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Biomedical subjects

O C Richards

Publications and source records attributed to O C Richards.

At least 37 records · Page 2Linked to original sources
PubMed1988

Expression of hepatitis A virus capsid sequences in insect cells.

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.

Animals↗
PubMed1988

Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli.

We have constructed a recombinant plasmid producing, in bacteria, a hepatitis A virus (HAV) capsid protein that may be useful as a subunit vaccine. HAV VP1 coding sequences were fused in-frame to NH2-terminal Escherichia coli TrpE coding sequences under the control of the tryptophan promoter. In the absence of exogenous tryptophan, E. coli containing this recombinant plasmid produced high levels of an 88-kilodalton fusion protein that was recognized in immunoblots by antibodies to TrpE and HAV. The TrpE/HAV VP1 protein was gel-purified and used to immunize rabbits. The resulting antiserum reacted with denatured HAV VP1 in immunoblots but did not react with intact virus. However, subsequent inoculation of an immunized animal with a subimmunogenic dose of inactivated, whole HAV resulted in the rapid appearance of a stable, virus-neutralizing antibody response.

Animals↗
PubMed1987

Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA.

The poliovirus polymerase 3D was synthesized in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA inserted into several plasmid expression vectors. Cleavage was accomplished by the action of viral protease 3C sequences expressed in the same bacteria, either from a second plasmid or from the same plasmid, cloned so as to produce contiguous sequences in the same protein. In the case of two plasmids, protease 3C functioned in trans to cleave the fusion protein at or very near the normal Gln/Gly cleavage site. When protease and polymerase sequences were produced in the same protein, the protease sequences acted in the precursor form to release the polymerase from itself. Thus, cleavage can occur to generate polymerase 3D both as an intermolecular reaction and, very likely, also as an intramolecular event.

Cloning, Molecular↗
PubMed1987

Host factor-induced template modification during synthesis of poliovirus RNA in vitro.

Poliovirus RNA polymerase, 3Dpol, transcribes poliovirus RNA in vitro in the presence of a host factor (HF) preparation from uninfected HeLa cells to yield heterogeneous-size product RNAs. The products include some molecules larger than the template that represent self-primed elongations of the template from a 3'-terminal hairpin. We showed that transcription proceeded through the formation of a modified RNA intermediate that was generated by nucleolytic cleavage of the template by HF in the absence of nucleoside triphosphates. Cleavage resulted in the loss of the original poly(A) 3' end and the generation of new, heterogeneous 3' ends that formed self-priming structures that could then be elongated by 3Dpol or reverse transcriptase. The two stages of the reaction, (i) cleavage to yield self-priming templates and (ii) subsequent chain elongation to yield heterogeneous-size products up to nearly dimer length, could be separated. RNAs whose original 3' ends were chemically oxidized so as to prevent chain elongation showed no reduction in template activity after preincubation with HF. We conclude that an HF preparation that contains a low level of nuclease activity is sufficient to activate RNA templates for transcription by 3Dpol to generate up to apparent dimer-length products. This reaction likely has little relevance to the mechanism of poliovirus RNA replication in vivo. It is likely that numerous other factors or activities, in addition to 3Dpol, would also result in transcription of poliovirus RNA in vitro.

Carrier Proteins↗
PubMed1987

Poliovirus snapback double-stranded RNA isolated from infected HeLa cells is deficient in poly(A).

A portion of poliovirus double-stranded RNA (25 to 50%) isolated from infected HeLa cells contains hairpin loops at one end of the duplex structure. These structures rapidly reformed double-stranded molecules after denaturation and appeared as molecules of up to two times genome length upon electrophoresis in denaturing agarose gels. A second form of poliovirus double-stranded RNA was readily denaturable into genome length strands. When the hairpin RNA was treated with S1 nuclease, subsequent denaturation resulted in formation of strands of up to genome length. Hairpin molecules contained very little, if any, poly(A) sequences, suggesting that the hairpin forms after nucleolytic removal of the 3' end of plus-strand templates. We conclude that the hairpin double-stranded RNA found in infected cells is likely generated by intracellular nicking and self-priming and that it does not represent an intermediate in the process of RNA replication.

HeLa Cells↗
PubMed1986

Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro.

