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

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Gene prediction and RFX transcriptional regulation analysis using comparative genomics

Date created: 
2011-02-23
Abstract: 

Regulatory Factor X (RFX) is a family of transcription factors (TF) that is conserved in all metazoans, in some fungi, and in only a few single-cellular organisms. Seven members are found in mammals, nine in fishes, three in fruit flies, and a single member in nematodes and fungi. RFX is involved in many different roles in humans, but a particular function that is conserved in many metazoans is its regulation of ciliogenesis. Probing over 150 genomes for the presence of RFX and ciliary genes led to the understanding of how RFX-cilia regulatory interaction occurred in evolution. Molecular phylogenetic analysis revealed that RFX is only found in metazoans, in some fungi, and in only one unicellular organism, Monosiga brevicollis. However, ciliary genes did not co-exist with RFX genes except in Allomyces macrogynus and Monosiga brevicollis. The data showed that RFX and cilia evolved independently until the time just before the establishment of metazoans. These results suggest that RFX TFs acquired the role of transcriptional regulation on ciliary genes before metazoans arose and such gain-in-function could be a driving force for metazoan evolution. RFX regulate genes via a regulatory motif called the X-box motif. My laboratory, as well as others, has identified novel RFX target genes in C. elegans. However, accumulating evidence suggest more RFX genes could be uncovered and some of these genes could be regulated by divergent X-box motifs. Additional RFX target genes with divergent X-box motifs were identified in C. elegans by first revising the gene set in C. briggsae, C. remanei, and C. brenneri using a novel homology-based gene finder, genBlastG. Comparing the four genomes with the revised gene set revealed promoter regions with conserved X-box motif in all species except in C. elegans. Detailed examination revealed divergent X-box motifs in these regions. Mutagenesis experiments in the region upstream of F25B4.2 showed that divergent X-box motifs could drive gene expression and may repress gene expression as well. This study provides a deeper understanding regarding the evolution and mechanism of a conserved and important transcription factor.

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

The protein kinase Nemo integrates specification, proliferation and morphogenesis to shape the Drosophila eye

Author: 
Date created: 
2011-01-07
Abstract: 

Development of multi-cellular organisms is orchestrated by genetic circuits, which control proliferation and differentiation. A complex interplay of inductive and inhibitory signals culminates in the expression of unique protein sets, which elicit a distinct cellular response and confer cellular identity. The relatively simple architecture of the Drosophila melanogaster eye provides a powerful model to explore the molecular and regulatory relationships that pattern a complex tissue. This work investigates novel roles for the serine-threonine kinase, Nemo (Nmo), in cell fate specification and morphogenesis of the Drosophila compound eye. Nmo is the founding member of the Nemo-Like Kinase (NLK) family, which regulates conserved signal cascades like Wnt and BMP across species. nmo mutants have developmental defects in processes including embryonic segmentation, wing and eye patterning. Here, I examine the molecular underpinnings of the nmo eye phenotype. nmo is dynamically expressed throughout eye development, suggesting previously uncharacterized functions. Genetic analyses reveal a dose-dependent requirement for Nmo in eye specification directed by the Retinal Determination Gene Network (RDGN). The RDGN comprises a conserved network of transcriptional regulators that is essential for eye development in Drosophila and vertebrates. Loss of nmo reduces the ability of the RD factors to induce ectopic eyes, while co-expressing Nmo synergistically enhances RD-mediated phenotypes. In biochemical assays, Nmo complexes with the RD components Sine oculis (So) and Eyes Absent (Eya). Eya is a novel substrate for Nmo’s kinase activity, and this regulatory interaction promotes Eya’s eye induction activity. Nmo additionally promotes So’s transcriptional activity in culture-based and in vivo assays. Genetic approaches are employed to elucidate the nature of the nmo small-eye phenotype. In genetic interaction studies, manipulating nmo levels modifies the size of the head and eye, indicating that nmo can generally affect proliferation. A requirement for Nmo in normal progression of the morphogenetic furrow, a wave of differentiation that moves anteriorly across the eye epithelium, is also described. As a consequence of retarded furrow movement, photoreceptor differentiation is delayed in nmo mutants. Together, these studies expand our understanding of the nmo eye phenotype and identify Nmo as a novel regulator of the RD signalling network.

