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

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The characterization of Lactate Dehydrogenase genes in rainbow smelt (Osmerus mordax)

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

Lactate Dehydrogenase isozymes (LDH-A, LDH-B and LDH-C) represent the classical example of a multi-gene system derived by successive gene duplications. By investigating the genes encoding the LDH isozymes in rainbow smelt, a diploid out-group of the tetraploid salmonids, I sought to gain insight into the effect of a whole genome duplication superimposed upon more ancient gene duplications. I isolated rainbow smelt BAC clones containing the LDH-A, LDH-B and LDH-C genes, made shotgun libraries of three representative BACs and annotated the sequences. I characterized the smelt LDH genes with respect to structure, tissue expression and genome organization. This information was used for comparative genomic analyses with the LDH genes from Atlantic salmon. There was no evidence for positive selection, an expectation of neo-functionalization, but different rates of amino acid substitutions between and within lineages were evident in the LDH-A and LDH-B salmonid duplicates. LDH-B1 and LDH-B2 in salmonids have experienced sub-functionalization.

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

Crystallographic analysis of the regulatory domain of human cardiac troponin C

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

The N-terminal regulatory domain of cardiac troponin C (N-cTnC) is an essential Ca2+ sensor to trigger muscle contraction. Using X-ray crystallography, the first crystal structure of wild type human N-cTnC was solved to 2.2 Å resolution. It revealed two novel features about this protein. First, each EF-hand binding loop coordinates two cadmium ions, even though one of the loops is normally inactive. One Cd2+ ion occupies the canonical ion binding site, whereas the other Cd2+ ion binds adjacent to it. Thus, N-cTnC is forced to adopt a very unusual conformation. Second, an additive, deoxycholic acid (DXC), was observed bound to the central hydrophobic cavity and between helix N and helix A. DXC has a similar chemical structure to Ca2+-sensitizing drugs that are used for treating troponin-related cardiovascular diseases. These new insights provide valuable information for the use of N-cTnC as a potential target for rational drug design.

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

Structural characterization of an RNA polymerase ribozyme and functional improvement by in vitro encapsulated selection

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

At the heart of the “RNA World” hypothesis is the requirement for an RNA polymerase ribozyme capable of replicating itself and other RNA molecules. The most recent variant, isolated from multiple generations of in vitro selection, is the B6.61 RNA polymerase ribozyme that is capable of extending an RNA primer in a template-dependent fashion by 20 nucleotides. It contains a 5' ligase core domain that confers phosphodiester bond formation ability and a poorly understood 3' accessory domain that is proposed to attribute its polymerization ability. In my research presented in this thesis, we study the structure of the accessory domain of B6.61 by mutagenesis, chemical probing and crosslinking experiments and provide molecular constraints that are useful for modeling the three-dimensional structure of the ribozyme. Secondly, I have used in vitro encapsulated selection in an attempt to improve the overall processivity of B6.61.

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

Epitope-targeting strategy for a vaccine against human immunodeficiency virus type-1; peptide ligands for the broadly-neutralizing antibodies b12, 2F5 and 2G12

Author: 
Date created: 
2005
Abstract: 

The research presented here is part of an ongoing effort to develop an epitopetargeted vaccine against the Human Immunodeficiency Virus type 1. We have used phage-displayed peptide libraries, in conjunction with other biochemical and structural techniques, to generate and characterize peptides specific for the broadly-neutralizing, human monoclonal antibodies (Mab) b12,2F5 and 2G12. First, peptide ligands for Mab b12 were isolated. One peptide, B2.1, was characterized in detail. The crystal structure of B2.1 in complex with b12 Fab was solved and compared with a model of the antibody docked to gp120. B2.1 was used to immunize mice, seeking to generate a b12-like, neutralizing antibody response. Minimal structural resemblance between the putative b12 epitope and B2.1 peptide was observed, indicating different mechanisms of binding to b12. Moreover, the anti B2.1 response from the immunized mice did not cross-react with gp120, further indicating this difference. Second, we demonstrate that Mab 2F5, which binds to the sequence ELDKWA on gp41, selects peptides with multiple, different amino acid sequences flanking its threeresidues DKW native core epitope. High-affinity binding was achieved by peptides with unrelated flanking sequences C-terminal to the core epitope, with the nature and location of critical binding residues differing between peptides. Our results implicate multiple mechanisms of binding, and suggest the existence of two distinctive functional regions in the 2F5 paratope, one that is DKW-specific and one that is multi-specific. Finally, a peptide ligand specific for the carbohydrate-binding Mab 2G12 was isolated, characterized and optimized. The peptide competes with carbohydrate ligands for binding to 2G12. The crystal structure of the 2G12-peptide complex, and its comparison with the 2G 1 2-Man9GlcNAc2 complex, shows that the peptide is not a mimic of Man9GlcNAc2. They occupy separate sites in the antibody's paratope, with minimal overlaping, therefore the peptide and Man,GlcNAc, make a different set of contacts with the Mab. In summary, we have observed that peptide mimicry of the b12,2F5 and 2G12 epitopes is essentially functional, not structural. Our results indicate that these ligands can be used as antibody-specific markers, and that most likely, conventional immunizations with so-called peptide mimics will not elicit these antibodies.

