Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase in the Immune Response

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 M column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of ³H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12–13 S was most active as a template. It was followed by a fraction corresponding to 6–7 S. Sucrose gradient analysis of the ³H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Ribonucleic Acid Polymerase Catalyzing Synthesis of Double-stranded Arbovirus Ribonucleic Acid

The large-particle fraction from the cytoplasm of chick embryo fibroblasts infected with Semliki Forest virus was found to catalyze the incorporation of the 5'-triphosphates of guanosine, adenine, cytidine, and uridine into an acid-insoluble alkali-labile product. The conditions affecting the preparation and assay of this enzyme were investigated. The ribonucleic acid (RNA) polymerase was not present in uninfected cells, and it appeared in infected cells at the time of rapid viral RNA synthesis. The polymerase was found to catalyze the synthesis of a species of RNA which was resistant to ribonuclease and which exhibited the sedimentation properties, buoyant density, and thermal transition temperature of the double-stranded RNA found in vivo in chick cells infected with Semliki forest virus. Attempts to demonstrate that the reaction product of this enzyme also included single-stranded viral RNA were not successful. Although other interpretations are possible, these results give some support to the suggestion that more than one enzyme may be involved in the replication of viral RNA.     

Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response.

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Reovirus-induced Ribonucleic Acid Polymerase

A virus-induced ribonucleic acid (RNA) polymerase activity was found in L cells infected with type 3 reovirus. Most of the enzyme is associated with the "large particle" fraction of the infected cells. The enzyme first appeared at 3 to 5 hr after infection and increased in amount until 7 to 9 hr. All four ribonucleoside triphosphates are incorporated in vitro into an acid-insoluble form by the enzyme. The major part of the product formed in vitro is a double-stranded RNA indistinguishable from viral RNA by electrophoresis on polyacrylamide gel. Approximately 40% of the product is a single-stranded RNA of relatively small molecular weight. More than 95% of the nucleotides incorporated into double-stranded RNA by the enzyme are bound in internal 3'-5'-phosphodiester linkages extending back from both 3'- and 5'-termini of the RNA strands.     

Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid

After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the (3)H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

Incorporation of Angustmycin C,a Nucleosidic Antibiotic,into Ribonucleic Acid in Escherichia coli

A nucleosidic antibiotic angustmycin C, which is an analogue of adenosine and which has an unusual hexose, psicofuranose, instead of ribose, was confirmed to be incorporated into RNA in Escherichia coli. After incubation of cells with tritiated angustmycin C, small but significant radioactivity was found in RNA fraction. When the RNA was hydrolyzed by an enzyme mixture of snake venom, pancreatic ribonuclease I and alkaline phosphatase, intact angustmycin C was recovered which indicated incorporation of the antibiotic into the polyribonucleotide chain of RNA.     

Ribonucleic acid in the immune response

Summary In the studies of experimental salmonellosis, immunization of mice with a live vaccine SER of S. enteritidis was found to be effective against further infection with virulent S. enteritidis 116-54. Macrophages obtained from the peritoneal cavity, subcutaneous tissue or liver of immunized mice inhibited intracellular growth of bacteria and resisted cell degeneration caused by engulfment of virulent 116-54 bacteria. This immunity was called cellular immunity.We discovered by chance in 1961 a transfer agent of immunity (TA) from the culture fluid of immunized macrophages. This agent is RNA in nature and can be extracted from the spleen, peritoneal exudate cells or the lymph node of immunized animals and is called immune (i) RNA. We could demonstrate antibody activity in macrophages treated in vitro or in vivo with iRNA by the immune adherence hemagglutination technique.Cellular immunity against tumor cells could be transferred in vitro or in vivo to lymphocytes through iRNA prepared from the spleen cells of syngeneic, allogeneic and xenogeneic animals immunized with the tumor cells.We prepared iRNA against antigens capable of inducing humoral antibody production in animals, i.e., RBCs, bacterial toxin, bacterial flagella and hapten-protein conjugates. Serum antibody was not demonstrated in recipient animals of iRNAs by single or repeated injections of these agents. However, in these animals an increase in the number of specific antibody-carrying cells was found as rosette-formers. It was found further that prior injection of iRNA could induce immunologic memory and produced a high titer of humoral antibody after a boosting stimulation with a small dose of the corresponding antigen. The required interval between the first iRNA and the second antigenic stimulation, and the minimal effective doses of iRNA and antigen are described.We studied the interaction of iRNA with either T- or B-cells and with both cells using adoptive transfer system, athymic nude mice and neonatally thymectomized (NT) mice. Immune RNAs against T-dependent and T-independent antigens could not induce the proliferation of antibody-carrying cells in cyclophosphamide-treated (B-cell depleted) mice. But these agents could induce the proliferation of rosette-formers, implying that iRNAs can replace some role of T-cells even against T-dependent antigens. B-cells can be directly activated by treatment with iRNA against both T-dependent and T-independent antigens, and they differentiated into rosette-formers.Passive transfers of iRNA were successful in establishing immunity against infection with S. enteritidis, or immunity to Salmonella flagella, RBCs and hapten-protein conjugates. The ability of iRNA to confer a secondary response of antibody formation is serially and passively transmissible in recipient animals. These facts suggest the presence of some mechanism that is responsible for the amplification of antigenic stimulation in the immune response. The RNA-dependent RNA polymerase and RNA-dependent DNA polymerase are presented and their role in the immune response is discussed.     

