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PubMed1967

Amino acid coding in Sarcina lutea and Saccharomyces cerevisiae.

Aminoacyl-tRNA's from Sarcina lutea were tested for incorporation into protein in a heterologous system from Escherichia coli or for biniding in a homologous system from Sarcina lutea. Aminoacyl-tRNA's from Saccharomyces cerevisiae were tested for biniding in a homologous Saccharomyces cerevisiae system. Synthetic polyribonucleotides were used as messengers. The code which exists in Sarcina lutea and Saccharomyces cerevisiae is the same as in Escherichia coli.

Amino Acids↗

PubMed1980

Modified polyriboinosinic-polyribocytidylic acid complex: modulation of toxicity for rabbits by alterations in components.

In an earlier study the physiological effects encountered in humans of a modified complex of polyriboinosinic acid-polyribocytidylic acid [poly(I)-poly(C)] with carboxymethylcellulose and poly-L-lysine [poly(ICLC) (9S)], namely, the induction of high titers of serum interferon along with fever and hypotension, were reproduced in rabbits. In the present studies, also in rabbits, we evaluated effects of complexes with homopolymer polyribonucleotide sedimentation coefficients decreasing from 9S to 6S and 4S. Modified complexes of carboxymethylcellulose and poly-L-lysine with decreasing molecular weights were also tested. In several studies diphenhydramine and indomethacin were administered concomitantly. These experiments showed that at a daily intravenous dose of 0.2 mg/kg the various preparations of poly(I)-poly(C) (9S) and poly(I)-poly(C) (6S) induced falls in blood pressure, but stabilized complexes of poly(I)-poly(C) (4S) did not. Alterations in the molecular weights of carboxymethylcellulose and poly-L-lysine n the modified complex and the concomitant administration of diphenhydramine did not influence the occurrence or severity of untoward reactions. In rabbits, poly(ICLC) (4S) along with indomethacin induced high titers of serum interferon without fever or hypotension.

Animals↗

PubMed1975

Application of the deoxyribonucleic acid/ribonucleic acid hybridization technique in Bdellovibrio as a model for studying ribonucleic acid turnover in host-parasite systems.

The kinetics of host ribonucleic acid (RNA) degradation and its resynthesis into Bdellovibrio-specific polyribonucleotides has been studied. The kinetics of RNA turnover was followed during a one-step synchronous growth cycle of Bdellovibrio growing within 32-po4-Labeled Escherichia coli host cells. The species of labeled RNA present at any given time was ascertained through the specificity of the deoxyribonuclei acid (DNA)/RNA hybridization technique. At nearsaturating levels of RNA and at zero time, 7% of the host DNA sequences and only 0.04% of the Bdellovibrio DNA became hybridized with 32-P-labeled host cell RNA (greater than 99% host specific). At the end of the burst, 98% of the labeled RNA sequences were specific for Bdellovibrio DNA. About 74% of the initial labeled host cell RNA became turned over into Bdellovibrio-specific sequences. We provide data indicating that host cell ribosomal RNA is assimilated by Bdellovibri. Degradation of host cell RNA occurs in a gradual fashion over most of the Bdellovibrio developmental growth cycle. This application of the DNA/RNA hybridization technique and its general concept should be of value in elucidating the kinetics of nucleic acid turnover in other types of host-parasite systems.

Bacteria↗

PubMed1971

Failure of synthetic polynucleotides to affect the immunogenicity of mycobacterial ribonucleic Acid and ribosomal protein preparations.

The synthetic polyribonucleotides adenylic acid (poly A), uridylic acid (poly U), cytidylic acid (poly C), and inosinic acid (poly I), whether single- or double-stranded (poly A:U, poly I:C), cannot replace mycobacterial ribonucleic acid (RNA) in the production of a high immune response in CF-1 mice against tuberculous disease. These conclusions are based on the results of several types of experiments. (i) Poly A and poly U, used either singly or in combination, did not increase the immunogenicity of mycobacterial RNA preparations whether emulsified in Freund's incomplete adjuvant (FIA) or not emulsified. (ii) Mycobacterial ribosomal protein, extracted with 2-chloroethanol, was not immunogenic; the addition of poly A:U to the protein did not produce an immune response and FIA did not affect these results. (iii) The RNA left after the protein was extracted was partially immunogenic when emulsified in FIA even though it was partially degraded. (iv) Mycobacterial RNA prepared with ethyl alcohol and partially degraded with ribonuclease had a significantly lower immunogenic activity, and the original higher immune response was not restored by the addition of poly A:U. (v) Mycobacterial RNA totally degraded by weak alkali was not immunogenic, the original immunogenic activity was not restored by the addition of poly A:U or poly I:C, and FIA again did not influence the results. These findings suggest that (i) protein, polypeptides, or other antigenic fragments, if present, are not the specific immunogens; and (ii) mycobacterial RNA is responsible for the high immunogenic activity of mycobacterial ribosomal and RNA preparations. In addition, since the double-stranded forms of these synthetic polynucleotides markedly potentiate the formation of circulating antibodies, these results also reemphasize the lack of correlation between conventional antibody formation and immunity against tuberculosis.

