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Anti-viral RNAi and its suppression in plants.Ciomperlik, Jessica J. 16 January 2010 (has links)
As a defense against viral infection, plants are thought to use RNA-induced
silencing complexes (RISCs) to target and cleave viral RNA. To counteract this, some
viruses have evolved proteins to inhibit RISC-mediated activity, thus ensuring
continued virulence. This research focused on the study and analysis of the anti-viral
RNAi response to various viruses in plants to gain an understanding of how the plant
defense operates on the molecular and biochemical levels. Nicotiana benthamiana plants
were infected with Tomato bushy stunt virus (TBSV) and Tobacco rattle virus (TRV).
These plants were subjected to column chromatography methods, and fractions
contained a virus-specific ribonuclease activity, co-eluting with small interfering RNAs
(siRNA), that was shown to be sensitive to inhibition with EDTA and enhanced by the
addition of divalent metal cations. This ribonuclease activity co-purified with proteins
that contained a domain from the hallmark RISC protein Argonaute family. To further
study host responses to viral infection, monocots were infected with Panicum mosaic
virus (PMV) and satellite panicum mosaic virus (SPMV) and also were subjected to
column chromatography following infection. Preliminary studies show that fractions
contained ribonuclease activity as well as siRNAs and proteins containing an Argonaute
domain. Additionally, silencing suppressors have been directly implicated in interfering
with RNAi pathways in plants. Studies involving Agrobacterium- and virus-vectored
cDNA to express green fluorescent protein (GFP) were used to establish that cointroduced
suppressors of RNAi can extend the production of a foreign protein for
enhancement of biotechnological applications. It was found that the hordeivirus protein
?b contributes to enhancement of expression for the foreign protein GFP early in the infection, while the potyvirus protein HcPro and tombusvirus protein P19 enhance and
extend protein production later in the infection.
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Elucidating the Functions of Proteins Up-regulated During Diapause in Artemia franciscana Using RNAiKing, Allison M. 08 February 2013 (has links)
Diapause embryos of the animal extremophile Artemia franciscana, a crustacean, are metabolically dormant and exceptionally tolerant to stressors such as heat and anoxia, characteristics thought to depend on the protective activity of molecular chaperones. RNAi methodology was developed and used to knock down individual molecular chaperones which are normally up-regulated during Artemia diapause. DsRNA and siRNAs injected into females were effective in knocking down proteins in embryos into the fifth release. Five proteins were knocked down including the ?-crystallin related small heat shock proteins (sHsps) p26, ArHsp21 and ArHsp22, artemin, a species-specific chaperone as well as p8 a transcription co-factor. The individual sHsps, artemin and p8 exhibited different roles during cyst development and diapause with only some of these proteins contributing to stress tolerance. p26, for example, enhances stress resistance in Artemia embryos, facilitates embryo development and prevents diapause termination indicated by spontaneous hatching. ArHsp21, another ?-crystallin type small heat shock protein contributes only slightly to freezing and desiccation stress and is not protective during heat stress. DsRNA specific to ArHsp22 is lethal to both male and female adults. Artemin contributes to stress tolerance but to a lesser extent than p26. Artemin also extended the period of time over which cysts were released. Cysts that did not contain p8 were also less stress resistant than those that did contain p8 and hatched upon release 10% for the time, suggesting an important role in diapause. By revealing separate and novel roles for molecular chaperones this work contributes substantially to our understanding of diapause, an important, phylogenetically widespread developmental process.
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Identification of non-essential host genes required for PrP106-126 mediated neurotoxicityStobart, Michael 31 August 2011 (has links)
Prion diseases are invariably fatal proteinaceous neurodegenerative disorders of the central nervous system. The infectious agent is the host encoded prion protein which has undergone a post-translational refolding from a predominantly alpha-helical to highly beta-sheet containing structure. The mechanism of prion-induced neurotoxicity remains elusive in large part due to the absence of a sufficiently neurotoxic cell culture assay. A modern technique for identifying previously unrecognized mediators of a biological pathway is to screen a commercially available library of gene silencing molecules targeting all known open reading frames. Synthetic gene silencing molecules, such as short hairpin RNA (shRNA), employ the endogenous gene silencing pathway to inhibit protein synthesis. To date, no publication has described the implementation of a large-scale library to screen for genetic mediators of prion neurotoxicity.
