The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
David C Baulcombe - One of the best experts on this subject based on the ideXlab platform.
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rna dna interactions and dna methylation in post transcriptional Gene Silencing
The Plant Cell, 1999Co-Authors: Louise Jones, Andrew Hamilton, Olivier Voinnet, Carole L Thomas, Andrew J Maule, David C BaulcombeAbstract:Post-transcriptional Gene Silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transGene encoding the green fluorescent protein (GFP). Gene Silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transGene. In each instance, homology-based, RNA-directed methylation was associated with Silencing. When the GFP-transcribed region was targeted, PTGS affected both transGene and viral RNA levels. When methylation was targeted to a promoter region, transGene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the Gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X-rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transGene associated with systemic spread of a Gene-Silencing signal from the infiltrated part of the plant. This transGene methylation was not affected when systemic PTGS was blocked by suppressors of Silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epiGenetic model of PTGS in which transGene methylation is associated with an RNA-DNA interaction that ensures that PTGS is maintained.
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suppression of Gene Silencing a General strategy used by diverse dna and rna viruses of plants
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Olivier Voinnet, Yvonne Pinto, David C BaulcombeAbstract:In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional Gene Silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transGene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of Gene Silencing have distinct modes of action, are targeted against distinct components of the host Gene-Silencing machinery, and that there is dynamic evolution of the host and viral components associated with the Gene-Silencing mechanism.
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Gene Silencing without dna rna mediated cross protection between viruses
The Plant Cell, 1999Co-Authors: Frank Ratcliff, Stuart A. Macfarlane, David C BaulcombeAbstract:Previously, it was shown that the upper leaves of plants infected with nepoviruses and caulimoviruses are symptom free and contain reduced levels of virus. These leaves are said to be recovered. Recovery is associated with RNA-mediated cross-protection against secondary virus infection. Here, by analyzing plants infected with viruses that are quite distinct from the nepovirus or caulimovirus groups, we demonstrate that this RNA-mediated defense is a General response to virus infection. Upon infection with a tobravirus, plants exhibited RNA-mediated cross-protection and recovery, as occurs in nepovirus-infected plants. However, upon infection with a potexvirus, plants exhibited RNA-mediated cross-protection without recovery. In both instances, a transient Gene expression assay showed that RNA-mediated cross-protection was functionally equivalent to post-transcriptional Gene Silencing. Combined, these data provide direct evidence that post-transcriptional Gene Silencing of nuclear Genes is a manifestation of a natural defense mechanism that is induced by a wide range of viruses.
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fast forward Genetics based on virus induced Gene Silencing
Current Opinion in Plant Biology, 1999Co-Authors: David C BaulcombeAbstract:Gene expression in plants can be suppressed in a sequence-specific manner by infection with virus vectors carrying fragments of host Genes. Recent developments have revealed that the mechanism of this Gene Silencing is based on an RNA-mediated defence against viruses. It has also emerged that a related mechanism is involved in the post-transcriptional Silencing that accounts for between line variation in transGene expression and cosuppresion of transGenes and endogenous Genes. The technology of virus-induced Gene Silencing is being refined and adapted as a high throughput procedure for functional genomics in plants.
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systemic signalling in Gene Silencing
Nature, 1997Co-Authors: Olivier Voinnet, David C BaulcombeAbstract:Gene Silencing in plants is a Genetic control mechanism implicated in virus resistance1,2, genome maintenance3 and developmental control4. We describe here our recent discovery that there is a systemic signal that can mediate Gene Silencing. From the Gene-specificity of the systemic Silencing, we infer that the signal molecule is likely to be a nucleic acid.
Giuseppe Macino - One of the best experts on this subject based on the ideXlab platform.
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redundancy of the two dicer Genes in transGene induced posttranscriptional Gene Silencing in neurospora crassa
Molecular and Cellular Biology, 2004Co-Authors: Caterina Catalanotto, Giuseppe Macino, Massimiliano Pallotta, Paul Refalo, Matthew S Sachs, Laurence Vayssie, Carlo CogoniAbstract:RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent Gene Silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transGenes induces sequence-specific mRNA degradation. These phenomena share a common Genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both Silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like Genes by mutation in the fungus Neurospora crassa eliminates transGene-induced Gene Silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like Genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in Gene Silencing induced by transGenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of Silencing.
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involvement of small rnas and role of the qde Genes in the Gene Silencing pathway in neurospora
Genes & Development, 2002Co-Authors: Caterina Catalanotto, Giuseppe Macino, Gianluca Azzalin, Carlo CogoniAbstract:Small RNA molecules have been found to be specifically associated with posttranscriptional Gene Silencing (PTGS) in both plants and animals. Here, we find that small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 Genes previously shown to be essential for Gene Silencing, but does not depend on a functional qde-2, indicating that this Gene is involved in a downstream step of the Gene Silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules, suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation.
