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E M Scolnick

Publications and source records attributed to E M Scolnick.

At least 73 records · Page 4Linked to original sources
PubMed1983

Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus.

When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells.

Autoradiography↗
PubMed1982

Mechanism of activation of a human oncogene.

The oncogene of the human EJ bladder carcinoma cell lines arose via alteration of a cellular proto-oncogene. Experiments are presented that localize the genetic lesion that led to activation of the oncogene. The lesion has no affect on levels of expression of the oncogene. Instead, it affects the structure of the oncogene-encoded protein.

Base Sequence↗
PubMed1982

Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus.

A normal human gene homologous to the p21 ras oncogene of Harvey murine sarcoma virus induced oncogenic transformation and high p21 ras levels in murine fibroblasts when this gene was ligated to a control element (the long terminal repeat) from a murine or feline retrovirus. These results indicate that high levels of a gene product encoded by a normal human oncogene can induce tumorigenic transformation.

Animals↗
PubMed1982

The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid.

We have found that the transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus all contain tightly bound lipid. This modification could play a role in the binding of these proteins to cellular membranes. The lipid associated with p60src, the transforming protein of Rous sarcoma virus, is located in the NH2-terminal domain of the polypeptide. This is the region of the protein that has been shown previously to participate in binding the protein to membranes. Two mature forms of p21, the transforming protein of Harvey sarcoma virus, contain lipid. Lipid is not, however, associated with newly synthesized p21. While mature p60src and p21 are bound to cellular membranes, the newly synthesized forms of these proteins are not. The posttranslational addition of lipid may therefore be the means by which these proteins acquire an affinity for membranes.

Abelson murine leukemia virus↗
PubMed1982

Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses.

Harvey and Kirsten murine sarcoma viruses each encode a structurally and functionally related 21-kilodalton protein (p21), which is the transforming protein of each virus. Using probes from the 0.9-kilobase (kb) p21-coding region of each virus (called v-Ha-ras and v-Ki-ras, respectively), we have molecularly cloned from normal human genomic DNA the sequences that hybridize to these probes. Four evolutionarily divergent restriction endonuclease fragments were isolated. Two hybridized preferentially to v-Ha-ras and were designated human c-Ha-ras1 and c-Ha-ras2; two hybridized preferentially to v-Ki-ras and were called c-Ki-ras1 and c-Ki-ras2. Human c-Ha-ras1 contained 0.9 kb of sequence homologous with v-Ha-ras interspersed with three intervening sequences; this gene was closely related to a previously cloned rat c-Ha-ras gene that also contained intervening sequences. Human c-Ha-ras2 was more divergent from v-Ha-ras and also hybridized poorly to human c-Ha-ras1. One c-Ki-ras gene contained 0.9 kb homologous to v-Ki-ras and had one intervening sequence, whereas the other contained only 0.3 kb homologous to v-Ki-ras. The results indicated that human DNA contains several copies of the c-ras family and that c-Ha-ras1 (with intervening sequences) was more highly conserved evolutionarily than was c-Ha-ras2.

Base Sequence↗
PubMed1982

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.

Amino Acid Sequence↗
PubMed1982

Envelope gene sequences which encode the gp52 protein of spleen focus-forming virus are required for the induction of erythroid cell proliferation.

A series of insertion-deletion mutants was constructed in a molecularly cloned DNA copy of the Friend strain of spleen focus-forming virus (SFFV). The mutants were produced by inserting a synthetic oligonucleotide linker containing the recognition sequence of SalI endonuclease into several different locations of the SFFV DNA. Three classes of mutants were isolated: insertion-deletion mutants in the 5' half of the SFFV genome, in the long terminal repeat of the SFFV genome, and in the env gene of the SFFV genome. The env gene mutant has a deletion of sequences shared in common between the env gene of SFFV and the env genes of mink cell focus-inducing murine leukemia viruses. From analyses of the biological activity of the various mutants and a biologically active subgenomic SFFV DNA fragment described herein, we can deduce that the coding sequence encompassing the env gene of SFFV is required for the biological activity. This region, required for the pathogenic phenotype, cannot be larger than 1.5 kilobase pairs, a size only slightly more than that sufficient to encode the nonglycosylated precursor of the gp52 env gene product.

Base Sequence↗
PubMed1982

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.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

Molecular cloning of biologically active proviral DNA of the anemia-inducing strain of spleen focus-forming virus.

Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.

Anemia↗
PubMed1982

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein.

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

Animals↗
PubMed1981

The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes.

The Harvey and Kirsten strains of murine sarcoma virus encode enzymatically and serologically related p21 src proteins which are required for virally mediated cellular transformation. The genes in each virus encoding p21 show such extensive divergence from each other that cloned probes from these genes detect distinct sets of cellular genes in the DNA from several vertebrate species. These data suggest that cellular p21 sarc genes constitute a divergent family of vertebrate genes that can regulate the growth of cells.

Animals↗