Concerns for the use of continuous cell lines and certain promoters for producing vaccine antigens by recombinant technology.
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Antigens↗
Biomedical subjects
R W Ellis
Publications and source records attributed to R W Ellis.
Identification and structure of the gene encoding gpII, a major glycoprotein of varicella-zoster virus.
The genome of varicella-zoster virus (VZV) encodes three major families of glycoproteins (gpI, gpII, and gpIII). mRNA from VZV-infected cells was hybrid selected using a library of VZV recombinant plasmids and translated in vitro; polypeptide products were immunoprecipitated by polyclonal monospecific guinea pig antibodies to gpII. The mRNA encoding a 100-kD polypeptide precipitable by anti-gpII antibodies mapped to the HindIII D fragment near the center of the UL region. DNA sequence analysis of this region of the VZV genome revealed a 2.6-kbp open reading frame (ORF) potentially encoding a 98-kDa polypeptide possessing the characteristics of a glycoprotein. The 100-kDa polypeptide was specified by mRNA isolated by hybrid selection using a plasmid containing part of the 2.6-kbp ORF, and immunoprecipitation of this protein by anti-gpII antibodies and by convalescent zoster serum was blocked specifically by purified gpII. We conclude that the 2.6-kbp ORF encodes gpII. The imputed primary amino acid sequence of gpII shows a high degree of homology to that of herpes simplex virus type 1 (HSV-1) gB, a result consistent with the equivalent map locations of the respective genes in the HSV and VZV genomes and with the recently reported serological cross-reactivity of HSV-1 gB and VZV gpII. Unlike the mature gene products of gB, those of gpII have been described as a pair of glycoproteins with approximate molecular weights of 60 kDa in reducing gels, products of a single glycoprotein species with approximate mol mass of 125-140 kDa in nonreducing gels. Amino-terminal sequences of purified gpII were determined and compared to the imputed amino acid sequence. This comparison implies that the primary translational product is cleaved approximately into halves in vivo and suggests that mature gpII is a disulfide-linked heterodimer.
Amino Acid Sequence↗
Purification of individual varicella-zoster virus (VZV) glycoproteins gpI, gpII, and gpIII and their use in ELISA for detection of VZV glycoprotein-specific antibodies.
We have utilized monoclonal antibodies in immune affinity chromatography to purify each of the 3 major glycoproteins of varicella-zoster virus (VZV), gpI, gpII, and gpIII, in immunologically active form. Upon injection into guinea pigs, each preparation elicited the production of specific antibodies capable of immunoprecipitating the homologous glycoprotein and of neutralizing VZV infectivity in vitro. Also, total glycoproteins from VZV-infected cells have been purified by lectin affinity chromatography. Each of the individual purified glycoproteins, as well as total VZV glycoproteins and appropriate uninfected cell protein controls, have been employed as solid-phase reagents in enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies directed against specific VZV glycoproteins. The specificity of the purified glycoproteins as ELISA reagents was verified by the ability of individual monoclonal antibodies to bind specifically to individual glycoprotein preparations. We have demonstrated the utility of the glycoprotein-specific ELISA by detecting antibodies in sera from post-zoster and post-varicella patients. The assay detects antibodies directed against each of the 3 major glycoproteins and is sensitive enough to detect antibodies in a 1:320 000 dilution of some sera. This assay, as well as the purified individual glycoproteins per se, should prove to be very useful reagents in understanding the role of each of gpI, gpII, and gpIII in immunity to VZV.
Animals↗
Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae.
