Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 205-207). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 77-85). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 205-207). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 77-85). Also available on the Internet.

The advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory. Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures. Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila. This dissertation includes previously published, co-authored material

Temperature-sensitivity (TS) is often used as a way to attenuate microorganisms to convert them into live vaccines. Studies indicate that live vaccines are often necessary for the complete clearance of certain pathogenic organisms. In this work we explore the use of TS genetic circuits that express lethal genes for their potential utility as a widely applicable approach to TS attenuation. Here, we use restriction endonucleases as the lethal gene products. We tested different combinations of TS repressors and cognate promoters controlling the expression of genes encoding restriction endonucleases inserted at four different non-essential sites in the Escherichia coli chromosome. We found that the presence of the restriction endonuclease genes did not affect the viability of the host strains at the permissive temperature, but that expression of the genes at elevated temperatures killed the strains to varying extents. The location of the genetic circuit cassette in the chromosome was critical, and insertion at the ycgH site led to minimal cell death. Induction of the TS circuit in a growing culture led to a pre-mature leveling off of the optical density, and a shift in the number of cells that could exclude a dye that indicated cell viability. Incubation of cells initially grown at low temperature and then suspended in phosphate buffered saline at high temperature, led to about 100-fold loss of cell viability per day compared to minimal loss of viability for the parental strain. The Dual strain containing two different genetic circuits was found to have reduced escape frequency compared to single circuit strains. However, strains carrying either one or two TS lethal circuits could generate mutants that survived high temperature. These mutants included start codon deletions as well as upstream deletions of the TetRD1 encoding gene as well as complete deletions of the lethal gene circuits. / Graduate

Restriction endonucleases are a group of enzymes that cleave DNA at or around specific sequences, which are typically palindromic. A fosmid library was constructed from a metagenome isolated from soil from the Kogelberg Nature Reserve, Western Cape and was functionally screened for restriction endonucleases. Next-generation (NGS) Illumina sequencing technology was used to identify putative endonucleases. The sequence data generated was assembled and analysed using CLC Bio Genomics Workbench and bioinformatics tools (NCBI BLAST, REBASE and MG-RAST). Using these tools, genes encoding restriction-modification systems and endonuclease homologues were discovered. Three genes were identified and were recombinantly produced in Rosetta™ (DE3) pLysS and purified with IMAC using Ni-TED resin and subsequently characterised. These three genes were selected based on the identity percentage when compared to sequences on the NCBI database. Production of Endo8 was scaled up using 2 l fermenter and the purification done using ÄKTA Avant 150 FPLC using a HiScale 50 column packed with Ni-TED resin and the total amount of protein achieved was 58.82 mg.g-1. The productivity achieved at 17 hours (8 h harvest) was 2-fold greater than at 12 hours. Endonuclease activity of endo8 and endo52 was tested, both exhibited strong non-specific activity at 37 °C with an incubation period of 30 min. This work demonstrates that environmental soil samples are a valuable source for discovery of novel enzymes and also the utility of functional metagenomics to discover and purify these enzymes. These endonucleases may contribute to the next generation of reagent enzymes for molecular biology research. / Chemistry / M. Sc. (Life Sciences)

