Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 95-109).

Thesis (Ph. D.)--University of Washington, 1989. / Vita. Includes bibliographical references (leaves [83]-90).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 107-118).

Mok Yu-Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (Leaves 195-201). / Abstract --- p.i / Acknowledgements --- p.iii / List of Abbreviations --- p.iv / Table of contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The need to increase the specificity and variety of restriction endonucleases --- p.1 / Chapter 1.2 --- Classification of methods used for increasing the specificity and variety of restriction endonculeases --- p.2 / Chapter 1.3 --- Isolation and characterization of restriction endonucleases from natural sources --- p.3 / Chapter 1.4 --- Modification of DNA substrate to produce new cleavage specificities --- p.6 / Chapter 1.4.1 --- Methylation of the DNA substrate --- p.6 / Chapter 1.4.1.1 --- Achilles' hell cleavage-The use of canonical methylation to produce novel specificities --- p.10 / Chapter 1.4.1.2 --- Cross protection-The use of non-canonical methylation to generate new cleavage specificity --- p.14 / Chapter 1.4.1.2.1 --- Recognition sequence of a restriction endonuclease and a methylase partially overlap --- p.14 / Chapter 1.4.1.2.2 --- Methylase recognizing a subset of the degenerate sequence of the restriction endonuclease --- p.16 / Chapter 1.4.1.2.3 --- Methylase-limited partial digestion --- p.16 / Chapter 1.4.1.3 --- The use of methylation dependent restriction endonucleases and methylases to generate new specificity --- p.17 / Chapter 1.4.1.4 --- Sequential double-methylation-A two step methylation procedure to generate new specificities --- p.20 / Chapter 1.4.2 --- The generation of a universal restriction endonuclease by combining a Type IIS restriction enzyme moiety and an oligonucleotide adaptor --- p.22 / Chapter 1.4.2.1 --- General principle for generating a universal restriction endonuclease --- p.22 / Chapter 1.4.2.2 --- Factors that affect the cleavage efficiency of universal restriction endonuclease --- p.25 / Chapter 1.4.2.3 --- Modifications and potential applications of the universal restriction endonuclease --- p.29 / Chapter 1.4.3 --- DNA triple helix formation-enhance restriction enzyme specificity by site-specific inhibition of restriction/modification enzymes --- p.32 / Chapter 1.5 --- Modification of the cleaving agent to produce new specificities --- p.36 / Chapter 1.5.1 --- Sequence-specific artificial endonucleases --- p.36 / Chapter 1.5.1.1 --- Oligonucleotides as sequence-specific ligand --- p.37 / Chapter 1.5.1.2 --- Protein or peptide as sequence-specific ligand --- p.40 / Chapter 1.5.1.3 --- General limitations and applications of artificial endonucleases --- p.42 / Chapter 1.5.2 --- Molecular cloning and protein engineering of the restriction-modification system of bacteria --- p.43 / Chapter 1.5.2.1 --- Molecular cloning of the bacterial restriction-modification systems --- p.43 / Chapter 1.5.2.1.1 --- The strategies used to clone and screen restriction-modification systems --- p.45 / Chapter 1.5.2.2 --- Protein engineering of the restriction-modification systems of bacteria --- p.50 / Chapter 1.5.2.2.1 --- Pre-requisites for protein engineering on the restriction-modification systems --- p.51 / Chapter 1.5.2.2.2 --- Effects of protein engineering on the activity and specificity of restriction endonuclease and methylase --- p.53 / Chapter 1.6 --- Variation of restriction endonuclease specificity by altering the reaction condition --- p.56 / Chapter 1.6.1 --- Effects of organic solvents --- p.57 / Chapter 1.6.2 --- Effects of pH and ionic environment on restriction endonuclease specificity --- p.58 / Chapter 1.6.3 --- Remarks on the use of star activity to introduce new specificity --- p.59 / Chapter 1.7 --- Aim of study --- p.59 / Chapter Chapter 2 --- Purification and characterization of thermophilic restriction endonucleases from soil Bacillus spp / Chapter 2.1 --- Materials and methods --- p.61 / Chapter 2.1.