Molecular Biology and Biochemistry - Theses, Dissertations, and other Required Graduate Degree Essays

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A light-harvesting catalytic DNA for thymine dimer repair

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Date created: 
2005
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)
Abstract: 

Since the discovery of ribozymes in the 198O's, there have been many examples of catalytic DNA or RNA that can catalyze a variety of reactions, including phosphodiester bond cleavage, porphyrin metallation and nucleotide synthesis. A novel catalytic DNA (DNAzyme) was generated by in vitro selection that could perform a photochemical reaction, the photoreversal of thymine dimers upon irradiation of light. Photolyase enzymes found in nature catalyse the photorepair of thymine dimers with the aid of sensitizers, such as FADH and tryptophan. Curiously, one DNAzyme, UVlC, could repair thymine dimers without the aid of photosensitizers. UV 1 C was found to be active when using light that was significantly red-shifted relative to that of normal DNA absorbance. Using spectroscopic techniques, it was determined that the red-shifted action spectrum and rate enhancement profile were caused by the presence of a guanine quadruplex in the catalytic core. Further characterization of UV1 C revealed interesting information on the folding, substrate specificity and enzyme mechanism. Using sequence mutant constructs, it was determined that the 3'-binding arm of the enzyme could be removed with very little effect on repair rates. Cross-linking reactions determined that the thymine dimer was situated in close proximity, or stacked with the putative guanine quartet. Interestingly, substrates containing uracil dimers with deoxyribose sugar rings were efficiently repaired by UVl C, whereas thymine dimer substrates containing ribosyl sugar rings were not. Oxidative damage to guanines was not detected, which implied that a fast rate of back electron transfer may have occured after the thymine dimer photosplitting process. DNA "aptamers" (binders) were generated by in vitro selection that could simultaneously bind to the electron transfer protein cytochrome c, and to the small metalloporphyrin, hemin. Aptarners selected contained guanine-rich sequences located centrally with respect to the overall sequence. Chemical probing analysis determined that a central guanine-quadruplex formed that was responsible for binding to hemin. Through footprinting analysis, the binding site for cytochrome c on the DNA was determined. The ternary complex represents a potential model for the study of electron transfer between artificial and naturally-occurring heme electron transfer systems.

Document type: 
Thesis

Recoding of translation in turtle mitochondrial genomes: Programmed frameshift mutations and evidence of a modified genetic code

File(s): 
Date created: 
2006
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)
Abstract: 

A +1 frameshift insertion has been documented in the mitochondrial gene nad3 in some birds and reptiles. By sequencing the polyadenylated mRNA in the chicken (Gallus gallus), we have shown that the extra nucleotide is transcribed and is present in the mature mRNA. Evidence from other animal mitochondrial genomes has led us to hypothesize that certain mitochondrial translation systems have the ability to tolerate frameshift insertions using programmed translational frameshifting. To investigate this, we sequenced the mitochondrial genome of the red-eared slider turtle (Trachemys scripta), where both the common nad3 frameshift insertion and a novel site in nad4l were found. Sequencing the region surrounding the insertion in nad3 in a number of other turtles and tortoises revealed general mitochondrial +1 programmed frameshift site features as well as the apparent redefinition of a stop codon in Parker’s sideneck turtle (Macrochelodina parkeri), the first known example of this in vertebrate mitochondria.

Document type: 
Thesis

Characterization of putative regulators of the small GTPase, Rac, in Drosophila

Author: 
File(s): 
Date created: 
2004
Department: 
Theses (Dept. of Molecular Biology and Biochemistry) / Simon Fraser University
Thesis type: 
Thesis (Ph.D.)
Document type: 
Thesis

Crystallographic analysis of Escherichia coli type I signal peptidase in complex with peptide-based inhibitors

Author: 
File(s): 
Date created: 
2007
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)
Abstract: 

Escherichia coli type I signal peptidase (SPase I) is a membrane-bound endopeptidase that cleaves amino-terminal signal peptides from secreted proteins and some membrane proteins. To contribute to the structure-based design effort for developing a novel class of antibiotics, the X-ray crystal structures of peptide-based inhibitors with SPase I have been solved. Nine complexes of SPase I with inhibitor were co-crystallized, five of which diffracted to 2.6 Å or better. Two crystal structures were solved using molecular replacement and refined. Crystallographic analysis of these two SPase I-inhibitor complexes will be useful for evaluating and optimizing these novel antibiotic drug candidates. The crystal contacts and structural variations of the SPase I molecules from each SPase I crystal structure solved to date were analyzed and compared in a first step towards the design of new constructs of SPase I that may crystallize more readily and in a more ordered fashion.

Document type: 
Thesis

Characterization of exogenous and endogenous plant small RNAs: discovery of a ubiquitous terminal modification.

