Thesis (M.Sc.)(Genetics--University of Pretoria, 2005. / Title from opening screen (viewed March 28, 2006). Summaries in English and Afrikaans. Includes bibliographical references.

From Summary: A survey of collagenolytic aerobic bacteria from cured hides yielded three strains of Bacillus and eight of Achromobacter which degraded collagen at 0.4 M NaCl. Achromobacter sp. 2 was chosen for genetic studies due to its high collagenolytic activity and the lack of genetic information on Achromobacter. Four temperate bacteriophages specific for Achromobacter sp. 2 were isolated and their relationships studied. The phages caused lysogenic conversion resulting in the inability of lysogens to adsorb phage. Achromobacter sp. 2 was shown to be a cryptic lysogen as it was not immune to superinfection but had a very low rate of spontaneous induction which could be increased with mutagens. It is proposed that the cryptic lysogeny of this strain is maintained by a defective excision mechanism and the mode of prophage integration in the host chromosome. DNA extracted from phage α3a was used to transfect spheroplasts. The optimal conditions for the development of competence for transfection were determined. The presence of nuclease-attack on phage DNA under conditions of prolonged incubation of DNA and spheroplasts was proposed. A method for extracting Achromobacter DNA was devised which yielded purified, undegraded DNA, but it was not possible to transform Achromobacter sp. 2 with this DNA. The a phages were used to transduce a number of genetic markers into Achromobacter auxotrophs. The transduct ants had the ability to release the cryptic α3 prophage at a high rate while maintaining their sensitivity to homologous phage infection. It is proposed that this is due to complementation between the cryptic prophage and the residual phage functions in the transducing particles. The transductants segregated auxotrophs with a probability of 10⁻³ per cell per generation. It appears that an unusual system of generalised transduction is operating whereby the transducing particles contain both phage and bacterial DNA which is incorporated into the recipient genome by a single recombination event yielding unstable transductants. In a study on induction of Escherichia coli (λ), carcinogenic nitrosamines were shown to be inducers of phage development. This provides a screening system for potentially harmful nitrosamines.

African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication. The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV. / Dissertation (MSc)--University of Pretoria, 2013. / Genetics / MSc / Unrestricted

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Until recently, complete understanding of the endogenous activity of pathologically relevant genes was out of reach and research was confined to in situ work, plasmid-based constructs and artificial model systems. The development and expansion of the CRISPR/Cas9 genome editing technique has enabled us to explore the molecular underpinnings of gene activation using the cell's own endogenous regulatory environment. In this work, we report on the development of a novel tool to monitor the endogenous activity of a causative gene in Parkinson's disease, a-synuclein. We use CRISPR/Cas9 to insert a highly sensitive engineered luciferase at the C-terminal of a-synuclein and assessed its responses to stimuli. Our system responds to epigenetic stimuli, which was unable to be recapitulated by previously available gene activity assays. After development of a sensitive detection tool for epigenetic stimuli, we focused on developed a modular suite of epigenetic writers and erasers by modification of the SunTag protein tagging system and used catalytically dead Cas9 (dCas9) to direct our modular epigenetic toolkit to individual genes. We show that our toolkit of epigenetic effectors successfully writes epigenetic information in a site-specific manner. Using the sensitive a-synuclein reporter we previously developed, we screen the promoter region of this pathologically relevant gene at high resolution and identify the most effective areas for epigenetic intervention in this cell line. These tools allow us to dissect and understand the endogenous regulatory mechanisms of almost any gene targetable by Cas9 in ways that were not previously available may prove to be an effective strategy for persistently altering pathologic transcriptional activity. This system offers a strong tool for to dissect and understand underlying epigenetic architecture and opens potential new avenues for therapeutic strategies for various disease conditions.

