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

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Identification of genes involved in heat stress in arctic charr

Author: 
Date created: 
2011-12-16
Abstract: 

I set out to identify candidate genes that can be used to develop genetic markers associated with Upper Temperature Tolerance (UTT) for use in a genomics-assisted broodstock program for Arctic charr (Salvelinus alpinus) and for examining wild populations at risk due to climate change. This was accomplished using genomic resources available for Atlantic salmon (Salmo salar), which allowed me to identify and examine genomic regions and specific genes of interest. In addition, I conducted expression profiling of Arctic charr exposed to acute and chronic thermal stress. Using comparative genomics, I identified several Atlantic salmon fingerprint scaffolds containing markers associated with UTT in Arctic charr and rainbow trout (Oncorhynchus mykiss). One of these was fully sequenced using 454 GS FLX next-generation sequencing and annotated, which identified nine genes in the putative Quantitative Trait Locus (QTL) region of the Atlantic salmon genome. This analysis also provided evidence that the 454 sequencing technology was suitable for partial assembly and gene annotation, but not for de novo whole genome sequencing of a complex salmonid genome. Next, I conducted expression profiling of phenotypically tolerant and intolerant Arctic charr. The differentially expressed genes were compared with those identified within the UTT QTL sequenced previously, which suggested COUP-TFII as a particularly interesting candidate gene. Heat shock proteins (Hsps) and hemoglobins were also significantly associated with acute thermal stress. Concurrently, I performed expression profiling of Arctic charr exposed to moderate, chronic temperature stress that mimicked a more realistic situation. Again, Hsps were identified in the thermal stress response, as well as ribosomal proteins, which were up-regulated throughout the exposure and the recovery period. Finally, I identified and fully annotated all of the hemoglobin genes in Atlantic salmon. This identified substantially more hemoglobin genes in this species than in any other fish analyzed to date, and included several non-Bohr beta hemoglobins, which may be used in emergency response situations. Combined, the findings of my research have substantial implications for developing a temperature tolerant Arctic charr broodstock and for examining wild populations of salmonids for responses to temperature stress brought by climate change.

Document type: 
Thesis
File(s): 
Supervisor(s): 
William S. Davidson
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Computational ortholog prediction: evaluating use cases and improving high-throughput performance

Date created: 
2013-03-08
Abstract: 

Orthologs are genes that diverged from an ancestral gene when the species diverged. High-throughput computational methods for ortholog prediction are a key component of many computational biology analyses. A fundamental premise in these analyses is that orthologs (when predicted correctly) are functionally equivalent and can be used to transfer gene annotations across species. Currently, many existing ortholog prediction methods generate a sizeable number of incorrect ortholog predictions, especially in cases of complex gene evolution. My thesis examines the functional equivalence hypothesis further and presents one solution that increases the precision of ortholog prediction. To examine the use of orthologs in computational analysis, I conducted and evaluated three projects that employ ortholog prediction in distinct ways. In these projects, orthologs were used to (1) identify conserved, unique genes in metazoan species, (2) validate predicted gene regulatory modules in Pseudomonas aeruginosa, and (3) construct a transcriptional regulatory network in Aspergillus fumigatus. I identified factors affecting ortholog prediction in these specific use cases, demonstrating how successive gene duplications, incomplete genomes and rapid evolution of gene regulation can impact the results for such analyses. To improve ortholog prediction, I evaluated and augmented an existing method called Ortholuge. Ortholuge is a computational method that increases the precision of ortholog prediction in a high-throughput setting. I evaluated the performance of Ortholuge, showing that its approach of classifying orthologs based on their relative phylogenetic divergence does identify orthologs that are more functionally equivalent. I compared Ortholuge to contemporary methods QuartetS and OMA, and showed that Ortholuge consistently identifies functionally-equivalent orthologs across a range of taxonomic distances. I also further developed Ortholuge’s functionality by reducing run-time, increasing accuracy and improving usability through a number of modifications. Lastly, to make Ortholuge results available to the research community, I developed a database of Ortholuge ortholog predictions for bacteria and archaea species. This online database provides high-level visualization of orthologs and the ability to easily run complex queries to retrieve genes that are shared or unique between specified taxa. Overall, this work contributes an enhanced method for precise high-throughput ortholog identification and increases our understanding of the functional equivalences between orthologs.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Fiona Brinkman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

The evolution of mitochondrial genome structure and function in insects

Date created: 
2005
Abstract: 