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

Ribozyme structure studied by recombination based evolution and biochemical methods

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

Ribozyme (catalytic RNA) evolution by in vitro selection is one of the powerful evolutionary systems used to study fundamental mechanisms of molecular evolution. In Chapter 1, I discussed the existence of ribozymes in random pools and showed that step-wise pathways, in which mutation and recombination were fundamental evolutionary mechanisms, were more likely applied to evolve complex ribozymes. In chapter 2, I reviewed some types of mappings between RNA sequence space, shape space and function space, together with some common methods used to discover them. RNA modularity, which was discovered in many natural and artificial ribozymes, was the intrinsic architecture for the evolutions of complex ribozymes by recombination. In Chapter 3, cooperating with Leslie Cheng, I revealed the modularity of B6.61, one of the RNA polymerase ribozymes that might have played critical roles in a putative “RNA World”. We found that the two modular domains of B6.61, the ligase core and the accessory domain, could cooperate in cis, in trans, and when they were hybridized with distinct orientations. We also determined the core motif sequences, the important structure-elements of the accessory domain, and its tertiary interactions with the ligase core by chemical probing, mutagenesis and crosslinking studies. Some three-dimensional models of the accessory domain were then built up for the initial attempt to map the structure-function relationship of B6.61. In Chapters 4 and 5, pre-existing RNA motif sequence(s) were randomly reassembled into nonhomologous random recombination (NRR) pools. In Chapter 4 a NRR pool with a diversity of 108 was generated from a nucleotide synthase ribozyme. The pool contained molecules with insertions, deletions, translocations, and inversions. A following in vitro selection quickly isolated the same functional ribozymes with a broad range of length and led to the rapid unbiased discovery of their core motif by phylogenetic analysis. In Chapter 5, a similar NRR pool derived from eight nucleotide synthase ribozymes was generated and subjected to in vitro selection for RNA ligase ribozymes, resulting in a new but uncharacterized functional RNA. Both studies in Chapters 4 and 5 experimentally demonstrated the power of NRR in shaping the evolution of functional RNAs.

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

Molecular identification of let-56 and characterization of its regulatory elements in Caenorhabditis elegans

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

let-56 was generated by EMS mutagenesis in a screen for essential genes in the unc-22(IV) region of C. elegans. The let-56 locus is the largest developmentally required mutagenic target in the area. By sequencing seven of twelve alleles, I have determined the molecular identity of let-56 to be ZK829.4, encoding glutamate dehydrogenase. GDH catalyzes the reversible formation of α-ketoglutarate and ammonia from glutamate, and mutations in GDH are associated with hyperinsulinism/hyperammonemia syndrome in humans. To gain better understanding of GDH activity, I have investigated mechanisms of its regulation. By analyzing let-56’s 5’ promoter region, I have identified a 31bp window containing a transcription factor binding site. Using chimeric GFP reporter constructs, I have also demonstrated the importance of let-56’s 3’ UTR in downregulating gene expression. In addition, by mimicking a mutation found in individuals with HI/HA, I have shown that C. elegans GDH also plays a role in insulin regulation.

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

DNA microarrays on agarose-coated glass slides for plant pathogen identification

Author: 
Date created: 
2005
Abstract: 

Agarose-coated glass slides were used as a platform for microarray analysis applied to plant pathogen identification. The agarose substrate combines the desirable features of fluorescence detection and high DNA immobilization capacity, in contrast to nylon membranes, which are unsuitable for fluorescent detection, and standard glass microarray slides, which have a low capacity for immobilization. Oligonucleotide probes were immobilized on the agarose substrate, then hybridized to fluorescently labeled sample DNA. Agarose concentration and hybridizing DNA length affected hybridization efficiency. Probes arrayed on the agarose distinguished Didymella bryoniae and Botrytis cinerea from each other with no cross reaction. No interference from other common greenhouse plant pathogens was found. Results compared favorably with those obtained on nylon membranes, and surpassed those achieved on the commercially available glass substrate. Agarose-coated slides are easily produced, and with the use of a manual arrayer, are an inexpensive alternative to commercial microarrays for small scale applications.

