Three behavioural mutants (fla-8, mot-6 and cheL) generated by transposon Tn5 mutagenesis and localised on cosmid pDUB1905 were studied. The cosmid pDUB1905, from a representative genomic library of the Agrobacterium tumefaciens C58C1 chromosome has previously been partly mapped and found to contain genes concerned with flagella. In this study a region of 5860 nucleotides from a 12 kb BamHl fragment of cosmid pDUB1905 was sequenced completely in both directions. Homology searches of this sequence with sequence databases and other computer analysis revealed two flagellar-related genes (flhA and fliR), a chemotactic gene (cheL) and four open reading frames (orfX, orfW, orfY and orfZ) with no significant sequence identity to any open reading frame in databases. A putative promoter-like sequence was also found upstream of orfZ. The FlhA and FliR are the inner members of type III flagellum-specific export apparatus which are responsible for delivering the flagellar subunits lacking a signal peptide leader to the surface of the cell. These have counterparts in the type III secretion proteins system responsible for transporting pathogen proteins to host cells. The function of CheL has not yet been identified. Three ORFs have chaperone characteristics. A mutant was created by insertion of a neomycin resistance cassette in the fliR homologue to determine the effects of the gene on motility. Phenotype analysis of the mutant showed no flagella and motility with small swarming pattern comparing to wild type, indicating that fliR is indeed a flagellar gene. In this study two more members of flagellum-specific export, a chemotactic gene, three open reading frames which could have specific chaperon activity, and an unknown open reading frame were identified in A. tumefaciens C58C'.

Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins, which are proposed to have roles in essential trace metal homoeostasis and in the detoxification of metal ions. The genes encoding MTs have been isolated from a wide range of eukaryotes, although MT genes have not previously been isolated from prokaryotes. The polymerase chain reaction (PGR) was initially used to isolate a prokaryotic MT gene fragment from Synechococcus PCC 6301. PGR fragments were amplified using inosine-containing primers designed from the amino acid sequence of a prokaryotic MT. Subsequent cloning and nucleotide sequence analysis revealed that the deduced amino acid sequence of the PGR product corresponded to the amino acid sequence of the prokaryotic MT. The amplified product was thus part of the gene encoding the MT, and was designated smtA. The same primers used in the initial amplification were subsequently utilised for anchored PGR, to amplify the remainder of the coding region and the 3' and 5' flanking regions of the smtA gene. A genomic library was produced from Synechococcus PGG 7942 DNA and screened using the PGR products described above as probes. A genomic clone was isolated, nucleotide sequence analysis revealed the structure of the smt locus, two open reading frames, smtA and smtB, arranged in a divergent orientation about the smt operator/promoter region. The operator/promoter region contains the transcriptional and translational signals for the two genes and three regions that are candidate sites for interaction of regulatory proteins. The transcript start sites of the two genes were mapped within the operator/promoter region by primer extension analysis. An increase in the relative abundance of transcripts of both smt genes was studied in response to various metal ions in a series of northern blots. Inhibitor studies confirmed that the smtA gene is regulated at the transcriptional level. The 5' flanking region of the smtA gene conferred metal specific induction of the reporter gene lacZ. SmtB has sequence similarity to several prokaryotic regulatory proteins and contains a putative helix-turn-helix structural domain. Deletion analysis suggests that SmtB is a repressor of smtA expression. Subsequent work has confirmed that SmtB is a trans-acting repressor of expression from the smt operator/promoter.

No description available.