Reovirus Replicase-Directed Synthesis of Double-Stranded Ribonucleic Acid

After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand (³H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of ³H-cytidine triphosphate (or ³H-uridine triphosphate) and adenine triphosphate-α-³²P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3′-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of ³H to ³²P in these fragments indicated that RNA synthesis proceeded from the 5′ to 3′ terminus.     

A particulate DNA polymerase activity in adult rat brain

A particulate fraction of adult rat brain (sucrose buoyant density 1.24 gm/ml) catalyzed the incorporation of [³H]dTTP into an acid-insoluble product in an endogenously templated reaction sensitive to ribonuclease pretreatment. Upon fractionation, this activity was identified in the cerebellum, pons, frontal lobes and base. The DNA polymerase present in these brain fractions exhibited a strong preference for the synthetic template dT_12–18·poly rA rather than dT_12–18·poly dA; dT₁₀ was completely inactive. Purification and equilibrium Cs₂SO₄ gradient centrifugation of the [³H]DNA product-endogenous template complex suggested that RNA was serving as primer for endogenous DNA synthesis.     

Ribonucleic Acid Synthesis in Plant Mitochondria: Effects of Some Inhibitors and Cyclic Nucleotides

Mitochondria isolated from 48-h germinating Vigna sinensis (L.) Savi can incorporate [³H]uridine into acid-insoluble material. The incorporation is highly sensitive to rifampicin and partially so to ethidium bromide, two specific inhibitors of template function. The inhibitory effect of rifampicin can be partly counteracted by cyclic 3′:5′-AMP but not by cyclic 3′:5′-GMP, if they are allowed to interact with the synthetic system before the treatment with rifampicin. This indicates that cyclic AMP and rifampicin compete for a common site on the RNA polymerase responsible for DNA-dependent RNA synthesis. Inhibition by ethidium bromide is unaffected by prior nucleotide interaction with the system.     

Ribonucleic Acid Polymerase Induced in Cells Infected with Sendai Virus

A ribonucleic acid (RNA)-dependent RNA polymerase was induced in chick embryo fibroblast cells after infection with Sendai virus (parainfluenza 1 virus). The enzyme was associated with the microsomal fraction of infected cells and reached maximum detectable activity at 18 hr after virus infection. The activity of the enzyme in vitro was dependent on the presence of added magnesium ions and all four nucleoside triphosphates and was not inhibited by actinomycin D. The RNA synthesized by the enzyme in vitro was sensitive to ribonuclease and consisted of a complex mixture of RNA species including 34S, 24S, and 18S components. Similar RNA components were detected in the microsomal fraction of Sendai virus-infected cells by labeling with (3)H-uridine from 17 to 18 hr postinfection in the presence of actinomycin D. Of the RNA synthesized by Sendai virus-induced RNA polymerase in vitro, 98% became insensitive to ribonuclease after annealing with RNA extracted from purified Sendai virus particles.     

Ribonucleic Acid Transcriptases in Sendai Virions and Infected Cells

Sendai virions contain an enzyme which catalyzes the incorporation of ribonucleotides into ribonucleic acid (RNA). Enzyme activity was optimal at pH 8.0 and 28 C; otherwise conditions were similar to those reported for Newcastle disease virion (NDV) RNA polymerase. The initial rate of RNA synthesis by the Sendai virion enzyme was about 10 pmoles per mg of protein per hr, but after 3 hr of incubation the rate increased about fivefold. The virion enzyme was compared with an RNA polymerase in the microsomal fraction of infected cells. Both enzymes made predominantly single-stranded RNA which was complementary in base sequences to 50S virion RNA. Most of the RNA synthesized by the virion polymerase sedimented at 16S, but the product of the microsomal enzyme sedimented at about 8S.     

Protein and Ribonucleic Acid Synthesis by Plant Mitochondrial Systems

The incorporation of ¹⁴C-labelled phenylalanine into proteins of the mitochondrial systems obtained from 48-h germinating seeds of Vigna sinensis (L.) Savi can be stimulated by polyuridylic acid [poly (U)] and depressed by rifampicin, which is, however, ineffective if poly (U) is allowed to interact with the incorporating system before the antibiotic has access to it. A system consisting of a mitochondrial S-100 fraction and ribosomes from the same source with other cofactors can bring about polymerization of phenylalanine. The incorporation of ¹⁴C-labelled uracil into RNA by the plant mitochondria is greatly dependent on the exogenous addition of adenine, guanine, cytosine and also on 5-phosphoribosyl-l-pyrophosphate (5-PRPP). It is greatly suppressed by rifampicin and ethidium bromide.     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.     

