGACCCAGCGGCCCTCATGCTGTAAG TCTTCCGCAG GCCGCAGGCGAGGGCAACM6GCCAt 1299 GGGTCTGGGCTTATGCCMAAATTCTGGGTTGACACAsTAGCTCCAGGGCAGGATGGGGTCCATGGAGAGAGCTGCCCTGAGCAGGCCGGAGCGGTGACCCAGGGGCCTAGTTCTTCTTGTCT 1412 CCAGTGTCCTATGG6AGAMGCTAT TMAGCCCTTAAGTGTTGTTAGGTTGTTAACCTTGAGACATACTGGGG GTCTGGA AGTCTGAGCTAAT6GCTCTGATTMCAGTGGTAM 1541 AGGAGTGAACCTACTTMAAAAGAGTGGCCTGTACTT M AGATGGCCAGGCCCAGCTCCCTATTATTTTGGTM GCAGCTGAGGAAGGGAGCCTCCAGCATGGG TGTTACCTGATCCA GTCAGGGAGMCTTACCCAGAGAGCAGAGCACCCCUTCCTCCTCAGAGTCCTCTCTGATCCAGTCCCTCTGTCCCCAG EXON Giy Val Glu Asp Ala Phe Tyr Thr Leu Vol GGT GTG GAG CAT GCC TTC TAC ACA CTA GTA Arg Glu Ile Arg Gln is Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu Ser Giy Pro Ciy Cys Met Ser Cys Lys Cys Val Leu Ser CGT GAC ATT CGC CAG CAT AAA CTG CGG AAA CTG MC CCG CCT GAT GAG AGT GGC CTT GCC TGC ATG AGC TGC MG TGT GTG CTG TCC TGACA CTAGGTGAGGCAMGGACCAGCCAACATCTGGGGCAMTGGCCTCAGCTAGCCAGATGMCTTCATATCCACTM ATGTCCCTGCTCCCCCAATTCTGCCAATCCCCCTGCCTGCAGGCAT 1985 CAGTCATGTCCMGTGCCCGTCCCGCAGGCTCAAGGACGAGGAGGTGCCGGATGCAGGGAG GG GAGGTGCTGTC GUGGGGkGAGATGGAGCC AGTTCAA 6 TGGTGGACAGTGTGCCAAGCCTTCGCATGGACAATTGAACAGACTGTCATGAACTATCCCGTTGCCACGCCACCCAAGTCCTCCGCCCCCTCTCAGCTCCCTTGGGCGC PolyA CTATGAGGGCACATGTTGMTCACAGTAAATTATTTSATGGACTTUACTTUTCTCTCGGCTGG... FIG. 1. Nucleotide sequence of the coding region of rat c--ras 1. (A). Restriction map of the c-ras-1-coding region and the strategy for determining the nucleotide sequence. The numbering indicates the distance (in base pairs) from the start of the BamI cloning linker. Recognition sites for restriction endonucleases that recognize six base pair sites in the c-ras_1 gene are indicated above the map. DNA digested with the appropriate restriction enzyme was end-labeled at its 5' or 3' terminus. The arrows below the map indicate the direction and extent of sequence determined for each fragment. (B). DNA sequence of the 2.3-kilobase BamI insert of the LXB3 clone containing the c-ras_1-coding sequences. The amino acid sequences encoded by exons of c_ras_1 are shown above the DNA sequences. A putative splice acceptor site in the 5'-noncoding region is underlined. Amino acid positions that differ from those of v_ras p21 are boxed. 216

3 178 RUTA ET AL. first exon. As is true of the human c-ras-1 gene, the rat c-ras 1 p21-encoding sequences were represented in four exons. Appropriate donor and acceptor splice sites were present at the exon borders. The sequence data predicted that the rat c-ras_1 gene encodes a protein of 189 amino acids, which is the same size as the viral ras and the human c-ras_1 p21 proteins (1, 6, 17). Indeed, the amino acid sequence of the p21 proteins encoded by the rat and human c-ras_1 genes were identical (Fig. 1 [1, 17]). This is consistent with the observation that ras genes are highly conserved in evolution (5, 16). In addition, the viral ras p21 protein also' shows a very striking similarity to the rat c-ras_1 protein. Only three base pair differences existed between the viral and rat cellular coding sequence. Two of the base pair differences (base pairs 194 and 527) resulted in amino acid differences, while the other base pair change was silent (base pair 1626). The expressed base pair differences resulted in altered amino acids that occurred at positions 12 and 59 in the protein. Mutations at amino acids 12 and 59 have been shown to play a role in activating the human c-ras-1 protein. For the reasons discussed below and elsewhere (17, 18, 24), we suggest that both of the amino acid changes play a role in activating the viral p21 protein. It is interesting that the viral and rat p21 proteins were identical between amino acids 165 and 184, although these sequences are apparently not required for transformation (29). We did not detect any promoterlike sequences in the 161 nucleotides upstream of the ATG start codon. owever, we noted that homology between the rat c-ras_1 gene and v-ras began to diverge at approximately nucleotide -4 upstream of the ATG initiation codon. We suggest that splicing may occur at this site and that a fifth noncoding exon may exist