The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

The over-expression of Down syndrome cell adhesion molecules (DSCAMs) is partially responsible for the mental retardation associated with Down syndrome. Previous work in our lab showed that a DSCAM homolog in C. elegans, Y32G9A.8, is expressed at all developmental stages and appears to be crucial for survival. In an effort to map the expression pattern, I used the Genome Sciences Centre’s primer design program (http://elegans.bcgsc.bc.ca/gfp_primers/) to design a GFP promoter fusion product that was used to monitor gene expression. The results indicate that Y32G9A.8 is expressed in the animal’s gut, suggesting that it may function in the worm’s innate immune response. I also designed a primer set to amplify the Y32G9A.8 transcript. RT-PCR of the entire Y32G9A.8 coding region resulted in a single product; there appears to be no alternative splicing. Although this gene shows homology to other N-CAMS, results indicate that this gene may function in the innate immune system of C. elegans.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

The green fluorescent protein chromophore exhibits remarkably different properties upon removal from the protective beta-barrel. This work focuses on the synthesis of these chromophores as wells studying the photophysics as to why they readily deactivate. Following these initial discoveries, these chromophores can be applied to many different environments providing a fluorescence "turn-on" and thus proving to be applicable in a number of different environments and fields.

Sugarcane holds great potential as a biofactory for the tailored production of novel products of commercial value. In many cases however, the accumulation of an alien product within the cytoplasm interferes with essential cell metabolism. To avoid potential interference, targeting the accumulation of biofactory products into vacuoles may be beneficial. Vacuoles represent one endpoint of the plant endomembrane system where proteins destined for inclusion must contain appropriate targeting peptide signals. However, targeting peptide signals used previously to direct heterologous proteins to the vacuole have not yet been shown to function efficiently in sugarcane. The emphasis of the work described in this thesis was first to characterise the diversity of vacuole types in selected sugarcane tissues, and second to identify and test the function of putative vacuolar targeting signals identified in vacuolar proteins of sugarcane. In order to investigate vacuole diversity in sugarcane cells, a series of membrane-permeable fluorescent probes were used to assess both the acidity and proteolytic properties of vacuolar compartments. It is clear that even from early in development, large vacuoles filled most of the volume of storage parenchyma cells within the developing sugarcane stem. These vacuoles were intensely acidic and contained active aminopeptidases. In leaf cells, vacuoles labelled by chromogenic indicators and enzyme substrates appear much more diverse in pH and proteolytic intensity owing to the multiple functions that leaf cells participate in. As the predominant sugarcane vacuole in vegetative tissues appears to be proteolytic, sugarcane sequences showing homology to proteases and protease inhibitors in other plant species were aligned and compared to identify potential vacuolar targeting signals. This analysis revealed the presence of several candidate vacuolar targeting motifs which displayed high conservation across plant homologues. One such motif, represented by the sequence IRLPS, was identified in the N-terminal region of a legumain protein from sugarcane, which was homologous to known vacuolar processing enzymes in other species. To test the efficacy of the legumain targeting signal and to compare with other motifs, a series of GFP reporter constructs was synthesised and expressed in sugarcane. The sugarcane legumain vacuole targeting signal was particularly efficient at directing an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP fusion proteins in the vacuole appeared to be dependent on cell type, suggesting that the vacuolar environment can vary in its ability to degrade introduced proteins. The legumain vacuole targeting signal was further tested for its ability to direct an avidin protein and a fructosyltransferase enzyme into the lytic vacuole of transgenic sugarcane plants. Avidin, derived initially from chicken egg white, is a glycoprotein that displays a high affinity to the vitamin biotin. For this reason it has been investigated for use in sugarcane as a biocontrol agent against cane grub species. For the production of avidin in planta careful targeting to an appropriate subcellular location is required to avoid a detrimental depletion of available plant cell biotin reserves. When the legumain targeting signal was fused to avidin and expressed as a stably integrated transgene, the avidin protein was detected by immunoblotting but appeared to be proteolytically cleaved within the lytic vacuole in all sugarcane tissues analysed. These plants were phenotypically indistinguishable from controls, indicating that avidin did not appreciably deplete cellular biotin reserves while in transit through the endomembrane system. In contrast, when avidin was designed for either retention in the endoplasmic reticulum or for transit to a different type of vacuole using a heterologous targeting signal, stably transformed plants exhibited a biotin deficient phenotype. This suggests that the legumain vacuole targeting signal was efficient at directing heterologous proteins to a lytic type vacuole where they can be degraded and inactivated if susceptible to proteolysis. When the fructosyltransferase (ftf) gene from Streptococcus salivarius was stably transformed into sugarcane and directed into the lytic vacuole using the legumain vacuole targeting signal, no fructan product could be detected. The low pH and proteolytic environment of this vacuole together with low expression of this bacterial transgene most likely resulted in minimal Ftf activity. Taken together, evidence is presented that the legumain vacuolar targeting signal functions efficiently in directing transgene products such as GFP, avidin and a fructosyltransferase enzyme into a lytic type vacuole. This vacuole has been demonstrated to be both acidic and proteolytic and therefore strategies to improve the stability of heterologous proteins targeted to this vacuolar environment are required and may be specific to the product in question.

abstract: Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging. / Dissertation/Thesis / Ph.D. Chemistry 2012