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At least 919 records · Page 51Linked to original sources
PubMed1989

Reconstitution of template-dependent in vitro transcriptase activity of a yeast double-stranded RNA virus.

Isolated mature L-A viral particles from yeast have a transcriptase activity that uses endogenous L-A double-stranded RNA (dsRNA) as template. We have previously demonstrated that empty particles derived from mature L-A viral particles have replicase activity capable of synthesizing minus strand single-stranded RNA (ssRNA) on an added plus strand ssRNA template to form dsRNA. We report here that empty particles also have transcriptase activity that uses added viral dsRNA as template. The newly synthesized ssRNA was the plus strand, and some of these transcripts were converted to the dsRNA form by the replicase activity associated with the empty particles. This transcriptase activity, however, required a much higher concentration of polyethylene glycol than that used previously for the replicase activity. The mode of transcription was conservative. The enzyme transcribed ssRNA from L-A, M1, or X (a deletion mutant of L-A) dsRNAs but not from other yeast dsRNAs (L-BC, T, or W), bacteriophage Phi6 dsRNAs, or animal rotavirus dsRNAs, indicating the same template specificity as that expected for the in vivo reaction. This assay system, and the replicase assay system, will allow us to study in vitro all the enzymatic reactions essential for the viral replication cycle.

Base Sequence↗
PubMed1989

Vaccinia specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate.

Mouse L-cells infected with vaccinia virus produce a specific kinase inhibitory factor (SKIF) which inhibits the activation of the interferon-induced, double-stranded (ds)RNA-dependent, eukaryotic initiation factor (eIF)-2 alpha-specific protein kinase in L-cell extracts (Whitaker-Dowling, P., and Younger, J. S., (1984) Virology 137, 171). The effects of a partially purified preparation of SKIF have been examined in cell-free extracts of rabbit reticulocytes. Both the phosphorylation state of eIF-2 and protein synthetic activity have been determined. SKIF inhibits the phosphorylation of the alpha subunit of eIF-2 by dsRNA-dependent eIF-2 alpha-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sensitive kinase. In addition to its effects on eIF-2 alpha-PKds activity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, SKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct correlation between the effects of SKIF on eIF-2 alpha phosphorylation and on protein synthetic activity and demonstrate the specificity of SKIF. The results also show that SKIF does not abolish dsRNA sensitivity, but increases the concentration of dsRNA required to activate the kinase and phosphorylate eIF-2.

Animals↗
PubMedDate not supplied

Electropherotypic analysis of rotaviruses isolated from turkeys.

From 1980 to 1984, several flocks of turkeys in Minnesota exhibiting signs of clinical enteritis were examined for viruses. Electron microscopic (EM) examination of fecal specimens from 35 flocks revealed the presence of rotavirus particles. Rotaviruses were successfully isolated in cell cultures from only 24 of these positive fecal specimens. Double-stranded RNA (dsRNA) preparations made from these 24 cell-culture isolates and from the remaining 11 fecal samples that were rotavirus-positive on EM examination were analyzed by polyacrylamide gel electrophoresis for genetic differences in their genomes. The study revealed eight distinct electropherotypes among the rotavirus dsRNA preparations. Atypical dsRNA migration patterns were recognized only in preparations of dsRNA from fecal materials.

Animals↗
PubMed1988

[Pharmacological analysis of the effect of natural double-helical nucleic acids on the detoxifying function of the liver].

The effect of interferon inductors i.e. double stranded RNAs from S. cerevisiae and phage F6 on the liver detoxicating function was studied on noninbred albino mice. The liver detoxicating function was tested by duration of hexenal sleep. It was shown that intraperitoneal administration of the yeast and phage RNAs in doses of 1/5 LD50 for three times led to increasing of the narcotic sleep duration in the animals by 65 and 207 per cent, respectively. The effect was of the dose-dependent nature. The doses not inducing reliable inhibition of hexenal metabolism were equal to 1/10 LD50 for the yeast dsRNA and 1/27 LD50 for the phage dsRNA. The inhibitory effect of the dsRNAs was retained for 2-3 days after discontinuation of the drug use. When the dsRNAs were administered simultaneously with nembutal, an inductor of the liver microsomal enzymes, the dsRNAs eliminated its inducing effect. Simultaneous administration of alpha-tocopherol lowered the dsRNA effect on hexenal metabolism. The findings suggested that the dsRNA inhibitory effect on the liver detoxicating function was grounded on the mechanisms associated with inhibition of syntheses and activation of lipid peroxidation specific of the monooxygenase system under the action of the dsRNAs.