File(s): 
Date created: 
2007
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)
Abstract: 

RNA silencing is gene regulatory process mediated by 21 to 25 nt RNA which originate from longer single- or double-stranded RNA transcripts. After synthesis, the 21 to 25 nt RNAs are incorporated into argonaute containing RNA-protein complexes acting either pre- or post-transcriptionally – silencing heterochromatin or silencing RNA transcripts, respectively. Plants can be infected with viral satellites, which are small infectious agents comprised of 200-400 nt single-stranded RNA genomes. These satellite genomes lack obvious protein coding regions and adopt characteristic secondary structures. Viral satellites depend on helper viruses for replication as the latter provide the RNA-dependent RNA polymerase. One viral satellite system is the Cucumber Mosaic Virus (CMV) and its Y-Satellite RNA (Y-Sat). This helper virus (CMV) and satellite (Y-Sat) pair can infect a range of plants including tobacco, causes particular tobacco strains to yellow. In a recent study, Wang et al. (PNAS 101:3275-3280) demonstrated that symptoms of the CMV Y-Sat were no longer evident despite an accumulation of Y-Sat RNA. This symptom suppression was due to an endogenously expressed strong RNA silencing suppressor called P1/HC-Pro. I cloned and sequenced 698 small RNA from infected and non-infected plants. Analyzing the cloning data I observed a sharp discrepancy between the size distribution of cloned small RNAs in the histogram versus the autoradiogram of 5' radiolabelled RNA in P1/HC-Pro(+) CMV/Y-Sat(+) plants. Using enzymatic assays and a new band-shift assay I proved that viral small RNA in P1/HC-Pro expressing plants were not modified on the 2'-hydroxyl of their 3' terminus, but commonly cloned endogenous micro RNAs were modified. The analysis of these results was greatly facilitated by my software called Ebbie, an automated sequencing data analysis pipeline using a dynamic web page, which assists the process of annotating cloned small RNAs. These findings imply a separation between the multiple small RNA biogenesis pathways for RNA silencing in plants via sub-cellular localization and/or recruiting distinct sets of proteins by each pathway. My research also showed that all small RNAs in plants have a 3' terminal modification, a significant difference between animal and plant RNA silencing pathways.

Document type: 
Thesis

RNA-mediated chemistries: a case of replication and capping

Author: 
File(s): 
Date created: 
2007
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)
Abstract: 

Our current understanding of biology suggests that early life relied predominantly on RNA for both catalysis and the storage of genetic information. Tremendous efforts have been undertaken to confirm that the catalytic abilities of RNA can sustain this “RNA World”. However, the ability of ribozymes to utilize certain chemistries known to be critical for present-day biology, and hence likely to be important for ribo-organisms, has yet to be demonstrated. Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry at phosphorous reaction centres. I have isolated and characterized an RNA-capping ribozyme that retains stereochemistry during the synthesis of a 5¢-5¢ RNA cap. Stereochemical data determined using thio-phosphate modifications, together with an early rate-limiting step, suggest that this ribozyme utilizes two distinct inverting chemical steps - proceeding via a ribozyme-covalent intermediate - during catalysis. A ribo-organism, like contemporary organisms, would have required ribozymes to replicate an RNA genome within some sort of primordial compartment. To provide support for this requirement, I have isolated an improved RNA polymerase ribozyme, referred to as B6.61, from a mutagenized pool containing ~9 ´ 1014 different sequences using a novel large-scale in vitro compartmentalization system. B6.61 polymerized all tested primer-template (PT) complexes faster than its parent, the Round-18 polymerase ribozyme. For one PT complex a rate enhancement of more than 80 fold was observed for extensions longer than one helical turn. The new variant also exhibited improved fidelity on a number of templates. Most interestingly, B6.61 was found to copy one PT complex by almost two complete turns of an RNA helix. To further study RNA-mediated replication, I investigated the role of nucleic acid structure during the course of oligonucleotide extension by T7 RNA polymerase. In addition to normal transcription, the enzyme can produce anomalous transcripts in the absence of a promoter. I have found oligonucleotides that are able to form transient unimolecular loop structures closed by as little as one base-pair to be viable substrates. This intermittent extension process was found to be quite efficient, and adds to the understanding of viral RNA replicative strategies.

Document type: 
Thesis

The sodium-calcium exchanger (NCX): temperature adaptation and evolutionary history

Author: 
File(s): 
Date created: 
2007
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)
Abstract: 

The sodium-calcium exchanger (NCX) is an important regulator of intracellular calcium and is highly conserved across species. NCX among different species that live in diverse environments demonstrate adaptation to different conditions while maintaining a relatively high degree of identity. For further understanding of NCX temperature sensitivity, we characterized NCX gene sequences from a wide variety of genomes for analyses. However, these analyses did not lead to specific predictions of temperature sensitivity among the various homologs of NCX. By comparing NCX orthologs with different temperature dependencies yet with high genotype conservation, ten amino acids were predicted as being primarily responsible for the variation in phenotype. Mutation of these ten amino acids and activity measurement over a range of temperatures resulted in a significant change in its temperature sensitivity. Further work to elucidate the changes in NCX function at different temperatures is required to establish the specific mechanisms underlying NCX temperature dependence.

Document type: 
Thesis