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

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Homology and evidence-based genome annotation of Caenorhabditis species

Author: 
Date created: 
2010-12-02
Abstract: 

I have re-annotated the genomes of four sister species of the model organism Caenorhabditis elegans: C. briggsae, C. remanei, C. brenneri, and C. japonica, using a homology-based gene predictor genBlastG. Compared to the existing gene sets of these four species, genes of the revised gene sets demonstrate higher conservation with their orthologs in C. elegans as well as more ortholog pairs. I have validated and further revised the C. briggsae gene set, through next-generation short read sequencing of the transcriptome. In the revised C. briggsae gene set (23,159 genes), 7,347 genes (33.9% of all genes with introns) have all introns confirmed. Of all introns in the revised gene set (103,083), 62,727 (60.9%) are confirmed. Additionally, I have identified 1,034 operons in C. briggsae, with 532 (51.4%) perfectly conserved in C. elegans. This study sets up a solid platform for comparative genomics analysis and evolutionary studies of Caenorhabditis species.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Jack Chen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Role of sulfated glycosaminoglycans and cell-associated proteoglycans in oral-aboral patterning and gastrulation of the Strongylocentrotus purpuratus urchin embryo

Date created: 
2010-11-09
Abstract: 

Glycosaminoglycans are a heavily sulfated component of the extracellular matrix (ECM) implicated in a variety of cell signaling events involved in patterning of embryos. Embryos of the sea urchin Strongylocentrotus purpuratus were exposed to several inhibitors that disrupt glycosaminoglycan function during development. Treatment with chlorate, a general inhibitor of sulfation that leads to undersulfated glycosaminoglycans, reduced sulfation of the urchin blastocoelar ECM. It also prevented correct specification of the oral-aboral axis and mouth formation, resulting in a radialized phenotype characterized by the lack of an oral field, incomplete gastrulation and formation of multiple skeletal spicule rudiments. Oral markers were initially expressed in most of the prospective ectoderm of chlorate-treated early blastulae, but then declined as aboral markers became expressed throughout most of the ectoderm. Nodal expression in the presumptive oral field is necessary and sufficient to specify the oral-aboral axis in urchins. Several lines of evidence suggest a deregulation of Nodal signaling is involved in the radialization caused by chlorate. Radial embryos resemble those in which Nodal expression was knocked down. Chlorate disrupted localized nodal expression in oral ectoderm, even when applied after the oral-aboral axis is specified and expression of other oral markers is resistant to treatment. Intriguingly, treatment of embryos with a sub-threshold dose of a Nodal receptor inhibitor rescued the radialization caused by low concentrations of chlorate.Glycosaminoglycans are typically found in association with core proteins as proteoglycans. Three cell-associated proteoglycans with possible roles in the regulation of cell signaling events leading to oral-aboral axis specification were identified in the urchin genome. These proteoglycans of the syndecan and glypican families are expressed in distinct but overlapping radially symmetrical patterns in the urchin embryo. Chlorate treatment upregulated proteoglycan expression, especially of glypicans. Our results indicate important roles for sulfated glycosaminoglycans (and possibly proteoglycans) in Nodal signaling and oral-aboral axial patterning, and in the cellular processes necessary for archenteron extension and mouth formation during gastrulation. We propose that interaction of the Nodal ligand with sulfated glycosaminoglycans limits its diffusion, and is required to specify an oral field in the urchin embryo and organize the oral-aboral axis.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Bruce P. Brandhorst
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Mechanosensory cells and swimming behaviour of embryos of the sea urchin Strongylocentrotus purpuratus.

Author: 
Date created: 
2010-08-18
Abstract: 

Recent sequencing of the genome of the sea urchin Strongylocentrotus purpuratus identified Usher syndrome genes involved in photo- and mechanosensation in vertebrate retina and inner ear as well as the TRPA1 ion channel often involved in mechanosensation. Sensory cells have not been definitively identified in sea urchins despite the known photo- and mechanosensory capabilities of adults, larvae and embryos. To identify sensory cells in embryos and early larvae expression of Usher and TRPA1 genes was investigated, but did not identify candidate sensory cells. Some Usher genes were expressed in neurogenic ectoderm of embryos. Screening for cells having putative mechanosensory channels was performed using cationic fluorescent dyes and identified several types, including blastocoelar cells. Temporal expression of the TRPA1 gene and inhibition of swimming activity by ion channel inhibitors suggest its involvement in control of swimming. This study provides evidence that mechanosensory channels modulate swimming behaviour of embryos and early larvae.

