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At least 109 records · Page 6Linked to original sources
PubMed2000

Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo.

We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.

Animals↗
PubMed2003

Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

Animals↗
PubMed1980

Virus-specific effects of interferon in embryonal carcinoma cells.

Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).

2',5'-Oligoadenylate Synthetase↗
PubMed2004

Complexity of dsRNA mycovirus isolated from Fusarium graminearum.

Fusarium graminearum is the causal agent of a serious scab disease of small grains in Korea. We screened 827 isolates of F. graminearum from diseased barley and maize and tested for the presence of double-stranded RNA (dsRNA) mycovirus. Of them, 19 isolates contained various sizes of dsRNAs. A dsRNA associated with pronounced morphological changes including reduction in mycelial growth, increase in dark orange to red pigmentation, reduced sporulation and virulence was previously observed in nine dsRNA-containing Fusarium isolates (Chu et al., Appl Env Microbiol 68, 2529-2534, 2002). Ten additional isolates were found infected with dsRNA mycoviruses. These mycoviruses contain 2-4 different segments of dsRNAs with the size-range of approximately 1.7-10 kbp in length. The presence of dsRNAs did not affect colony morphology and were transmissible through conidia and ascospore with incidence of 30-100%. Interestingly, dsRNA mycovirus found in F. graminearum isolates, JB33 and JNKY19, that show the pattern of mixed infection of two different viruses were transmitted to all progeny conidia and ascospores. These results indicate that there is genomic diversity of dsRNA mycoviruses that infect F. graminearum isolates and that impact of virus infection on host's morphology and virulence is determined by the interaction between dsRNAs and the fungal host, not by the mere presence of the dsRNAs.

Amino Acid Sequence↗
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↗
PubMed2004

DNA interference: a simple and efficient gene-silencing system for high-throughput functional analysis in the fern adiantum.

RNA interference (RNAi) has become a powerful tool for determining gene function and is used in a wide variety of organisms. Since it is necessary to generate double-stranded RNA (dsRNA) as an inducer for RNAi, preparation of RNAi-inducing constructs is somewhat cumbersome and time consuming, especially for the thousands of genes used in a genome-wide analysis. To overcome these problems, we have developed a more convenient gene-silencing method in the fern Adiantum using double-stranded DNA (dsDNA) as a model system for functional analysis in plants. Delivery of dsDNA fragments homologous to an endogenous gene into gametophytic cells can induce sequence-specific gene silencing. As it only requires dsDNA fragments homologous to a target gene, PCR-amplified fragments are enough to trigger gene silencing. Maximum gene silencing efficiencies of >90% have been achieved for transformed plants. In addition, simultaneous transfer of dsDNA fragments corresponding to multiple genes still has a silencing effect for individual genes. We term this approach 'DNA interference'.

Adiantum↗
PubMed2000

Viral double-stranded RNA, cytokines, and the flu.

The symptoms of the flu, such as fever, drowsiness, and malaise, are the sole means by which this common clinical syndrome is defined. The syndrome is usually the first clinical manifestation of both acute bacterial and viral infections. In the case of acute bacterial infections, several proinflammatory cytokines induced by bacterial products have been implicated as the causative agents of the flu syndrome. Viruses induce similar cytokines to bacteria, plus substantial amounts of interferon-alpha (IFN-alpha), although the direct association of these cytokines with the viral flu syndrome is less clear. Furthermore, the viral inducer(s) of cytokines has not been defined. The best candidate cytokine inducer associated with a majority of viral infections is virus-associated double-stranded RNA (dsRNA). This review examines the essential physical properties of toxic dsRNA, the cytokines induced by it, its viral and cellular sources, evidence for its presence in infected cells, its quantities in normal and infected cells, its cytotoxic mechanisms, and its cell-penetration properties. Toxic effects of viruses and dsRNA are compared. Energetics and extraction artifact issues are also discussed. Whereas most research on dsRNA toxicity has employed synthetic dsRNA, studies with virus-associated dsRNA are featured when available. Finally, a model for how viral dsRNA might initiate systemic disease is presented.

Animals↗
PubMed1979

Evidence for secondary structure in poliovirus virion RNA demonstrated by antibodies against double-stranded RNA.

Poliovirus particle RNA has been considered to have little secondary structure. Specific binding of poliovirion RNA by antibodies against double-stranded RNA (dsRNA) has been confirmed and further characterized by a radioimmunoassay using Staphylococcus aureus protein A to precipitate the nucleic acid-antibody complex. Competitive binding studies between virion single-stranded RNA (ssRNA) and poly(I).poly(C) demonstrated that the dsRNA effectively inhibited binding of radiolabelled poliovirion RNA by the anti-dsRNA antibodies but the virion RNA was a poor competitor of radiolabelled ds RNA. This indicates that both RNAs reacted with the same species of antibodies in the sera, but avidity of the antibodies for dsRNA was greater than for the poliovirion RNA.