<p>The Wnt signaling pathway plays a vital role in a multitude of cellular processes across a breadth of multicellular organisms, from simple nematode roundworms such as the Caenorhabditis species to humans. Through the study of this evolutionarily conserved signal transduction pathway in a simple model organism, a greater understanding of the regulation and function of the Wnt pathway can be gained. Emerging research is highlighting the promiscuous nature of Wnt signaling within the network of signaling pathways involved in C. elegans development and aging processes. However, impeding the study of the diverse roles of Wnt signaling in the nematode is the fact that little is known about regulation of key Wnt pathway components in the nematode species, and only a handful of downstream Wnt pathway target genes have been identified. We have used parallel genetic and genomic approaches to elucidate transcriptional targets of the Wnt pathway in C. elegans and the related species C. briggsae. This analysis has uncovered an array of putative Wnt pathway gene targets including pry-1/Axin, a key regulatory component of the pathway. Furthermore, our genome-wide search for Wnt targets has revealed a novel interaction between the Wnt and Hedgehog signaling pathways that is conserved between these two nematode species.</p> / Master of Science (MSc)

Chan, Wai Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 140-157). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.1 / List of Tables --- p.4 / List of Figures --- p.5 / List of Abbreviations --- p.8 / Chapter 1. --- Introduction --- p.10 / Chapter 1.1 --- Biology of metals --- p.10 / Chapter 1.2 --- Metal detoxification systems --- p.11 / Chapter 1.3 --- Metallothionein --- p.13 / Chapter 1.4 --- Classification of MTs --- p.15 / Chapter 1.5 --- Biological roles of MT --- p.15 / Chapter 1.5.1 --- Homeostasis of essential transition metal ion --- p.15 / Chapter 1.5.2 --- Detoxification of non-essential heavy metal ion --- p.17 / Chapter 1.5.3 --- Protection against oxidative stress --- p.18 / Chapter 1.5.4 --- Role in neurodegenerative diseases --- p.19 / Chapter 1.6 --- Molecular biology of MT --- p.19 / Chapter 1.6.1 --- MT gene structure --- p.19 / Chapter 1.6.2 --- MT gene regulation --- p.21 / Chapter 1.7 --- MRE binding transcription factor-1 (MTF-1) --- p.30 / Chapter 1.8 --- Activation of MTF-1 --- p.31 / Chapter 1.9 --- Target genes of MTF-1 --- p.32 / Chapter 1.10 --- Fish MT gene and MTF-1 --- p.33 / Chapter 1.11 --- Tilapia --- p.39 / Chapter 1.12 --- Study of tilapia MT --- p.41 / Chapter 1.13 --- Aims and rationale of study --- p.43 / Chapter 2. --- Materials and Methods --- p.45 / Chapter 2.1 --- Cloning of tilapia MT gene 5'-flanking region --- p.45 / Chapter 2.1.1 --- Animals --- p.45 / Chapter 2.1.2 --- Preparation of tilapia genomic DNA --- p.45 / Chapter 2.1.3 --- DNA walking --- p.45 / Chapter 2.1.4 --- Amplification of whole tiMT gene --- p.50 / Chapter 2.2 --- Determination of transcription start site --- p.51 / Chapter 2.2.1 --- Total RNA extraction --- p.51 / Chapter 2.2.2 --- Rapid amplification of 5，complementary DNA ends (5' RACE) --- p.52 / Chapter 2.3 --- Transient transfection assay --- p.54 / Chapter 2.3.1 --- Cell culture --- p.54 / Chapter 2.3.2 --- Construction of pGL3-tiMT deletion mutants --- p.54 / Chapter 2.3.3 --- Preparation of heavy metal solutions --- p.56 / Chapter 2.3.4 --- Determination of heavy metal ion toxicities by alamarBlue´ёØ assay --- p.56 / Chapter 2.3.5 --- Transient transfection of plasmids to Hepa-T1 cells --- p.56 / Chapter 2.3.6 --- Metal ions treatment and study of tiMT promoter activities --- p.57 / Chapter 2.3.7 --- Transient gene expression studies of deletion mutants of tiMT promoter --- p.57 / Chapter 2.4 --- Site-directed mutagenesis of tiMT promoter --- p.58 / Chapter 2.4.1 --- Polymerase chain reaction (PCR)-based site-directed mutagenesis --- p.58 / Chapter 2.4.2 --- Transient transfection of plasmids to Hepa-T1 cells and study of tiMT promoter activities --- p.62 / Chapter 2.5 --- Electrophoretic mobility shift assay (EMSA) --- p.63 / Chapter 2.5.1 --- Extract preparation --- p.63 / Chapter 2.5.2 --- Preparation of radiolabeled tiMRE oligonucleotides --- p.63 / Chapter 2.5.3 --- Electrophoretic mobility shift assay (EMSA) --- p.64 / Chapter 3. --- Results --- p.66 / Chapter 3.1 --- "Cloning of tilapia MT (tiMT) gene 5,-flanking region and amplification of whole tiMT gene" --- p.66 / Chapter 3.2 --- Determination of transcription start site --- p.69 / Chapter 3.3 --- Cloning of tiMT promoter fragment into reporter vector --- p.72 / Chapter 3.4 --- Determination of heavy metal ion toxicities by alamarBlue´ёØ assay --- p.72 / Chapter 3.5 --- Study of tiMT promoter activities by heavy metal ions exposure..… --- p.72 / Chapter 3.6 --- Cloning of deletion mutants of tiMT promoter --- p.79 / Chapter 3.7 --- Transient gene expression studies of deletion mutants of tiMT promoter --- p.80 / Chapter 3.8 --- Cloning of mutants with site-directed mutagenesis in tiMT promoter --- p.88 / Chapter 3.9 --- Site-directed mutagenesis of tiMT promoter --- p.92 / Chapter 3.10 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.97 / Chapter 4. --- Discussion --- p.102 / Chapter 4.1 --- Tilapia MT gene --- p.102 / Chapter 4.2 --- Resistance of tilapia to heavy metal ions --- p.107 / Chapter 4.3 --- Functional analysis of tiMT gene promoter by transient transfection --- p.111 / Chapter 4.4 --- DNA binding of metal responsive transcription factor in Hepa-T1 cells --- p.121 / Chapter 4.5 --- Conclusion --- p.138 / References --- p.140

