Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...

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Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.

The concentration of RNA (Schneider procedure, 1945) and the dry weight of the developing stages of the brine shrimp Artemia salina were measured during the first 63 days of growth of the organism in the laboratory; RNA and the dry weight of starved stage V Calanus plumchrus were followed for 20 days. The same techniques were applied to the developmental stages of several species of zooplankton obtained during the course of one year from Saanich Inlet, Vancouver Island, B.C. The data collected from the laboratory and the field were used to test the validity of the RNA/Growth relationship proposed by Sutcliffe (1965) as a means to predict the rate of increase in dry weight (growth rate) of such organisms. Errors in the estimation of RNA occured. These resulted from chromogenic materials other than the pentose sugars ribose and desoxyribose being present in the hot PCA extracts of zooplanktonic organisms. Detection of the interfering chromogens was possible by examining the absorption spectra of the hot PCA extracts treated with orcinol between the wave-lengths 550 to 700 mμ. An attempt has been made to minimize such errors by calculating the amount of RNA from the difference in absorbance recorded at 670 mμ minus that at 615 mμ. Correlation of growth predicted from RNA values and the observed increase in dry weight of the organisms was greatest during their most rapid period of development. During other periods of development, RNA values were generally in excess of requirements for the observed increase in dry weight and may represent metabolic processes other than those associated with this increase. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Ribonucleic acid (RNA) from calf spleen tissue was isolated and purified by a modified Kirby phenol procedure. The absorption maximum of the isolate occurred at 260 mµ indicating the presence of nucleic acids and the absorption minima recorded at 230 mµ and 280 mµ indicated the absence of peptides and proteins. Colorimetric analyses indicated the presence of RNA and the absence of peptide, protein, DNA and carbohydrate contamination. Chromatographic analysis indicated the absence of carbohydrate contamination only after the purification with 2-methoxyethanol. The spleen RNA prepared by the phenol method was undegraded and demonstrated three distinct molecular species when analysed with the ultracentrifuge; a 27S fraction, an 18S fraction and an 8S fraction. Competent early gastrula ectoderm and embryos of Xenopus laevis exposed to undegraded spleen RNA demonstrated no tissue-specific induction. However, in both the in vitro and in vivo experimental series an enhancement of development was observed. A possible explanation of this phenomena was discussed. / Science, Faculty of / Zoology, Department of / Graduate

Frequent subgraph mining is a useful method for extracting biologically relevant patterns from a set of graphs or a single large graph. Here, the graph represents all possible RNA structures and interactions. Patterns that are significantly more frequent in this graph over a random graph are extracted. We hypothesize that these patterns are most likely to represent a biological mechanisms. The graph representation used is a directed dual graph, extended to handle intermolecular interactions. The graph is sampled for subgraphs, which are labeled using a canonical labeling method and counted. The resulting patterns are compared to those created from a randomized dataset and scored. The algorithm was applied to the mitochondrial genome of the kinetoplastid species Trypanosoma brucei. This species has a unique RNA editing mechanism that has been well studied, making it a good model organism to test RiboFSM. The most significant patterns contain two stem-loops, indicative of gRNA, and represent interactions of these structures with target mRNA.

The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein. / Land and Food Systems, Faculty of / Graduate

A biochemical approach was used to detect the appearance of the heavy chain of skeletal myosin (HCSM) and myosin mRNA during the early development of Xenopus laevis embryos. An antibody against the HCSM of adult X. laevis muscles was biochemically characterized and shown to be monospecific. This anti-myosin antibody reacted with embryonic polysomes synthesizing myosin and with tadpole tail myosin. This indicates that the myosins of adult muscles, early embryonic muscles and tadpole tails are sufficiently homologous to share some antigenic determinants. Polysomes from various stages of X. laevis embryogenesis were reacted with the anti-myosin antibody. Analysis of these reactions showed that myosin synthesis begins in stage 20 embryos, in which about 7 somites have segregated. The RNA from stage 12, stage 16/17 and stage 20 embryos was then analyzed for the presence of the heavy chain myosin mRNA in order to determine whether the synthesis of myosin is under translational or transcriptional control. Total RNA preparations from staged embryos were fractionated on oligo(dT)-cellulose columns and fractions that did and did not bind were translated in a wheat germ cell-free protein synthesizing system. The translational products were precipitated with the anti-myosin antibody and characterized biochemically. Myosin mRNA was detected by this method in stage 16/17 embryos. We conclude that somite segregation results in the appearance of new myosin mRNA molecules in X. laevis embryos. It seems likely, by all the evidence considered, that a large pool of untranslated myosin mRNA molecules is not responsible for muscle myosin synthesis. Therefore, the synthesis of certain proteins in early development is under transcriptional control. / Science, Faculty of / Zoology, Department of / Unknown

