Effect of protein phosphorylation on the activity of RNA polymerase in streptomycetes

RNA polymerase was isolated fromStreptomyces granaticolor and protein kinase was partially purified fromStreptomyces albus. When RNA polymerase was treated with protein kinasein vitro the activity of RNA polymerase was markedly enhanced. Furthermore, a protein ofM=65 kDa was isolated which, after being phosphorylated, stimulated RNA polymerase activityin vitro. Because neither the β-subunits nor the α-subunits of RNA polymerase were phosphorylated it is assumed that phosphorylation of the 65 kDa protein may regulate the activity of RNA polymerase in streptomycetes.     

Some perspectives on the transfer of cell-mediated immunity by immune-RNA

Summary Ribonucleic acid extracts of lymphoid cells from immune hosts were used to transfer in vivo and in vitro cell-mediated immune reactivity to a variety of antigens. The in vivo immune responses transferred by RNA included the delayed cutaneous hypersensitivity reaction to fungal and chemically-defined antigens and the tumor-rejection reaction to guinea pig hepatoma antigens. The in vitro immune responses transferred by RNA included macrophage migration inhibition by fungal, chemically-defined, and tumor antigens. The transfer activity of RNA preparations was contained in the 8 s to 18 s species of RNA and was sensitive to RNAse but not to DNase or trypsin. Antigen was not detectable in the RNA preparations and appeared to have no role in the transfer activity. Syngeneic, allogeneic, or xenogeneic sources of RNA could transfer immune reactivity. In each system tested, the transfer of cell-mediated reactivity by RNA was specific for the antigen used to sensitize the RNA donor. The potential use of RNA-mediated transfer of immunity is discussed.     

Synthesis of the Oligoribonucleotides Incorporating 8-Oxo-Guanosine and Evaluation of their Base Pairing Properties

6-O-7-N-Bis(diphenylcarbamoyl)-2-N-phenoxyacetyl-5′-O-dimethoxytrityl-2′-O-{[(triisopropyl- silyl)oxy]methyl}-8-oxoguanosine-3′-yl-β-cyanoethyl-N,N-diisopropylphosphoramidite (5) was synt- hesized as a new phosphoramidite precursor unit for the synthesis of RNA. Compound 5 was successfully incorporated into the middle of the RNA sequences, and the synthesized RNAs were identified by MALDI-TOF mass measurements. Their properties were evaluated for formation of the RNA duplex and RNA/DNA heteroduplex. ORNs 1 and 4 containing 8-oxo-G can form base pairs with rC or dC in an anti conformation, while it can also interact with rA or dA in a syn conformation in the RNA duplex or RNA/DNA heteroduplex. The described synthetic method is therefore a useful procedure for the synthesis of ORN containing 8-oxo-G and contributes to the study of 8-oxo-G in RNA.     

Related base sequences in the DNA of simple and complex organisms. III. Variability in the base sequence of the reduplicated genes for ribosomal RNA in the rabbit

A comparative study has been made of the arrangement of base sequences in the ribosomal RNA cistrons of Escherichia coliand rabbit DNA. This was accomplished by examination of the thermal stability profiles of DNA/RNA hybrids formed by the two types of ribosomal RNA under various conditions. The thermal stabilities of ribosomal RNA hybrids of rabbit origin are more dependent on the conditions of reaction during the formation and are always lower than those of E. coli RNA. It is concluded that the rabbit ribosomal RNA hybrids are formed mainly from mismatching between RNA molecules and DNA sites other than those from which they were transcribed. Thus, the cluster of ribosomal RNA cistrons in a mammalian DNA, representing a historical series of tandem duplications, exhibits intercistronic base sequence divergence. This research was supported by a research grant from the National Science Foundation (GB 6099) and a predoctoral traineeship (to R.L.M.) from the U.S. Public Health Service.     

