Further genetic localization of the transforming sequences of the p21 v-ras gene of Harvey murine sarcoma virus.

The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment, DNA sequence analysis has found a single open reading frame large enough to encode the viral p21 (R. Dhar, R. W. Ellis, T. Y. Shih, S. Oroszlan, B. Shapiro, J. Maizel, D. Lowy, and E. M. Scolnick, Science 217:934-937, 1982). There are three potential in-frame ATG initiation codons at the 5' end of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient for synthesis of the viral p21 and for cellular transformation.     

An altered DNA sequence encompassing the ras gene of Harvey murine sarcoma virus.

The DNA fragment encompassing the ras gene of Harvey murine sarcoma virus was sequenced and assigned the coding region of a transforming protein, p21, to the sequence. Examination of nucleotide sequence, taken together with the result of analysis of the ras mRNAs (1), has revealed that p21 is encoded from a continuous coding region starting with the 5' proximal initiation codon but not a processed protein. However, there were found several differences between the sequence published by Dhar et. al. (2) and ours, including 9 deletions, 7 substitutions and 2 insertions of nucleotides in the published sequence of 997 nucleotides in length. Among these, one of the substitutions occurring in the coding region resulted in amino acid replacement of glycine by alanine at position 122 of p21. The evidences are presented with some of actual gel autoradiographs.     

Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus.

Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation-competent deletion mutant from which amino acids 166-175 had been deleted. The Ser-186 mutant was defective for transformation. The p21s encoded by the Ser-186 mutant and by the previously described transformation-defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.     

Assignment of murine cellular Harvey ras gene to chromosome 7

Mouse-Chinese hamster somatic cell hybrids containing various combinations of mouse chromosomes were analyzed for the presence of the mouse c-Ha-ras (1) sequences after restriction endonuclease digestion and hybridization with a 32P-labeled Ha-ras specific probe according to the procedure of Southern (2). The presence of the mouse c-Ha-ras containing fragment was correlated with the presence of mouse chromosome 7 in the hybrids.     

Monoclonal antibodies to feline sarcoma virus gag and fes gene translational products

A series of hybridomas have been isolated which produce monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strains of FeSV. Within these are representatives of several immunoglobulin classes including IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM. Antibody produced by one hybridoma recognizes immunologic determinants localized within an FeLV gag gene structural component (p15) common to polyproteins encoded by all three FeSV isolates whereas antibody produced by a second is specific for p30 determinants unique to P170^gag-fms. Additional hybridomas secrete antibody directed against v-fes-encoded determinants common to the Gardner and Snyder-Theilen FeSV-encoded polyproteins. GA P110^gag-fes and ST P85^gag-fes immunoprecipitated by antibody directed against p15 exhibit tyrosine-specific protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components.     

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.     

Functional organization of the Harvey murine sarcoma virus genome.

The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.     

Nucleotide sequence of the transforming gene of m1 murine sarcoma virus.

The v-mosm1 nucleotide sequence codes for a protein that is 376 amino acids long. Although the N-terminus is homologous with that of the v-mos124 protein, the C-terminus is substantially different from the C-termini of all other examined mos proteins, suggesting that this region is nonessential and perhaps cleaved. Overall, v-mosm1 has greater homology with c-mos than does v-mos124, but mutually exclusive differences between c-mos and each of the v-mos genes preclude linear descent and suggest a common ancestral murine sarcoma virus.     

Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus

The p21 transforming protein coded for by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV) migrates as a doublet band between 21,000 and 23,000 daltons during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lower band of the doublet is designated p21, and the upper band is designated pp21 since it comigrates with the phosphorylated form of p21. By pulse-labeling with [35S] methionine, we detected a p21 precursor, pro-p21, which migrated as if it was approximately 1,000 daltons larger than p21. The precursor-product relationship was established by pulse-chase experiments with [25S] methionine in the presence of 100 micrograms of cycloheximide per ml, which inhibited all de novo protein biosynthesis. Within 4 h, pro-p21 was completely chased into p21, and during the next 24 h pp21 accumulated. Thus, formation of pp21 from p21 did not require de novo protein synthesis. By subcellular fractionation into cytosol amd membrane fractions, we found that pro-p21 was synthesized in a non-membrane-bound state and that shortly after its complete synthesis, the p21 product was associated with the membrane fraction. By selective cleavage of p21 at a unique aspartic acid-proline residue with 70% formic acid or with Staphylococcus aureus V8 protease, we found that the intramolecular site of pro-p21 processing was located in the C-terminal portion of the pro-p21 molecule. The possibilities that the precursor was involved in the assembly of p21 into the plasma membrane and, alternatively, that the processing was a step in the activation of p21 biochemical activities are discussed.     

