Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.     

Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family.

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.     

Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1

We constructed lambda recombinants containing the Harvey murine sarcoma virus genome and the thymidine kinase (tk) gene of herpes simplex virus type 1 linked to each other. The tk gene was located in a position downstream from both the long terminal repeat and the src gene of Harvey murine sarcoma virus. The DNAs of the lambda recombinants were used to transfect NIH3T3 mouse fibroblasts in order to obtain Harvey murine sarcoma virus DNA-induced foci of transformed cells. The transformed foci were superinfected with a helper-independent retrovirus, and new individual retrovirus were isolated from the superinfected foci. The new viruses could induce focus formation on NIH3T3 cells and could convert NIH3T3(TK-) cells into TK+ cells by carrying the herpes simplex virus type 1 tk gene into the TK- cells. From virus-infected cells, we isolated nonproducer foci on NIH3T3 cells and TK+ transformants on NIH3T3(TK-) cells containing one such new viral genome coding for the dual properties. The new retroviral sequence in the nonproducer cells could be rescued into virus particles at high titers by superinfection with a helper-independent retrovirus. A hybridization analysis indicated that the recombinant virus contained both the Harvey murine sarcoma virus src sequence and the tk gene sequence in a single RNA species approximately 4.9 kilobases long. We concluded that retroviruses can be used as true vectors for genes other than genes that lead to oncogenesis.     

Viral Harvey ras p21 expressed in Escherichia coli purifies as a binary one-to-one complex with GDP

The ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation. Viral Harvey ras p21, cloned into Escherichia coli HB101 lambda/pRAS1, has been purified to homogeneity by a three-step procedure. The purified E. coli p21 can be bound by a monoclonal antibody to viral Harvey ras p21 and has an amino acid composition consistent with that predicted from its DNA sequence. Purified E. coli p21 has been shown by HPLC analysis on an ion-exchange column to contain near stoichiometric amounts of GDP. This noncovalently associated GDP is seen in the UV absorption spectrum of the purified protein. The noncovalently associated GDP acts as a competitive inhibitor of the interaction of added guanine nucleotides with p21.     

Nucleotide sequence of the two rat cellular rasH genes.

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-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-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.     

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.     

Markedly elevated levels of an endogenous sarc protein in a hemopoietic precursor cell line.

The src gene product of Harvey murine sarcoma virus is a 21,000-dalton guanine nucleotide-binding protein. We have recently shown that a wide variety of vertebrate cell strains and cell lines express much lower levels of an endogenous p21 immunologically related to the Harvey murine sarcoma virus-coded p21. In this report, we have examined the levels of endogenous p21 in a unique hemopoietic precursor cell line, 416B, which was originally described as a continuous cell line of a hemopoietic stem cell, CFU-S. The currently available 416B cells express markedly elevated levels of endogenous p21. The level of endogenous p21 in the 416B cells is 5- to 10-fold higher than the level of p21 in Harvey murine sarcoma virus-infected cells and more than 100 times higher than the level of endogenous p21 that we have observed in a variety of other fresh or cultured cells. The results indicate that marked regulation of the levels of an endogenous sarc gene product can occur, and speculation about a possible role for endogenous p21 in normal hemopoietic stem cells is discussed.     

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.     

Direct identification of palmitic acid as the lipid attached to p21ras.

p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K-ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid.     

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.     

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.     

Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes.