As a model system for the study of factors affecting gene expression, hepatitis B virus core antigen (HBcAg) has been expressed in the yeast Saccharomyces cerevisiae. The singularly high levels of expression achieved are approx. 40% of the soluble yeast protein. The HBcAg polypeptides are present as 28-nm particles which are morphologically indistinguishable from HBcAg particles in human plasma and are highly immunogenic in mice. The plasmid construction employed to achieve these very high levels of expression utilizes the constitutively active yeast promoter from the GAP491 gene which is fused in a way that all non-translated sequences flanking the HBcAg coding region are yeast-derived. Hybrid constructions containing 3'-nontranslated viral DNA (yeast 5') or 5'-nontranslated viral DNA (yeast 3') as well as a construction with both 5'- and 3'-nontranslated viral DNA also have been made. A comparison of these constructions for levels of HBcAg expression indicates that the strongest contributor to the high levels of protein is the presence of 5'-flanking sequences which are yeast-derived; secondarily, a significant improvement can be achieved if the 3'-flanking sequences also are yeast-derived. The high abundance of HBcAg in the highest producer is explicable in part on the basis of the very high stability in yeast cells of HBcAg polypeptides. Analysis of the HBcAg coding sequence reveals a very low index of codon bias for S. cerevisiae, largely discounting codon usage as a contributor to the high level of protein obtained.
Genes↗
Production and immunological analysis of recombinant hepatitis B vaccine.
The synthesis of the hepatitis B surface antigen (HBsAG) in cells of Saccharomyces cerevisiae and its subsequent isolation, purification and analysis is described. The final, purified HBsAg particle exhibits close structural and biochemical similarities to particles derived from the plasma of chronically infected humans. Particles of yeast and human origin have been found, by chimpanzee efficacy studies and by various in vitro analyses, to be immunologically equivalent. The antigenic expression of a determinant-specific epitopes, as measured by antibody binding to synthetic peptides, has also been shown to be equivalent.
Animals↗
New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products.
The accumulation of recent data concerning the reactivity of monoclonal antibodies with particular varicella-zoster virus (VZV) glycoproteins and the mapping of several of their respective genes on the VZV genome has led to a unified nomenclature for the glycoprotein genes of VZV and their mature glycosylated products. Homologs to herpes simplex virus glycoprotein genes are noted.
Genes, Viral↗
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.
Amino Acid Sequence↗
Assignment of three rat cellular RAS oncogenes to chromosomes 1, 4, and X.
Mouse hepatoma-rat hepatocyte hybrids that segregate rat chromosomes were used to determine the chromosomal localization of rat cellular RAS genes. The cellular KRAS gene, homologous to the Kirsten sarcoma virus oncogene was mapped to rat chromosome 4, a chromosome that is often present in three copies in rat neurogenic tumor cells and transformed glial cells. The rat cellular HRAS-1 gene, homologous to the Harvey sarcoma virus oncogene was assigned to chromosome 1, whereas its intron-less counterpart HRAS-2 was mapped to the X chromosome. Since the human HRAS-2 also resides on the X chromosome, it appears that the cellular HRAS-2 gene (or pseudogene) conserved its chromosomal localization during mammalian evolution.
Animals↗
Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC.
The genome of varicella-zoster virus (VZV) encodes at least three major glycoprotein genes. Among viral gene products, the gC gene products are the most abundant glycoproteins and induce a substantial humoral immune response (Keller et al., J. Virol. 52:293-297, 1984). We utilized two independent approaches to map the gC gene. Small fragments of randomly digested VZV DNA were inserted into a bacterial expression vector. Bacterial colonies transformed by this vector library were screened serologically for antigen expression with monoclonal antibodies to gC. Hybridization of the plasmid DNA from a gC antigen-positive clone revealed homology to the 3' end of the VZV Us segment. In addition, mRNA from VZV-infected cells was hybrid selected by a set of VZV DNA recombinant plasmids and translated in vitro, and polypeptide products were immunoprecipitated by convalescent zoster serum or by monoclonal antibodies to gC. This analysis revealed that the mRNA encoding a 70,000-dalton polypeptide precipitable by anti-gC antibodies mapped to the HindIII C fragment, which circumscribes the entire Us region. We conclude that the VZV gC glycoprotein gene maps to the 3' end of the Us region and is expressed as a 70,000-dalton primary translational product. These results are consistent with the recently reported DNA sequence of Us (A.J. Davison, EMBO J. 2:2203-2209, 1983). Furthermore, glycosylation appears not to be required for a predominant portion of the antigenicity of gC glycoproteins. We also report the tentative map assignments for eight other VZV primary translational products.
