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

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Characterization of HAM-1 in the asymmetric division of C. elegans neuroblasts

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

HAM-1 (HSN abnormal migration) is a novel protein implicated in asymmetric cell division (ACD) of many C. elegans neuroblasts. HAM-1 is localized at cell peripheries and becomes asymmetrically distributed during mitosis. However, little is known about how it regulates ACDs. I have analyzed a series of protein truncations to determine sequences important for HAM-1 function and localization. Results indicate that the C-terminus is essential for function and N-terminal sequences are required for peripheral association. HAM-1 was also observed in the nucleus, a finding not previously reported. Deletion of a predicted nuclear localization signal partially disrupted nuclear targeting and function of the protein. This and other bioinformatics analyses suggest a possible nuclear role for HAM-1. Previous studies have proposed potential interactions between HAM-1 and the Wnt pathway. In this study, mutations in ham-1 and several Wnt signalling components were shown to produce opposing defects in the lineage generating the PLM neuron.

Document type: 
Thesis

Cofactors and co-chaperones of the chaperonin CCT: mechanistic insights and in vivo relevance

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

Protein folding is the essential process by which a linear chain of amino acids folds upon itself to adopt a defined three-dimensional structure. All proteins must undergo folding to be functional and while the amino acid sequence dictates the tertiary structure, in the crowded cellular environment the folding process is not always spontaneous. To circumvent this problem proteins called molecular chaperones have evolved to stabilize non-folded polypeptides and facilitate their transformation to the folded state. This thesis revolves around a molecular chaperone called CCT, which uses a barrel-shaped structure to bind non-native proteins and sequester them in a protected environment to allow folding. My work has focused on two proteins that co-operate with CCT in the cell to promote efficient folding of its substrates. The first, called prefoldin (PFD), is another molecular chaperone that binds non-native polypeptides and delivers them to CCT for completion of folding. The second is a family of proteins called phosducin-like proteins (PhLPs), which bind CCT and affect its ability to fold substrates. Here we show that PFD uses long coiled-coil tentacles to grasp substrates using interhelical-hydrophobic residues at the very tips. We find that the general properties of a coiled-coil are sufficient to confer some chaperone activity indicating the importance of this super-secondary structure to PFD function. We also find that archaeal PFD can alter its shape to accommodate substrates of different sizes, but that in most cases a large proportion of the substrate protrudes from the PFD cavity. We also show that the mechanism of PhLP-mediated CCT regulation involves PhLP binding to CCT-substrate complexes and slowing of ATP hydrolysis. In yeast the PhLP homologues Plp1p and Plp2p both affect cytoskeletal function but Plp2p, which is essential, also appears to affect the cell cycle. Finally, we use a genomic approach to suggest novel cellular roles for the chaperonin CCT in pathways such as septin ring assembly. Altogether these studies illuminate the role of CCT co-chaperones (PFD) and cofactors (PhLP) in modulating the chaperonin’s function and open up new research prospects by identifying novel genetic interactors of CCT.

Document type: 
Thesis

DNA Helix-stack Switching as the Basis for the Design of Versatile Deoxyribonsensors

File(s): 
Date created: 
2004
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)
Abstract: 

The charge conduction properties of deoxyribonucleic acids (DNA) can be harnessed for monitoring the binding of a ligand to its receptor. Here, we show how DNA-based sensors (deoxyribosensors) consisting of a photo-activated oxidant tethered to a receptor-containing DNA molecule can be used to monitor the presence of a ligand. Phosphorescence measurements, reflective of changes in charge conduction to a targeted region in these deoxyribosensors are made in the presence and absence of the test ligand. The deoxyribosensors described here, exploit established rules for DNA helical stacking in three-way junctions and use previously selected aptamer sequences as receptors for target analytes. More specifically, a systematic investigation outlining the characteristics of a deoxyribosensor for the detection of the amino acid derivative, argininamide are presented. These results suggest a general design for deoxyribosensors for any molecular ligand for which an aptamer sequence can be obtained. Also, a new methodology for investigating helical stacking of nucleic acids of unknown tertiary structure, such as DNAzymes or RNAzymes is discussed.

Document type: 
Thesis

In-vitro selection of charge conducting DNA sequences

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

Double helical DNA transports electrical charge, via hole transport, over distances over 2008, (58 base pairs). Because the exact mechanism of hole transport is under debate, an alternative method to the current 'rational' design approach used by investigators may shed new light on the problem. In vitro selection was used to isolate DNA sequences most efficient at hole conduction out of a random pool. Several selections were carried out, each providing valuable information for subsequent selection design. Tests carried out on the Round 7 of the SYB series confirmed that selection superior conductors was indeed occurring. 93 clones sequenced from Round 7 were analyzed against 89 clones from the initial round and found to have statistically elevated levels of specific purine motifs segregated to only one specific strand of the duplex. These sequences, termed 'transport permissive candidate motifs', suggested that Phononassisted Polaron-like hole hopping occurs in duplex DNA.