Cancer is a devastating disease for human society with thousands of deaths and estimated new cases every year around the globe. Intensive research efforts on understanding the disease progression and determining effective diagnostics and therapeutics have been employed for over one hundred years. Throughout this time, and in particular during the last two decades, computational-based methods have gained increasing importance in cancer biology research by providing significant advantages in the analysis and interpretation of high-throughput data at the molecular and genomic levels. More specifically, after completion of the Human Genome Project in 2003, and with the Cancer Human Genome Project underway, high-throughput biological assays (e.g., microarray chips, next generation sequencing machines) have supplied researchers thousands of measurements per experimental sample. The massive amount of related data has oftentimes been challenging to interpret and translate, particularly in cancer biology and therapeutics. This thesis reports the results of three independent studies in which high-throughput gene expression is computationally analyzed to address longstanding issues in cancer biology. Two of the studies utilize data from ovarian cancer patients while the third involves data collected from pancreatic cancer patients. In Chapter 1, I address the importance of personalized profiling in pancreatic cancer ; in Chapter 2 the role of cancer stroma in the progression of ovarian cancer and in Chapter 3 evidence for the role of epithelial-to-mesenchymal transition (EMT) in ovarian cancer metastasis. More specifically, Chapter 1 emphasizes the power of personalized molecular profiling in unmasking unique gene expression signatures that correspond to each individual patient. These individual expression patterns (individual profiling), which may be overlooked by the traditional methods of gene signatures enriched in groups of afflicted individuals (group profiling), can provide valuable information for more successful targeted therapies. In order to address this issue in pancreatic cancer, comparisons of the most significantly differentially expressed genes and functional pathways were performed between cancer and control patient samples as determined by group vs. personalized analyses. There was little to no overlap between genes/pathways identified by group analyses relative to those identified by personalized analyses. These results indicated that personalized and not group molecular profiling is the most appropriate approach for the identification of putative candidates for targeted gene therapy of pancreatic and perhaps other cancers with heterogeneous molecular etiology. Chapter 2, also with strong implications on personalized molecular profiling, unveils the functional variability of the tumor microenvironment among ovarian cancer patients. The purpose of this study was to investigate the process of microenvironmental stroma activation in human ovarian cancer by molecular analysis of matched sets of cancer and surrounding stroma tissues from individual patients. Expression patterns of genes encoding signaling molecules and compatible receptors in the cancer stroma and cancer epithelia samples indicated the existence of two sub-groups of cancer stroma with different propensities to support tumor growth. These results demonstrated that functionally significant variability exists among ovarian cancer patients in the ability of the microenvironment to modulate cancer development. Chapter 3 aims to uncover the molecular mechanisms that underlie the metastatic process with the hope that such knowledge may lead to more effective therapeutic treatments. For this purpose, pathological and molecular analyses were conducted in 14 matched sets of primary and metastatic samples from late staged ovarian cancer patients. Pathological examination revealed no morphological differences between any of the primary and metastatic samples. In contrast, gene expression analyses identified two distinct groups of patient samples. One group displayed essentially identical expression patterns to primary samples isolated from the same patients. The second group displayed expression patterns significantly different from primary samples isolated from the same patients. Predominant among the differentially expressed genes characterizing this second class of metastatic samples were genes previously associated with epithelial-to-mesenchymal transtion (EMT). These results supported a role of EMT in at least some ovarian cancer metastases and demonstrated that indistinguishable morphologies between primary and metastatic cancer samples is not sufficient evidence to negate the role of EMT in the metastatic process. / The data related to the ovarian cancer work discussed in this dissertation are available at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38666

The shikimate pathway connects primary metabolism with the biosynthesis of the three aromatic amino acids (phenylalanine, tyrosine and tryptophan), which are essential protein building blocks. This pathway also provides precursors for a wide array of plant secondary metabolites with adaptive functions in plant adaptation and defense. The third and fourth steps of the shikimate pathway (the conversion of shikimate from 3-dehydroquinate via 3-dehydroshikimate) are catalyzed by a bi-functional enzyme called 3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH). DQD/SDHs have been biochemically characterized in a few plant species including Arabidopsis thaliana, Solanum lycopersicum and Nicotiana tabacum. The embryo-lethal phenotype of Arabidopsis null mutants lacking DQD/SDH highlights a critical role of shikimate in primary metabolism. Quinate shares high structural similarity with shikimate and is an important secondary metabolite present in many plant species. Quinate and its derivatives (e.g. chlorogenic acid) serve important functions in plant defense due to their astringent (i.e. bitterness) and antimicrobial properties. Quinate can be derived from 3-dehydroquinate, and this reaction is catalyzed by quinate dehydrogense (QDH), the reaction mechanism of which resembles that of SDH. With a functional genomics approach, I demonstrated that two of the five poplar putative DQD/SDHs (Poptr1 and Poptr5, poplar DQD/SDH1 and 2) have exclusive specificity for shikimate, while the other three (Poptr2 to Poptr4, poplar QDH1 to 3) are involved in quinate biosynthesis. Phylogenetic reconstruction of the DQD/SDH/QDH superfamily has identified two distinct clades in seed plants that may act preferentially on either shikimate or quinate, whereas lineages that have diverged prior to the angiosperm/gymnosperm split, only have a single copy DQD/SDH. An evolutionary analysis was carried out, and the sequence of the immediate pre-duplication ancestral DQD/SDH (>300MYA) was estimated and reconstructed. Protein structure modelling and in vitro biochemical characterization of the ancestral recombinant protein was performed along with some extant members of this family (pre-duplication representatives: Rhodopirellula baltica (Rhoba), Chlamydomonas reinhardtii (Chlre), Physcomitrella patens (Phypa) and Selaginella moellendorffii (Selmo); post-duplication species: Pinus taeda (Pinta1 & Pinta2) and Populus trichocarpa (Poptr1 & Poptr3). Together, the results indicate that quinate biosynthetic activity was gained prior to duplication and remained low until it became beneficial and favored by selection. The optimization of quinate biosynthetic activity was at the expense of losing some primary shikimate biosynthetic function creating a pleiotropic conflict. This was then resolved by gene duplication and further specialization leading to genes encoding specialized enzymes (either SDH or QDH). Diversification of the DQD/SDH/QDH superfamily likely occurred through sub-functionalization via a mechanism described as “Escape from Adaptive Conflict.” / Graduate / 0307 / guojia@uvic.ca