Cloning, Molecular↗
Breast cancer and lactic acidosis. An unusual metabolic complication.
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Acidosis↗
Chromosome assignments of four mouse cellular homologs of sarcoma and leukemia virus oncogenes.
Molecular probes for the oncogenes of Rous sarcoma virus (v-src), avian myeloblastosis virus (v-myb), Kirsten murine sarcoma virus (v-Ki-ras), and Harvey murine sarcoma virus (v-Ha-ras) were hybridized to the DNA from mouse-Chinese hamster somatic cell hybrids. The v-src, v-myb, v-Ki-ras, and v-Ha-ras genes each detected one or a few homologous mouse DNA fragments whose segregation was analyzed in cell hybrids. Mouse cellular homologs c-src, c-Ki-ras, c-Ha-ras, and c-myb segregated concordantly with chromosomes 2, 6, 7, and 10, respectively. Comparison with the known locations of human c-src (chromosome 20) and human c-Ha-ras1 (chromosome 11 short arm) suggests that the human and mouse homologs of these two viral oncogenes reside in conserved linkage groups. The c-Ki-ras gene on mouse chromosome 6 might reside also in a conserved linkage group, along with glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase. However, direct confirmation of this suggestion must await a demonstration that c-Ki-ras on mouse chromosome 6 is homologous to c-Ki-ras2 on the short arm of human chromosome 12.
Animals↗
Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.
The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.
Amino Acid Sequence↗
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.
Animals↗
Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes.
Varicella-zoster virus (VZV) codes for approximately eight glycosylated polypeptides in infected cell cultures and in virions. To determine the number of serologically distinct glycoprotein gene products encoded by VZV, we have developed murine monoclonal antibodies to purified virions. Of 10 monoclonal antibodies which can immunoprecipitate intracellular VZV antigens and virion glycoproteins, 1 (termed gA) reacted with gp105, 1 (termed gB) reacted with gp115 (intracellular only), gp62, and gp57, and 8 (termed gC) reacted with gp92, gp83, gp52, and gp45. The anti-gA monoclonal antibody neutralized VZV infectivity in the absence of complement. All eight anti-gC monoclonal antibodies neutralized only in the presence of complement. An anti-gB monoclonal antibody obtained from another laboratory also neutralizes in the absence of complement. Since the above reactivities account for all major detectable VZV glycoprotein species, the data strongly suggest that VZV has three major glycoprotein genes which encode glycosylated polypeptides with neutralization epitopes.
Animals↗
Regional localization of two human cellular Kirsten ras genes on chromosomes 6 and 12.
Human cellular Kirsten ras1 and ras2 genes were localized to chromosomes 6p23 ----q12 and 12p12 .05----pter, respectively, using human-rodent cell hybrids. Thus, the short arms of human chromosomes 11 (encoding lactate dehydrogenase-A and the proto-oncogene c-Ha- ras1 ) and 12 (encoding lactate dehydrogenase B and c-Ki- ras2 ) share at least two pairs of genes that probably evolved from common ancestral genes.
Animals↗
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.
Animals↗
Expression of endogenous p21 ras genes.
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Animals↗
Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus.
Harvey murine sarcoma virus is a retrovirus which transforms cells by means of a single virally encoded protein called p21 has. We have determined the nucleotide sequence of 1.0 kilobase in the 5' half of the viral genome which encompasses the has coding sequences and its associated regulatory signals. The nucleotide sequence has identified the amino acid sequence of two additional overlapping polypeptides which share their reading frames and the carboxyl termini with p21 but which contain additional NH2-terminal amino acids.
Amino Acid Sequence↗