Resting complexes of the persistent yeast 20S RNA Narnavirus consist solely of the 20S RNA viral genome and its RNA polymerase p91

The positive strand 20S RNA narnavirus persistently infects Saccharomyces cerevisiae. The 20S RNA genome has a single gene that encodes the RNA‐dependent RNA polymerase (p91). 20S RNA forms ribonucleoprotein resting complexes (RNPs) with p91 and resides in the cytoplasm. Here we found no host proteins stoichiometrically associated with the RNP by pull‐down experiments. Furthermore, 20S RNA, when expressed from a vector in Escherichia coli, formed RNPs with p91 in the absence of yeast proteins. This interaction required the 3′ cis signal for complex formation. Moreover, when 23S RNA, the genome of another narnavirus, was expressed in E. coli, it also formed RNPs with its RNA polymerase p104. Finally, when both RNAs were expressed in the same E. coli cell, they formed RNPs only with their cognate RNA polymerases. These results altogether indicate that narnaviruses RNPs consist of only the viral genomes and their cognate RNA polymerases. Because the copy number of the RNPs can be induced almost equivalent to those of rRNAs in some yeast strains, the absence of host proteins may alleviate the burden on the host by not sequestering proteins into the RNPs. It may also contribute to the persistent infection of narnaviruses by decreasing their visibility.     

Localization of the 5S RNA genes in Zea mays by RNA-DNA hybridization in situ

5S RNA was extracted from Zea mays tissue and iodinated in vitro with ¹²⁵I to a high specific activity. Acrylamide gel electrophoresis of the ¹²⁵I-5S RNA, 11/2 weeks after iodination demonstrated that most of the 5S RNA molecules were degraded to half-size or smaller. In situ hybridization with this iodinated RNA to pachytene microsporocyte chromosomes showed that the 5S RNA cistrons are located near the end of the long arm of chromosome 2. No obvious association of the 5S locus with the nucleolus was seen during pachytene or later stages.     

Secondary structure of the autoregulatory mRNA binding site of ribosomal protein L1

Summary The secondary structure of the autoregulatory mRNA binding site of Escherichia coli ribosomal protein L1 has been studies using enzymatic methods. The control region of the E. coli L11 operon was cloned into a vector under control of the Salmonella phage SP6 promoter, and RNA transcribed using SP6 RNA polymerase. The secondary structure of this RNA was probed using structure-specific nucleases, and by comparison of the data with computer predictions of RNA folding, secondary structural features were deduced. The proposed model is consistent with elements of some previously proposed models, but differs in other features. Finally, secondary structure information was obtained from two mutant mRNAs and the structural features correlated with observed phenotypes of the mutants.Abbreviations MB mung bean nuclease - V₁ cobra venom nuclease - sss single-strand-specific - dss double-strand-specific     

Ribonucleic Acid Dependent Ribonucleic Acid Replicase in the Immune Response

An immune ribonucleic acid (iRNA) preparation was made using phenol extracts of spleens of mice previously immunized with Salmonella tennessee flagella. An enzyme, also prepared from the spleens of these mice, induced the incorporation of ³H-UTP into the acid-insoluble fraction in a cell-free system in the presence of this RNA. The enzyme activity could be demonstrated from the spleens of immunized mice but not from normal ones, and this activity was also inhibited by two derivatives of rifamycin. Treatment with ribonuclease or heating at alkaline pH resulted in a loss of activity in added RNA. The ³H-uridine-labeled product was found resistant to ribonuclease treatment but became sensitive when the product was subjected to heat treatment. However, actinomycin D, mitomycin C or bleomycin A₂ did not inactivate the enzyme activity. These results suggest that this enzyme induces the incorporation of UTP into the acid-insoluble fraction using iRNA as a template and the product may be a newly synthesized RNA which forms a hybrid with iRNA. This enzyme activity may play a role in the antibody formation process, and may account for the in vivo replication of iRNA by this enzyme, viz., probably an RNA-dependent RNA replicase.     

Molecular cloning of the ribosomal RNA genes of the photosynthetic bacterium Rhodopseudomonas capsulata

Summary Chromosomal segments of Rhodopseudomonas capsulata carrying the ribosomal operons and cloned with the cosmid vector pHC79 have been identified by cross hybridization with ³²P-ATP labeled rRNAs. At least seven rRNA operons are present in the R. capsulata chromosome. By R-loop analyses of DNA-RNA hybrids, two distinct loop structures of sizes 1.50 kb and 2.52 kb corresponding to the 16S and 23S RNA molecules, respectively, were detected. Intact 23S RNA molecules can be isolated from R. capsulata ribosomes by sucrose density centrifugation. However, fragmentation of the 23S RNA molecule into a 16S-like molecule was observed during gel electrophoresis. Restriction mapping and hybridization of a 9 kb PstI fragment that contained one copy of the rRNA operon showed the following sequence of the RNA genes in R. capsulata 16S, 23S, and 5S. A spacer region of 0.91 kb was found between the 16S and the 23S RNA genes.     