This project was aimed at developing a cell culture model of acute prion neurotoxicity and screening a library of shRNA molecules in order to identify previously unrecognized gene targets essential to prion-induced neurotoxicity. Using a fragment of the prion protein (PrP106-126 peptide) to mimic prion neurotoxicity, human neuroblastoma cells transduced with a retroviral shRNA library were screened for resistance. Involvement of a subset of library identified gene targets in prion disease was assessed in vivo by quantitative real-time PCR (qPCR) analysis. Validation of the protection conferred by reducing expression of a gene target of interest was accomplished using individual lentiviral vectors expressing shRNA.
Of the approximately 54,000 shRNA sequences screened, 80 different shRNA sequences recovered from neurotoxic prion peptide-resistant cells were considered to be of interest. Of these, 49 corresponding gene targets were assessed in vivo by qPCR with the majority demonstrating significant differential expression in brains of prion infected mice. Validation of the protection conferred from knockdown of two identified genes, abcb4 and ube2cbp, was completed. Knockdown of either gene imparted significant protection against prion-induced neurotoxicity, with qPCR analysis confirming significantly reduced mRNA transcript levels. Overall, the validity of the novel assay system developed has been demonstrated, and the first comprehensive list of gene candidates involved in mediating acute prion neurotoxicity has been determined.
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Identification of non-essential host genes required for PrP106-126 mediated neurotoxicityStobart, Michael 31 August 2011 (has links)
Prion diseases are invariably fatal proteinaceous neurodegenerative disorders of the central nervous system. The infectious agent is the host encoded prion protein which has undergone a post-translational refolding from a predominantly alpha-helical to highly beta-sheet containing structure. The mechanism of prion-induced neurotoxicity remains elusive in large part due to the absence of a sufficiently neurotoxic cell culture assay. A modern technique for identifying previously unrecognized mediators of a biological pathway is to screen a commercially available library of gene silencing molecules targeting all known open reading frames. Synthetic gene silencing molecules, such as short hairpin RNA (shRNA), employ the endogenous gene silencing pathway to inhibit protein synthesis. To date, no publication has described the implementation of a large-scale library to screen for genetic mediators of prion neurotoxicity.
This project was aimed at developing a cell culture model of acute prion neurotoxicity and screening a library of shRNA molecules in order to identify previously unrecognized gene targets essential to prion-induced neurotoxicity. Using a fragment of the prion protein (PrP106-126 peptide) to mimic prion neurotoxicity, human neuroblastoma cells transduced with a retroviral shRNA library were screened for resistance. Involvement of a subset of library identified gene targets in prion disease was assessed in vivo by quantitative real-time PCR (qPCR) analysis. Validation of the protection conferred by reducing expression of a gene target of interest was accomplished using individual lentiviral vectors expressing shRNA.
Of the approximately 54,000 shRNA sequences screened, 80 different shRNA sequences recovered from neurotoxic prion peptide-resistant cells were considered to be of interest. Of these, 49 corresponding gene targets were assessed in vivo by qPCR with the majority demonstrating significant differential expression in brains of prion infected mice. Validation of the protection conferred from knockdown of two identified genes, abcb4 and ube2cbp, was completed. Knockdown of either gene imparted significant protection against prion-induced neurotoxicity, with qPCR analysis confirming significantly reduced mRNA transcript levels. Overall, the validity of the novel assay system developed has been demonstrated, and the first comprehensive list of gene candidates involved in mediating acute prion neurotoxicity has been determined.
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Biological Confinement of Zebrafish Using RNAiBoratynska, Susan January 2015 (has links)
The increasing demand for fish in the food industry has resulted in the extensive overfishing of wild fisheries. In efforts to alleviate the demand from the food industry, genetically modified (GM) fish were developed possessing traits such as larger mass, faster growth, and increased resistance to disease. However, the greater fitness advantage of GM fish presents potential risks for wild type populations in the event of release or escape from a confined fish facility. In addition to physical barriers, it is critical to develop a genetic mechanism in order to ensure that the spread of GM transgene to the natural populations does not occur.