Journal Article↗

PubMed1994

Characterization and pathological significance of monoclonal DNA-binding antibodies from mice with experimental malaria infection.

Malaria infection is accompanied by the production of a number of autoantibodies, including some that react with DNA. Epidemiological evidence implicates these in the nephritides that arise in human quartan malaria and in experimental malaria infections in mice. Through parallels with the involvement of DNA-reactive antibodies in the autoimmune syndrome systemic lupus erythematosus, a role for DNA-reactive antibodies in forming phlogistic immune deposits in the kidneys is implied. To more fully understand the relationship between antibodies of this specificity made in malaria and systemic lupus erythematosus, we prepared monoclonal DNA-reactive antibodies from BALB/c mice infected with Plasmodium berghei (clone RC) and compared their properties with those of other antibodies previously isolated from lupous MRL/Mp lpr/lpr and (NZB x NZW)F1 mice. Antibodies from malarial mice were all immunoglobulin M class and bound to single-stranded but not double-stranded DNA in an enzyme-linked immunosorbent assay. They also reacted with synthetic polyribonucleotides in the enzyme-linked immunosorbent assay and with parasitized erythrocytes and parasite pigment in kidney sections. None of the antibodies from lupous mice had identical specificities. The potential involvement of the DNA-reactive antibodies in malarial nephritis was demonstrated, by use of immunocytochemical methods, on the basis of their binding to existing immune deposits in kidney sections from malarial mice, a similar property having been previously demonstrated for antibodies from lupous mice. Furthermore, antibodies from malarial mice expressed public idiotypes, notably Id.V-88, which is a member of the Id.16/6 family, commonly found on DNA-reactive antibodies in lupus and other infectious and connective tissue diseases. This study indicates that DNA-reactive antibodies in malaria have immunochemical properties similar but not identical to those of such antibodies in systemic lupus erythematosus and that they have the potential to participate in the formation of immune deposits in nephritic malarial kidneys.

Animals↗

PubMed1970

Uptake of synthetic polynucleotides by competent cells of Bacillus subtilis.

A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with transforming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides employed and the amino acids whose incorporation was stimulated.

Amino Acids↗

PubMed1975

Characterization of polyadenylate from the fungus Trichoderma viride.

Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic acid of the deuteromyceteous fungus Trichoderma viride. Similar findings in two other groups of the true fungi raise the possibility that short polyadenylate sequences may be a feature of these relatively simple organisms.

Adenine Nucleotides↗

PubMed1967

Characterization of deoxyribonucleic acids from streptomycetes and nocardiae.

The relationships among selected streptomycetes, nocardiae, and mycobacteria have been determined, based upon the base composition of their deoxyribonucleic acid (DNA) and upon the ability of their denatured DNA to anneal with single-stranded reference DNA. The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 73 mole% guanine + cytosine (% GC). Moreover, the streptomycetes examined showed 37 to 88% homology with the Streptomyces venezuelae and S. rimosus reference DNA. The nocardial and mycobacterial DNA both contained 62 to 69% GC. The nocardial strains studied fell into either a 62 to 64% GC group or a 68 to 69% GC group, indicating that they should not be assigned to a single species. The nocardiae having 68 to 69% GC showed 24 to 44% homology with S. venezuelae reference DNA. In competition experiments, wherein unlabeled heterologous DNA interfered with binding of labeled homologous DNA, the nocardial DNA with 68 to 69% GC showed a greater degree of homology with the streptomycetes than did the nocardial DNA with 62 to 64% GC. In addition, the DNA from spores of S. venezuelae was cursorily examined, and interactions between S. venezuelae denatured DNA and polyribonucleotides were sought. The buoyant density of the DNA from S. venezuelae spores was distinctly less than that from mycelia. Moreover, denatured S. venezuelae DNA formed a dense complex with polyriboguanylate.