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

Computational prediction and characterization of genomic islands: insights into bacterial pathogenicity

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

Genomic islands (GIs), including pathogenicity islands, are commonly defined as clusters of genes in prokaryotic genomes that have probable horizontal origins. These genetic elements have been associated with rapid adaptations in prokaryotes that are of medical, economical or environmental importance, such as pathogen virulence, antibiotic resistance, symbiotic interactions, and notable secondary metabolic capabilities. As the number of genomic sequences increases, the impact of GIs in prokaryotic evolution has become more apparent and detecting these regions using bioinformatics approaches has become an integral part of studying microbial evolution and function. In this dissertation, I describe a novel comparative genomics approach for identifying GIs, called IslandPick, and the application of this method to construct robust datasets that were used to test the accuracy of several previously published GI prediction programs. In addition, I will discuss the features of a new GI web resource, called IslandViewer, which integrates the most accurate GI predictors currently available. Further, the role of several GI and prophage regions and their involvement in virulence in an epidemic Pseudomonas aeruginosa strain that infects cystic fibrosis patients will be described; as well as an observation that recently discovered phage defence elements, CRISPRs, are over-represented within GIs.

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

The Fate of Duplicated Regions of the Atlantic Salmon (Salmo salar) Genome

Author: 
Date created: 
2004
Abstract: 

Gene and genome duplications have played a major role in vertebrate evolution. Salmonids provide a useful resource for studying the consequences of these events as their common ancestor underwent a genome duplication between 25 and 120 million years ago. To understand how a genome reorganizes itself to cope with duplicated chromosomes and the importance of gene duplications for evolution and adaptation, homeologous regions of the Atlantic salmon genome were identified and studied within a large insert, genomic BAC library; these BACs contain the metallothionein loci, a gene known to have remained in duplicate since the tetraploidization event. A BAC from each region was subsequently shotgun subcloned and sequenced. Sequence analysis revealed the presence of 10 genes, retaining their collinearity between the BACs, although pseudogenization events have occurred in one of the duplicate loci in two instances. Comparative genomic analysis revealed the existence of extraordinary conservation of synteny over time.

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

A comprehensive study of ribozyme-mediated nucleotide and sugar chemistries

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

The ability of RNA catalysts (ribozymes) to synthesize nucleotides as basic building blocks for the assembly of larger RNA polymers is an important aspect of the 'RNA World' hypothesis. To examine the ability of RNAs to catalyze this chemistry, we performed an in vitro selection in my first project and successfully isolated ribozymes capable of synthesizing a purine nucleotide (6-thioguanosine, 6SG) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine (6SGua). Compared to the previously selected pyrimidine synthase ribozymes, these ribozymes are 50-100 times more efficient. In a continuation of this work, we next deduced the secondary structure of two purine synthase ribozymes by performing a re-selection on two truncated pools for reactivity with 6SGua. Interestingly we were able to isolate for functional sequences that were ~2.5 fold shorter than their full-length parental ribozymes, and with much simpler secondary structure relative to the pyrimidine nucleotide synthase ribozymes. The ability of RNAs to synthesize both purine and pyrimidine nucleotides strongly suggests that RNA could have preceded proteins in a hypothetical RNA based metabolism. While the ribozymes isolated from our selection were successful in catalyzing purine nucleotide synthesis, they do require a pre-activated sugar substrate. This is not highly plausible in the RNA world since PRPP readily undergoes hydrolysis. To examine this phenomenon, in our third project we implemented a selection using a RNA pool tethered to ribose-5-phosphate and selected them for reactivity with 6SGua. After six selection rounds, a single RNA sequence dominated the selection. Interestingly, this ribozyme can produce three different products: two when tethered with PR and a third product when tethered with PRPP, which was determined to be 6SG. Our results suggest that this highly versatile ribozyme is capable of recognizing two similar substrates for the synthesis of different products, and thus demonstrates the promiscuous potential of ribozymes upon encountering of an alternative substrate.

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