Animals↗
PubMed1988

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity.

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

[Toxic properties of the double-stranded RNA of virus-like particles from Saccharomyces cerevisiae killer yeasts].

The toxic properties of dsRNA isolated from S. cerevisiae were studied on noninbred albino mice. The preparation was administered intraperitoneally. The lots of the yeast dsRNA consisted of highly and moderately toxic compounds. The toxicity level was directly associated with the content of the dsRNA in the preparation. Administration of the preparation in doses equal to the maximum tolerance dose and 1/5 of LD50 had no significant effect on the motor activity, temperature and weight of the animals. However, it induced marked morphological impairments in the gastrointestinal tract, liver and kidneys and pronounced shifts in the hematological indices. 1/50 of LD50 had no significant effect on the physiological, hematological and morphological indices of the organs. No capacity for the yeast dsRNA cumulation in the host was observed.

Animals↗
PubMed1985

[Antitumor activity of Ca-double-stranded RNA from sodium nucleinate].

Ca-dsRNA introduced in the concentration of 5 micrograms per mouse 24 hours before the MG22a mouse hepatoma transplantation causes the 70% inhibition of the hepatoma growth. The sodium nucleinate used in the considerably higher concentration had a less effect. The sodium form of dsRNA also had a less antitumour effect comparing with its calcium form. The calcium form of dsRNA proves to be relatively stable and physiologically active form of dsRNA during its parenteral administration.

Animals↗
PubMed1984

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells.

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.

Animals↗
PubMedDate not supplied

[Thermodynamic characteristics of nucleic acid complexes with silver ions].

By means of mixing reaction calorimetry the enthalpy of the complexes formation between Ag+ ions and DNA and dsRNA was measured. It was shown that Ag+ ions are able to form two types of complexes (I and II) with dsRNA. Using the method of the competitive reaction with chloride ions the stability constants of complex formation were obtained for dsRNA-Ag+ complexes for different temperatures. These measurements gave the delta H and delta S values for both complexes: delta HI = -74,9 +/- 7,1 kjouls/mol, delta SI = -100.0 +/- 25.0 jouls/mol deg; delta HII = -39,8 +/- 4,2 kjouls/mol, delta SII = +2 +/- 14 jouls/mol deg. The calorimetric results of delta H determination are the same within the limits of experimental errors. The enthalpy term of dsRNA-Ag+ complexes proved to bring the main contribution into the free energy of complex formation.

Animals↗
PubMedDate not supplied

[Cloning in Escherichia coli cells of mouse DNA fragments homologous to two main species of long hairpin regions of pre-mRNA].

Clones of plasmid pBR322, containing fragments of mouse DNA were obtained. DNA from many of these clones (at least 15%) was able to hybridize with double-stranded hairpin-like regions of pre-mRNA (dsRNA-B). It was shown by hybridization of dsRNA-B with clone DNA, that there are two abundant species of sequences in the population of these molecules designated as B1 and B2. They are 40-50% and 20-25% of all the molecule of dsRNA-B, respectively. The size of DNA sequences complementary to dsRNA is about 200--400 base pairs. The melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher but still the number of substitutions does not exceed 10% of bases. The sequence B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.

Animals↗
PubMed1997

Cloning, sequencing and expression of a full-length cDNA copy of the M1 double-stranded RNA virus from the yeast, Saccharomyces cerevisiae.

