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1.
Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3′-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed “degradative PCR” and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3′-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila. 相似文献
2.
Molecular Weight Determination of Sendai RNA by Dimethyl Sulfoxide Gradient Sedimentation 总被引:12,自引:10,他引:2
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The molecular weight of the large RNA of Sendai virus has been determined by sedimentation analysis in sucrose gradients containing 99% dimethyl sulfoxide (DMSO) to be 2.3 × 106. Sendai RNA recovered from 99% DMSO was found to cosediment with nondenatured Sendai RNA at 46 to 48s in ordinary sucrose gradients. The molecular weight value of 2.3 × 106 is considerably smaller than the estimates of 6 × 106 to 7 × 106 determined under nondenaturing conditions, suggesting a unique structure for Sendai RNA. 相似文献
3.
4.
Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola 总被引:2,自引:0,他引:2
Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-32P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria. 相似文献
5.
Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 106 r γ-radiation (60Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 106 r but not in suspensions exposed to 8 × 106 r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage. 相似文献
6.
Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 106 r γ-radiation (60Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 106 r but not in suspensions exposed to 8 × 106 r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage. 相似文献
7.
Electron Microscopy of Viral RNA: Molecular Weight Determination of Bacterial and Animal Virus RNAs 总被引:4,自引:3,他引:1
A method for preparation of single-stranded RNA for electron microscopy determination of molecular weight is reported. The method uses treatment with formaldehyde at elevated temperatures to remove secondary structure and spreading in a protein monolayer from 50% formamide onto a 50% formamide hypophase. Molecular weights were determined for some bacterial and animal viruses, for which conflicting values had been reported earlier. Molecular weights determined by the method, using Escherichia coli large subunit rRNA for a standard (1.1 × 106), are as follows: E. coli small subunit rRNA, 0.53 × 106; coliphage f2-RNA, 1.3 × 106; Qβ-RNA, 1.55 × 106; and Newcastle disease virus RNA, 5.78 × 106. 相似文献
8.
Comparison of the ribonucleic Acid subunits of reovirus, cytoplasmic polyhedrosis virus, and wound tumor virus 总被引:8,自引:6,他引:2
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Double-stranded ribonucleic acid (RNA) from intact cytoplasmic polynedrosis virus (CPV) and wound tumor virus (WTV) was analyzed by polyacrylamide gel electrophoresis. Using RNA from type 3 reovirus as a standard, it was calculated that CPV-RNA consisted of 9 subunits corresponding to a molecular weight of 12.7 × 106 and WTV-RNA consisted of 12 subunits corresponding to a molecular weight of 15.5 × 106. 相似文献
9.
Corantin Maurin Zhiguo He Marielle Mentek Paul Verhoeven Sylvie Pillet Thomas Bourlet Franoise Rogues Jean Loup Pugniet Thierry Peyragrosse Marion Barallon Chantal Perrache Ins Aouimeur Sophie Acquart Sandrine Ninotta Marc Baudhuin Bertrand Vabres Sylvain Poinard Philippe Gain Gilles Thuret 《PLoS medicine》2022,19(3)
10.
The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus 总被引:2,自引:0,他引:2
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Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 106 was calculated from the sedimentation coefficient, S°20,w = 86.2S, the diffusion coefficient, D20,w = 1.55 × 10-7 cm2/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 106 atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described. 相似文献
11.
The Gilchrist and M-33 strains of rubella virus exposed in the frozen state to 137Ce or 60Co were inactivated exponentially according to “one hit” kinetics. There was no difference in the radiosensitivity of the two strains. Experimental D37 values for both strains ranged from 1.9 × 105 to 2.9 × 105 rads, and computed radiosensitive molecular weights ranged from 2.6 × 106 to 4.0 × 106 daltons. 相似文献
12.