by Chu Ka-yi. / Thesis submitted in: October 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 160-174). / Abstracts in English and Chinese. / ABSTRACT (English) --- p.i / ABSTRACT (Chinese) --- p.iii / ACKNOWLEDGMENT --- p.v / LIST OF CONTENTS --- p.vi / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xii / ABBREVIATIONS --- p.xiv / TERMS --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Taxonomy of Acinetobacter - historical and current --- p.1 / Chapter 1.2 --- Ecology and clinical significance of Acinetobacter --- p.5 / Chapter 1.3 --- General identification and typing methods for Acinetobacter species / Chapter 1.3.1 --- Identification at species level --- p.9 / Chapter 1.3.2 --- Identification at strain level --- p.11 / Chapter 1.4 --- Methods used in this study for characterization of Acinetobacter species / Chapter 1.4.1 --- Amplified ribosomal DNA restriction analysis (ARDRA) --- p.14 / Chapter 1.4.2 --- tDNA spacer fingerprinting (tDNA) --- p.15 / Chapter 1.4.3 --- Fluorescent amplified fragment length polymorphism (FAFLP) --- p.16 / Chapter 1.4.4 --- Phenotypic methods including carbon utilization tests --- p.20 / Chapter 1.5 --- Objectives --- p.25 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.27 / Chapter 2.1 --- Bacterial strains and isolates --- p.27 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Antimicrobial agents and chemicals --- p.30 / Chapter 2.2.2 --- "Carbohydrates, enzymes and other materials" --- p.32 / Chapter 2.2.3 --- Commercial media and media prepared manually --- p.33 / Chapter 2.2.4 --- "Buffers, solutions and list of instruments" --- p.35 / Chapter 2.3 --- General Bacteriological Techniques / Chapter 2.3.1 --- Isolation of acinetobacters --- p.38 / Chapter 2.3.2 --- Routine bacteriological identification --- p.39 / Chapter 2.4 --- General Molecular Biology Techniques / Chapter 2.4.1 --- DNA isolation --- p.40 / Chapter 2.4.2 --- Transformation --- p.41 / Chapter 2.4.3 --- Agarose gel electrophoresis --- p.43 / Chapter 2.5 --- Genospeciation of acinetobacters by Amplified Ribosomal Restriction DNA Analysis (ARDRA) --- p.44 / Chapter 2.6 --- Characterization of ARDRA unclassifiable acinetobacters (AUA) by Phenotypic methods / Chapter 2.6.1 --- Temperature tolerance tests --- p.47 / Chapter 2.6.2 --- Carbon utilization tests --- p.47 / Chapter 2.6.3 --- Gelatin and hemolysis tests --- p.48 / Chapter 2.6.4 --- Minimum Inhibitory Concentration (MIC) --- p.49 / Chapter 2.7 --- Characterization of AUA by tDNA spacer fingerprinting (tDNA) method --- p.51 / Chapter 2.8 --- Characterization of AUA by Fluorescent Amplified Fragment Length Polymorphism analysis (FAFLP) --- p.55 / Chapter 2.9 --- Relatedness study of isolates within the same AUA group by Enterobacterial Repetitive Intergenic Consensus (ERIC) typing method --- p.58 / Chapter CHAPTER 3 --- COLLECTION OF UNCLASSIFIABLE ACINETOBACTERS by ARDRA (AUA) METHOD --- p.59 / Chapter 3.1 --- Results / Chapter 3.1.1 --- Isolation and genospeciation of acinetobacters from hospital environments and raw food --- p.59 / Chapter 3.1.2 --- Collection of ARDRA unclassifiable acinetobacters (AUA) --- p.63 / Chapter 3.2 --- Discussion / Chapter 3.2.1 --- Limitations and merits of ARDRA method --- p.68 / Chapter 3.2.2 --- Potential significance of the representative AUA groups --- p.71 / Chapter CHAPTER 4 --- CHARACTERIZATION OF ARDRA UNCLASSIFIABLE ACINETOBACTERS (AUA) BY tDNA SPACER (tDNA) FINGERPRINTING METHOD --- p.72 / Chapter 4.1 --- Results / Chapter 4.1.1 --- Assessment of reproducibility --- p.72 / Chapter 4.1.2 --- Construction of the database with the reference strains --- p.75 / Chapter 4.1.3 --- Characterization of the representative AUA groups --- p.78 / Chapter 4.2 --- Discussion / Chapter 4.2.1 --- Evaluation of the reproducibility and discriminatory power --- p.89 / Chapter 4.2.2 --- Possible genospeciation of the representative AUA groups --- p.92 / Chapter 4.2.3 --- Limitations and merits --- p.96 / Chapter CHAPTER 5 --- CHARACTERIZATION OF ARDRA UNCLASSIFIABLE ACINETOBACTERS (AUA) BY FLUORESCENT AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (FAFLP) METHOD --- p.98 / Chapter 5.1 --- Results / Chapter 5.1.1 --- Assessment of robustness and reproducibility --- p.98 / Chapter 5.1.2 --- Construction of the database with the reference strains --- p.104 / Chapter 5.1.2 --- Characterization of the representative AUA groups --- p.108 / Chapter 5.2 --- Discussion / Chapter 5.2.1 --- "Evaluation of robustness, reproducibility and discriminatory power" --- p.116 / Chapter 5.2.2 --- Possible genospeciation of the representative AUA groups --- p.120 / Chapter 5.2.3 --- Merits and limitations --- p.122 / Chapter CHAPTER 6 --- CHARACTERIZATION OF ARDRA UNCLASSIFABLE ACINETOBACTERS (AUA) BY PHENOTYPIC METHODS --- p.125 / Chapter 6.1 --- Results Characterization of the representative AUA groups --- p.125 / Chapter 6.2 --- Discussion / Chapter 6.2.1 --- Possible genospeciation of the representative AUA groups --- p.134 / Chapter 6.2.2 --- Limitations in classification of Acinetobacter species at genomic species level --- p.135 / Chapter CHAPTER 7 --- RELATEDNESS OF ISOLATES WITHIN THE SAME AUA GROUPS --- p.139 / Chapter 7.1 --- Results Typing results of the studied AUA groups by ERIC method --- p.139 / Chapter 7.2 --- Discussion Relatedness of the isolates within the same AUA group --- p.146 / Chapter CHAPTER 8 --- GENERAL DISCUSSION --- p.148 / Chapter 8.1 --- Possible genospeciation of the representative AUA groups --- p.150 / Chapter 8.2 --- "Comparison of ARDRA, tDNA fingerprinting, FAFLP and phenotypic methods" --- p.154 / Chapter 8.3 --- Conclusion and significance of the AUA groups studied --- p.158 / Chapter 8.4 --- Future work --- p.159 / REFERENCES --- p.160 / APPENDIX --- p.176