RNA interference (RNAi) is an endogenous mechanism used to regulate gene expression by destroying targeted mRNA molecules. Manipulation of this biological process has been successfully utilized to knockdown specific genes through the introduction of synthetic transgenes in organisms such as C. elegans, M. musculus, and D. melanogaster. Although the use of RNAi as a biological tool is still relatively new in zebrafish, recent work has explored elements of this mechanism allowing for greater knockdown efficiencies.
The deadend (dnd) gene is required for primordial germ cell (PGC) development and survival. Previous studies have shown that zebrafish dnd knockouts develop into sterile adults without disrupting somatic development. In efforts to induce sterility in zebrafish, short hairpin RNA (shRNA) constructs targeting dnd were designed to exploit the endogenous RNAi pathway. Upon qualitative analysis in transient and transgenic zebrafish subjected to the synthetic RNAi construct, a reduction in the germ cell population at early stages of development was observed. However, quantification of dnd mRNA in fish from the same time points did not show significant changes in expression levels compared to their wildtype counterparts. Adult fish subjected to the transgene construct produced viable gametes.
The use of RNAi as a tool for bioconfinement relies on sterility among all individuals subjected to the shRNA bearing transgene. Based on the results obtained, the verdict is still unclear as to whether shRNA is a viable mechanism for large scale bioconfinement.
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An Approach to Genetic Silencing of Ricin in Castor (Ricinus Communis L.)Barnes, Daniel Joseph 13 December 2014 (has links)
Castor (Ricinus communis L.) is a high-yielding oilseed crop native to tropical Africa. The seed contains ~60% oil by weight, yielding approximately 1,200 kg of oil per hectare. The oil is composed of ~90% ricinoleic acid, a unique hydroxylatty acid. Its unique composition provides castor oil with distinctive characteristics important for industrial use. Unfortunately, this valuable oilseed has not been widely cultivated in the United States since 1972, due in part to the presence of ricin in the seed. Ricin is a highly toxic lectin found in the endosperm of mature castor seed. This project sought to silence ricin production through the introduction of an RNAi element into the castor genome. The RNAi vector (pC1-RKO) containing a segment of ricin mRNA and its inverted repeat separated by a chalcone synthase A intron from pFGC5941 enclosed in a pCambia1301 backbone was created, verified via sequencing, and transformed into Agrobacterium tumefaciens for castor transformation. Fungal contamination was a serious concern; successful disinfestation used a 10-minute wash with 0.1% mercuric chloride (w/v). Media supplemented with 6-benzylaminopurine generated healthier shoots from embryo axes dissected from mature seed compared to thidiazuron-treated mesocotyls dissected from mature seed. Short treatments of thidiazuron on 6-benzylaminopurine initiated shoot cultures showed greater shoot proliferation on embryo axes dissected from mature seed. Rooting occurred with incubation on half-strength medium containing naphthaleneacetic acid or indole-3-butyric acid; however, naphthaleneacetic acid produced hardier roots which better survived acclimatization. Inoculation of embryo axis explants after 2 days pre-culture improved survivability. Likewise, transformations using A. tumefaciens cultures of 0.5 O.D.600 and lower did not lead to downstream bacterial contamination. The pCambia1304 vector was used as a test plasmid for refinement of the transformation protocol. Of the 870 pCambia1304 inoculation explants, 2 survived hygromycin screening and showed gusA activity. Of the 2,500 pC1-RKO inoculated explants, 6 survived hygromycin selection and rooted. Further analysis via PCR, end-point RT-PCR, and Western and dot-blotting showed these to be non-transformed and ricin content unaffected.
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Developing RNAi therapy For DYT1 dystoniaMartin, Janine Nicole 01 May 2011 (has links)
DYT1 dystonia is an early onset central nervous system-based movement disorder characterized by uncontrolled sustained muscle contractions that can lead to debilitating abnormal postures. Though a genetic mutation in the gene TOR1A is responsible for most DYT1 cases, the low penetrance of the disease implicates additional genetic and environmental modifiers. Current therapeutic options for DYT1 dystonia are limited to symptomatic treatments with variable effectiveness. Currently, the underlying pathogenesis of this disease and the role of torsinA (torA), the protein product of TOR1A, in the development of this disease have yet to be established.