Ingle J 《Plant physiology》1968,43(9):1448-1454
The chloroplast ribosomal-RNAs (1.1 × 106 and 0.56 × 106 mol wt) are synthesized in the normal ratio of 2:1. The non-ribosomal distribution observed after extraction and fractionation results from the lability of the 1.1 × 106 component, and a correction for this breakdown can be applied in certain cases. Newly synthesized 1.1 × 106 RNA is more stable than the older accumulated 1.1 × 106 RNA. Accumulation of the chloroplast RNA during growth of radish cotyledons occurs at a later time than the accumulation of cytoplasmic RNA, and its turnover is much less than that of the cytoplasmic ribosomal-RNA. 相似文献
13.
Effects of Actinomycin D and Ultraviolet and Ionizing Radiation on Pichinde Virus 总被引:8,自引:7,他引:1
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Michael F. Carter Frederick A. Murphy J. Pierre Brunschwig Christine Noonan William E. Rawls 《Journal of virology》1973,12(1):33-38
Actinomycin D (0.05 μg/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with 3H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 × 106 to 8 × 106 daltons. This value is greater than the 3.2 × 106 daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods. 相似文献
14.
Gene Expression During the Development of Bacillus subtilis Bacteriophage φ29 II. Resolution of Viral-Specific Ribonucleic Acid Molecules
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The ribonucleic acid (RNA) specified by bacteriophage 29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 × 106, 0.44 × 106, 0.37 × 106, 0.25 × 106, 0.09 × 106, and 0.04 × 106, accounting for 35% of the coding capacity of 29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 × 106 were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 × 106) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 × 106, 0.93 × 106, and 0.07 × 106, appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins. 相似文献
15.
Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes
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70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D37 values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 105 and 1.4 × 105, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 105) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome. 相似文献
16.
Isolation and Characterization of an RNA-Dependent RNA Polymerase from Black Beetle Virus-Infected Drosophila melanogaster Cells 总被引:8,自引:7,他引:1
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Crude lysates of black beetle virus (BBV)-infected cells of Drosophila melanogaster contain an RNA-dependent RNA polymerase activity not detectable in uninfected cells. The activity (designated BBV polymerase) sedimented at 20,000 × g, indicating an association with particulate material. It was solubilized from the pellet by sonication in a magnesium-deficient buffer. Differential centrifugation resulted in a 43-fold purification with 84% recovery of polymerase activity. The effects of divalent and monovalent cations, time, temperature, and pH on the activity of the partially purified polymerase were examined. RNA synthesis was not stimulated by the addition of exogenous BBV RNA, suggesting that an enzyme-template complex existed. Analysis of the RNA products of the RNA polymerase reaction indicated that full-length “negative” strand BBV RNAs were synthesized. 相似文献
17.
Jamison RM 《Journal of virology》1969,4(6):904-906
Purified preparations of echovirus 22 were examined in the electron microscope. The virus was found to possess 32 capsomers arranged at the vertices of either a pentakis dodecahedron or a rhombic triacontahedron. The size of the virions ranges from 22 × 10−3 to 32 × 10−3 μm with a mean of 27 × 10−3 μm and a mode of 28 × 10−3 μm. 相似文献
18.
Aalto AP Sarin LP van Dijk AA Saarma M Poranen MM Arumäe U Bamford DH 《RNA (New York, N.Y.)》2007,13(3):422-429
The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons. 相似文献
19.
Hans Verkerke Caitlin Naylor Lennart Zabeau Jan Tavernier William A. Petri Jr Chelsea Marie 《PloS one》2014,9(4)
Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: ka 1.76×106±0.193×106 M−1 s−1, kd 1.21×10−4±0.707×10−4 s−1, KD 6.47×10−11±3.30×10−11 M; Q223R: ka 1.75×106±0.0245×106 M−1 s−1, kd 1.47×10−4±0.0505×10−4 s−1, KD 8.43×10−11±0.407×10−11 M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies. 相似文献
20.
Melanson DL 《Plant physiology》1978,62(5):761-765