The poliovirus RNA polymerase, 3Dpol, was used to synthesize RNA in vitro in the presence of a host factor preparation from uninfected HeLa cells and poliovirion RNA as the template. The transcription products included molecules approximately twice the length of the template, apparently resulting from hairpin formation and template-directed elongation, as previously reported (D. C. Young, D. M. Tuschall, and J. B. Flanegan, J. Virol. 54:256-264, 1985). Other polyadenylated template RNAs also yielded products that were twice the length of the template. The polarity of the products synthesized from plus-strand poliovirus RNA template was analyzed by Southern blotting using labeled product RNA to probe single-stranded poliovirus DNAs cloned into M13 vectors. The results demonstrated that host factor-mediated polymerase products contain newly synthesized plus-strand sequences as well as the expected minus-strand sequences. Polymerase products primed with oligo(U) were all of minus-strand polarity.

Base Sequence↗
PubMed1984

Two forms of poliovirus VPg result from amino acid modification of a single viral protein.

The protein (VPg) covalently attached to the 5' terminus of poliovirus RNA has been reported to resolve into two forms, separable by electrofocusing, yet the viral gene sequence predicts only one apparent gene for VPg. The two VPg species were separated and analyzed for structural differences. The protein contains no phosphorylated amino acid residues other than the junction tyrosine linked to the nucleic acid moiety. After isolation, the acidic form of VPg remains stable, but the more basic form again generates a distribution of both species. This suggests some lability or modification of at least one amino acid residue postsynthesis. The position of the alteration in the protein was localized to the amino-terminal tryptic peptide, containing nine amino acids.

Amino Acid Sequence↗
PubMed1984

Structure of poliovirus replicative intermediate RNA. Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative.

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4'-aminomethyl-4,5',8-trimethylpsoralen. After purification from infected cells, undenatured RI appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [3H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.

Base Composition↗
PubMed1983

Analysis of Theiler's virus isolates from persistently infected mouse nervous tissue.

The DA strain of Theiler's virus causes a chronic progressive demyelination in mice following intracerebral inoculation. Virus was isolated from chronically infected mice, and then grown in cell culture, and the isolates were compared with the parent virus used for inoculation. No defective interfering particles or temperature-sensitive virus were recovered, and capsid proteins appeared identical by SDS-PAGE. One of three isolates had evidence of genomic mutation by Tl ribonuclease oligonucleotide fingerprinting. The significance of these findings with regard to the generation and maintenance of persistence and to adaptation to cell culture is discussed. Also of interest was the marked difference between the DA fingerprint and that of GD VII, a serologically related strain with different biological activity.

Animals↗
PubMed1982

Characterization of a cell culture persistently infected with the DA strain of Theiler's murine encephalomyelitis virus.

We established a persistent infection in L 929 cells with the DA strain of Theiler's murine encephalomyelitis virus. Our studies showed that only a small number of cells in the cultures contained infectious virus or viral antigen. A role for interferon in the maintenance of persistence was suggested. Viral isolates from the cultures were not temperature sensitive, nor did they contain viral capsid polypeptide mutations or defective interfering particles. T1 oligonucleotide maps showed evidence of mutation in two of three isolates.

Animals↗
PubMed1981

Two forms of VPg on poliovirus RNAs.

The protein (VPg) linked to the 5' termini of poliovirus RNAs resolved into two species when subjected to non-equilibrium electrofocusing. The differently charged forms of VPg were not due to protein phosphorylation nor to variability of the number of phosphate residues associated with the nucleotide moiety remaining after RNase digestion of the nucleoprotein. Single-stranded viral RNA isolated from mature virions contained predominantly the more basic form of VPg, whereas unpackaged single-stranded RNa remaining in cells at the end of the virus replication cycle contained predominantly the more acidic form of VPg. Replicative-form (RF) molecules also contained both species of VPg, with the more acidic form representing the major species. Both plus and minus RNA strands in RF had similar VPg compositions; however, there appeared to be a strongly selective loss of VPg from only the minus strand in RF, particularly at late times postinfection.

HeLa Cells↗
PubMed1980

Heterogeneity of the 3' end of minus-strand RNA in the poliovirus replicative form.