Cytosine↗

PubMed2006

Production of monoclonal antibodies to Tropheryma whipplei and identification of recognized epitopes by two-dimensional electrophoresis and mass spectrometry.

Tropheryma whipplei, the agent of Whipple's disease, is a gram-positive rod-shaped bacterium that belongs to the group of actinobacteria. In order to produce monoclonal antibodies (MAbs) against this bacterium, we inoculated mice with two different strains, Slow2 and Endo5. We produced 13 and 10 MAbs against Slow2 and Endo5, respectively. Nine of the Slow2 MAbs and seven of the Endo5 MAbs recognized a 58-kDa epitope. In addition, three other Endo5 MAbs detected a unique 84-kDa epitope. These MAbs were species specific, as they did not react with a selection of 22 different bacterial species, but they were not strain specific, as they did react with six other strains of T. whipplei. Two-dimensional gel electrophoresis (2-DE) was combined with mass spectrometry (MS) to identify the 58-kDa and 84-kDa epitopes recognized by MAbs. After trypsin in-gel digestion of the spot, the 58-kDa protein was identified as an ATP synthase F1 complex beta chain, whereas the 84-kDa protein was identified as a polyribonucleotide nucleotidyltransferase by MS with matrix-assisted laser desorption ionization-time of flight. In an in vitro model, one of these MAbs allowed good detection of T. whipplei in stool samples, contrary to a rabbit polyclonal antibody, which led to high fluorescent background. In the prospective studies, the produced MAb will be tested for detection of T. whipplei in clinical samples, and the gene coding for identified 58-kDa and 84-kDa antigens will be tentatively cloned and then tested for its use in a diagnostic enzyme-linked immunosorbent assay for Whipple's disease.

Actinobacteria↗

PubMed1973

Stimulation of Sendai virion transcriptase by polyanions.

Exogenous polyribonucleotides stimulated the ribonucleic acid (RNA) transcriptase in Sendai virions. Added yeast RNA, polyadenylic acid, or polycytidylic acid increased incorporation of (3)H-guanosine monophosphate as much as fivefold. The products of stimulated reactions were virus-specific as determined by hybridization with Sendai virion RNA, but they sedimented more slowly (13s) than the product of an unstimulated reaction (16s). The stimulating activity was nondialyzable and heat stable, but was abolished by alkaline hydrolysis. Nucleoside monophosphates, individually or in combination, were ineffective, confirming the requirement for a polymer. Among other substances tested for effects on Sendai virion transcriptase, polyaspartic acid and polyglutamic acid stimulated the enzyme; polyinosinic acid, polyuridylic acid, and polyamines had no effect; and dextran sulfate and polyvinyl sulfate were inhibitory.

Animals↗

PubMed1973

Effects of polyadenylic acids on functions of murine RNA tumor viruses.

Single-stranded polyribonucleotides, which competitively inhibit murine RNA tumor virus reverse transcriptase in vitro, were tested as inhibitors of various virus functions in cell culture. The compounds had two concentration-dependent effects. At high concentrations (100 mug/ml), both poly(adenylic acid) [poly(A)] and poly(2'-O-methyladenylic acid) [poly(Am)] inhibited the uptake of radioactively labeled leukemia virus by Swiss mouse embryo cells, but neither had a similar effect on Sindbis virus adsorption. At low concentrations (10 mug/ml), poly(Am) did not inhibit the uptake of leukemia virus but did inhibit virus replication by 85%; in contrast, the replication of Sendai virus and Sindbis virus was not inhibited significantly at this concentration. Both compounds were effective only when added prior to or early during virus infection. Poly(Am) was a much more effective inhibitor than poly(A), probably due to the nuclease resistance of the former compound. Poly(Am) at 5 mug/ml also inhibited transformation of 3T3 cells by Moloney sarcoma virus. However, neither poly(A) nor poly(Am) at high concentrations inhibited the activation of endogenous leukemia virus by iododeoxyuridine in AKR mouse embryo cells. These results suggest that virus reverse transcriptase plays an essential role in both the replication of exogenous murine leukemia viruses and the transformation of cells by murine sarcoma viruses but probably has no role in the activation of endogenous leukemia virus.

Adenosine Monophosphate↗

PubMed1974

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. IX. Template topography in the early region of simian virus 40.