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

The intrinsically disordered nuclear localization signal and phosphorylation segments distinguish the membrane binding affinity of two cytidylyltransferase isoforms

Date created: 
2010-12-03
Abstract: 

Membrane phosphatidylcholine (PC) homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP: phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane PC content by reversible membrane binding and activation. Two prominent isoforms, CCTα and β2, have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains, but have divergent and structurally disordered amino-terminal (N) and carboxy-terminal phosphorylation (P) regions. I found that the anionic membrane binding affinity of purified CCTβ2 was weaker than CCTα by at least an order of magnitude. Using chimeric CCTs, insertion/deletion mutants and truncated CCTs I showed that the stronger affinity of CCTα can be attributed in large part to the secondary electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured region of region N of CCTα, but lacking in CCTβ2. The membrane partitioning of CCTβ2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCTα, and was elevated by incorporation of the NLS motif. Thus, the polybasic NLS can function as a secondary membrane-binding motif not only in vitro but also in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P truncated forms showed that the in vitro membrane affinity of CCTβ2 is more sensitive than CCTα to phosphorylation status, which antagonizes membrane binding of both isoforms. These data provide a model wherein the primary membrane binding motif, an amphipathic helical domain, works in collaboration with other intrinsically disordered segments, which modulate membrane binding strength. The NLS reinforces, while the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.

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

Sex determination in the genus Salmo

Author: 
Date created: 
2010-12-09
Abstract: 

Salmonids share a strictly genetic mechanism for sex determination with males being the heterogametic sex. FISH analysis using BACs containing sexlinked microsatellite markers in Atlantic salmon identified chromosome 2 as its sex chromosome. The same sized chromosome 2 pairs in both sexes suggesting that the sex chromosome in Atlantic salmon is not highly differentiated. Combining information from sex determination pathways in different phyla, I created a list of 14 candidate sex determining genes. Candidate gene mapping identified all gene locations. However, their positions rule them out as the master regulatory gene. Comparative genomic analysis shows that the closest related species brown trout sex linkage group (LG) is syntenic to Atlantic salmon autosomal LG 8. FISH analysis revealed the brown trout sex chromosomes as a pair of small submetacentric chromosomes. Although they are closely related, I predict that Atlantic salmon and brown trout are likely to have different sex determining genes.

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

Light-controlled regulation of eukaryotic gene expression using a diffusible effector

Author: 
Date created: 
2010-07-28
Abstract: 

This project focuses on the design of a small synthetic, trans-acting noncoding RNA to directly regulate gene expression by light irradiation in a eukaryotic system at the mRNA level. This trans-acting noncoding RNA is designed to incorporate an in vitro-selected small ligand-binding domain, called an aptamer domain. This aptamer domain selectively binds to one but not the other of the two isomers of a dihydropyrene photo-switch compound. Such an aptamer sequence allowed us to design an allosteric riboregulator, a RNA molecule with a regulatory function whose mode of activity depends on its photo-switch ligand binding to the aptamer domain, which in turn is influenced by light irradiation at an appropriate wavelength. This light-controlled riboregulator is referred to as an antiswitch. This antiswitch is specifically designed to control the expression of Green Fluorescent protein (GFP) in vivo in Saccharomyces cerevisiae as a model organism.

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

Characterization of nematode DAF-19 and human RFX6 transcription factors

Author: 
Date created: 
2010-07-16
Abstract: 

Regulatory factor X (RFX) transcription factors play an important role in regulating expression of ciliary genes associated with ciliopathies. However, the annotation of RFX genes may be incomplete and their function is not well understood. Here I describe two novel tissue-specific RFX genes in humans: RFX6 and RFX7. To study RFX genes in the model organism Caenorhabditis elegans, I undertook examining the expression of all four known isoforms of daf-19, the only RFX gene in C. elegans, by using Mos1 mediated Single Copy transgene Insertion (MosSCI) method. I discovered that both daf-19c and daf-19d isoforms are expressed in ciliated neurons and that their promoters are modular. In particular, daf-19c is expressed in all ciliated neurons while daf-19d in all but labial neurons. My analysis helped select suitable promoters for driving expression of RFX6 in ciliated neurons of C. elegans for testing its function in cilia.