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.61 / Chapter 2.1.1.1 --- Preparation of crude enzyme extract --- p.61 / Chapter 2.1.1.2 --- Purification of BsiB I and BsiE 1 --- p.63 / Chapter 2.1.1.3 --- Purification of BsiY I --- p.63 / Chapter 2.1.1.4 --- Preparation of BsiG I and BsiU I --- p.64 / Chapter 2.1.1.5 --- Concentration and storage of the purified restriction endonucleases --- p.64 / Chapter 2.1.1.6 --- Regeneration of the columns --- p.64 / Chapter 2.1.2 --- Characterization of restriction endonucleases --- p.65 / Chapter 2.1.2.1 --- Assay for the working temperature and ionic requirement for the restriction enzymes --- p.65 / Chapter 2.1.2.2 --- Unit determination of the restriction endonucleases --- p.66 / Chapter 2.1.2.3 --- Assay for the purities of restriction endonucleases --- p.66 / Chapter 2.1.2.4 --- Determination of recognition specificity --- p.67 / Chapter 2.1.2.5 --- Determination of the restriction endonuclease's sensitivity to dam and dcm methylation --- p.68 / Chapter 2.1.2.6 --- Determination of the cleavage specificities of restriction endonucleases --- p.70 / Chapter 2.1.2.7 --- Sequencing using Deaza dGTP --- p.73 / Chapter 2.2 --- Results --- p.73 / Chapter 2.2.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.73 / Chapter 2.2.1.1 --- Strain identification --- p.74 / Chapter 2.2.1.2 --- Elution properties of the restriction endonucleases from columns --- p.74 / Chapter 2.2.1.2.1 --- BsiB I --- p.74 / Chapter 2.2.1.2.2 --- BsiE I --- p.77 / Chapter 2.2.1.2.3 --- BsiY 1 --- p.78 / Chapter 2.2.1.3 --- The working digestion temperature and ionic strength requirement --- p.81 / Chapter 2.2.1.4 --- Unit determination --- p.82 / Chapter 2.2.1.5 --- Purities of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6 --- Recognition sites of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6.1 --- BsiB I --- p.83 / Chapter 2.2.1.6.2 --- BsiE I --- p.85 / Chapter 2.2.1.6.3 --- BsiY 1 --- p.87 / Chapter 2.2.1.6.4 --- BsiU I and BsiG I --- p.88 / Chapter 2.2.1.7 --- Sensitivity of restriction endonucleases to dam and dcm methylation --- p.90 / Chapter 2.2.1.8 --- Cleavage specificities of the purified restriction endonucleases --- p.91 / Chapter 2.2.1.8.1 --- BsiB I --- p.91 / Chapter 2.2.1.8.2 --- BsiE I --- p.92 / Chapter 2.2.1.8.3 --- BsiY I --- p.93 / Chapter 2.2.1.9 --- Sequencing of a wrongly sequenced site in pACYC177 using Deaza-dGTP --- p.94 / Chapter Chapter 3 --- The use of Xcm I and BsiY I as an universal restriction endonuclease / Chapter 3.1 --- Materials and methods --- p.98 / Chapter 3.1.1 --- Assay of universal restriction endonuclease using ss DNAs --- p.98 / Chapter 3.1.1.1 --- Annealing reaction between adaptors and ss DNAs --- p.99 / Chapter 3.1.1.2 --- Digestion of the annealed DNA complex --- p.100 / Chapter 3.1.1.3 --- Assay of the digested ss DNA on alkaline denaturing agarose gel --- p.100 / Chapter 3.1.2 --- Assay system involving 5' end-labelled oligonucleotide --- p.101 / Chapter 3.1.2.1 --- Purification of oligonucleotides using preparative polyacrylamide gel electrophoresis --- p.102 / Chapter 3.1.2.2 --- 5'end-labelling of the oligonucleotide DNA substrate --- p.104 / Chapter 3.1.2.3 --- The annealing between adaptors and oligonucleotide DNA substrate and the digestion condition --- p.104 / Chapter 3.1.2.4 --- Assay of the labelled oligonucleotides in polyacrylamide gel after digestion --- p.105 / Chapter 3.2 --- Results --- p.106 / Chapter 3.2.1 --- Xcm I adaptors #2 and #4 --- p.106 / Chapter 3.2.1.1 --- Assay conditions used for the universal restriction endonucleases --- p.107 / Chapter 3.2.1.1.1 --- Conditions used for hybridization --- p.107 / Chapter 3.2.1.1.2 --- Conditions used for digestion --- p.108 / Chapter 3.2.1.2 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #4 --- p.110 / Chapter 3.2.1.2.1 --- Methods used to optimize the hybridization process --- p.110 / Chapter 3.2.1.2.2 --- Methods used to relax the secondary DNA structures --- p.112 / Chapter 3.2.1.2.2.1 --- Linearization of M13mp7 with BamH I befor annealing the adaptor --- p.113 / Chapter 3.2.1.2.2.2 --- Relaxation of secondary structure using boiling and NaOH denaturation --- p.114 / Chapter 3.2.1.2.3 --- Methods used to optimize the digestion process --- p.115 / Chapter 3.2.1.2.3.1 --- Addition of BSA --- p.115 / Chapter 3.2.1.2.3.2 --- Addition of the restriction endonuclease in separate batches --- p.115 / Chapter 3.2.1.3 --- Digestion of ss M13mpl8 and ssM13mpl9 DNA using Xcm I adaptor #2 and adaptor #4 --- p.116 / Chapter 3.2.2 --- Xcm I adaptor #1 and #3 --- p.118 / Chapter 3.2.2.1 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #1 and adaptor #3 --- p.119 / Chapter 3.2.2.1.1 --- Methods used to relax the secondary structure --- p.119 / Chapter 3.2.2.1.1.1 --- Linearization of M13mp7 with BamH I before the annealing reaction --- p.120 / Chapter 3.2.2.1.1.2 --- Relaxation of secondary structure by NaOH denaturation --- p.121 / Chapter 3.2.2.1.1.3 --- Relaxation of secondary structure by adding DMSO and urea --- p.122 / Chapter 3.2.2.1.2 --- Methods used to optimize the digestion and hybridization processes --- p.123 / Chapter 3.2.2.1.2.1 --- Annealing of M13mp7 with a different amount of adaptor #3 and digesting the DNA complex with Xcm I at different temperatures --- p.123 / Chapter 3.2.2.1.2.2 --- Optimization of digestion by adding Xcm I in separate batches --- p.124 / Chapter 3.2.3 --- BsiY I adaptor --- p.124 / Chapter 3.2.3.1 --- Methods used to optimize the cleavage of M13mp7-BsiY I adaptor complex with BsiY I --- p.126 / Chapter 3.2.3.1.1 --- Optimization of hybridization using various concentrations of NaCl during the annealing reaction --- p.126 / Chapter 3.2.3.1.2 --- Optimization of digestion by binding BsiY I to the BsiY I adaptor before annealing --- p.127 / Chapter 3.2.4 --- The use of 5' end-labelled oligonucleotide DNA substrates for digestion with universal restriction endonuclease --- p.128 / Chapter Chapter 4 --- Molecular cloning of the BsiY I restriction-modification system / Chapter 4.1 --- Materials and methods --- p.132 / Chapter 4.1.1 --- Preparation of chromosomal DNA from BsiY I producing Bacillus stearothermophilus --- p.132 / Chapter 4.1.1.1 --- Restriction digestion of the chromosomal DNA --- p.134 / Chapter 4.1.1.2 --- Southern hybridization to locate the position of the DNA fragment coding for the restriction-modification system --- p.135 / Chapter 4.1.1.2.1 --- Southern transfer of DNA fragments onto nitro-cellulose paper --- p.135 / Chapter 4.1.1.2.2 --- Labelling of the probes by Nick-translation --- p.136 / Chapter 4.1.1.2.3 --- Hybridization of the nick-translated probes onto the DNA fragments fixed on NC paper --- p.137 / Chapter 4.1.2 --- Large-scale preparation of the cloning vector --- p.137 / Chapter 4.1.2.1 --- Restriction endonuclease digestion and dephosphorylation of the vector ´Ø.