Document type: 
Thesis

Computational prediction and comparative analysis of protein subcellular localization in bacteria

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

Predicting the subcellular localization of a protein is a critical step in processes ranging from genome annotation to drug and vaccine target discovery. Previously developed methods for localization prediction in bacteria exhibit poor predictive performance and are not conducive to the high-throughput analysis required in this era of genome-scale biological analysis. We therefore developed PSORTb, a hig h-precision, hig h-throug hput tool for the prediction of bacterial protein localization. PSORTb implements a multi-component approach to prediction, incorporating the detection of several sequence features known to influence subcellular localization. With a reported overall precision of 96%, it is the most precise method available and one of the most comprehensive methods capable of assigning a query protein to one or more of four Gram-positive or five Gram-negative localization sites. The PSORTb algorithm comprises a series of analytical steps, each step - or module - being an independent piece of software which scans the protein for the presence or absence of a particular sequence feature. Modules include: SCL-BLAST for homology-based detection, the HMMTOP transmembrane helix prediction tool, a signal peptide prediction tool, a series of frequent subsequence-based support vector machines, as well as motif and profile-matching modules. The modules return as output either a predicted localization site or - if the feature is not detected - a result of unknown'. The output is then integrated by a Bayesian network into a final prediction. Development of PSORTb also required the creation of PSORTdb, a database storing both known and predicted localization information for bacterial proteins. This is a valuable resource to both the localization prediction and microbial research communities, providing a source of training data for new predictive algorithms and acting as a discovery space. The release of PSORTb v.2.0 allowed us to carry out a number of analyses related to localization. We performed the first genome-wide computational and laboratory screen for Nterminal signal peptides in the opportunistic pathogen Pseudomonas aeruginosa, used PSORTb as a complement to laboratory-based high-throughput 2D gel studies of individual cellular compartments, and examined protein localization in a global context, revealing trends with implications for adaptive evolution in microbes.

Document type: 
Thesis

Repetitive landscape of the Atlantic salmon genome

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

The duplication of genes and genomes is considered a major force in evolution. Ohno, in 1970, theorized that with two copies of the genome, one copy would be under normal evolutionary constraints while the other copy could serve as raw material for evolution. Salmonids are a classical example of species containing duplicated genomes, and they offer an opportunity to investigate how such genomes undergo reorganization as they attain a stable diploid state. Repetitive elements play an important role in genome reorganization. Therefore, I investigated the repeat structure and organization of the Atlantic salmon genome. An analysis of the fingerprinted CHON-214 BACs classified the singletons, or those that contained few restriction sites, into three categories of repetitive structures. The first group contains histone genes in a tandemly repeating cluster of H4 - H2B - H1 - H2A - H3. A second group contains the ribosomal DNA (rDNA) cistron. Intriguingly, fluorescent in situ hybridization (FISH) analysis indicated that one of each of the duplicated histone and rDNA clusters was lost or rearranged in the genome. The remaining group of BACs contains novel repetitive sequences and tRNA clusters. The Atlantic salmon EST libraries and BAC-end sequences were data-mined for simple sequence repeats (SSRs) and 2,497 SSRs were recovered. 154 SSR loci gave clean PCR amplicons and 94 produced polymorphic banding patterns with eleven of the EST-SSRs indicating duplicated loci. These loci were mapped on the Atlantic salmon linkage map. Novel repetitive elements were detected in the sequences of Atlantic salmon BACs and ESTs. Using computational tools for data-mining, repetitive elements were identified and classified based on sequence similarity to other known repetitive elements such as SINES, LINES and retrotransposons. A repeat database that can be used to mask repetitive elements, is now available for the Atlantic salmon genome. A website was developed to host the Atlantic salmon linkage and physical maps, correlating them based on marker hybridization. A BAC annotation pipeline analyzes the BAC sequences for ORFs, transcript similarity and repetitive elements. Information generated from sequence annotation, microsatellite development and repetitive element identification provides essential resources for investigating salmonid genomes.