The objective of this thesis was to study whether heterocyclic dicationic compounds that are minor groove binders have the ability to inhibit the digestive properties of type II restriction enzymes which bind to the major groove of the DNA. If these compounds do possess the ability to inhibit restriction enzymes, then what factors influence their ability to inhibit the restriction enzymes? The methods used to study the interactions of DNA, compounds, and enzymes are gel electrophoresis, DNA thermal melting, and circular dichroism. The results from this project reveal that the minor grove binding compounds are able to inhibition type II restriction enzymes. The inhibition is heavily influenced by compound structure and the DNA binding sequence of the enzyme.

Restriction Modification (R-M) systems prevent the invasion of foreign genetic material into bacterial cells and are therefore important in maintaining the integrity of the host genome. The spread of antibiotic resistance, which is proposed to occur via the transfer of foreign genes to the bacterial genome, makes the subject of R-M systems extremely relevant. R-M systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Kennaway et al (2012) proposed that there is an evolutionary link between Types I and II. Comparing the structures of examples from two of the subfamilies of Type II systems (IIB and IIG) to those of Type I structures, similarities can be observed. Due to the fact that Type II R-M systems cut DNA at fixed positions, they can be used to obtain genetic material selectively. They have therefore proven to be invaluable in molecular biology. One aspect of this project aims to create a novel R-M system, a pseudo-Type II system, by removing the molecular motors from the restriction subunit of a Type I system and fusing the remaining nuclease domain to a known Type I methyltransferase (MTase). This will not only provide evidence to support the theory that evolution has produced a pared down form of the Type I systems in the Type II systems, but it may also become a useful biological tool. This thesis describes the several attempts at doing this and how the subsequent constructs were expressed, purified and assayed to varying degrees of success. An important characteristic of the Type I systems is their ability to methylate DNA, and it is the mechanism via which host DNA is protected from restriction. This is another subject investigated in this project. As with the nuclease activity of the Type I systems, the site at which DNA is methylated is dictated by the HsdS subunit. It is described here how this subunit can be altered to change the sequence of DNA that is recognised by the system. Again, using Type II system subtypes as a reference, various mutations were made to the HsdS subunit of an MTase from Staphylococcus aureus. This is in an effort to bring about a new mode of action, but also to provide further evidence for an evolutionary link between the two system types. The HsdM and HsdS subunits are expressed from two separate genes at the same locus. There is a frameshift between the genes where the start of the hsdS gene occurs a few base pairs upstream from the stop codon of the hsdM gene. This work shows that removing this frameshift creates an MS fusion product, and in vivo studies show that this product has methylase activity and can form an active restriction complex when the HsdR subunit is added. The product can also be over-expressed and purified, and shows in vitro restriction activity on addition of the HsdR subunit protein. The HsdS subunit is composed of two target recognition domains (TRDs), each dictating one part of the bipartite recognition sequence. These TRDs can be altered, bringing about a change in the sequence of DNA recognised by the enzyme. In this thesis, it is shown that the C-terminal TRD can be removed and that the subsequent “Half S” enzyme possesses both methylase and restriction activity in vivo and that its recognition sequence is different from that of the wild-type enzyme. After the successful creation of both “MS fusion” and “Half S” recombinant proteins of the Sau1, Type I system from a CC398 strain of Staphylococcus aureus, a further construct was produced. This possesses both in vivo and in vitro activity. The novel “M Half S Fusion” enzyme not only links the two aspects of this project but also creates a structure similar to some seen in the Type II systems. This shows that the Type I systems can be manipulated to change their mode of action but also supports the idea that Types I and II are evolutionarily linked. By making the alterations in a step-wise fashion identifies that these structural changes can create viable enzymes, and that they could have occurred through the process of evolution.