In the first part of this thesis we aimed to further understand the effects of the TOR1A mutation at the molecular, cellular and organismal level in order to identify disease associated biomarkers that can be later used to measure the effectiveness of novel therapies. We found that expression of mutant torsinA (torA(ÄE)) in a cellular and an animal model of DYT1 had no significant effect on global transcription, despite its interaction with nuclear envelope proteins. Recent research has unearthed a role for microRNAs (miRNAs) in neuronal development and maturation. Consequently we explored whether torA(ÄE) expression in murine neural tissue was associated with changes in miRNA expression in young DYT1 knockin (KI) mice. Since the primary sight of dysfunction is still being debated, we profiled miRNA expression of the two strongest candidates, the striatum and cerebellum, both of which have well established roles in the control and coordination of muscle movements. We have identified several microRNAs that were uniquely altered in either the striatum or cerebellum and further research will be conducted to determine their usability as disease biomarkers. Finally, we were unable to identify motor phenotypes in either a DYT1 (KI) mice or a novel DYT1 transgenic model in open field, rotarod or staircase forepaw reaching tests.
In the second part of this thesis we aimed to develop and evaluate the safety and efficacy of viral therapeutic RNAi constructs for in DYT1 murine models. DYT1 is an ideal candidate for this form of therapy due to its dominant inheritance, common mutation and potentially reversible phenotype. Virally delivered short-hairpin RNAs (shRNA) designed to knockdown torA(ÄE) in either an allele-specific or nonallele-specific manner were injected into the striatum of DYT1 transgenic or KI mice respectively. Unexpectedly, we found widespread lethal toxicity and behavioral abnormalities in mice injected with either therapeutic or control shRNAs that weren't observed in mice injected with no shRNAs. Further studies found that regions where toxic shRNAs were expressed corresponded with neuronal loss and glial activation. Finally, we found evidence that the severity of toxicity was influenced in part by the genetic background of the mice.
In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.
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The IgSF protein MDGA1 regulates morphology during a defined stage of placode-derived neuron maturation in developing chick cranial sensory gangliaSmith, Alexandra January 2015 (has links)
The developing distal cranial sensory ganglia of the chick present an interesting and tractable model for the study of general processes of neural development. While the early stages of placodal neurogenesis, including induction of the placodes and initiation of the neurogenic programme, have been extensively studied, little is known about the molecular mechanisms that regulate migration of placode-derived neuroblasts and their aggregation to form ganglia. These questions have been addressed in the context of the trigeminal ganglion, however it remains unclear whether these principles apply to the epibranchial ganglia, on which the work presented here is focussed. Molecules potentially involved in controlling placodal neuroblast migration in the epibranchial ganglia were identified through a comparative microarray screen carried out in the Begbie lab. A list of candidate genes implicated in a variety of different cellular was validated by determining expression patterns in the region of the epibranchial CSG by in situ hybridisation. These expression patterns showed that different genes were expressed by different populations within the migratory stream. This question was further addressed through the detailed analysis of the expression patterns of a panel of neuronal and neurogenic markers, leading to the finding that placodal neuroblasts appear to sequentially upregulate different groups of genes as they migrate away from the placode. Neuroblasts within the migratory stream can further be subdivided according to cell morphology, which was assessed through high resolution imaging of GFP-labelled placodal cells. Multipolar and bipolar cells were concentrated around two different regions of the migratory stream with multipolar cells localised near the placode and bipolar cells localised closer to the neural tube. Together these findings support the hypothesis that placodal neuroblasts mature as they migrate towards the site of ganglion aggregation. With this detailed description of the system in mind, the question of molecular control was addressed through the functional characterisation of a candidate gene identified in the original microarray screen. MDGA1, a GPI-anchored IgSF molecule that has been implicated in controlling radial migration of cortical neurons, was specifically expressed in the chick CSG at the relevant stages. RNAi-mediated knockdown and overexpression were used to test the function of MDGA1 in migrating placodal neuroblasts. These experiments showed that MDGA1 negatively regulates the formation and extension of neuronal projections in bipolar neuroblasts. With the mechanisms of MDGA1 function relying entirely on protein-protein interactions at the cell-surface, we then set out to identify and characterise potential MDGA1 binding partners. SPR binding experiments carried out in collaboration with the Aricescu lab revealed that MDGA1 interacts with the Neuroligin family of synaptic proteins. Recent evidence has shown that MDGA1 interacts in cis with NLGN2 in rat hippocampal neurons where it disrupts its interaction in trans with Neurexin1. Neuroligins and Neurexins function to stabilise dendritic filopodia by creating trans-synaptic adhesions and recruiting the synaptic apparatus. Having determined that both NLGN2 and NRXN1 are expressed in placode-derived neuroblasts of the CSG, we propose that these molecules play a role in the stabilisation and extension of neuronal projections in this system and that this function in modulated by MDGA1 function.