Animals↗
PubMed2004

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interference (RNAi). mRNA degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. In this chapter, we describe some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA and analysis of dsRNA, and target mRNA.

Animals↗
PubMed2004

Substrate specificities of bacterial and human AlkB proteins.

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.

AlkB Homolog 1, Histone H2a Dioxygenase↗
PubMed1986

Sequence of the M1-2 region of killer virus double-stranded RNA.

A full-length complementary DNA (cDNA) copy of the M1-2 region of the double-stranded genome of the yeast killer virus was synthesized by reverse transcription, utilizing the m in vitro transcript as template and synthetic primers for both strands. The sequence lacks any long open reading frames (ORFs). The internal portion of the M1-2 region includes the sequence that is linked to the subterminal 229 bases of the M1-1 homologous region in the S3 defective-interfering mutant of killer virus double-stranded RNA (dsRNA). Thus, the probable site at which the deletion occurred in S3 has been identified.

Base Sequence↗
PubMed2000

The silence of the genes.

About two years ago, it was recognized that introduction of double-stranded RNA (dsRNA) had a potent effect on gene expression, in particular on mRNA stability. Since then, this process has been found to occur in many different organisms, and to bear a strong resemblance to a previously recognized process in plants, called cosuppression. Both genetic and biochemical studies have started to unravel the mysteries of RNA interference: genes involved in this process are being identified and in vitro studies are giving the first hints of what is happening to both the dsRNA and the affected mRNA molecules after the introduction of the dsRNA.

Animals↗
PubMed1987

L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus- strand L-A single-stranded RNA (ssRNA). These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA). The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism. In this paper, we show that mutations in mak10 or the pet18 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro. The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles. Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction. Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.

Genotype↗
PubMed1998

Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA.

Protein interactions with double-stranded RNA (dsRNA) are critical for many cell processes; however, in contrast to protein-dsDNA interactions, surprisingly little is known about the molecular basis of protein-dsRNA interactions. A large and diverse class of proteins that bind dsRNA do so by utilizing an approximately 70 amino acid motif referred to as the dsRNA-binding domain (dsRBD). We have determined a 1.9 A resolution crystal structure of the second dsRBD of Xenopus laevis RNA-binding protein A complexed with dsRNA. The structure shows that the protein spans 16 bp of dsRNA, interacting with two successive minor grooves and across the intervening major groove on one face of a primarily A-form RNA helix. The nature of these interactions explains dsRBD specificity for dsRNA (over ssRNA or dsDNA) and the apparent lack of sequence specificity. Interestingly, the dsRBD fold resembles a portion of the conserved core structure of a family of polynucleotidyl transferases that includes RuvC, MuA transposase, retroviral integrase and RNase H. Structural comparisons of the dsRBD-dsRNA complex and models proposed for polynucleotidyl transferase-nucleic acid complexes suggest that similarities in nucleic acid binding also exist between these families of proteins.

Amino Acid Sequence↗
PubMed1994

Association of yeast viral 23 S RNA with its putative RNA-dependent RNA polymerase.

T double-stranded RNA (dsRNA) and its single-stranded RNA (ssRNA) counterpart, 23 S RNA, are present in some strains of the yeast Saccharomyces cerevisiae. 23 S RNA is identical with the T (+)-strand, and both are cytoplasmically inherited. The T (+)-strand encodes a protein of 104 kilodaltons (kDa) (p104) that has sequences diagnostic of RNA-dependent RNA polymerases from (+)-strand and double-stranded RNA viruses. Here we show that p104 is expressed only in yeast strains carrying T dsRNA (and 23 S RNA) and that the ability to produce p104 is co-transmitted from one strain to another by cytoplasmic mixing (cytoduction) along with T dsRNA. Neither T nor 23 S RNA is encapsidated into viral particles; however, the latter was found to be non-covalently associated with p104. This finding was based on the evidence that: (i) both 23 S RNA and p104 cosedimented in sucrose gradients and that (ii) 23 S RNA was immunoprecipitated by a p104-specific polyclonal antiserum. Consistent with this association, p104 shows single-stranded RNA-binding activity in Northwestern blots. We have localized the RNA-binding domain of p104 within its C terminus 234 amino acids.

Fungal Proteins↗
PubMed2000

dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference.

RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.

Animals↗
PubMed2006

[Construction of "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference].

OBJECTIVE: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs). METHODS: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing. RESULTS: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment. CONCLUSION: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.

Cloning, Molecular↗
PubMed2002

RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA.

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.

Acid Anhydride Hydrolases↗