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

Determining the correlation between structure and function for the Drosophila insulin receptor substrate chico

Date created: 
2010-08-09
Abstract: 

The Drosophila gene chico codes for the homolog of the vertebrate insulin receptor substrate (IRS) family of proteins. CHICO is phosphorylated by an activated insulin receptor and signals to several downstream signalling pathways through various predicted binding domains. To investigate how the structure of CHICO contributes to its function, an ethyl methanesulfonate (EMS) screen was performed to generate novel chico alleles. One novel allele, chico13063, was recovered out of 22,072 lines screened. This allele displayed numerous chico phenotypes including: reduced body size, weight, wing size, and developmental time delay. These phenotypes were stronger in the chico13063 allele than the null allele, chico1 and the deficiency, Df(2L)flp 147E. The chico13063 coding region was sequenced and no genetic lesions were found, suggesting the mutation is in the regulatory region. Future work would be to sequence putative regulatory regions of chico and examine the expression levels of CHICO in the chico13063 mutant.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Esther Verheyen
Eve Stringham
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Molecular identification and phenotypic characterization of let-65 (mars-1) in Caenorhabditis elegans.

Date created: 
2010-07-26
Abstract: 

let-65 was first identified in a screen for lethal mutations in C. elegans’unc-22 region. I have demonstrated the molecular identity of let-65 to be F58B3.5 (mars-1), which encodes a methionyl tRNA synthetase (MARS-1). To develop a deeper insight into MARS-1 activity, I experimentally confirmed, from its sub-cellular localization, that it is a cytoplasmic enzyme. I also attempted to determine the sub-cellular localization of every known C. elegans amino-acyl tRNA synthetase using computational methods and, in collaboration with WormBase, renamed the genes appropriately. In addition, I studied let-65 transcription regulation by analyzing its 5’ promoter containing region. Haplo-insufficiency phenotypes manifest as a consequence of reduction in copy number of genes that encode proteins involved in translation. I have investigated this in C. elegans. I used two alleles of each of let-65 (mars-1) and let-336 (rps-27) and examined putative haplo-insufficiency phenotypes for both these genes.

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

Identification, evolution and expression of three olfactory gene families in Atlantic salmon (Salmo salar)

Date created: 
2011-11-30
Abstract: 

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. To understand the molecular basis for imprinting and homing in Atlantic salmon (Salmo salar), it is important to identify and characterize the olfactory receptors in the Atlantic salmon genome. Aquatic chemical cues are received through the salmon nares and into the nasal cavity that contains a single olfactory organ, olfactory rosette. The olfactory rosette contains sensory neurons, which are thought to express only one olfactory receptor. In this study, three major superfamilies of fish olfactory receptors (MOR, ora and OlfC) were examined. To identify the olfactory genes in Atlantic salmon several genomic and bioinformatic techniques were used. First, an Atlantic salmon bacterial artificial chromosome (BAC) library was screened with probes designed from previously identified fish olfactory receptor sequences. Then a selected number of hybridization positive BACs containing olfactory receptors were shotgun cloned and sequenced. From these BAC sequences, two ora genes and 55 OlfC genes were identified in Atlantic salmon. The second technique used to identify olfactory receptors in Atlantic salmon was a bioinformatic approach that involved screening a 3-fold Atlantic salmon genome sequence for olfactory receptors. Using this approach, 24 MOR and the remaining five ora genes were identified, as well as another 24 partial genes or pseudogenes. As a first step to understand how olfactory receptors are involved in imprinting and homing, a suite of olfactory receptors were selected to examine the expression profiles of these genes across different life stages and life histories of wild Atlantic salmon from Newfoundland, Canada. Seven differentially expressed OlfC genes were identified in juvenile anadromous salmon compared to returning adult salmon. From this research, I hypothesize that OlfC genes may play an important role in the imprinting of home stream water olfactory cues in anadromous Atlantic salmon.

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

Generation and Analysis of Suppressors of Caenorhabditis Elegans Unc-2 Alleles

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2004
Document type: 
Thesis
File(s): 
Supervisor(s): 
David L. Baillie
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.