Characterization of the determinants of economically important phenotypes showing complex inheritance should lead to more effective use of genetic resources. This study was conducted to determine the number, genome location and effects of QTLs determining malting quality and agronomic traits in the two North American barley quality standards. Using a doubled haploid population of 140 lines from the cross of Harrington x Morex, agronomic phenotype and malting quality data sets from nine and eight environments, respectively, and a 107-marker linkage map, QTL analyses were performed using simple interval mapping and simplified composite interval mapping procedures. Thirty five QTLs were associated either across environments or in individual environments, with five grain and agronomic traits (yield, kernel plumpness, test weight, heading date and plant height). Thirteen QTLs were associated with five malting quality traits (grain protein percentage, soluble/total protein ratio, ��-amylase activity, diastatic power and malt extract percentage). QTLs for multiple traits were coincident. The loci controlling inflorescence type [vrsl on chromosome 2 (2H) and int-c on chromosome 4 (4H)] were coincident with QTLs affecting all traits except heading date and malt extract percentage. The largest effect QTLs -for yield, kernel plumpness test weight, plant height grain protein percentage, S/T ratio, and diastatic power- were coincident with the vrsl locus. QTL analyses were conducted separately for each sub-population (six-rowed and two-rowed). Ten new QTLs were detected in the sub-populations. There were significant interactions between the vrsl and int-c loci for plant height, grain protein percentage, and SIT protein ratio. Positive transgressive segregants were found for all agronomic traits. They were more prevalent in the six-rowed sub-population, indicating that more favorable alleles were fixed in the two-rowed parent. Results suggest that this mating of two parents representing different germplasm groups caused a disruption in the balance of traits involved in malting quality, which resulted in no progeny carrying all favorable alleles and therefore surpassing the quality of either parent. This study describes some of the genetic determinants of agronomic and malting quality traits in a two-rowed x six-rowed cross and it is a first step toward the further characterization and manipulation of these determinants. / Graduation date: 2001

Thesis (M.A.)--York University, 2001. / Typescript. Includes bibliographical references (leaves 257-270). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ67717.