The purpose of this study was to characterize recombinant plasraids carrying Drosophila melanogaster tRNA genes. The two groups of recombinant plasmids studied were those which carried tRNA₄Val genes and those with tRNA₄,₇Ser genes. pDt92 and pDt120, both tRNA₄Val gene-carrying plasmids, were characterized initially to determine the number of inserts they contained and the size of the inserts. For plasmids containing multiple inserts, the insert which carried the tRNA₄Val gene was also determined. These characteristics were studied by HindIII digestion of the plasmid DNA, agarose gel electrophoresis, Southern transfer onto nitrocellulose filters and hybridization to [¹²⁵I] tRNA₄Val. It was found that both, pDt92 and pDt120 contained two inserts each of sizes 0.5kb and 1.7kb,and 2.0kb and 5.*fkb respectively, with the 0.5kb and 2.0kb fragments carrying the tRNA₄Val genes. pDt92 and pDt120 then were recloned so as to contain only the fragments which carried the tRNA₄Val genes, namely the 0.5kb and 2.0kb fragment respectively. pDt92RC and pDt120RC plus three other tRNA₄,₇Ser gene containing plasmids, pDt16, pDt17RC and pDt27RC were further characterized by the technique of in situ hybridization to study the organization of these tRNA genes on the Drosophila genome. Four of these plasmids with the exception of pDt17RC hybridized to only one site on the Drosophila chromosome. Both, pDt92RC and pDt120RC hybridized to the 90BC site on the right arm of the third chromosome; pDt16 and pDt27RC hybridized to the 12DE site on the first or the X chromosome. pDt17RC on the other hand hybridized predominantly to the 12DE site and to a lesser extent to 2}E (2L), 56D (2R), 62D (3L) and 64D (3L) sites. These in situ hybridization results when studied together with those reported by Dunn et al. (1979b) show that genes for a single species of tRNA are located on more than one site on the Drosophila genome. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Au sein des cellules, les voies enzymatiques sont souvent organisées spatialement sous forme de complexes, sur des structures protéiques ou dans des micro-compartiments. Cette organisation spatiale aide au déroulement optimal des réactions enzymatiques en limitant les pertes d’intermédiaires métaboliques, en isolant les voies de signalisations et en augmentant le rendement des réactions enzymatiques. Dans ce travail de thèse nous avons étudié la possibilité de créer des outils permettant de contrôler et optimiser de novo l’organisation spatiale de voies métaboliques in vivo.Nous avons dessiné et assemblé des structures d’ARN non codants utilisées comme support pour organiser le métabolisme bactérien. Ces ARN s’assemblent spontanément in vivo en des structures à une ou deux dimensions avec des sites distincts d’attachement protéique. Nous démontrons l’utilité de cette approche via l’optimisation d’une voie enzymatique de synthèse de biohydrogène et démocratisons l’utilisation de ces structures d’ARN en développant un protocole simplifié. Nous étendons cette étude à d’autres stratégies d’organisation, notamment via l’ingénierie des cellules spécialisées dans la fixation de l’azote atmosphérique de la cyanobactérie Anabaena PCC7120, les hétérocystes. Ce travail de thèse ouvre de nouvelles portes à la biologie de synthèse à la biologie structurale et aux nanotechnologies / In cells bio-enzymatic pathways are often spatially organized into complexes, into organelles or onto protein scaffolds. Spatial organization limits diffusion and helps channels substrates between enzymatic cores, limiting competing reactions, insulating and increasing yields of sequential metabolic reactions. In this PhD thesis work, we engineered new tools to control the precise spatial organization of enzymes and increase the titer of specific pathways. We design and engineer “artificial organelles” made of assembling RNA nanostructures. These scaffolds are made out of assembling non-coding RNA molecules we specifically design to polymerize into multi-dimensional nanostructures inside bacterial cells. These structures have docking sites to target enzymes onto them and control their respective distance and stochiometry. We demonstrate the validity of our approach by optimizing and improving the production of biohydrogen and designing a protocol to simplify and standardize the use of RNA scaffold. Moreover, we develop a new synthetic biology “chassis” by developing strategies to engineer AnabaenaPCC7120 and control the spatial localization of metabolic pathway at the cellular level. By targeting specific enzymes into oxygen-depleting heterocysts, metabolic engineers can now implement oxygen-sensitive pathways into oxygen evolving cyanobacteria. This PhD work opens the door to an array of new applications spanning synthetic biology, structural biology to nanotechnology