Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

Development of a bacteriophage-based biopesticide for fire blight

Fire blight is an economically important disease of apples and pears that is caused by the bacterium Erwinia amylovora. Control of the disease depends on limiting primaly blosson1 infection in the spring, and rapidly removing infected tissue. The possibility of using phages to control E.amylovora populations has been suggested, but previous studies have. failed to show high treatment efficacies. This work describes the development of a phage-based biopesticide that controls E. amylovora populations under field conditions, and significantly reduces the incidence of fire blight. This work reports the first use ofPantoea agglomerans, a non-pathogenic relative ofE. amylovora, as a carrier for E. amylovora.phages. Its role is to support a replicating population of these phages on blossom surfaces during the period when the flowers are most susceptible to infection. Seven phages and one carrier isolate were selected for field trials from existing collections of 56 E. amylovora phages and 249 epiphytic orchard bacteria. Selection of the . /' phages and carrier was based on characteristics relevant to the production and field perfonnance of a biopesticide: host range, genetic diversity, growth under the conditions of large-scale production, and the ability to prevent E. amylovora from infecting pear blossoms. In planta assays showed that both the phages and the carrier make significant contributions to reducirig the development of fire blight symptoms in pear blossoms. Field-scale phage production and purification methods were developed based on the growth characteristics of the phages and bacteria in liquid culture, and on the survival of phages in various liquid media. Six of twelve phage-carrier biopesticide treatments caused statistically signiflcant reductions in disease incidence during orchard trials. Multiplex real-time PCR was used to simultaneously monitor the phage, carrier, and pathogen populations over the course of selected treatments. In all cases. the observed population dynamics of the biocontrol agents and the pathogen were consistent with the success or failure of each treatment to control disease incidence. In treatments exhibiting a significantly reduced incidel1ce of fire blight, the average blossom population ofE.amylovora had been reduced to pre-experiment epiphytic levels. In successful treatments the phages grew on the P. agglomerans carrier for 2 to 3 d after treatment application. The phages then grew preferentially on the pathogen, once it was introduced into this blossom ecosystem. The efficacy of the successful phage-based treatnlents was statistically similar to that of streptomycin, which is the most effective bactericide currently available for fire blight prevention. The in planta behaviour ofE. amylovora was compared to that ofErwinia pyrifoliae, a closely related species that causes fire blight-like synlptoms on pears in southeast Asia. Duplex real-time PCR was used to monitor the population dynamics of both species on single blossonls. E. amylovora exhibited a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae. The genome ofErwinia phage Ea21-4 was sequenced and annotated. Most of the 8-4.7 kB genome is substantially different from previously described sequences, though some regions are notably similar to Salmonella phage Felix 01 . Putative functions were assigned to approximately 30% of the predicted open reading frames based on amino acid sequence comparisons and N-terminal sequencing of structural proteins.

Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

Evolutionary synthesis of stochastic gene network models using feature-based search spaces

A feature-based fitness function is applied in a genetic programming system to synthesize stochastic gene regulatory network models whose behaviour is defined by a time course of protein expression levels. Typically, when targeting time series data, the fitness function is based on a sum-of-errors involving the values of the fluctuating signal. While this approach is successful in many instances, its performance can deteriorate in the presence of noise. This thesis explores a fitness measure determined from a set of statistical features characterizing the time series' sequence of values, rather than the actual values themselves. Through a series of experiments involving symbolic regression with added noise and gene regulatory network models based on the stochastic 'if-calculus, it is shown to successfully target oscillating and non-oscillating signals. This practical and versatile fitness function offers an alternate approach, worthy of consideration for use in algorithms that evaluate noisy or stochastic behaviour.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.