The DNAs of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses contain overlapping segments of the early region of wild-type SV40 DNA. The complementary DNA strands of these five viruses have been separated with synthetic polyribonucleotides in isopycnic cesium chloride gradients. The relative amounts of early and late SV40 template in the DNA of each virus were determined by RNA-DNA hybridization with late lytic SV40 RNA, which contains sequences complementary to both templates. From the distribution of early and late templates in the five overlapping SV40 segments, we conclude that either the entire early region of SV40 is symmetrically transcribed in vivo, or, more probably, that the early SV40 templates are not contiguous.

Adenoviridae↗

PubMed1974

Purification of avian myeloblastosis virus DNA polymerase by affinity chromatography on polycytidylate-agarose.

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.

Avian Leukosis Virus↗

PubMed1983

Genomic RNA of mengovirus. VI. Translation of its two cistrons in lysates of interferon-treated cells.

The addition of low levels (40 ng/ml) of the synthetic double-stranded polyribonucleotide poly I:C to lysates of interferon-treated L-cells resulted in a strong inhibition (70 to 75%) of the in vitro translation of mengovirus RNA. Under these conditions, the rates of incorporation of [35S]methionine or formyl-[35S]methionine were depressed to a comparable extent. The sequences of mengovirus RNA recognized by ribosomes of interferon-treated cells at initiation of translation were compared with those present in initiation complexes formed by ribosomes of untreated controls. Fingerprint analysis revealed that the same sequences of mengovirus RNA were protected against nuclease attack by the 80S and the 40S initiation complexes formed in vitro in lysates of control or interferon-treated L-cells. Mengovirus RNA-coded proteins were labeled at their N-terminal end with formyl-[35S]methionine and digested to completion with trypsin. The resulting fragments were separated by high-voltage paper electrophoresis. Two different formyl-[35S]methionine-labeled N termini were resolved. Further analyses supported the notion that the two radioactive peaks originated in the initiation of translation at two different sites. This pattern did not change when mengovirus RNA was translated in lysates of interferon-treated cells.

Animals↗

PubMed1985

MC29 virus-coded protein occurs as monomers and dimers in transformed cells.

The MC29 virus-coded protein p110gag-myc was found exclusively in the nucleus of transformed Japanese quail (Q8) cells, and time course experiments indicated that the protein had a half-life of about 30 min. When extracts of either Q8 or chicken embryo cells infected with MC29 virus were prepared with nondenaturing detergents and then sedimented in sucrose gradients, p110 was found in the fractions expected to contain monomers (5.9S), dimers (9.3S), or mixtures of the two. The same extracts treated with denaturing detergent (0.2% sodium dodecyl sulfate) exhibited p110 only in fractions expected for the monomeric protein, but beta-mercaptoethanol had no effect on the original distribution. Gradients prepared with 0.5 or 1.0 M NaCl failed to dissociate the faster-sedimenting form. No other protein or polyribonucleotide which could increase the sedimentation rate of p110 was found, and neither RNase nor DNase altered the sedimentation pattern of p110 in nondenatured extracts. A reassociation of monomeric p110 into dimers discernible by gel electrophoresis was demonstrated.

Animals↗

PubMed1986

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.

Aleutian Mink Disease↗

PubMed1970

Complementary Strands of Bacteriophage phi29 Deoxyribonucleic Acid: Preparative Separation and Transcription Studies.

Bacillus subtilis phage phi29 has a nonpermuted, duplex deoxyribonucleic acid (DNA) with cohesive ends and a molecular weight of 11 x 10(6). Denaturation of this DNA yielded two intact polynucleotide chains. Preferential binding of the polyribonucleotide polyuridylic-guanylic acid (poly UG) to the complementary strands of denatured phi29 DNA permitted separation of the strands in neutral CsCl gradients. In analytical CsCl density gradient centrifugation, the separated strands with poly UG appeared as two symmetrical bands, both heavier than the normal denatured DNA band. The strands differed in density by 11 mg/cc. Preparative separation of the phi29 DNA strands resulted in two fractions, heavy (H) and light (L). The H fraction was essentially free from L contamination, whereas L contained up to 25% of H, as determined both by rebanding the separated fractions in CsCl and by electron microscopic examination of self- and mixed-annealed fractions. Pulse-labeled ribonucleic acid (RNA) prepared at intervals after infection was hybridized with the self-annealed DNA strands. Preliminary experiments indicated that both strands of phi29 DNA are transcribed during the development of the virus. Early transcribed phi29-specific RNA hybridizes only with the L strand; at later times, transcription occurs from both the L and H strands.

Journal Article↗

PubMed1993

RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria.

The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses.

Adenosine Triphosphatases↗