In vitro translation of Harvey murine sarcoma virus RNA.

The viral RNA of the Harvey strain of murine sarcoma virus (Ha-SV), which does not encode for any known viral structural polypeptides, has been translated in a nuclease-digested, cell-free system. The major protein product of the in vitro translation reaction has a molecular weight of 21,000 and is initiated faithfully with [35S]formylmethionine from formyl-[35S]methionyl-tRNAFMET. This polypeptide is clearly distinct from the RNA of the Moloney strain of type C helper virus used to pseudotype the Ha-SV. The intensity of the 21,000-dalton polypeptide on gels correlates well to the concentration of Ha-SV RNA in different viral RNA preparations. These experiments indicate that a polypeptide marker for Ha-SV is now available for the first time. The possibility that this protein is the product of the rat portion of the Ha-SV genome is discussed.     

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.     

Nucleotide sequence analysis of the BALB/c murine sarcoma virus transforming gene.

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.     

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage λ Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.     

Inhibition of yeast adenylate cyclase by antibodies to ras p21.

Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.     

Association of p21ras with cellular polypeptides

p21ras specific antiserum was used to immunoprecipitate p21ras polypeptides from human A431 cells. In addition to p21ras, this antiserum precipitated a series of polypeptides with relative molecular weights of 150,000, 120,000, 105,000, and 50,000. The precipitation of these polypeptides was prevented by preincubation of the antiserum with an excess of purified Ras protein. These polypeptides do not share an epitope with p21ras, and two of them (120 and 150 kDa) copurify with a fraction of p21ras. The co-precipitation of p21ras with these polypeptides was detected in a variety of cell types. The pattern of the immunoprecipitates was consistently different in normal and ras-transformed cells. The 120- and 150-kDa polypeptides are phosphorylated on serine and threonine in A431 cells. Serum treatment resulted in a 2-fold increase in the phosphoserine content of the 120-kDa polypeptides.     

Activation of cellular p21ras by myristoylation

p21ras is palmitoylated on a cysteine residue near the C-terminus. Changing Cys-186 to Ser in oncogenic forms produces a non-palmitoylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. To examine whether palmitate acts in a general way to increase ras protein hydrophobicity, or is involved in more specific interactions between p21ras and membranes, we constructed genes that encode non-palmitoylated ras proteins containing myristic acid at their N-termini. Myristoylated, activated ras, without palmitate (61Leu/186Ser) exhibited both efficient membrane association and full transforming activity. Unexpectedly, we found that myristoylated forms of normal cellular ras were also potently transforming. Myristoylated c-ras retained the high GTP binding and GTPase characteristic of the cellular protein and, moreover, bound predominantly GDP in vivo. This implied that it continued to interact with GAP (GTPase-activating protein). While the membrane binding induced by myristate permitted transformation, only palmitate produced a normal (non-transforming) association of ras with membranes and must therefore regulate ras function by some unique property that myristate does not mimic. Myristoylation thus represents a novel mechanism by which the ras proto-oncogene protein can become transforming.     

Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras.

The Harvey murine sarcoma virus genome contains two rat-derived sets of genetic information recombined with the Moloney mouse leukemia virus. The rat sequences represent a ras oncogene and a rat VL30 element. The VL30 sequences have several discrete regions of similarity with retroviral sequences which were detected by searching a protein database for similarities with predicted polypeptide sequences from the VL30 regions. On the 5' side, the most similar sequences were those of feline sarcoma viruses; on the 3' side, murine leukemia viruses were the most similar. Some of the regions of similarity could also be detected directly by searching a nucleic acid sequence database with the viral DNA sequences. The most extensive region of similarity was that which corresponded to the endonuclease in the pol gene of a murine leukemia virus. The majority of the rat-derived sequences present in the Harvey sarcoma virus genome can now be attributed exclusively to ras or retrovirus- or retrotransposon-related sequences.     

Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities.

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation.     

Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.