Strains of the budding yeast, Saccharomyces cerevisiae, may contain one or more cytoplasmic viruses with double-stranded RNA (dsRNA) genomes. The killer phenomenon in yeast, in which one cell secretes a killer toxin that is lethal to another cell, is dependent upon the presence of the L-A and M1 dsRNA viruses. The L-A viral genome encodes proteins for the viral capsid, and for synthesis and encapsidation of single-stranded RNA replication cycle intermediates. The M1 virus depends upon the L-A-encoded proteins for its capsid and for the replication of its killer-toxin-encoding genome. A full-length cDNA clone of an M genome has been made from a single dsRNA molecule and shown to encode functional killer and killer-immunity functions. The sequence of the clone indicates minor differences from previously published sequences of parts of the M1 genome and of the complete genome of S14 (an internal deletion derivative of M1) but no unreported amino acid variants and no changes in putative secondary structures of the single-stranded RNA. A 118-nucleotide contiguous segment of the M1 genome has not previously been reported; 92 of those nucleotides comprise a segment of A nucleotides in the AU-rich bubble that follows the toxin-encoding reading frame.

Amino Acid Sequence↗
PubMed2000

Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila.

Posttranscriptional gene silencing (PTGS) induced by double-stranded RNA (dsRNA) is an intriguing phenomenon that has been observed in a variety of organisms, including Drosophila melanogaster. Although PTGS in Drosophila is typically observed following direct injection of the dsRNA into embryos, it is theoretically possible that the in vivo transcription of an inverted repeat transgene might also produce a dsRNA "hairpin" that is capable of triggering PTGS. Here we test this idea, and show that an expressed inverted repeat of a portion of the sex differentiation gene, transformer-2, (tra-2), driven by a GAL4-dependent promoter, does genetically repress the endogenous wild-type tra-2 function, producing a dominant loss-of-function mutant phenotype. Remarkably, this effect is temperature-sensitive, with phenotypic consequences seen at 29 degrees, but not at 22 degrees. Moreover, by altering the dosage of either the transgenes or the endogenous tra2(+) loci, one can vary the effect over a wide range of mutant phenotypes.

Animals↗
PubMed2002

Selection of small-molecule mediators of the RNA regulation of PKR, the RNA-dependent protein kinase.

The RNA-dependent protein kinase (PKR) is a component of the interferon antiviral response and a member of the class of RNA-binding proteins with a double-stranded RNA binding motif. PKR is activated when it binds to double-stranded RNA (dsRNA) or viral replicative intermediates that comprise dsRNA and this activation results in the inhibition of protein synthesis. Some viruses circumvent this activity through the synthesis of highly structured decoy RNAs that bind PKR and block activation. Small-molecule mediators of the binding of PKR to these RNA inhibitors would be useful tools to further define the importance of specific PKR-RNA complexes in vivo and may possess antiviral activity. Here we investigate the ability of a library of structurally diverse peptide-acridine conjugates (PACs) to target a complex formed between the dsRNA binding domain (dsRBD) of PKR and a viral RNA inhibitor. We used a novel screening method based on the cleavage of RNA ligands with ethylenediaminetetraacetic acid.Fe modified protein. The selection revealed a PAC (9-anilinoacridine-4-Hyp-Nap-Nap, where Hyp is trans-4-hydroxyproline and Nap is 1-napthylalanine), able to inhibit the binding of the PKR dsRBD to RNA with an IC(50) value of 10 +/- 5 microM. Furthermore, the structural requirements for inhibition by the selected PAC were substantiated in an independent PKR activation assay. We found that the potency of inhibition by an intercalating ligand can be increased by the introduction of a substituent that does not increase the overall charge of the molecule. This result is important for the design of inhibitors of PKR-RNA binding that function inside living cells.

Acridines↗
PubMed2005

Molecular mechanisms of RNAi: implications for development and disease.

Research over the past few years has led to dramatic new discoveries on the role of double-stranded RNA (dsRNA) in the cell. RNA duplexes have been shown to orchestrate epigenetic changes, repress translation, and direct mRNA degradation in a sequence-specific manner. These diverse effects of dsRNA on gene expression have been termed RNA interference (RNAi). In addition to playing a role in viral defense and silencing transposons, RNAi also has a critical function in a number of developmental processes in the embryo. In this review, we explore these roles and discuss the molecular mechanisms behind dsRNA-mediated gene silencing. Further, we address the use of RNAi as a tool to study gene function in biology, and as a strategy for treating human disease.

Animals↗
PubMed2006

Engineering eukaryotic signal transduction with RNAi: enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1.