In earlier studies, we molecularly cloned a normal cellular gene, c-rasH-1, homologous to the v-ras oncogene of Harvey murine sarcoma virus (v-rasH). By ligating a type c retroviral promotor to c-rasH-1, we could transform NIH 3T3 cells with the c-rasH-1 gene. The transformed cells contained high levels of a p21 protein coded for by the c-rasH-1 gene. In the current studies, we have purified extensively both v-rasH p21 and c-rasH p21 and compared the in vivo and in vitro biochemical properties of both these p21 molecules. The p21 proteins coded for by v-rasH and c-rasH-1 shared certain properties: each protein was synthesized as a precursor protein which subsequently became bound to the inner surface of the plasma membrane; each protein was associated with guanine nucleotide-binding activity, a property which copurified with p21 molecules on a high-pressure liquid chromatography molecular sizing column. In some other properties, the v-rasH and c-rasH p21 proteins differed. In vivo, approximately 20 to 30% of v-rasH p21 molecules were in the form of phosphothreonine-containing pp21 molecules, whereas in vivo only a minute fraction of c-rasH-1 p21 contained phosphate, and this phosphate was found on a serine residue. v-rasH pp21 molecules with an authentic phosphothreonine peptide could be synthesized in vitro in an autophosphorylation reaction in which the gamma phosphate of GTP was transferred to v-rasH p21. No autophosphorylating activity was associated with purified c-rasH-1 p21 in vitro. The results indicate a major qualitative difference between the p21 proteins coded for by v-rasH and c-rasH-1. The p21 coded for by a mouse-derived oncogenic virus, BALB murine sarcoma virus, resembled the p21 coded for by c-rasH-1 in that it bound guanine nucleotides but did not label appreciably with 32Pi. The forms of p21 coded for by other members of the ras gene family were compared, and the results indicate that the guanine nucleotide-binding activity is common to p21 molecules coded for by all known members of the ras gene family.     

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.     

Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals.

A family of normal vertebrate genes and oncogenes has been called the ras gene family. The name ras was assigned to this gene family based on the species of origin of the viral oncogenes of the rat-derived Harvey and Kirsten murine sarcoma viruses. There are now three known functional members of the ras gene family, and genes homologous to ras genes have been detected in the DNA of a wide variety of mammals and in Drosophila melanogaster. Prior experiments have detected proteins coded for by ras genes in a large number of normal cells, cell lines, and tumors. We report here the detection of ras-related proteins in D. melanogaster, a result predicted by the earlier detection of ras-related genes in the Drosophila genome. We also report for the first time the detection of ras-related proteins in a single-cell eucaryocyte, Saccharomyces cerevisiae. These proteins, approximately 30K in size, are recognized by both a monoclonal antibody which binds to the p21 coded for by mammalian ras genes and a polyclonal rat serum made by transplanting a v-Ha-ras-induced tumor in Osborne-Mendel rats. The p21 of v-Ha-ras and the 30K proteins from S. cerevisiae share methionine-labeled peptides as detected by two-dimensional tryptic peptide maps. The results indicate that S. cerevisiae synthesizes ras-related proteins. A genetic analysis of the function of these proteins for yeast cells may now be possible.     

Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays

Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family. We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products. Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines. One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21. Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin. These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product. The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material.     

The malignant histiocytosis sarcoma virus, a recombinant of Harvey murine sarcoma virus and Friend mink cell focus-forming virus, has acquired myeloid transformation specificity by alterations in the long terminal repeat.

The malignant histiocytosis sarcoma virus (MHSV), in contrast to other viruses with the ras oncogene, induces acute histiocytosis in newborn and adult mice. Molecular structure and function studies were initiated to determine the basis of its unique macrophage-transforming potential. Characterization of the genomic structure showed that the virus evolved by recombination of the Harvey murine sarcoma virus (Ha-MuSV) and a virus of the Friend-mink cell focus-forming virus family. Structural analysis of MHSV showed two regions of the genome that are basically different from the Ha-MuSV: (i) the ras gene, which is altered by a point mutation in codon 181 leading to a Cys----Ser substitution of the p21 protein, and (ii) the U3 region of the long terminal repeat, which is largely derived from F-MCFV and contains a deletion of one direct repeat as well as a duplication of an altered enhancer-like region. Biological studies of Ha-MuSV, MHSV, and recombinants between the two viruses show that the U3 region of the MHSV long terminal repeat is essential for the malignancy and specificity of the disease. A contributing role of the ras point mutation in determining macrophage specificity, however, cannot be excluded.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.