The mitochondrial genomes (mt-genomes) of animals are very compact in structure, encoding thirteen protein genes involved in the production of ATP, and the key components of the translation system to express these proteins. The mitochondrial expression system, which functions separately from that of the nucleus, shows characteristics of both prokaryotic and eukaryotic expression systems, and has diverged greatly from that currently observed in the closest living relatives of mitochondria, the a-proteobacteria. Current understanding of transcript maturation is that large multi-gene transcripts are processed by the removal of intervening tRNA genes, leaving behind RNA templates to be matured into the functional mRNAs and rRNAs. One of the most striking features of insect mt-genomes has been the apparent replacement of a start codon with a stop codon for the essential mitochondrial gene cytochrome c oxidase subunit 1 (coxl). When first observed in Drosophila, Clary and Wolstenholme proposed a highly unusual four-base "ATAA" start codon. With the expanded sampling of mitochondrial sequence across the various insect orders, the data does not support the use of this aberrant initiation for coxl. At the initiation of this study, the diversity of insect groups represented by complete mt-genome sequence was very poor. To address this deficit, I undertook sequencing projects to increase the number of insect orders represented in the mitochondrial sequence databases. I report the complete mt-genome sequences for two insects, the spittlebug Philaenus spumarius, and the giant stonefly Pteronarcys princeps. The sequences are annotated and compared to other insect mt-genomes in the sequence databases. I report the cDNA sequences of Drosophila melanogaster mitochondrial mRNAs, rRNA subunits, and a population of pre-mRNA molecules that are intermediates of the RNA processing system. Models to explain mitochondrial transcript maturation in light of these new observations are proposed. Comparative analyses were undertaken to apply the information gained from the mitochondrial transcripts of D. melanogaster to the mitochondrial structure and annotations of mt-genomes from the other insects. These analyses suggest a 5' specific modification to the tRNA punctuation model for insect mitochondria. This modification may represent a further evolutionary simplification of the mitochondrial expression system.

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

Investigation of the dimer interface of CTP:phosphocholine cytidylyltransferase and its modulation by lipids

Author: 
Date created: 
2005
Abstract: 

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme in phosphatidylcholine synthesis. CCT is an amphipathic homodimer, whose activity is regulated by membrane binding - a process resulting in conformational restructuring of the dimer interface. I used site-directed mutagenesis of domain N lysines in CCT, followed by lysine specific chemical cross-linking in the presence and absence of activating lipids, to locate sites of dimer interactions and to pinpoint where conformational rearrangement occurs. Analysis revealed that none of the lysines in the N-terminus are required for cross-linking. Thus lysinemediated cross-links involve other CCT domains. I found that CCT 236, despite lacking a membrane-binding domain, appears to undergo conformational changes that disrupt the dimer interface in the presence of activating lipids. The mechanism causing lipid-induced rearrangements of both CCT 367 and CCT 236 dimer interfaces remains unknown, but appear to be different. Moreover, the two CCTs are activated by different lipids suggesting different activation mechanisms.

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

Developing broodstock of Arctic Charr (Salvelinus alpinus L.)

Author: 
Date created: 
2004
Abstract: 

Most of the economically important traits in animal breeding programs are quantitative in nature. Detecting major genes and/or blocks of genes influencing these traits has been made possible by the availability of hypervariable DNA markers. In this study, phenotypic variations related to growth and body girth in the two domesticated strains of Arctic char (Salvelinus alpinus L.) at Icy Waters Ltd. (Whitehorse, Yukon, Canada) were examined and then quantitative trait loci for growth were identified using a genome wide scan approach. Twelve crosses involving the pure strains (Tree River and Yukon GoldTM), the reciprocal hybrids, and the reciprocal backcrosses were set up with ten families per cross. After 18 months of rearing in the hatchery environment under identical culture conditions, it was observed that backcrosses with a 75% Tree River genome contribution ((YGfxTRm)fxTRm) grew fastest and possessed greatest variance. A total of 198 highly polymorphic microsatellite markers, from various salmonid species, covering 41 linkage groups on the current Arctic charr linkage map were tested for a genome scan. Sixty two highly polymorphic markers were chosen to perform a genome wide scan on a hll-sib backcross family, namely 6-1 0, to detect genetic factors responsible for the variation of growth in Arctic charr. These markers cover 28 of the 46 linkage groups in the currently available, low-resolution genetic map of Arctic charr. Results from a transmission disequilibrium test (TDT) indicate a significant association (0.001

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

Deuterium NMR investigation on the effect of isopropyl myristate on the skin barrier using a model stratum corneum

Date created: 
2005
Abstract: 

The function of the skin as a barrier is found to be closely associated with lipids in the stratum corneum (SC). At physiological temperature, SC lipids form solid or crystalline domains which may contribute to skin impermeability. A 2 ~ - ~ ~ study has been conducted to observe the effect of Isopropyl Myristate (IPM), a skin penetration enhancer, on the physical properties of model SC (MSC). IPM was added to MSC comprising equimolar mixtures of N-palmitoyl D-erythro-sphingosine (Cerl6), cholesterol and palmitic acid (PA), containing either Cer 16-d31 or PA-d31. 2 ~ - ~ data showed that IPM had pronounced effects on the phase behaviour of MSC. Relative proportions of solid, liquid-crystalline and isotropic phases varied with temperature and IPM concentration. Further experiments, using synthesized chain-deuterated IPM, allowed the examination of the behaviour of IPM in MSC. These data, coupled with other studies, can help elucidate the effect of IPM on the integrity of the SC barrier.