The 3' terminus of the strand (minus strand) complementary to poliovirion RNA (plus strand) has been examined to see whether this sequence extends to the 5'-nucleotide terminus of the plus strand, or whether minus-strand synthesis terminates prematurely, perhaps due to the presence of a nonreplicated nucleotide primer for initiation of plus-strand synthesis. The 3' terminus was labeled with 32P using [5'-32P]pCp and RNA ligase, and complete RNase digests were performed with RNases A, T1, and U2. 32P-oligonucleotides were analyzed for size by polyacrylamide-urea gel electrophoresis. The major oligonucleotide products formed were consistent with the minus strand containing 3' ends complementary and flush with the 5' end of the plus strand. However, a variable proportion of the isolated minus strands from different preparations were heterogeneous in length and appeared to differ from each other by the presence of one, two, or three 3'-terminal A residues.

Base Sequence↗
PubMed1979

Strand-specific attachment of avidin-spheres to double-stranded poliovirus RNA.

Poliovirus-specific double-stranded RNA molecules containing covalently attached protein were coupled with a biotin ester through the protein moiety. Subsequent interaction of the RNA-biotin with avidin attached to electronopaque plastic spheres led to the formation of complexes that were easily visualized in the electron microscope. Avidinspheres were associated only with one end of the RNA-biotin molecules, as seen in the electron microscope. Avidin-sphere attachment to poliovirus double-stranded RNA is strand specific, as shown by molecular hybridization of strand-specific probes to the separated strands of denatured complexes. [3H]DNA complementary to polio virion RNA hybridized exclusively to the strands bearing associated spheres [(+) strands] whereas 125I-labeled virion RNA hybridized predominantly with strands without spheres [(-)strands]. This biotin-avidin labeling technique provides a means for the isolation of full-length poliovirus (-) strands and may provide a general means for isolation of double-stranded polynucleotides containing tightly attached protein.

Avidin↗
PubMed1978

DNA synthesis and DNA polymerase activity in Leydig cells of diethylstilbestrol-stimulated mouse testes.

Using a modification of the collagenase dispersion method of Dufau et al., we examined changes in DNA synthesis produced by estrogens in the interstitial cells of mice that develop malignant Leydig cell tumors after prolonged estrogen administration. Previous work in cryptorchid mice indicated that during continuous estrogen administration [3H]thymidine incorporation into DNA rises to a maximum in 3 to 4 days and then falls to approximately base levels within 2 to 3 weeks. This was confirmed both in Leydig cell concentrates of estrogen-treated mice after either injection with [3H]thymidine or incubation with [3H]thymidine in vitro. This DNA synthesis was blocked by hydroxyurea. DNA synthesis in cells of estrogen-treated BALB/c mice of the Huseby substrain, which have a high incidence of Leydig cell tumors, was 5 to 11 times that in untreated controls. Cells from estrogen-treated C3H/Bi mice, which have a low incidence of Leydig cell tumors, showed only a 2- to 3-fold increase. In the Huseby substrain the rise of DNA synthesis is a peak and subsequent recession were paralleled by a rise and fall in DNA polymerase alpha activity. DNA polymerase beta did not show this variation. In C3H/Bi mice, neither polymerase showed significant change. The evidence suggests that the early estrogen-stimulated DNA synthesis is probably replicative and is associated with increased DNA polymerase alpha activity.

Animals↗
PubMed1976

Isolation of a transcriptionally active chromosome from chloroplasts of Euglena gracilis.

A transcriptionally active chromosome has been isolated in highly purified form from choroplasts of Euglena gracilis, It contains chloroplast DNA, DNA-dependent RNA polymerase, and other proteins. Transcription occurs at low levels of endogenous DNA, and is indifferent to high levels of exogenous DNA. RNA chain elongation continues for several hours in vitro, and RNA chain initiation, determined by [gamma-32P]ATP incorporation, is continuous for at least 1 h in vitro. Maximal rates for RNA synthesis require only a divalent cation and the four ribonucleoside triphosphates. Apparent Km values for adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate are 4.0, 0.6, 2.5, and 2.3 muM, respectively. As would be expected for a DNA-dependent RNA polymerase, RNA synthesis is inhibited by actinomycin D. However, rifampicin and streptolydigin, inhibitors of procaryotic RNA synthesis, and alpha-amanitin, an inhibitor of eucaryotic nuclear RNA polymerases II and III, do not inhibt the RNA synthesis reaction. Heparin, which is a potent inhibitor of the initiation of RNA synthesis by a nontemplate bound RNA polymerase, also does not inhibit RNA synthesis. Isolation of transcriptionally active chromosomes should prove to be a useful method to study the mechanism of selective RNA transcription of eucaryotic chromosomes.

Aminoglycosides↗