(A) Photoregulation of DNA Functions by Cyclic Azobenzene-tethered Oligonucleotides (B) Site-specific Fluorescent Labeling of DNA using Inverse Electron Demand Diels-Alder Reaction between trans-Cyclooctene Derivatives and BODIPY-Tetrazine Adducts

(A) Most azobenzene-based photoswitches require UV light for photoisomerization, which limit their applications in biological systems due to possible photodamage. Cyclic azobenzene derivatives, on the other hand, can undergo cis-trans isomerization when exposed to visible light. A shortened synthetic scheme was developed for the preparation of a building block containing cyclic azobenzene and D-threoninol (cAB-Thr). trans-Cyclic azobenzene was found to thermally isomerize back to the cis-form in a temperature-dependent manner. cAB-Thr was transformed into the corresponding phosphoramidite and subsequently incorporated into oligonucleotides by solid phase synthesis. Melting temperature measurement suggested that incorporation of cis-cAB into oligonucleotides destabilizes DNA duplexes, these findings corroborate with circular dichroism measurement. Finally, Fluorescent Energy Resonance Transfer experiments indicated that trans-cAB can be accommodated in DNA duplexes. (B) Inverse Electron Demand Diels-Alder reactions (IEDDA) between trans-olefins and tetrazines provide a powerful alternative to existing ligation chemistries due to its fast reaction rate, bioorthogonality and mutual orthogonality with other click reactions. In this project, an attempt was pursued to synthesize trans-cyclooctene building blocks for oligonucleotide labeling by reacting with BODIPY-tetrazine. Rel-(1R-4E-pR)-cyclooct-4-enol and rel-(1R,8S,9S,4E)-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were synthesized and then transformed into the corresponding propargyl ether. Subsequent Sonogashira reactions between these propargylated compounds with DMT-protected 5-iododeoxyuridine failed to give the desired products. Finally a methodology was pursued for the synthesis of BODIPY-tetrazine conjugates that will be used in future IEDDA reactions with trans-cyclooctene modified oligonucleotides.

The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein.

Conformational analysis of antibody binding site using EDMA methods /

Euclidean distance matrix analysis (EDMA) methods are used to distinguish whether or not significant difference exists between conformational samples of antibody complementarity determining region (CDR) loops, isolated LI loop and LI in three-loop assembly (LI, L3 and H3) obtained from Monte Carlo simulation. After the significant difference is detected, the specific inter-Ca distance which contributes to the difference is identified using EDMA.The estimated and improved mean forms of the conformational samples of isolated LI loop and LI loop in three-loop assembly, CDR loops of antibody binding site, are described using EDMA and distance geometry (DGEOM). To the best of our knowledge, it is the first time the EDMA methods are used to analyze conformational samples of molecules obtained from Monte Carlo simulations. Therefore, validations of the EDMA methods using both positive control and negative control tests for the conformational samples of isolated LI loop and LI in three-loop assembly must be done. The EDMA-I bootstrap null hypothesis tests showed false positive results for the comparison of six samples of the isolated LI loop and true positive results for comparison of conformational samples of isolated LI loop and LI in three-loop assembly. The bootstrap confidence interval tests revealed true negative results for comparisons of six samples of the isolated LI loop, and false negative results for the conformational comparisons between isolated LI loop and LI in three-loop assembly. Different conformational sample sizes are further explored by combining the samples of isolated LI loop to increase the sample size, or by clustering the sample using self-organizing map (SOM) to narrow the conformational distribution of the samples being comparedmolecular conformations. However, there is no improvement made for both bootstrap null hypothesis and confidence interval tests. These results show that more work is required before EDMA methods can be used reliably as a method for comparison of samples obtained by Monte Carlo simulations.

Delineation of within-site terroir effects using soil and vine water measurements in Riesling vineyards within the Niagara Peninsula

The major focus of this dissertation was to explain terroir effects that impact wine varietal character and to elucidate potential determinants of terroir by testing vine water status (VWS) as the major factor of the terroir effect. It was hypothesized that consistent water status zones could be identified within vineyard sites, and, that differences in vine performance, fruit composition and wine sensory attributes could be related to VWS. To test this hypothesis, ten commercial Riesling vineyards representative of each Vintners Quality Alliance sub-appellation were selected. Vineyards were delineated using global positioning systems and 75 to 80 sentinel vines per vineyard were geo-referenced for data collection. During the 2005 to 2007 growing seasons, VWS measurements [midday leaf water potential ('l')] were collected from a subset of these sentinel vines. Data were collected on soil texture and composition, soil moisture, vine performance (yield components, vine size) and fruit composition. These variables were mapped using global information system (GIS) software and relationships between them were elucidated. Vines were categorized into "low" and "high" water status regions within each vineyard block and replicate wines were made from each. Many geospatial patterns and relationships were spatially and temporally stable within vineyards. Leaf'l' was temporally stable within vineyards despite different weather conditions during each growing season. Generally, spatial relationships between 'l', soil moisture, vine size, berry weight and yield were stable from year to year. Leaf", impacted fruit composition in several vineyards. Through sorting tasks and multidimensional scaling, wines of similar VWS had similar sensory properties. Descriptive analysis further indicated that VWS impacted wine sensory profiles, with similar attributes being different for wines from different water status zones. Vineyard designation had an effect on wine profiles, with certain sensory and chemical attributes being associated from different subappellations. However, wines were generally grouped in terms of their regional designation ('Lakeshore', 'Bench', 'Plains') within the Niagara Peninsula. Through multivariate analyses, specific sensory attributes, viticulture and chemical variables were associated with wines of different VWS. Vine water status was a major contributor to the terroir effect, as it had a major impact on vine size, berry weight and wine sensory characteristics.