… --- p.139 / Chapter 4.1.3 --- Ligation between vector and DNA inserts --- p.139 / Chapter 4.1.4 --- Transformation of the ligated DNA into competent cells --- p.140 / Chapter 4.1.4.1 --- Preparation of competent cells --- p.140 / Chapter 4.1.4.2 --- Transformation of the ligated vector and insert DNA into competent cells --- p.142 / Chapter 4.1.5 --- Rapid alkaline lysis method for screening transformants that contains an insert --- p.143 / Chapter 4.1.6 --- Preparation of the genomic library and its plasmid DNA --- p.144 / Chapter 4.1.7 --- Screening procedures used to clone the BsiY I restriction-modification system --- p.144 / Chapter 4.1.7.1 --- In vitro selection using Hungarian Trick --- p.145 / Chapter 4.1.7.2 --- In vivo selection using the host strain AP1-200 and AP1-200-9 --- p.145 / Chapter 4.1.7.2.1 --- Preparation of competent AP1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.7.2.2 --- Transformation of the genomic library plasmid into competent AP 1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.8 --- Assay of BsiY I restriction endonuclease and methylase activities in the suspecting clones --- p.147 / Chapter 4.1.8.1 --- Assay to BsiY I methylase activity - resistance of the plasmid to BsiY I digestion --- p.147 / Chapter 4.1.8.2 --- Assay of BsiY I methylase activity - ability to incorporate H3-methyl group from H3-SAM into DNA substrate molecules --- p.148 / Chapter 4.1.8.3 --- Assay of BsiY I restriction endonuclease activity - ability of crude enzyme extract to cleave DNA --- p.149 / Chapter 4.2 --- Results --- p.150 / Chapter 4.2.1 --- Construction of the BamH I genomic library --- p.150 / Chapter 4.2.1.1 --- Vector and insert used --- p.150 / Chapter 4.2.1.2 --- Optimization of the ligation and transformation process --- p.151 / Chapter 4.2.1.3 --- Preparation of the BamH I library --- p.153 / Chapter 4.2.1.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.155 / Chapter 4.2.1.4.1 --- The Hungarian Trick --- p.155 / Chapter 4.2.1.4.2 --- Screening of the restriction-modification system using the strains API-200 and AP1-200-9 --- p.159 / Chapter 4.2.2 --- Construction of the Hind III library --- p.161 / Chapter 4.2.2.1 --- Vector and insert used --- p.161 / Chapter 4.2.2.2 --- Optimization of the ligation and transformation process --- p.162 / Chapter 4.2.2.3 --- Preparation of the Hind III library --- p.164 / Chapter 4.2.2.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.165 / Chapter 4.2.2.4.1 --- The Hungarian Trick --- p.165 / Chapter 4.2.2.4.2 --- Screening of the restriction-modification system using the strain AP1-200 and AP1-200-9 --- p.168 / Chapter 4.2.2.5 --- Assay of methylase activity using H3-SAM --- p.170 / Chapter 4.2.3 --- The use of Southern blotting and hybridization to find if two available probes have homology to the BsiY I restriction-modification system --- p.173 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Purification and characterization of restriction endonucleases from Bacillus spp --- p.176 / Chapter 5.1.1 --- Methods used to purify the restriction endonuclease --- p.177 / Chapter 5.1.2 --- Characterization of the restriction endonucleases --- p.179 / Chapter 5.1.2.1 --- Determination of the purities of the purified restriction endonucleases --- p.179 / Chapter 5.1.2.2 --- Determination of the recognition site --- p.179 / Chapter 5.1.2.3 --- Determination of the cleavage site --- p.180 / Chapter 5.1.2.4 --- Sequencing using Deaza-dGTP --- p.181 / Chapter 5.2 --- The use of Xcm I and BsiY I as universal restriction endonucleases --- p.182 / Chapter 5.2.1 --- The adverse effects of hair-pin loop on the cleavage with universal restriction enzymes --- p.183 / Chapter 5.3 --- Molecular cloning of the BsiY I restriction-modification system --- p.187 / Chapter 5.3.1 --- Construction of the genomic library --- p.187 / Chapter 5.3.1.1 --- Preparation of the insert and vector --- p.188 / Chapter 5.3.1.2 --- Optimization of the ligation and transformation