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post transcriptional Gene Silencing across kingdoms
Current Opinion in Genetics & Development, 2000Co-Authors: Carlo Cogoni, Giuseppe MacinoAbstract:Post-transcriptional Gene Silencing (PTGS) as a consequence of the introduction of either transGenes or double-stranded RNA molecules has been found to occur in a number of species. In the past year, studies in different systems have greatly enhanced our understanding of the molecular mechanisms of these phenomena. The ubiquitous presence of PTGS in both the plant and animal kingdoms and the finding of common Genetic mechanisms suggest that PTGS is a universal Gene-regulation system fundamental in biological processes such as protection against viruses and transposons.
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Gene Silencing in worms and fungi
Nature, 2000Co-Authors: Caterina Catalanotto, Giuseppe Macino, Gianluca Azzalin, Carlo CogoniAbstract:The introduction into cells of foreign nucleic acid molecules can induce sequence-specific Gene Silencing in some organisms. Here we show that two distantly related organisms, the nematode Caenorhabditis elegans and the fungus Neurospora crassa, which have quite different mechanisms of Gene Silencing, both use a similar protein to control the process. This suggests that they may share an ancestral mechanism that evolved to protect the genome against invasion by foreign DNA.
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posttranscriptional Gene Silencing in neurospora by a recq dna helicase
Science, 1999Co-Authors: Carlo Cogoni, Giuseppe MacinoAbstract:The phenomenon of posttranscriptional Gene Silencing (PTGS), which occurs when a transGene is introduced into a cell, is poorly understood. Here, the qde-3 Gene, which is required for the activation and maintenance of Gene Silencing in the fungus Neurospora crassa, was isolated. Sequence analysis revealed that the qde-3 Gene belongs to the RecQ DNA helicase family. The QDE3 protein may function in the DNA-DNA interaction between introduced transGenes or with an endogenous Gene required for Gene-Silencing activation. In animals, Genes that are homologous to RecQ protein, such as the human Genes for Bloom's syndrome and Werner's syndrome, may also function in PTGS.
Carlo Cogoni - One of the best experts on this subject based on the ideXlab platform.
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redundancy of the two dicer Genes in transGene induced posttranscriptional Gene Silencing in neurospora crassa
Molecular and Cellular Biology, 2004Co-Authors: Caterina Catalanotto, Giuseppe Macino, Massimiliano Pallotta, Paul Refalo, Matthew S Sachs, Laurence Vayssie, Carlo CogoniAbstract:RNA interference (RNAi) in animals, cosuppression in plants, and quelling in fungi are homology-dependent Gene Silencing mechanisms in which the introduction of either double-stranded RNA (dsRNA) or transGenes induces sequence-specific mRNA degradation. These phenomena share a common Genetic and mechanistic basis. The accumulation of short interfering RNA (siRNA) molecules that guide sequence-specific mRNA degradation is a common feature in both Silencing mechanisms, as is the component of the RNase complex involved in mRNA cleavage. During RNAi in animal cells, dsRNA is processed into siRNA by an RNase III enzyme called Dicer. Here we show that elimination of the activity of two Dicer-like Genes by mutation in the fungus Neurospora crassa eliminates transGene-induced Gene Silencing (quelling) and the processing of dsRNA to an siRNA form. The two Dicer-like Genes appear redundant because single mutants are quelling proficient. This first demonstration of the involvement of Dicer in Gene Silencing induced by transGenes supports a model by which a dsRNA produced by the activity of cellular RNA-dependent RNA polymerases on transgenic transcripts is an essential intermediate of Silencing.
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involvement of small rnas and role of the qde Genes in the Gene Silencing pathway in neurospora
Genes & Development, 2002Co-Authors: Caterina Catalanotto, Giuseppe Macino, Gianluca Azzalin, Carlo CogoniAbstract:Small RNA molecules have been found to be specifically associated with posttranscriptional Gene Silencing (PTGS) in both plants and animals. Here, we find that small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 Genes previously shown to be essential for Gene Silencing, but does not depend on a functional qde-2, indicating that this Gene is involved in a downstream step of the Gene Silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules, suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation.
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post transcriptional Gene Silencing across kingdoms
Current Opinion in Genetics & Development, 2000Co-Authors: Carlo Cogoni, Giuseppe MacinoAbstract:Post-transcriptional Gene Silencing (PTGS) as a consequence of the introduction of either transGenes or double-stranded RNA molecules has been found to occur in a number of species. In the past year, studies in different systems have greatly enhanced our understanding of the molecular mechanisms of these phenomena. The ubiquitous presence of PTGS in both the plant and animal kingdoms and the finding of common Genetic mechanisms suggest that PTGS is a universal Gene-regulation system fundamental in biological processes such as protection against viruses and transposons.
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Gene Silencing in worms and fungi
Nature, 2000Co-Authors: Caterina Catalanotto, Giuseppe Macino, Gianluca Azzalin, Carlo CogoniAbstract:The introduction into cells of foreign nucleic acid molecules can induce sequence-specific Gene Silencing in some organisms. Here we show that two distantly related organisms, the nematode Caenorhabditis elegans and the fungus Neurospora crassa, which have quite different mechanisms of Gene Silencing, both use a similar protein to control the process. This suggests that they may share an ancestral mechanism that evolved to protect the genome against invasion by foreign DNA.
