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R Ralston

Publications and source records attributed to R Ralston.

33 records · Page 2Linked to original sources
PubMed1992

Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells.

Truncated and full-length versions of the hepatitis C virus protein domain encoding a presumptive envelope glycoprotein designated E2/NS1 were stably expressed in CHO cell lines. Characterization of the processing events involved in the maturation of E2/NS1 revealed that a high-mannose form resident in the endoplasmic reticulum was the most abundant form detected intracellularly. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that the E2/NS1 glycoprotein resided in the endoplasmic reticulum. The full-length form of E2/NS1 appeared to be cell-associated and could not be detected as a secreted product. C-terminal truncated molecules could be detected in the extracellular media as fully processed glycoproteins containing terminal sialic acid additions. These truncated glycoproteins are predicted to be biologically relevant targets of the host immune response and are therefore potential subunit vaccine candidates.

Animals↗
PubMed1991

Complementation of transforming domains in E1a/myc chimaeras.

The myc oncogene is functionally similar to adenovirus E1a in its ability to collaborate with activated ras oncogenes to transform primary fibroblasts. The transforming functions of E1a and myc have been mapped to two distinct regions in each protein. I investigated the functional similarities between E1a and myc by constructing E1a/myc chimaeras to discover whether the individual transforming domains of E1a could complement individual myc-transforming domains. Transformation assays in rat embryo fibroblasts demonstrated that the N-terminal transforming domain of E1a (CR1) could complement the C-terminal transforming domain of myc in cis, and that the reciprocal chimaera (N-terminal myc/C-terminal E1a) was also active. Chimaeras constructed using domains from transformation-defective mutants of either E1a or myc were inactive, indicating that both E1a and myc domains contribute to function. These experiments suggest that transformation by myc and E1a may involve interactions with common substrates.

Adenoviridae↗
PubMed1985

The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor.

We have observed a modification of the cellular protein kinase pp60c-src, elicited in murine 3T3 fibroblasts by platelet-derived growth factor (PDGF). The modification occurred rapidly after addition of PDGF to the culture medium and was first detected as a reduction in the electrophoretic mobility of a portion of the pp60c-src molecules. A similarly modified form of the viral homologue pp60v-src occurs in vivo in the absence of stimulation by PDGF. The occurrence of modified forms of both pp60c-src and pp60v-src was associated with a novel phosphorylation at tyrosine in the amino-terminal domains of the proteins. The time-course and dose-response for this modification of pp60c-src paralleled PDGF-induced increases in phosphorylation of pp36, a major cellular substrate for several tyrosine-specific protein kinases. In parallel experiments, treatment of cells with PDGF increased the kinase activity of pp60c-src in an immunocomplex assay. These results suggest pp60c-src may play a role in the mitogenic response to PDGF.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53.

Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.

Amino Acid Sequence↗
PubMed1984

The membrane glycoprotein encoded by the retroviral oncogene v-erb-B is structurally related to tyrosine-specific protein kinases.

Avian erythroblastosis virus can transform both fibroblasts and erythroid cells to neoplastic growth. A locus within the virus genome, v-erb B, encodes a membrane glycoprotein essential for the oncogenic properties of the virus. No biochemical function has until now been attributed to the v-erb B protein. We report here that the v-erb B glycoprotein shares strong structural homologies with the tyrosine-specific protein kinases encoded by certain retroviral oncogenes. The patterns of amino acid conservation between the tyrosine-specific protein kinases and the v-erb B protein, and between the v-erb B protein and the catalytic subunit of bovine protein kinase, suggest a possible functional as well as structural relatedness.

Alpharetrovirus↗
PubMed1983

The p36 substrate of tyrosine-specific protein kinases co-localizes with non-erythrocyte alpha-spectrin antigen, p230, in surface lamina of cultured fibroblasts.

Biochemical and immunofluorescence studies have demonstrated that p36, a major substrate for the tyrosine-specific protein kinases induced by several sarcoma viruses and epidermal growth factor, is associated with plasma membranes and detergent-resistant cytoskeletal structures of cultured cells. We have used here polyclonal antisera and monoclonal antibodies in indirect immunofluorescence microscopy to study the subcellular location of p36 and the p230, which is a subplasmalemmal polypeptide showing immunologic cross-reactivity with erythrocyte alpha-spectrin. Both p36 and p230 showed a diffuse distribution in fixed and permeabilized cells and were localized in a surface lamina-like network in Triton-extracted cells. In double-staining experiments, an extensive co-distribution between these proteins was seen in detergent-treated cultured fibroblasts. These results, together with our previous work, suggest that the p36 protein is an integral part of the detergent-resistant proteinaceous network at the cytoplasmic face of the plasma membrane.

Animals↗
PubMed1983

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S.A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.

Animals↗
PubMed1978

Protein synthesis in rabbit reticulocytes XXI. Purification and properties of a protein factor (Co-EIF-1) which stimulates Met-tRNAf binding to EIF-1.

The peptide chain initiation factor, Co-EIF-1 has been purified to homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous preparation gives a single protein band corresponding to a molecular weight of approximately 20,000. In the crude preparation, Co-EIF-1 exists in two molecular forms: Co-EIF-1H (Mr = 200,000) and Co-EIF-1L (Mr = 20,000). Both forms are equally active in all the reactions studied. Upon heating, the heavy form (Co-EIF-1H) is completely converted into the light form (Co-EIF-1L). Radioactively labeled [14C]Co-EIF-1 was prepared by reductive alkylation using [14C]formaldehyde and borohydride. [14C]Methyl-Co-EIF-1 binds specifically to EIF-1; EIF-1.[14C]Co-EIF-1 complex was analyzed by gel (Sephadex G-100) filtration. EIF-1.Co-EIF-1 complex is distinctly more stable towards heat than EIF-1 alone and the quarternary complex, Met-tRNAf.EIF-1.Co-EIF-1.GTP is more resistant to aurintricarboxylic acid than the ternary complex, Met-tRNAf.EIF-1.GTP. Both the quarternary complex, Met-tRNAf.EIF-1.Co-EIF-1.GTP, and the ternary complex, Met-tRNAf.EIF-1.GTP, are equally sensitive to Mg2+ in the presence of EIF-2 (TDF). In the presence of Co-EIF-1, the initial rate of Met-tRNAf binding to 40 S ribosomes was significantly increased.

Animals↗
PubMed1978

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet.40S ribosomal subunit complexes.

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.

Adenosine Triphosphate↗
PubMed1976

Heat stroke.

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Heat Exhaustion↗
PubMedDate not supplied

The protein products of the myc and myb oncogenes and adenovirus E1a are structurally related.

Structural and functional homologies have been found among proteins encoded by several retroviral oncogenes, demonstrating the existence of families of these genes. Because the retroviral oncogenes have cellular homologues, the existence of similar families among these 'cellular oncogenes' is also implied (for a review, see ref. 2). Cellular genes belonging to these families have been found in such evolutionarily distant species as humans, fruit flies, nematodes and brewer's yeast (E. Scolnick and S. Reed, personal communications), consistent with the hypothesis that these genes have evolved from a small number of ancestral sequences. We extend these observations by showing here that the proteins encoded by the oncogenes myc, myb and adenovirus E1a are structurally related. Our findings suggest that oncogenes of RNA and DNA tumour viruses may in at least some instances share evolutionary origins and function according to common principles.

Adenoviridae↗