Effect of Tobacco Mosaic Virus Infection on Amino Acid Incorporation into Isolated Mitochondria from Tobacco Leaves

Mitochondria isolated from tobacco leaves incorporated ¹⁴C-leucine into the protein and the rate was enhanced by tobacco mosaic virus (TMV) infection as compared with noninfected level. In vitro amino acid incorporation by mitochondria required adenosine triphosphate (ATP), Mg²⁺, and KC1 and the energy sources from oxidative phosphorylation as well as from ATP-generating system. This incorporation was inhibited by ribonuclease (RNase), deoxyribonuclease (DNase), actinomycin D, mitomycin C, puromycin, and chloramphenicol added in the reaction medium. The pretreatment of the mitochondria with DNase and actinomycin D reduced the rate of incorporation. The mitochondria incorporated ³H-guanosine triphosphate (GTP) and this activity was blocked by actinomycin D. The presence in this system of 15,000 g supernatant cell sap fraction or bacterial contamination was carefully checked obtaining a negative result. The reaction product into which ^l4C-amino acids incorporated was solubilized by trypsin. The nature of the amino acid incorporating activity of isolated mitochondria obtained from TMV-infected tobacco leaves is discussed.     

Ribonucleic acid synthesis by chromatin isolated from Phaseolus aureus Roxb.

Chromatin was isolated from the hypocotyls of Phaseolus aureus Roxb. and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity in vitro. The molecular size of the RNA product, measured by polyacrylamide gel electrophoresis, was found to be much smaller than that known to be synthesised in vivo and was affected by the assay temperature. Although conventional enzyme assays provided no evidence for the presence of ribonuclease in chromatin, a more sensitive technique revealed sufficient ribonuclease activity to degrade high-molecular weight RNA to smaller fragments. The inclusion of unlabelled exogenous RNA in the media for chromatin preparation and RNA polymerase assay substantially increased the molecular-weight of the RNA products synthesised in vitro.Abbreviations TCA trichloroacetic acid - UTP uridine-5-triphosphate - UMP uridine-5-monophosphate - SOS sodium dodecyl sulphate     

Isolation of Messenger Ribonucleic Acid for Myosin Heavy Chain

A chicken embryonic polysome fraction that contains 50–60 monoribosomes and synthesizes the heavy chains of myosin is separated from other polysomes of smaller sizes by centrifugation through two cycles of discontinuous and continuous sucrose gradients. The unique properties of the polyadenylic acid segment present at the 3′-end of eukaryotic messenger RNA (mRNA) were used to purify the mRNA for myosin heavy chain from the phenol-extracted total RNA obtained from this polysome fraction. The total RNA was filtered thro ugh millipore filters resulting in partition of the riboscmal RNA (rRNA) and mRNA species. This millipore-bound RNA fraction, which consists of the mRNA and some ribosomal RNAs, was eluted from the filters with sodium dodecyl sulfate (SDS). Subsequent chromatography of this fraction on a cellulose column gave two well-separated peaks: an unadsorbed peak of ribosomal RNAs which was eluted with buffers of high ionic strength and an adsorbed peak of mRNA which was eluted only with a buffer of low ionic strength. Polyacrylamide gel electrophoresis of the mRNA peak fraction showed a single band with no detectable amounts of other RNAs, the mRNA migrating slower than 28S rRNA. The product of in vitro translation of the purified mRNA using a homologous cell-free system was identified as the myosin heavy chain by the following criteria: coprecipitation with carrier myosin at low ionic strength; elution properties on DEAE-cellulose column; and comigration with the heavy chain in polyacrylamide gel electrophoresis. In order to demonstrate the fidelity of translation of the mRNA, ¹⁴C-labeled products of the in vitro translation were copurified with unlabeled myosin heavy chains added as a carrier. The mixture of polypeptides was then cleaved with CNBr and the resulting peptides were separated by molecular sieving. The correlation between the radioactivity and the UV absorbance in the separated peptides indicates that total synthesis of the myosin heavy chain was achieved.     

Ribonucleic Acid, Deoxyribonucleic Acid, and Protein Content of Cells of Different Ages of Mycobacterium tuberculosis and the Relationship to Immunogenicity

The amount of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) was determined in pellicle cultures of different ages of the H37Ra strain of Mycobacterium tuberculosis, grown on a synthetic medium. We found that the highest content of RNA and protein was present in 2-week-old cultures, indicating that these cells were in the logarithmic phase of growth. DNA content was highest at 1 and 2 weeks. The amount of all three compounds then decreased about 50% during the following 6 weeks. Two-week-old cells should therefore be used for preparation of the immunogenic ribosomal fraction. The optimal concentration of zinc chloride increased RNA and protein synthesis, and also improved the appearance of the pellicle growth. Two-week-old cells, which contained the largest amount of RNA and protein, immunized mice significantly better than older cells. Since protein and DNA are not involved in the production of immunity, a correlation could be made between amount of RNA and the capacity of viable H37Ra cells to immunize mice. The immunizing capacity of these cells was not affected by ribonuclease, probably because the ribonuclease did not penetrate into the whole cells.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.