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posttranscriptional Gene Silencing in neurospora by a recq dna helicase
Science, 1999Co-Authors: Carlo Cogoni, Giuseppe MacinoAbstract:The phenomenon of posttranscriptional Gene Silencing (PTGS), which occurs when a transGene is introduced into a cell, is poorly understood. Here, the qde-3 Gene, which is required for the activation and maintenance of Gene Silencing in the fungus Neurospora crassa, was isolated. Sequence analysis revealed that the qde-3 Gene belongs to the RecQ DNA helicase family. The QDE3 protein may function in the DNA-DNA interaction between introduced transGenes or with an endogenous Gene required for Gene-Silencing activation. In animals, Genes that are homologous to RecQ protein, such as the human Genes for Bloom's syndrome and Werner's syndrome, may also function in PTGS.
Jorgen Kjems - One of the best experts on this subject based on the ideXlab platform.
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the influence of polymeric properties on chitosan sirna nanoparticle formulation and Gene Silencing
Biomaterials, 2007Co-Authors: Xiudong Liu, Kenneth A Howard, Mingdong Dong, Morten Ostergaard Andersen, Ulrik Lytt Rahbek, Mads Groenvold Johnsen, Ole C Hansen, Flemming Besenbacher, Jorgen KjemsAbstract:Abstract We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for Gene Silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro Gene Silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight ( M w ) and degree of deacetylation (DD). High M w and DD chitosan resulted in the formation of discrete stable nanoparticles ∼200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low M w (∼10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher M w (64.8–170 kDa) and DD (∼80%) showed greater Gene Silencing ranging between 45% and 65%. The highest Gene Silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher M w (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of ∼200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of Gene Silencing using chitosan/siRNA nanoparticles.
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the influence of polymeric properties on chitosan sirna nanoparticle formulation and Gene Silencing
Biomaterials, 2007Co-Authors: Xiudong Liu, Kenneth A Howard, Mingdong Dong, Morten Ostergaard Andersen, Ulrik Lytt Rahbek, Mads Groenvold Johnsen, Ole C Hansen, Flemming Besenbacher, Jorgen KjemsAbstract:We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for Gene Silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro Gene Silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight (Mw) and degree of deacetylation (DD). High Mw and DD chitosan resulted in the formation of discrete stable nanoparticles approximately 200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low Mw (approximately 10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher Mw (64.8-170 kDa) and DD (approximately 80%) showed greater Gene Silencing ranging between 45% and 65%. The highest Gene Silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher Mw (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of approximately 200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of Gene Silencing using chitosan/siRNA nanoparticles.
Kirankumar S Mysore - One of the best experts on this subject based on the ideXlab platform.
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tobacco rattle virus based virus induced Gene Silencing in nicotiana benthamiana
Nature Protocols, 2014Co-Authors: Muthappa Senthilkumar, Kirankumar S MysoreAbstract:Tobacco rattle virus (TRV)-based virus-induced Gene Silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant Gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional Gene Silencing (PTGS), the TRV2 genome is Genetically modified to carry a fragment of the target Gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target Gene Silencing occurs in the newly developed noninoculated leaves within 2-3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other Gene Silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.
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A systematic study to determine the extent of Gene Silencing in Nicotiana benthamiana and other Solanaceae species when heterologous Gene sequences are used for virus-induced Gene Silencing.
The New phytologist, 2007Co-Authors: Muthappa Senthil-kumar, Li Kang, Ramanna Hema, Ajith Anand, Makarla Udayakumar, Kirankumar S MysoreAbstract:Summary • Virus-induced Gene Silencing (VIGS) is a rapid and robust method for determining and studying the function of plant Genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous Genes and their endogenous orthologs for Silencing. • Two approaches were used. First, the extent of phytoene desaturase (PDS) Gene Silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS Gene in N. benthamiana. • The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous Gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between Gene Silencing efficiency and percentage homology of the heterologous Gene sequence with the endogenous Gene sequence. • It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous Gene sequences is not absolutely required for Gene Silencing.
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computational estimation and experimental verification of off target Silencing during posttranscriptional Gene Silencing in plants
Plant Physiology, 2006Co-Authors: Ping Xu, Yuanji Zhang, Marilyn J. Roossinck, Li Kang, Kirankumar S MysoreAbstract:Successful application of posttranscriptional Gene Silencing (PTGS) for Gene function study in both plants and animals depends on high target specificity and Silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50% to 70% of Gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available Web-based computational tool called siRNA Scan to identify potential off-targets during PTGS. Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative reverse transcription-PCR. Up to 50% of the predicted off-target Genes tested in plants were actually silenced when tested experimentally. Our results suggest that a high risk of off-target Gene Silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target Gene Silencing in both plants and animals.