Development of quantitative liquid competition radioimmunoassays for the ras oncogene and proto-oncogene p21 products.

The ras gene family of rodents and humans is highly conserved and consists of several distinct genes, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. This gene family mediates transformation via (1) a point-mutation resulting in the change of one amino acid in the 21 kDA ras gene product (p21) or (2) increased expression of ras p21. Group-specific, type-selective and interspecies indirect binding liquid competition radioimmunoassays (RIAs), capable of providing truly quantitative analyses of the 21 ras oncogene and proto-oncogene products, have been developed. Using purified recombinant ras p21 from Escherichia coli expressing the full-length T24 mutant human Harvey-ras gene protein product as a standard in these RIAs, we have defined the absolute numbers of pg, fM and molecules of ras p21 in: (1) E. coli expressing the point-mutated or proto-ras p21 and (2) mammalian cell lines of human and murine origin. Two of the RIAs developed can be termed group-specific in that they have the ability to detect the point-mutated and proto forms of all 3 human ras genes (Harvey, Kirsten, and neuroblastoma), while the third RIA is type-selective, since it detects an antigenic determinant located primarily on the Harvey ras p21. All 3 RIAs are interspecies-specific since they are able to detect ras p21 in rodent as well as human cells. The adaptability of the RIAs to various assay conditions and ease of methodology make these immunoassays applicable to the study of several parameters associated with ras p21 expression. These assays, used in conjunction with specific cDNA probes to identify specific ras proto-oncogenes or point-mutated oncogenes being expressed, now provide truly quantitative analysis of ras p21 in mammalian cells to further the study of the association between ras p21 expression and 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.

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.

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.

Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus.

The purified p21src protein of Harvey sarcoma virus shows a guanine nucleotide-binding activity and, in addition, at elevated temperature an autophosphorylating activity at a threonine residue using as phosphoryl donor GTP or dGTP but not ATP or dATP. These biochemical activities are unique among those associated with transforming proteins of RNA-containing or DNA-containing tumour viruses.

p21 of Kirsten murine sarcoma virus is thermolabile in a viral mutant temperature sensitive for the maintenance of transformation.

We have recently described an intracellular protein, p21, in nonproducer cells transformed by either the Kirsten (Ki-MSV) or Harvey (Ha-MSV) strain of murine sarcoma virus (Shih et al., Virology, in press). The p21 is phosphorylated and has been shown to be coded for by either Ki-MSV or Ha-MSV. In this report, we compare the thermal stability of the newly synthesized [35S]methionine-labeled p21 in cells transformed by the wild-type Ki-MSV or by a mutant of Ki-MSV (ts 371) which is temperature sensitive in a viral function required for the maintenance of several properties of the transformed phenotype. The immunoprecipitability of the p21 coded for by the ts 371 Ki-MSV was markedly more thermolabile than the p21 of the wild-type Ki-MSV when the cell extracts are heated in vitro. The present finding suggests that the p21 is required for the maintenance of transformation induced by Ki-MSV.

Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus.

A similar protein of 21,000 MW (p21) coded for by Harvey or Kirsten murine sarcoma virus has been identified in nonproducer cells transformed by these two viruses. Antisera prepared from rats bearing tumors induced by syngeneic transplantation of NRK cells transformed by Harvey murine sarcoma virus (Ha-MuSV) specifically precipitated the Ha-MuSV p21 from a nonproducer Balb/c mouse cell and a nonproducer dog cell transformed by Ha-MuSV. The same antisera also precipitated a similar protein, Ki-MuSV p21, from a nonproducer mink cell transformed by Kirsten murine sarcoma virus (Ki-MuSV). Both the p21 of Ha-MuSV and of Ki-MuSV are phosphoproteins. Previous studies have reported a virus-specific p21 polypeptide from translation of Ha-MuSV RNA in cell-free protein synthesis systems (W. P. Parks and E. M. Scolnick, 1977, J. Virol. 22, 711-719; T. Y. Shih, D. R. Williams, M. O. Weeks, J. M. Maryak, W. C. Vass, and E. M. Scolnick, 1978, J. Virol 27, 45-55). This p21 protein was specifically precipitated by the same anti-tumor sera. Similarly, a p21 polypeptide translated from Ki-MuSV RNA was also specifically precipitated by the antitumor sera. Therefore, it is concluded that the p21 of Ha-MuSV and Ki-MuSV are homologous proteins coded for bv homologous sequences found in the recombinant genomes of Ha-MuSV and Ki-MuSV.