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Armet transcript knockdown in Tribolium castaneumBechard, Jarrod January 1900 (has links)
Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet has been found in mammalian systems to be a bi-functional protein that is secreted extracellularly and is also found in the endoplasmic reticulum. It has been shown to be a neurotrophic factor and also a member of the unfolded protein response. Transcript knockdown of Armet via RNA interference in late instar larvae of Tribolium castaneum produces a fatal phenotype during eclosion from pupa to adult. Initial observations of pupae cuticle indicate disorganization of cuticles in insects with the Armet transcript knocked down. Here I expand studies on the effects of dsArmet RNA injection; both in a wild type strain and a fluorescent strain of Tribolium, and discuss possible mechanisms for the fatal phenotype.
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Evolution of Dipteran Argonaute genes through duplication, selection and functional specialisationLewis, Samuel Howard January 2016 (has links)
The RNA interference (RNAi) mechanism is a conserved system of nucleic acid manipulation, based on the interaction between small RNA guide molecules and Argonaute effector proteins. RNAi pathways are found in the vast majority of eukaryotes, and have diversified into a broad array of functions including gene regulation, antiviral immunity and transposable element (TE) suppression. Many of these functional innovations coincide with duplication of Argonaute genes, suggesting that gene duplication may be a key driving force in the diversification of RNAi. However, few studies have explicitly investigated Argonaute evolution after duplication. In this thesis, I focused on the impact of gene duplication on the evolution of Argonaute genes. Argonaute genes in different species exhibit a broad array of functions; however, most of our knowledge of Argonaute function in the arthropods is based on studies in D. melanogaster. To compare the rate of duplication and its evolutionary effect between different Argonaute subclades, I quantified gene turnover rates and evolutionary rate change in Argonaute genes from 86 Dipteran species (Chapter 2). I find that duplication rate varies widely between subclades and lineages, and that duplication drives an increase in evolutionary rate, suggesting that functional divergence after Argonaute duplication is prevalent throughout the Diptera. In the obscura group of Drosophila I identified a series of recent duplications of Argonaute2 (Ago2), which has antiviral and anti-TE functions in D. melanogaster. To quantify the extent of functional divergence between these paralogues, I measured the expression of paralogues from three species (D. subobscura, D. obscura and D. pseudoobscura), in different tissues and under viral challenge (Chapter 3). I find that the majority of Ago2 paralogues have specialised to a derived testis-specific role, potentially to suppress TE activity or meiotic drive. While CRISPR-Cas9 mediated knockout of these genes ultimately proved unsuccessful (Chapter 5), the selective importance of their derived function is suggested by its multiple independent origins. Functional novelty, as appears to have evolved in the obscura group Ago2 paralogues, is often driven by strong selection. To quantify the evolutionary rate and positive selection on these paralogues, I gathered intraspecies polymorphism data for all paralogues in D. subobscura, D. obscura and D. pseudoobscura, combining this with publicly-available population genomic data for D. pseudoobscura (Chapter 4). I find that the majority of paralogues in all species have extremely low diversity, indicative of recent selection, and identify recent selective sweeps on three paralogues in D. pseudoobscura. This suggests that the majority of Ago2 paralogues in the obscura group are evolving under strong positive selection. In this thesis I have aimed to quantify the effect of gene duplication on Argonaute evolution. I find that Argonaute genes duplicate frequently in some lineages, resulting in the evolution of derived functions that may be driven by positive selection. This suggests that functional diversification is prevalent in eukaryotic RNAi, and is likely to coincide with expansion of the Argonaute gene family.
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