upstream of the region of c-ras_1 incorporated in our clone. Divergence at this site is also found in the human gene (1, CCCCCCCCCCCCCCCCCGGCGCCCGCAGCCCGCAGCCCAGGCGGCGCCGGCCGCGGACGG 7 AGCCCATGCGCCGACCCGGTCGGCGCCCGTCCACGCGCCCCGCCCTGCCCCGGCCCCGGC 14 CCCGGGGGCAGTCGCGCCAGCAAGCGGTGGGGCAGGAGCTCCTGGATTGGCAGCCCCTGT 21 Met Thr Glu Tyr Lys Leu Vai Vai Vai Gly Al ij1gly AGAAGCG ATG ACA GM TAC MG CTT GTG GTG GTG GGC GCT 8GGC 259 Val Gly Lys Ser Ala Leu Thr Ile Gin Lou Ile Gln Asn is Phe GTG GGA AAG AMT GCC CTG ACC ATC CAG CTG ATC CAM MC CAT m 294 Vai Asp Glu Tyr Asp SiThr Ile Glu Asp Ser Tyr Arg Lys Gin GTG GAC GAG TAT GAT ACT ATA GAG GAC TCC TAC CGG AM CAG 349 Vai Val Ile Asp Gly Glu Thr Cys Leu i Asp Ile Leu Asp Thr GTA GTC ATT GAT GGG GAG ACG TGT TTA GAC ATC TTA GACACA 394 A a Gly Gln Glu Glu Tyr Ser Ala Met Ar9 Asp GIn Tyr Met Arg C GGT CMG MG AG TAT ATGCC ATG CGG GAC CAG TAC ATC CGC 449 Thr Gly JR Gly ACA GGG M GGC 461 FIG. 2. A partial DNA sequence analysis of the c-ras-2 gene contained in the GAB clone. The amino acid sequences predicted by the c-ras-2 sequence are shown above the DNA sequence. The sequence begins approximately 2 nucleotides upstream of the ATG codon of c-ras-2. Amino acid positions that differ from those of v-ras p21 are boxed. TABLE 1. Gene MOL. CELL. BIOL. Differences in the p21 protein-coding sequences of c-ras_j, c-ras-2, and v-rasa Amino acid at position: v-ras Arg Pro Leu Thr Glu c-ras - Gly Pro Leu Ala Glu c-ras-2 Gly Ser Met Ala Lys athe comparison between c-ras_1 and v-ras shows only two differences out of 189 amino acids. The comparison between c-ras-2 and v-ras shows five differences out of the 77 codons that were compared. We wish to correct the previously published sequence of v-ras. This gene encodes an ala nine at amino acid position 122 and not the previously published glycine. 17). We were able to detect a potential polyadenylation signal at nucleotides 213 to Sequencing of rat c-ras"-2. In Fig. 2 is presented a partial sequence analysis of the rat c-ras-2 gene. Our results are consistent with previous heteroduplex studies that the c-ras_ 2 gene apparently lacks introns and is colinear with the viral gene. The putative coding sequence of the c-ras-2 gene began with the ATG codon at -nucleotide 118 (Fig. 2). We detected five bases pair differences over the first 231 base pairs that would result in a predicted five amino acid difference between v-ras p21 and the c-ras-2 gene product over the first 77 amino acids (Fig. 2 and Table 1). Of most importance, it is significant that the codons for amino acids 12 and 59 of c-ras-2 were the same as those for c-ras_1. We conclude that the amino acids encoded by v-ras at positions 12 and 59 are not present in the homologous rat cellular genes but were acquired by the virus. We also note that c-ras-2 shows a greater extent of homology with v-ras in the upstream noncoding region, with divergence occurring at approximately nucleotide 25. The properties of c-ras-2 are consistent with the idea that c-ras-2 is a nonfunctional pseudogene. ras-related pseudogenes have also been detected in other species (14). Consistent with this idea'and with the idea that v-ras was derived from c-ras_1, the c-ras-2 gene contained multiple lesions that prevented its activation (see below). Activation of c-ras-2. To determine whether c-ras-2 can be activated to induce transformation, we engineered a series of recombinations between c-ras-2 and v-ras. These recombinants were analyzed for their ability to induce morphological transformation in the NI 3T3 cell transfection assay. A summary of the clones and their transforming ability is shown in Fig. 3. When the c-ras-2 gene was ligated to the -MuSV promoter, the resulting recombinant was unable to induce transformation of NI 3T3 cells (Fig. 3). This suggests that c-ras-2 contains one or more defects that prevent its activation. In contrast, the c-ras_1 gene efficiently transformed NI 3T3 cells when ligated to the -MuSV promoter (3, 4). To localize the lesions in c-ras-2, we made additional clones. Clone 3 replaced the carboxy-terminal 185 amino acids of v-ras with the corresponding c-ras-2 sequence. This clone also failed to induce transformation in the NI 3T3 cell assay. This result suggests that c-ras-2 contains a lesion in its coding or 3'-noncoding region. This lesion might be due to one or more of the predicted amino acid differences between c-ras-2 and v-ras (Table 1). In addition, when the 5'-noncoding portion of c-ras-2 was substituted for the corresponding v-ras region (Fig. 3, clone 4), transformation was not detected. Thus, the c-ras_