Comparison of the genomic organization of Kirsten and Harvey sarcoma viruses.

Current studies were undertaken to compare the genomes of Kirsten murine sarcoma virus (Ki-MuSV), Harvey murine sarcoma virus (Ha-MuSV), and the replication-defective endogenous rat virus to understand the function of these viral RNAs. Genome organization and sequence homology were studied by fingerprinting large RNase T1-resistant oligonucleotides and by cross-protecting homologous oligonucleotides against RNase A and T1 digestion with complementary DNA prepared from each of the other viral RNA. Ki-MuSV and Ha-MuSV were found to share an extensive series of rat-derived oligonucleotides begining ca. 1 kilobase (kb) from the 3' end and extending to within 1.5 kb of the 5'end of Ki-MuSV RNA. The total map distance covered in ca. 5.5 kb. The eight oligonucleotides covering the 1.5 kb at the 5' end of Ki-MuSV RNA were not found in Ha-MuSV RNA. Five out of these eight oligonucleotides, however, could be designated with certainty to be of rat virus origin. Since Ha-MuSV is 6.5 kb in size and Ki-MuSV is 8 kb in size, the major difference between them is the 1.5 kb from the replication-defective endogenous rat virus sequences at the 5' end of Ki-MuSV not present in Ha-MuSV. Consistent with the difference in the genome structure, these two sarcoma viral RNA'S yielded distinct major translation products in cell-free systems, I.E., A 50,000-dalton polypeptide (P50) from Ki-MuSV and a 22,000-dalton polypeptide (p22) from Ha-MuSV. These polypeptides may provide the necessary protein makers for identifying in vivo virus-coded proteins.

Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus.

The site of recombination of a mink cell focus-inducing strain (Mo-MuLV83) derived from an ecotropic Moloney murine leukemia virus (Mo-MuLV) was mapped by fingerprint analysis of the large RNase T1-resistant oligonucleotides, employing a two-dimensional gel electrophoresis method. Mo-MuLV83, in contrast to the ecotropic Mo-MuLV, demonstrated a broadened host range, i.e., growth not only on mouse cells but also on mink cells, and recombination involved the env gene function. The genomic RNA of these two viruses shared 42 out of a total of 51 to 53 large T1 oligonucleotides (81%) and possessed a similar subunit size of 36S. Most of these T1 oligonucleotides were mapped in their relative order to the 3' polyadenylic acid end of the viral RNA molecules. There were 10 common oligonucleotides immediately next to the 3' termini. A cluster of 7 (in Mo-MuLV83) or 10 (in Mo-MuLV) unique T1 oligonucleotides were mapped next to the common sequences at the 3' end, and they all appeared concomitantly in a polyadenylic acid-containing RNA fraction with a sedimentation coefficient slightly larger than 18S. Therefore, the env gene of Mo-MuLV was situated at a location approximately 2,000 to 4,000 nucleotides from the 3' end of the genomic RNA, and the gene order of Mo-MuLV appeared to be similar to that of the more rigorously determined avian oncornaviruses. cDNA(SFFV) specific for the xenotropic sequences in the spleen focus-forming virus RNA hybridized to the cluster of unique oligonucleotides of Mo-MuLV83 RNA. This suggests that the loci of recombination involve the homologous env gene region of a xenotropic virus.