RNAi has been useful in the study of biochemical pathways, but has not been widely used as a tool in metabolic engineering. The work described here makes use of double-stranded RNA (dsRNA) for the post-transcriptional gene silencing of TSC1 in Drosophila S2 cells. TSC1 downregulates the insulin-mediated signal transduction pathway, and serves as a metabolic control to guard against cellular overproliferation and tumorogenesis in both flies and mammals. By silencing TSC1 with in vitro-synthesized dsRNA, we have created a tunable and specific metabolic "throttle" that, like insulin, apparently increases the specific growth rate of S2 cells in a dose-dependent manner. This "throttle," augments the benefits of insulin addition while apparently avoiding deleterious and pleiotropic effects which can lead to lysis. During the period wherein dsRNA was active, cell growth rate was increased by 11% by the addition of 15 microg/mL dsTSC1 and by over 20% by the addition of 30 microg/mL dsTSC1. Additionally, synthesis of recombinant green fluorescent protein (GFP) was increased nearly 50% in a stable S2 cell line inducibly expressing GFP. Accordingly, we have "tuned" a normally tumorogenic pathway in animals into an advantage for both growth and recombinant product synthesis in cell culture. Potential applications for improving eukaryotic cell culture are anticipated.

Animals↗
PubMed1996

Induction of CD14 expression in Lpsn, Lpsd and tumor necrosis factor receptor-deficient mice.

The involvement of CD14 in lipopolysaccharide (LPS) recognition and signaling has been demonstrated in several studies. For this reason, we investigated whether the resistance of Lpsd mice to LPS might be related to an impaired CD14 expression. We compared the in vivo and in vitro expression of CD14 in Lpsn (LPS sensitive) and Lpsd mice, and its modulation by LPS, killed gram-negative and gram-positive bacteria and double-stranded (ds)RNA. Untreated Lpsn and Lpsd cultured macrophages (M phi), expressed similar amounts of CD14 mRNA and membrane-bound (m)CD14. LPS enhanced CD14 expression only in Lpsn M phi, while all bacteria, or dsRNA, enhanced CD14 in Lpsn and Lpsd M phi. Similarly, in vivo administration of LPS induced or enhanced CD14 mRNA in different organs of Lpsn mice only, while bacteria or dsRNA in both types of mouse. Furthermore, exogenous recombinant tumor necrosis factor (TNF) induced in vivo and in vitro enhanced CD14 expression in Lpsn, Lpsd and also in TNF receptor 2-deficient (TNFR2-/-) mice, but failed to do so in TNFR1-/- mice, showing that TNFR1 mediates the effect of TNF on CD14. However, LPS, bacteria and dsRNA induced CD14 in both TNFR2-/- and TNFR1-/- mice to a similar extent, revealing that this induction does not require TNF signaling.

Animals↗
PubMed2005

The binding affinity of double-stranded RNA motifs to HIV-1 Tat protein affects transactivation and the neutralizing capacity of anti-Tat antibodies elicited after intranasal immunization.

In this study we examined the hypothesis that the binding affinity of two double-stranded (ds) RNA motifs to HIV-1 Tat protein might affect transactivation and the type of anti-Tat immune responses. Using surface plasmon resonance technology we demonstrated the capacity of the poly(A):poly(U) (pA:pU) motif to bind with high affinity to a totally synthetic Tat protein and to inhibit more efficiently the Tat/transactivation response element (TAR) RNA interaction as compared to the poly(I):poly(C) (pI:pC) motif. Furthermore, the pA:pU motif was tenfold more effective in inhibiting Tat-driven transactivation than the pI:pC motif. Following intranasal immunization of mice, both dsRNA motifs enhanced the antibody (serum and mucosal) and cellular responses to Tat. However, only the serum samples of mice immunized with Tat + pI:pC inhibited Tat-driven transactivation. The profile of serum antibody subclasses together with the secreted cytokines by Tat-stimulated splenocyte cultures indicated that both dsRNA motifs favored the induction of a balanced Th1 and Th2 immune response. The demonstration in this study that two dsRNA motifs had a marked effect on Tat/TAR RNA interaction and on the neutralizing capacity of anti-Tat specific antibody responses highlights their potential for biological applications and the importance of selecting the appropriate motif as an adjuvant for vaccine design.

Administration, Intranasal↗
PubMed1984

Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced. These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation. Potential glycosylation sites in the preprotoxin peptide are identified. Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor.

Amino Acid Sequence↗