4 VOL. 6, 1986 RAT CELLULAR ras GENE NUCLEOTIDE SEQUENCE 179 aon Foci per g 1 i p2r 11 E 2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4 -U --;, 7r---- E S 3,6 FIG. 3. Activation of c-ras-2. Solid lines represent that region of the construct composed of viral ras sequences, while the dashed line represents that region of the construct composed of c-ras-2 sequences. Abbreviations: E, an EcoRI restriction site; S, a SacII restriction site (Fig. 2, nucleotides 61 to 67);, a indill restriction site (Fig. 2, nucleotides 23 to 236); LTR, long terminal repeat. 2 gene contains a second lesion in its 5'-noncoding region that prevents its activation. This lesion must lie upstream of the SacII site, because clone 5, which contains a small region of the 5' sequence of c-ras-2, was able to substitute for the corresponding v-ras region. Our data demonstrate that c-ras-2 contains multiple lesions that prevent its activation, although results of our experiments did not determine whether the defects in c-ras-2 occur at the level of mrna expression or translation or in the putative protein product. DISCUSSION We determined the nucleotide sequence of the rat cellular c-ras-1 gene, as well as a portion of the c-ras-2 gene. The predicted amino acid sequence of the p21 protein encoded by the c-ras_1 gene was identical to the sequence of the human p21 protein (1, 17). This striking conservation of ras genes is consistent with observations that ras genes are highly conserved in evolution as they are detected in S. cerevisiae, and are likely to play a central role in the eucaryotic cell cycle (5, 16). Indeed, the ras p21 proteins have several properties similar to G proteins that act at the cell surface to transduce signals to critical cell pathways (1). Although we cannot unambiguously conclude whether v-ras was derived from c-ras_1 or c-ras-2 or a more complicated event involving both genes, results of our studies are more consistent with the idea that v-ras was derived from the c-ras_1 gene. First, the c-ras-1 gene can be activated to transform NI 3T3 cells by ligation to a viral promoter (3, 4). In contrast, the c-ras-2 gene contains at least two lesions that prevent its activation (Fig. 3). Second, the v-ras gene more closely resembles the c-ras_1 gene in the predicted sequence of their p21 proteins, differing only at amino acids 12 and 59 (Fig. 1 and Table 1). In contrast, the c-ras-2 gene, which has not been shown to encode a protein, differs in the sequence of its predicted product from v-ras p21 in at least five amino acid positions (Table 1). Thus, for the v-ras_coding region to have been derived from c-ras_1 it would have had to have undergone only two mutations, whereas the v-ras gene would have had to have undergone at least five mutations to have been derived from c-ras-2. Finally, the structure of c-ras-2, which is colinear with the viral sequence but differs at numerous amino acids, is consistent with that of a pseudogene. We suggest that both the v-ras gene and the c-ras-2 gene were derived in a 44 similar manner from a processed mrna of c-ras_1. This model would account for the homology in the 5'-noncoding region of v-ras and c-ras-2 and would predict that there is a fifth 5'-noncoding exon in the c-ras_1 gene that contains these sequences. We compared the nucleotide sequences of the rat and human c-ras_1 genes. The coding sequences of these genes exhibited an 88% homology with 57 differences due to third base pair changes. Interestingly, there were two regions of homology in the