processes --- p.188 / Chapter 5.3.2 --- Screening strategies used to clone the BsiY I restriction-modification system --- p.189 / Chapter 5.3.2.1 --- The Hungarian Trick --- p.189 / Chapter 5.3.2.2 --- Screening using the strains AP1-200 and AP1-200-9 cells --- p.191 / Chapter 5.3.3 --- Assay of the gene products from the cloned restriction-modification system --- p.192 / Chapter 5.3.3.1 --- Methylase activity --- p.192 / Chapter 5.3.3.2 --- Restriction endonuclease activity --- p.193 / Chapter 5.4 --- Future prospects --- p.193 / References --- p.195 / Appendix --- p.201

Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.

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

No description available.

Bacterial type I restriction-modification systems are composed of three different subunits: one HsdS subunit is required for identification of target sequence and anchoring the enzyme complex on DNA; two HsdM subunits in the methyl-transferase complex serve for host genome modification accomplishing a protective function against self-degradation; two HsdR (or motor) subunits house ATP-dependent translocation and consequent cleavage of double stranded DNA activities. The crystal structure of the 120 kDa HsdR subunit of the Type I restriction-modification system EcoR124I in complex with ATP was recently reported. HsdR is organized into four approximately globular structural domains in a nearly square-planar arrangement: the N-terminal endonuclease domain, the RecA-like helicase domains 1 and 2 and the C-terminal helical domain. The near-planar arrangement of globular domains creates prominent grooves between each domain pair. The two helicase-like domains form a canonical helicase cleft in which double-stranded B-form DNA can be accommodated without steric clash. The helical domain, probably involved in complex assembly, exhibits only a few specific interactions with helicase 2 domain. Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been yet structurally investigated. Here we propose a translocation cycle of the restriction-modification system EcoR124I based on analysis of available crystal structures of superfamily 2 helicases, strutural modeling and complementary biochemical characterization of mutations introduced in sites potentially inportant for translocation in the HsdR motor subunit. Also a role of the extended region of the helicase motif III in ATPase activity of EcoR124I was probed.

>Magister Scientiae - MSc / The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint. / The National Research Foundation and the Norwegian Research Council

The mitochondrial DNA restriction fragment patterns of killer whales (Orcinus orca) were investigated in order to determine the level of genetic differentiation that exists between killer whales from various geographic locations. Twenty one killer whales were examined, seventeen of which were captive killer whales that originated from the North Atlantic and Northeast Pacific Oceans. Two were captive-born animals and two were killer whales that stranded along the Northeast Pacific coast. DNA was extracted from blood and/or tissue samples, cleaved with a variety of restriction endonucleases and the DNA fragments were separated by horizontal agarose gel electrophoresis. The DNA was then transferred to nylon membranes and the killer whale mitochondrial DNA was visualized by hybridization to the complete mitochondrial DNA genome of Commerson's dolphin (Cephalorhynchus commersonii). The resultant restriction fragment patterns were analyzed to determine whether mitochondrial DNA variation was present between killer whales from different geographic regions or between communities and pods of killer whales from the same geographic location.