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

Interactions between photoswitches and nucleic acids and the selection of nucleotide synthase ribozymes

Date created: 
2005
Abstract: 

The RNA World hypothesis suggests prebiotic enzymes were RNA molecules (ribozymes) rather than proteins. Previously, in vitro selection identified a ribozyme that synthesized the glycosidic bond in a pyrimidine nucleotide. A selection to minimize size and increase catalytic efficiency was concluded and kinetic behaviour of one isolate was examined. In preparation for two new selections for purine and pyridine nucleotide synthase ribozymes, constructs representing theoretical end products were synthesized and used to test the feasibility of the selective procedure. Dithienylethenes are molecules that interconvert between two isomers (each with unique properties) in response to different light stimuli. It was our interest to control DNA binding and RNA cleavage abilities of novel switches by light exposure. One switch successfully bound DNA, with each isomer displaying the same association constant. Another project featured a histidinefunctionalized switch to mimic RNase A. However, no significant difference in cleavage was observed between the two isomers.

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

Identification of cis-acting regulatory elements using orthology biased Gibbs sampling

Date created: 
2005
Abstract: 

Many computational pattern searching tools for the discovery of novel, common regulatory elements between co-expressed genes have been developed over the last ten years. However, few approaches attempt to incorporate valuable additional information, such as inter-species conservation, into the prediction process. Orthology biased Gibbs sampler (OrBS) is an expansion on the Gibbs sampler motif discovery approach. I introduce dynamic motif width prediction, a novel convergence detection approach, and a scoring function that incorporates cross-species sequence conservation. The algorithm was refined using the Caenorhabditis elegans X box element and is shown to successfully identify the element in sequence sets with only 33% of X box regulated genes. Using the reported X box consensus, I successfully identified additional genes, like the C. elegans orthologue to human BBS4. OrBS was less successful in the identification of the other C. elegans regulatory elements, such as the PHA-4 binding site and the UNC-86 binding site.

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

NMR studies of membrane-binding peptides in monoolein cubic phases

Date created: 
2005
Abstract: 

Membrane proteins are of particular interest to structural biologists since it is believed that these proteins comprise approximately thirty percent of the proteins encoded for by genomes. Despite the biological significance of these proteins, the atomic reso1utio:n structures of membrane proteins are being solved at a much slower rate than their soluble counterparts. This is primarily due to difficulties that arise in maintaining the lipid interactions required to retain the structural and functional integrity of the proteins during structural studies. One: approach to resolving this problem would be to develop alternative membrane-mimetic environments for structural studies of membrane proteins. The cubic phases formed by mixtures of lipids and water have been identified as such an environment. In recent years cubic phases have been used to crystallize membrane proteins for structural studies using X-ray crystallographic techniques. This application of cubic phases, along with information provided from previous studies, indicated that cubic phases possessed the necessary properties to make them ideal membrane-mimetic environments for solution NMR studies of membrane proteins. I investigated the suitability of the cubic phases formed by mixtures of monoolein and water as membrane-mimetic environments for the solution NMR study of incorporated membrane proteins. For my studies I used two transmembrane peptides (WNALAAVMAAVA&AAGKSKSKS and alamethicin), and one membrane surface-associating peptide (methionine-enkephalin), as models of membrane proteins. In the NMR spectra collected on the transmembrane peptides, only the residues found in the interfacial regions of the cubic phase were observed, whereas all of the residues of the membrane surface-associating peptide were observed. In order to gain insights into the behaviour of the incorporated peptides, the diffusion rates of lipid, peptide and water molecules were measured in peptide-containing cubic phases using :solution NMR techniques. The data that I have collected provide the first examples of solution 'NMR spectra collected on peptides incorporated into a lipid cubic phase. The NMR spectra that were obtained suggest that the monoo1ein:water cubic phase may be a suitable membrane-mimetic environment for the study of membrane surface-associating peptides and proteins, and the inter-helical loops of integral membrane proteins.

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

The interferon-inducible protein 16 is a single-stranded nucleic acid binding protein with unwinding and endonuclease activities

Author: 
Date created: 
2010
Abstract: 

IFI16 belongs to a protein family (HIN200) that contains a characteristic 200-amino-acid protein domain with unknown function. Members of this family were initially described as transcriptional regulators but IFI16 is also involved in other processes such as DNA repair and programmed cell death. To gain further insights into the biochemical mechanisms underlying these functions we made bioinformatics predictions followed by biochemical validation. We predicted and verified experimentally that the 200-amino-acid domain of IFI16 is structurally and functionally similar to replication protein A (RPA), a protein that removes secondary structure on single-stranded DNA during DNA replication and repair. In addition, I found that IFI16 can unwind double-stranded DNA and has an endonuclease activity. This later unexpected discovery reveals a new function for IFI16. Finally, I further discuss the implications of this discovery in regard to the current knowledge about the function of this protein as well as new directions of studies.

Document type: 
Thesis
File(s): 
Supervisor(s): 
D
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)