noncoding sequences of these genes. The first region was located immediately downstream of the third coding exon and was dispersed over rat gene nucleotides 955 to 13 (Fig. 1). The second region of homology was located downstream of the termination codon and was dispersed over rat gene nucleotides 182 to 214 (Fig. 1). The functional significance of these sequences is not yet known, but such sequences may play a role in controlling gene expression. Activation of c-ras. Two mechanisms have been described for the activation of ras cellular proto-oncogenes. One mechanism is based on the enhanced expression of the cellular ras gene. These studies involve the ligation of cellular proto-oncogenes to a viral promoter (3, 4). Both the rat and human c-ras_1 genes can be activated in this manner, and the resulting constructs induced foci in the NI 3T3 transfection assay. Cells transformed by these activated genes synthesize high levels of p21. Thus, transformation can occur because of increased synthesis of a normal p21 protein. Recent studies of the EJ/T24 bladder carcinoma cell line demonstrated a second mechanism for the activation of proto-oncogenes. Single amino acid alterations at critical positions in the c-ras_1 gene can activate the p21 protein (8, 18, 24). The viral ras gene contains an arginine at amino acid 12 in place of the normal glycine (Fig. 1 and Table 1). These results suggest that alteration of amino acid 12 in v-ras plays a role in the activation of the viral p21 protein. The second amino acid difference between the viral p21 and the c-ras_1 p21 occurred at amino acid 59. The viral protein encoded a threonine at position 59, whereas c-ras-1 encoded an alanine (Fig. 1). The viral p21 protein is phosphorylated at threonine 59 (19, 2). In addition, results of mutagenic studies suggest that alteration of amino acid 59 may activate the p21 protein in the NI 3T3 transfection assay (8). The most striking result of this study is the close similarity between the c-ras_1 and v-ras-coding sequences. Only two amino acid differences exist between v-ras p21 and c-ras_1 p21. Both of the differences can activate the p21 protein and have similar effects on the biochemical properties of the protein (9, 23, 25). Although one might have predicted that transduction of c-ras_1 into a retrovirus would be sufficient to induce transformation, -MuSV underwent two additional mutational events that activated the p21 protein. We suggest that during the initial generation or subsequent passaging of -MuSV the virus was selected to be highly transforming and that this selection resulted in a virus that contained multiple activating lesions, each of which increased the transforming potential of the viral ras gene. Results of recent studies have suggested that p21 proteins are similar to G proteins and may serve to transduce signals form the cell surface to a critical target at the plasma membrane (1). Consistent with this idea, it has been shown that the normal p21 protein can bind and hydrolyze GTP, while p21 proteins with alterations at amino acids 12, 59, or

5 171 RUTA ET AL. 61 have an impaired ability to hydrolyze GTP (9, 23, 25). The -MuSV p21 protein which contains alterations of both amino acids 12 and 59 has an impaired ability to hydrolyze GTP (9, 23, 25). We are currently performing mutagenic studies to identify the structural and functional properties of p21. LITERATURE CITED 1. Capon, D. J., E. Y. Chen, A. D. Levinson, P.. Seeburg, and D. V. Goeddel Complete nucleotide sequence analysis of the T24 human bladder carcinoma oncogene and its normal homologue. Nature (London) 32: Capon, D. J., P.. Seeburg, J. P. McGrath, J. S. ayfick, U. Edman, A. D. Levinson, and D. V. Goeddel Activation of Ki-ras 2 gene in human colon and lung carcinomas by two different point mutations. Nature (London) 34: Chang, E.., M. E. Furth, E. M. Scolnick, and D. R. Lowy Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of arvey murine sarcoma virus. Nature (London) 297: Defeo, D., M. A. Gonda,. A. 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