Lipid based non-viral delivery systems are potentially of great importance to the development of an effective and versatile therapeutic gene treatment. Many difficulties are faced in designing efficient lipid based gene delivery systems and addressing the problem of endosomal escape is one key area where efficiency could be greatly improved Molecules aimed at inducing aggregate disruption in response to changes in pH, or metal ion concentration, have been synthesised. Series of compounds were prepared based on cationic 5-alkyl-2-methylaminoalkyl pyridine amphiphiles and amphiphilic C(_8)-C(_16) EDTA dialkylamides. The critical aggregation concentration of each species was measured and a series of experiments undertaken, designed to assess the extent of pH, or metal ion concentration, induced aggregate disruption effects. The experiments were carried out by including the molecules to be examined as small mole percent co-aggregates in micellar and liposome model systems. Each group of compounds appears to exhibit disruption effects on the more strongly ordered bilayer membrane systems, with the EDTA based compounds displaying the most consistent pH dependent disruption. However, the more dynamic micellar aggregate models were less sensitive to disruption using the particular observation method employed.

The aim of this work was to isolate the gene encoding a bifunctional a-amylase inhibitor/endochitinase protein from the seeds of Coix lachryma-jobi, a tropical cereal. Prior to this study, it had been demonstrated that this bifunctional protein had anti-insect and possibly anti-fungal properties. Consequently the gene could potentially be used to confer insect and fungal resistance in transgenic plants. A multifunctional approach was undertaken to isolate the a-amylase inhibitor/endochitinase cDNA and genomic sequences, involving three main strategies. Immunoscreening a Coix cDNA expression library with antibodies raised against a wheat germ endochitinase protein resulted in the isolation of three immunopositive clones. These cDNA's, were sequenced and one characterised as a seed storage protein, named a-coixin. Despite extensive searches of the appropriate databases, the function of the other two are as yet unknown. Another strategy was the production of polyclonal antibodies, raised against a glutathione S- transferase-a-amylase inhibitor fusion peptide. It was envisaged that these antibodies could be used to isolate the gene of interest following immunoscreening of the Coix cDNA expression library. Polyclonal antibodies were successfully elicited against the glutathione S- transferase moiety, but could not detect the a-amylase inhibitor protein when assayed. Using the polymerase chain reaction, amplification of the a-amylase inhibitor coding sequence was attempted from Coix genomic DNA, cDNA and a Coix seed cDNA library. PGR product were successfully amplified from genomic DNA and the cDNA library. Further characterisation of these product revealed that they were a result of non specific amplifications. Further work required to isolate the a-amylase inhibitor gene is discussed.

No description available.

Differentiation and gene expression in the oomycete Achlya ambisexualis ( strain E87) were studied during three responses, sexual antheridium formation, asexual sporangium formation and heat shock. Polyacrylamide gel electrophoresis of proteins labelled in vivo and in cell free systems directed by poly(A) mRNA showed that during formation of antheridia only one change was detectable in the proteins synthesized. No detectable changes were observable in the translatable poly(A) RNA population indicating that post-translational events may be required for differentiation. During early sporangium formation several proteins showed changes in relative rates of synthesis. These correlate with similar changes in cell free translation products suggesting a transcriptional level of regulation. Nucleic acid hybridization analyses of mRNA populations indicated that many new sequences appear late and accumulate in the spore. These late transcripts are probably utilized during spore germination. During heat shock several proteins showed rapid increases in rates of synthesis; these seem to be controlled at the level of transcription.

Transport, a common cause of stress in livestock, has been documented to increase cortisol, and epinephrine in goats. However, little is known about the timing of changes in the immune system in these stressed animals. The objective of this study was to determine whether expression of immune-related genes changes in goats that are exposed to transport stress. In this study, 15 Spanish-Boer goats ranging from 3 to 4 yrs of age were transported for 12 h. Goats were divided into 5 groups of 3 and placed in 1.219 m x 1.219 m pens. Blood samples were collected via jugular veni-puncture from each animal at 0 h, 3 h, 6 h, 9 h, and 12 h of transport, plasma and leukocytes were harvested for cortisol analysis and PCR analysis for gene expression. Data was analyzed using trailer location (group) as the experimental unit in a mixed model, repeated measures analysis of variance with compound symmetry and autoregressive covariance structures, depending on the best fit for each model. Percent weight losses were analyzed using a diagonal covariance mixed model. Hourly temperature humidity index (THI) values inside the trailer and from the shade were analyzed using a two-independent sample T-test. Cortisol concentrations were significantly elevated during transport (P<.049), indicating that goats experienced stressful events during hours of transport. Cortisol concentrations peaked after 6 hours, and returned to near basal concentrations after 12 h of transport. There was an overall trend for greater expression of many of the genes of interest to increase expression after 12 h of transport, but none were significantly different from pre-transport expression values. Overall, the data suggests that the goats transported during this study experienced transport stress, as indicated by the elevation in cortisol concentrations, but did not have significant changes in expression of the immune-related genes after 12 h of transport.