by Lee Kai Fai, Calvin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 159-167). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iv / ABBREVIATIONS --- p.xi / Chapter CHAPTER ONE --- General Introduction --- p.1 / Chapter 1.1 --- The Phenomenon of Host-controlled Restriction --- p.1 / Chapter 1.2 --- Classification of Restriction and Modification Systems --- p.2 / Chapter 1.2.1 --- Type I Restriction-Modification Systems --- p.2 / Chapter 1.2.2 --- Type II Restriction-Modification Systems --- p.3 / Chapter 1.2.3 --- Type III Restriction-Modification Systems --- p.4 / Chapter 1.2.4 --- Type IV Restriction-Modification Systems --- p.5 / Chapter 1.3 --- Occurrence of Restriction-Modification Systems --- p.6 / Chapter 1.4 --- Effect of Methylation --- p.7 / Chapter 1.5 --- Alternation of Recognition Specificities --- p.7 / Chapter 1.5.1 --- Cross Protection by DNA Methyltransferase --- p.8 / Chapter 1.5.2 --- A-Assisted Restriction Endonuclease (RARE) Cleavage --- p.9 / Chapter 1.5.3 --- Site-specific Cleavage mediated by Triple-helix formation --- p.9 / Chapter 1.5.4 --- Site-specific Cleavage of Duplex DNA with a λ repressor- Staphylococcal Nuclease Hybrid --- p.10 / Chapter 1.5.5 --- Achilles' heel Cleavage --- p.10 / Chapter 1.5.6 --- Chimeric Restriction Endonuclease --- p.11 / Chapter 1.6 --- Cloning of Restriction and Modification Systems --- p.11 / Chapter 1.6.1 --- Selection based on Modification --- p.11 / Chapter 1.6.2 --- Other Cloning Strategies --- p.12 / Chapter 1.6.2.1 --- Sub-Cloning of Plasmids --- p.12 / Chapter 1.6.2.2 --- Selection based on Restriction --- p.13 / Chapter 1.6.2.3 --- Multi-step Cloning --- p.13 / Chapter 1.6.2.4 --- Cloning in AP1-200 and AP1-200-9 strain --- p.13 / Chapter 1.6.2.5 --- Direct Cloning of Restriction gene by 'endo-blue' method --- p.14 / Chapter 1.7 --- Genetic Location of Restriction-Modification Systems --- p.14 / Chapter 1.8 --- Sequences of Restriction-Modification Systems --- p.15 / Chapter 1.9 --- Catalytic Properties of Type II Restriction-Modification Systems --- p.17 / Chapter 1.10 --- Crystallography of Type II Restriction and Modification Enzymes --- p.19 / Chapter 1.11 --- Evolution of Type II Restriction and Modification Enzymes --- p.22 / Chapter 1.12 --- Aim of Study --- p.23 / Chapter CHAPTER TWO --- Materials and Methods --- p.24 / Chapter 2.1 --- Bacterial Strains --- p.24 / Chapter 2.2 --- General Techniques --- p.25 / Chapter 2.2.1 --- Phenol/Chloroform Extraction --- p.25 / Chapter 2.2.2 --- Ethanol Precipitation --- p.25 / Chapter 2.2.3 --- Spectrophotometry --- p.25 / Chapter 2.2.4 --- Restriction digestion of DNA --- p.26 / Chapter 2.2.5 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.2.6 --- Recovery of DNA fragment from Agarose gel --- p.26 / Chapter 2.2.7 --- Minipreparation of Plasmid --- p.27 / Chapter 2.2.8 --- Large-Scale Preparation of Plasmid DNA --- p.28 / Chapter 2.2.8A --- By Equilibrium Centrifugation in Cesium Chloride- Ethidium Bromide Gradient --- p.28 / Chapter 2.2.8B --- By Using Qiagen-tip 100 Cartridge --- p.29 / Chapter 2.2.9 --- Preparation of Competent Cells --- p.30 / Chapter 2.2.10 --- Transformation of Competent Cells --- p.31 / Chapter 2.2.11 --- Screening of Recombinant Plasmids --- p.32 / Chapter 2.2.11A --- Using Selective media --- p.32 / Chapter 2.2.11B --- Rapid Alkaline Lysis Method --- p.32 / Chapter 2.2.12 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.13 --- Size Exclusion Chromatography --- p.34 / Chapter 2.2.14 --- Electroblotting of Protein on Polyvinylidene Difluoride (PVDF) membrane --- p.35 / Chapter 2.2.15 --- Isoelectric Focusing (EEF) --- p.36 / Chapter 2.2.16 --- Protein Assay --- p.37 / Chapter 2.3 --- DNA Sequencing --- p.37 / Chapter 2.3.1 --- Isolation of a template DNA --- p.38 / Chapter 2.3.2 --- DNA Denaturation and Annealing Reaction --- p.38 / Chapter 2.3.3 --- Labeling and Termination Reaction --- p.38 / Chapter 2.3.4 --- DNA Sequencing Electrophoresis --- p.39 / Chapter 2.3.5 --- Autoradiography --- p.40 / Chapter CHAPTER THREE --- Purification and Characterization of Restriction Endonuclease from Escherichia coli HK31 --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Preparation of Crude enzyme Extract --- p.42 / Chapter 3.2.2 --- Purification of R.EcoHK31I --- p.42 / Chapter 3.2.3 --- Characterization of Restriction endonuclease --- p.43 / Chapter 3.2.3.1 --- Enzyme Activity assay --- p.43 / Chapter 3.2.3.2 --- "Optimal pH, Temperature, Metal Ion and Salt concentration of R.EcoHK31I" --- p.43 / Chapter 3.2.3.3 --- Assay for the Purity of R.EcoHK31I --- p.43 / Chapter 3.2.3.4 --- Determination of Recognition Specificity --- p.44 / Chapter 3.2.3.5 --- Determination of the Cleavage Specificity --- p.44 / Chapter 3.3 --- Results and Discussion --- p.45 / Chapter 3.3.1 --- Purification ofR.EcoHK31I from Escherichia coli HK31 --- p.45 / Chapter 3.3.2 --- "Optimal pH,Temperature, Metal ions and Salt concentration of R.EcoHK31I" --- p.46 / Chapter 3.3.3 --- Unit Definition --- p.51 / Chapter 3.3.4 --- Purity of the R.EcoHK31I --- p.51 / Chapter 3.3.5 --- Recognition Site of the R.EcoHK31I --- p.51 / Chapter 3.3.6 --- Sensitivity of the R.EcoHK31I to dcm Methylation --- p.52 / Chapter 3.3.7 --- Cleavage Specificity of R.EcoHK31I --- p.52 / Chapter CHAPTER FOUR --- Cloning of EcoEK31I Restriction and Modification (R-M) System from Escherichia coli HK31 --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and Methods --- p.58 / Chapter 4.2.1 --- Extraction of genomic DNA from E. coli HK31 --- p.58 / Chapter 4.2.2 --- Extraction of Extra-Chromosomal DNA from E. coli HK31 --- p.59 / Chapter 4.2.3 --- Restriction Digestion of the Total DNA --- p.59 / Chapter 4.2.4 --- Preparation of Linearized and Dephosphorylated Vector --- p.60 / Chapter 4.2.5 --- Fill-in Reaction --- p.60 / Chapter 4.2.6 --- Ligation between Vector and Digested Chromosomal DNA --- p.61 / Chapter 4.2.7 --- Selection of Clones Harboring Methyltransferase gene --- p.61 / Chapter 4.2.8 --- Screening of the Survival Clones --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Construction of Genomic Libraries --- p.62 / Chapter 4.3.2 --- Selection of the Methyltransferase Gene --- p.66 / Chapter 4.3.3 --- In vitro Detection of R.EcoHK31I activity --- p.67 / Chapter 4.3.4 --- Functional Localization of EcoHK31I --- p.67 / Chapter 4.3.5 --- Subcloning of the Complete EcoHK31I R-M System --- p.72 / Chapter 4.4 --- Discussion --- p.72 / Chapter 4.4.1 --- Construction of Genomic Libraries --- p.72 / Chapter 4.4.2 --- Cloning of EcoHK31I Restriction and Modification System --- p.75 / Chapter 4.4.2.1 --- Selecting Endonuclease --- p.75 / Chapter 4.4.2.2 --- Detection of Restriction Endonuclease Activity --- p.76 / Chapter 4.4.3 --- Functional Localization of the R-M System --- p.76 / Chapter CHAPTER FIVE --- The Nucleotide Sequences of the EcoHK31I R-M System --- p.78 / Chapter 5.1 --- Introduction --- p.78 / Chapter 5.2 --- Materials and Methods --- p.79 / Chapter 5.2.1 --- Sequencing Strategies --- p.79 / Chapter 5.2.2 --- DNA Sequencing --- p.80 / Chapter 5.2.3 --- Sequence Analysis --- p.80 / Chapter 5.3 --- Results and Discussion --- p.80 / Chapter 5.3.1 --- Nucleotide Sequences and Deduced Amino Acid sequences --- p.80 / Chapter 5.3.2 --- Comparison of Amino Acid Sequences --- p.85 / Chapter CHAPTER SIX --- Purification and Characterization of EcoHK31I Methyltransferase from E. coli K802 [pEcoHK31E] --- p.91 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.92 / Chapter 6.2.1 --- Preparation of Crude enzyme Extract --- p.92 / Chapter 6.2.2 --- Purification of M.EcoHK31I --- p.92 / Chapter 6.2.3 --- Characterization of EcoHK31I Methyltransferase --- p.93 / Chapter 6.2.3.1 --- Enzyme Activity assay --- p.93 / Chapter 6.2.3.2 --- Determination of Methylation specificity --- p.93 / Chapter 6.2.3.3 --- Determination of Molecular weight of M.EcoHK31I --- p.94 / Chapter 6.2.3.4 --- Determination ofM.EcoHK31I Kinetics --- p.94 / Chapter 6.3 --- Results and Discussion --- p.96 / Chapter 6.3.1 --- Purification of EcoHK31I Methyltransferase --- p.96 / Chapter 6.3.2 --- M.EcoHK31I Modification Specificity --- p.99 / Chapter 6.3.3 --- "Determination of Molecular Weight ofM,EcoHK31I" --- p.99 / Chapter 6.3.4 --- Catalytic Properties of EcoHK31I Methyltransferase --- p.103 / Chapter 6.3.5 --- A Novel m5C-MTase M.EcoHK31I --- p.103 / Chapter CHAPTER SEVEN --- Over-expression and Characterization of EcoHK31I Restriction and Modification Enzymes --- p.106 / Chapter 7.1 --- Introduction --- p.106 / Chapter 7.1.1 --- Expression Vector pTrc series --- p.107 / Chapter 7.1.2 --- Expression Vector pET series --- p.107 / Chapter 7.2 --- Materials and Methods --- p.109 / Chapter 7.2.1 --- General technique --- p.109 / Chapter 7.2.2 --- Polymerase Chain Reaction --- p.110 / Chapter 7.2.3 --- Construction of plysSM13 --- p.110 / Chapter 7.2.4 --- Construction of pTrc99A-R36 --- p.110 / Chapter 7.2.5 --- Construction of pET3a-M38 --- p.111 / Chapter 7.2.6 --- Construction of pET3a-C23 --- p.111 / Chapter 7.2.7 --- Expression of Recombinant Proteins in E. coli hosts --- p.115 / Chapter 7.2.8 --- Purification of Recombinant R.EcoHK31I --- p.115 / Chapter 7.2.9 --- Determination of Molecular Weight of Recombinant R. EcoHK31I --- p.115 / Chapter 7.2.10 --- Polyclonal Antibodies against R.EcoHK31I --- p.116 / Chapter 7.2.11 --- Western Blotting --- p.116 / Chapter 7.2.12 --- Purification of Recombinant M.EcoHK31I polypeptide α --- p.117 / Chapter 7.2.13 --- Purification of Recombinant M.EcoHK31I polypeptide β --- p.118 / Chapter 7.2.14 --- In vitro Complementation Methylation Activity --- p.118 / Chapter 7.2.15 --- Incorporation of [3H]-AdoMet to non-methylated Lambda DNA --- p.119 / Chapter 7.3 --- Results and Discussion --- p.119 / Chapter 7.3.1 --- Expression of Recombinant R. EcoHK31I --- p.119 / Chapter 7.3.2 --- Purification and Characterization of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.1 --- Purification of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.2 --- Characterization of Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.1 --- Molecular Weight and Isoelectric point of the Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.2 --- Antibodies to Recombinant R.EcoHK31I --- p.125 / Chapter 7.3.3 --- Expression and Purification of M.EcoHK31Ipolypeptide α --- p.127 / Chapter 7.3.4 --- Expression and Purification of M.EcoHK31I polypeptide β --- p.127 / Chapter 7.3.5 --- Characterization of M.EcoHK31I polypeptides a and β --- p.129 / Chapter 7.3.5.1 --- Molecular Weight Determination --- p.129 / Chapter 7.3.5.2 --- Isoelectric Point Determination --- p.132 / Chapter 7.3.5.3 --- In vivo and in vitro Methylation Activity --- p.132 / Chapter CHAPTER EIGHT --- Generation and Activity Assay of Q193G Mutein of M.EcoHK31I Polypeptide a --- p.138 / Chapter 8.1 --- Introduction --- p.138 / Chapter 8.2 --- Materials and Methods --- p.139 / Chapter 8.2.1 --- Construction of pET3a-M38 (Q193G) --- p.139 / Chapter 8.2.2 --- Expression and Purification of Q193G protein --- p.140 / Chapter 8.2.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.140 / Chapter 8.3 --- Results and Discussion --- p.145 / Chapter 8.3.1 --- "Construction, Expression and Purification of Q193G Mutein" --- p.145 / Chapter 8.3.2 --- Determination of Molecular Weight and Isoelectric point of Q193G --- p.145 / Chapter 8.3.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.145 / Chapter 8.3.4 --- Recognition Specificity of Q193G Mutein --- p.147 / Chapter CHAPTER NINE --- General Discussion --- p.151 / REFERENCES --- p.159 / APPENDIX A --- p.168
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318320 |
| Date | January 1995 |
| Contributors | Lee, Kai Fai Calvin., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xi, 169 leaves : ill. (some mounted col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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