Detection and characterization of defective interfering RNAs associated with the cocksfoot mottle sobemovirus

A new RNA of about 900 nt was found in the virions of cocksfoot mottle virus (CfMV) and in infected plants by RNA hybridization and RT-PCR. Structural features suggested that this RNA is a defective interfering RNA (diRNA). The CfMV diRNA was shown to consist of a 35-nt 5′-terminal genomic region, which formed a hairpin, and a 3′-terminal genomic region, which included the coat protein (CP) gene lacking the first 120 nt.In vitro translation of the diRNA started at the third Met codon to produce truncated CP. The CfMV diRNA was assumed totrans-activate synthesis of the CP subgenomic RNA (sgRNA).     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

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.     

Studies on the nature and role of polyadenylated RNA in spore development of Bacillus subtilis

Summary cDNA probes synthesized on poly(A)RNAs isolated from sporulating cells of Bacillus subtilis were used for hybridization studies with RNAs derived from cells at different stages of growth and sporulation. It was shown that these cDNAs hybridized only to RNA from sporulating cells. No hybridization was observed if total RNA isolated from vegetative cells or from stationary phase cells of a zero stage asporogenic mutant was used. The hybridization studies also indicate that at each sporulation stage different poly(A)RNA species are synthesized. Furthermore, the hybridization kinetics have clearly demonstrated the existence of three distinct abundance classes of poly(A)RNA similar to those observed in eukaryotic cells. BamHI endonuclease restriction fragments of B. subtilis DNA that were found to hybridize to labeled poly(A)RNA were ligated to the pHV33 vector and hybrid clones that hybridized efficiently to poly(A)RNA were selected. Among these, three have been found to carry the spoOB gene.These results strongly suggest that the appearance of poly(A)RNA can be correlated to the expression of spore genes.     

RNA synthesis in the cells of the apical meristem of Sinapis alba during transition from the vegetative to the reproductive condition

Summary Vegetative plants of Sinapis alba, a long-day species, were induced to flower by exposure to a single 20-hr long day. RNA synthesis in the apical meristem of vegetative (control) and induced plants was investigated by using ³H-uridine and autoradiography of sections.Light-microscope autoradiographs showed a sharp increase in total RNA synthesis per cell in induced meristems. This increase occurred as early as 18 hr after the start of the long day, i.e. at the presumed time of the arrival of the floral stimulus at the meristem. At the same time, electron-microscope autoradiographs showed that there were changes in the pattern of RNA synthesis in the meristematic cells. The ratio of the number of grains in the nucleus to that in the cytoplasm slightly decreased and the ratio of the number of grains in the chromatin to that in the nucleolus greatly increased.Experiments with 2-thiouracil (2-TU), a pyrimidine analogue which was shown to inhibit RNA synthesis in Sinapis, indicated that this compound was most inhibitory to floral induction between the 12th and the 20th hour after the start of the long day, i.e. at the same time as important quantitative and qualitative changes in RNA synthesis were detected in induced meristems by autoradiographic methods. It was thus assumed that 2-TU inhibits floral induction via its effect on these (or on one of these) changes.     

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.     

Studies on the biosynthesis of TMV

Summary Heating of the TMV replicative form (RF) above a certain temperature (T _m)causes a sharp shift from RNase resistance to sensitivity. The T _mwas determined at different salt concentrations and in the presence of formamide.The kinetics of the annealing reaction between TMV RNA and its complementary RNA was studied, and the rate constant was estimated. Under the chosen conditions which are appropriate for annealing, no dissociation of double-stranded TMV RNA was detected. The kinetic data permitted a maximum estimate of the equilibrium constant of the annealing (or dissociation) reaction.     

Characterization of 6S RNA in the Lyme disease spirochete

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.