Document type: 
Thesis

Evolution of mitochondrial DNA in the genus Salmo

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

The mitochondrial genome (mtDNA) is a valuable source of data for evolutionary studies because of its small size, lack of recombination and its higher rate of accepted mutations than nuclear coding sequences. All salmonid rnitochondrial genomes are -16.7 Kb in size and identical in their genome organization. PCR amplification with thirty-three conserved primer pairs and subsequent direct sequencing was used to obtain whole mitochondrial genome sequences from fourteen Atlantic salmon (Salmo snlar) samples. The nucleotide and amino acid sequences were aligned and compared with those of a sister species, brown trout (Salmo trutta) to study the mode and tempo of mtDNA evolution. Varying percent sequence divergence was observed in different parts of the genome suggesting that different constraints operate across the genome. Further, by measuring the amount of variation in the Atlantic salmon from different geographical locations, previous hypotheses regarding the structuring of Atlantic salmon populations were confirmed.

Document type: 
Thesis

Pinguid, the drosophila homolog of huntingtin interacting protein 14, encodes an essential protein involved in TGF beta signalling

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

To elucidate the cellular function of Huntingtin interacting protein 14 (Hipl4), a genetic approach using Drosophila was initiated. The gene encoding Pinguid was identified as the closest Drosophila homolog to human Hip14 with 44% identity and 59% similarity. A P-element insertion in the 5' UTR of pinguid was used for carrying out a transposase mediated P-element excision screen. Subsequent genetic and molecular experiments confirmed that pinguid encodes an essential gene. Five pinguid alleles were identified: three excision alleles and two EMS-induced alleles. No visible defects were observed in the mutants since the morphology and patterning of the embryos appeared normal, CNS development appeared wild type, and pharate adults had no visible external defects. However, the ectopic expression of pinguid identified genetic interactions with components of the TGFP signalling pathway. Strong genetic interactions were seen with short gastrulation, crossveinless 2, baboon, and the ecdysone receptor.

Document type: 
Thesis

Analysis of antibody reactivities in lysozyme-immunized mice with peptide ligands: A model for epitope-targeted vaccine design

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

Many viruses evade antibody (Ab)-mediated clearance through the presentation of immunodominant, highly variable epitopes, and the masking of conserved ones. It has been proposed that vaccines incorporating peptides that cross-react with conserved epitopes could be used to target the production of Abs that protect against a range of viral isolates. For this thesis the concept of epitope-targeted vaccine design is explored with the model protein hen egg lysozyme (HEL). The goals of the work are to develop peptide markers for murine anti-HEL Abs that can also be used as immunogen to amplify Ab production against target HEL epitopes. Three types of peptides are analyzed: (i) a peptide selected from a phagedisplayed random peptide library (RPL) with the anti-HEL monoclonal Ab D1.3, (ii) peptides identified from RPLs with polyclonal (pc) Abs from the sera of HEL-immunized mice, and, (iii) peptides derived from the linear sequence of HEL. A peptide selected with Dl .3 Ab shares critical binding residues in common with its cognate discontinuous epitope but showed no reactivity with anti-HEL sera. A prime-boost immunization study, in which mice were primed with HEL to produce anti-HEL pcAbs, and boosted with the peptide to amplify the production of Dl .3 or Dl .3-like Abs, did not yield crossreactive Abs. Thus, Dl .3 Ab may not have been produced in the priming immunization because it is a rare specificity in the immune response to HEL. A peptide marker for an anti-HEL Ab commonly produced amongst mice was identified in a RPL screening with anti-HEL pcAbs. The commonly reactive peptide was also used in ELISPOT analysis to characterize epitope-specific B cell responses to HEL; the peptide-reactive cells represented less than 1% of IgG-producing B cells. The peptide was not recognized by anti-HEL IgG from rabbit, however, emphasizing the limitations of using animal models for developing epitope-targeted vaccines. Lastly, a set of 10- mer overlapping HEL peptides showed common patterns of reactivity with different murine anti- HEL sera. These findings indicate that common HEL epitopes are targeted amongst mice in the humoral immune response to HEL and that a prime-boost immunization strategy may succeed with a commonly reactive peptide marker.

Document type: 
Thesis

A serotonin-dependent deoxyribozyme that uses light to repair thymine dimers in DNA

Author: 
File(s): 
Date created: 
2005
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)
Abstract: 

Thymine dimers are the most common lesion formed in DNA as a result of exposure to ultraviolet (UV) light. Naturally occurring protein enzymes are known which repair these lesions in different ways. It was hypothesized that in early Earth it may have been RNA that played the functional role, which proteins play today. Also in early Earth it was suspected that the intensity of UV radiation was strong, which could produce dinier products within RNA. A previous in vitro selection has been performed to determine whether DNA was capable of catalyzing a photochemical reaction to reverse this damage. This selection yielded two different deoxyribozymes able to catalyze thymine dimer repair in DNA. The deoxyribozyme SerolC, the topic of this thesis, uses light and serotonin as a cofactor to repair the thymine dimers. Characterization of SerolC provided hypotheses for structure, the proposed reaction mechanism, and aspects of substrate specificity.

Document type: 
Thesis