Document type: 
Thesis
Supervisor(s): 
Bruce P. Brandhorst
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Application of next-generation sequencing for comparative transcriptome analysis

Author: 
Date created: 
2010-06-14
Abstract: 

I have used novel whole transcriptome sequence data generated from massively parallel high-throughput next generation sequencing technologies, namely 454 pyrosequencing and Illumina sequencing, to perform comparative transcriptome analyses of C. elegans populations in specific biological conditions and developmental stages. Firstly, I have conducted transcriptome profiling of C. elegans in its first larval (L1) stage using data generated from the Roche 454 sequencing platform. I have used this data to refine gene structures, identify putative novel transcripts, and characterize the L1 specific transcriptome. Secondly, I have taken Illumina paired-end sequencing transcriptome data generated from starved and fed populations of C. elegans in the L1 stage and investigated metabolic and developmental gene expression changes that are specific to this developmental stage. Additionally I have taken advantage of this paired-end data to perform de novo assembly of the short tag sequences to identify novel transcript structures with confirmed splice sites, providing greater confidence in novel gene structure discovery. Lastly, I have investigated global transcriptional responses to oxidative stress in aging defective aak-2 mutants. This analysis has led to the identification of potential downstream targets of the AMP-activated protein kinase, AAK-2, which is known to be involved in stress resistance and lifespan control in C. elegans. This study demonstrates the impact of new high-throughput sequencing technologies on genomic analysis, providing valuable insight into transcriptome complexity as a result of developmental, metabolic, and mutational effects in C. elegans. The approaches I have described here can be applied to transcriptome analyses of more complex organisms ultimately leading to investigation of transcriptional changes in human diseases.

Document type: 
Thesis
Supervisor(s): 
David Baillie
Steven Jones
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Mechanism of silencing the catalytic domain by the regulatory membrane lipid binding domain of an amphitropic cytidylyltransferase

Date created: 
2011-06-01
Abstract: 

CTP: phosphocholine cytidylyltransferase (CT) is an auto-regulated, homodimeric enzyme that catalyzes the rate-limiting step in the synthesis of phosphatidylcholine (PC). CT is activated by binding to PC-depleted membranes using a lipid-induced amphipathic helix (domain M). This same M domain functions to silence catalysis when the enzyme is not membrane-engaged. While the structure of CT’s catalytic domain has recently been solved (PDB, 3HL4), if and how domain M makes contact with it to silence catalysis remains a mystery. To identify contact sites between domain M and other CT domains, I constructed single cysteine substitutions along domain M and conjugated each site to a sulfhydryl-reactive, biotinylated, benzophenone, BBP. After photo-cross-linking and trypsin digestion, the cross-linked peptides were affinity-purified and identified by mass spectrometry. Each domain M-conjugated site forged cross-links to the same set of catalytic domain peptides, which flank one side of the active site, and these contacts were broken upon membrane insertion of domain M. I then targeted the most conserved region in domain M for mutagenesis. Mutation of F289 and F293 to aspartates obliterated cross-links between the catalytic domain and the BBP-labeled site at neighboring Cys-301. However, loss of this contact did not relieve inhibition of catalysis, suggesting that this site is but one of several cooperating inhibitory segments in the M domain. Evidence for a partially disordered domain M which acquires order upon contact with the catalytic domain was obtained by fluorescence anisotropy, monitoring Oregon Green conjugated to sites in three distinct CT domains. All domain M sites had similar anisotropy values intermediate between the rigid catalytic domain and the very flexible C-terminal tail. Urea denaturation analysis suggested a weakly folded structure for domain M. Breaking a contact between M and C by the F → D mutation dramatically lowered the anisotropy of that sub-region and its resistance to unfolding by urea. These results support an emerging model in which silencing by domain M is achieved through multiple transient, alternating, contacts with the catalytic domain. Contacts with the catalytic domain may introduce ordering into an otherwise flexible domain M. These inhibitory contacts are replaced with protein-lipid interaction upon activation.

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

Structural and catalytic properties of DNA/RNA-heme complexes

Date created: 
2011-05-26
Abstract: 

DNA and RNA G-quadruplexes have been shown to catalyze 1-electron oxidation reactions when bound to heme. In this work, we set out to explore the fundamental nature of G-quadruplexes that allow for heme binding, and the potential of nucleoheme complexes in catalyzing other heme-related enzymatic reactions. A combination of spectroscopic and gel-based tools was used to observe structural features of nucleoheme complexes. We determined that the in vitro selected heme apatamer, PS2.M, is highly polymorphic and predominantly parallel-stranded. The ability of many parallel G-quadruplexes to bind heme and perform peroxidase reactions led us to believe that some nucleoheme complexes may be capable of catalyzing oxygen transfer or 2-electron oxidation reactions. By using gas chromatography and high performance liquid chromatography analysis, we determined that nucleoheme complexes were capable of oxidizing electron-rich substrates: thioanisole, indole, and styrene. This discovery has expanded the catalytic repertoire of nucleic acids, which leads to questions about the consequences and potential of this catalytic ability.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Dipankar Sen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Mapping putative regulatory regions using Histone H3 Lysine 4 monomethylation marks in breast cancer cell lines.

Date created: 
2011-02-23
Abstract: 

Breast cancer is the most frequently diagnosed cancer in women. In cancer, tumour cells accumulate changes over time that allow them to replicate indenitely. These changes can be mutations to DNA and also epigenetic modications. This study looks at a histone modication, H3K4me1, in multiple breast cancer cell lines. It has been found that the regions between flanking H3K4me1 peaks, referred to as "valleys", are enriched for bound transcription factors. Multiple cell lines were used to form functional groups (luminal vs. basal cell lines and tumourigenic vs. a non-tumourigenic match control) in which to look for concordance of valleys. In addition, overexpressed genes in a functional group, as determined by RNA-seq, were correlated with associated uniquely marked valleys. A motif analysis was done on the valley sequences using MEME and STAMP to yield putative transcription factor binding sites. This analysis yielded some known and putative tumour suppressors and oncogenic factors.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Steven Jones
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Molecular identification of let-336 and analysis of ionizing radiation induced lethal mutations within caenorhabditis elegans eT1(III;V)

Date created: 
2011-04-20
Abstract: 

let-336 was one of 194 EMS induced lethal mutations generated within the eT1(III;V) balanced region on the left arm of Caenorhabditis elegans LGV. Using data obtained from complementation tests with the deficiency strain hDf36, sequencing candidate genes within this region, as well as performing a fosmid rescue, I determined the molecular identity of let-336 to be F56E10.4 (rps-27). Using eT1(III;V) as a mutational capture system, I screened 13,218 individuals and determined the spontaneous lethal mutation rate within the balanced region to be 0.03%. Furthermore, I analyzed the lethal mutation response of C. elegans when exposed to low doses of ionizing radiation. Exposure to a cumulative low dose of X-ray radiation was shown to produce less mutations when compared to an equivalent single high dose exposure. Increasing the time between exposures, irradiating dauers, as well as administering a low dose before a second high dose resulted in less lethals being produced.

Document type: 
Thesis
File(s): 
Supervisor(s): 
David Baillie
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

The regulation and function of Hu-li tai shao (Hts) at the Drosophila neuromuscular junction

Date created: 
2011-04-15
Abstract: 

Hu-li tai shao (Hts) is the Drosophila homolog of mammalian adducin, a cytoskeletal protein that regulates the submembranous actin-spectrin network. Potential upstream regulatory proteins that alter the distribution or function of Hts at neuromuscular junctions (NMJ) were evaluated, along with putative interacting proteins and phosphoinositides. Muscle-specific RNAi knockdown of the regulatory PKA subunit or conventional PKC altered immunoreactivity against phosphorylated Hts at the NMJ, suggesting that these kinases are involved in Hts phosphorylation. Hts was required for proper assembly of the spectrin cytoskeleton at the NMJ, as changes in hts expression levels strongly disrupted alpha-Spectrin organization. Hts immunoreactivity colocalized with a GFP reporter for phosphatidylinositol-(4,5)-bisphosphate, which Hts could potentially interact with via its conserved MARCKS-homology domain. The transmembrane engulfment receptor draper genetically interacted with hts. These results highlight many avenues by which Hts may be exerting its influence on NMJ development, and open up worthwhile possibilities for future studies.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Charles Krieger
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

The regulatory domain of CTP: phosphocholine cytidylyltransferase (CCT): structure, membrane interactions, and similarity to α-synuclein.

Author: 
Date created: 
2011-03-31
Abstract: 

α-Synuclein, a synaptic vesicle protein whose mis-folding is linked to Parkinson’s Disease, and CTP:phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme in phosphatidylcholine synthesis, share a common structure and function. Both contain lipid-inducible, amphipathic helical (AH) membrane binding motifs followed by unstructured acidic tails. Circular dichroism and tryptophan fluorescence studies comparing membrane-binding properties showed that α-synuclein has weaker affinity for anionic phospholipids than CCT tail regions, and a more stringent lipid compositional selectivity for binding. This may reflect the lower propensity of α-synuclein for helical conformation. However, both proteins can penetrate the lipid bilayer to a similar depth. A phospho-mimic version of the CCT-tail had lower α-helicity than wildtype CCT upon membrane binding, suggesting phosphorylation affects the secondary structure of the adjacent AH domain. These studies provide a foundation for exploring the usefulness of CCT-tails to promote the α-helical character of α-synuclein, thus promoting its non-toxic folding pathway.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Rosemary Cornell
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.