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

by Henry, Kam-yin Cheung. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 119-126.

by Liang Yiu-hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 104-117). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.iii / AMINO ACIDS SHORTHAND --- p.v / TABLE OF CONTENTS --- p.vi-x / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- The Discovery of IGFs --- p.1 / Chapter 1.3 --- The Growth Promoting Actions of IGFs --- p.3 / Chapter 1.4 --- Molecular Biology of IGFs in Mammals --- p.6 / Chapter 1.4.1 --- IGF Genes and Transcripts --- p.6 / Chapter 1.4.2 --- Regulation of IGF Gene Expression --- p.8 / Chapter 1.5 --- IGF Binding Proteins --- p.11 / Chapter 1.5.1 --- Regulation of IGF Action by IGF Binding Proteins --- p.11 / Chapter 1.6 --- The Insulin and IGF Receptors --- p.13 / Chapter 1.6.1 --- IGF-I Receptor --- p.13 / Chapter 1.6.2 --- IGF-II Receptor --- p.13 / Chapter 1.6.3 --- Insulin/IGF-I Hybrid Receptor --- p.15 / Chapter 1.7 --- IGF in Mammalian Fetal Growth --- p.17 / Chapter 1.8 --- The Role of IGFs in Fish --- p.19 / Chapter 1.9 --- Aims of the Present Study --- p.26 / Chapter CHAPTER 2 --- GENERAL METHODOLOGY / Chapter 2.1 --- Materials --- p.27 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Gene Clean --- p.32 / Chapter 2.2.1A --- Gene Clean by Glassmilk Method --- p.32 / Chapter 2.2.1B --- Gene Clean by Sephaglas´ёØ BandPrep Kit --- p.32 / Chapter 2.2.2 --- Preparation of Radioactive Nucleic Acid Probes --- p.33 / Chapter 2.2.3 --- Sephadex G-50 Spun-column Chromatography --- p.33 / Chapter 2.2.4 --- Small Scale Alkali Preparation of Plasmid DNA --- p.34 / Chapter 2.2.5 --- Large Scale Preparation of Plasmid DNA 36 - using Wizard Maxiprep Kit (Promega) / Chapter 2.2.6 --- DNA Sequencing using T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.37 / Chapter 2.2.7 --- Restriction Enzyme Digestion --- p.38 / Chapter 2.2.8 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.9 --- Dephosphorylation of Linearized Plasmid DNA --- p.39 / Chapter 2.2.10 --- Ligation of Foreign DNA with Linearized Plasmid --- p.40 / Chapter 2.2.11 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.40 / Chapter 2.2.12 --- Blotting : Transfer of DNA to Nylon Membrane --- p.41 / Chapter 2.2.12A --- Capillary Transfer of DNA to Nylon Membrane in 10X SSC --- p.41 / Chapter 2.2.12B --- Capillary Transfer of DNA to Nylon Membrane under Alkaline Condition --- p.42 / Chapter CHAPTER 3 --- SCREENING OF COMMON CARP LIVER cDNA LIBRARY / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Methods --- p.48 / Chapter 3.2.2.1 --- Preparation of the Plating Host --- p.48 / Chapter 3.2.2.2 --- Phage Titering --- p.48 / Chapter 3.2.2.3 --- Primary Screening of Common Carp Liver cDNA Library --- p.48 / Chapter 3.2.2.4 --- Purification of the Positive Clone --- p.49 / Chapter 3.2.2.5 --- Checking the Insert Size of the Positive Clone --- p.50 / Chapter 2.2.2.6 --- In vivo Excision to Release Phagemid from the Phage vector --- p.51 / Chapter 3.2.2.7 --- Plasmid Minipreparation of the Positive Clone --- p.52 / Chapter 3.2.2.8 --- Restriction Enzyme Digestion to Release the Insert --- p.52 / Chapter 3.2.2.9 --- Large Scale Plasmid Preparation of the Positive Clone --- p.53 / Chapter 3.2.2.10 --- DNA Sequencing of the Positive Clone --- p.53 / Chapter 3.2.2.11 --- Restriction Mapping of the Positive Clone --- p.53 / Chapter 3.2.2.12 --- Subcloning of the Positive Clone into Plasmid Vectors --- p.53 / Chapter 3.2.2.13 --- DNA Sequencing of the Subclones --- p.54 / Chapter 3.3 --- Results and Discussion --- p.55 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.2 --- Materials and Methods --- p.71 / Chapter 4.2.1 --- Materials --- p.71 / Chapter 4.2.2 --- Methods --- p.72 / Chapter 4.2.2.1 --- Tissue Preparation --- p.72 / Chapter 4.2.2.2 --- Total RNA Extraction --- p.72 / Chapter 4.2.2.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.73 / Chapter 4.2.2.4 --- First Strand cDNA Synthesis --- p.74 / Chapter 4.2.2.5 --- IGF-I Specific PCR --- p.75 / Chapter 4.2.2.6 --- Preparation of Carp IGF Conserve Region --- p.75 / Chapter 4.2.2.7 --- Southern Hybridization of PCR Products --- p.76 / Chapter 4.3 --- Results and Discussion --- p.76 / Chapter CHAPTER 5 --- GENOMIC SOUTHERN ANALYSIS / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and Methods --- p.81 / Chapter 5.2.1 --- Materials --- p.81 / Chapter 5.2.2 --- Methods --- p.82 / Chapter 5.2.2.1 --- Preparation of Genomic DNA from Carp Testis --- p.82 / Chapter 5.2.2.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.82 / Chapter 5.2.2.3 --- Southern Blotting of the Digested Genomic DNA --- p.83 / Chapter 5.2.2.4 --- Preparation of the Carp IGF-I Specific Probe --- p.83 / Chapter 5.2.2.5 --- Genomic Southern Hybridization --- p.83 / Chapter 5.3 --- Results and Discussion --- p.84 / Chapter CHAPTER 6 --- THE SEARCH OF OTHER IGF cDNA SUBTYPES IN COMMON CARP / Chapter 6.1 --- Introduction --- p.88 / Chapter 6.2 --- Materials and Methods --- p.89 / Chapter 6.2.1 --- Materials --- p.89 / Chapter 6.2.2 --- Methods --- p.91 / Chapter 6.2.2.1 --- Screening using a Conserve Region cDNA Probe of Carp IGF-I --- p.91 / Chapter 6.2.2.2 --- PCR using IGF-I Specific Primers --- p.92 / Chapter 6.2.2.3 --- PCR Using T3 and T7 Primers --- p.92 / Chapter 6.2.2.4 --- Southern Blot Analysis of T3 and T7 PCR Products of cDNA Insert --- p.93 / Chapter 6.2.2.5 --- DNA Sequencing of Positive Clones --- p.94 / Chapter 6.3 --- Results and Discussion --- p.94 / Chapter CHAPTER 7 --- GENERAL DISCUSSION AND CONCLUSION --- p.99 / REFERENCES --- p.104-117

by Lau Kwok Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 126-134). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iv / Abbreviations --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / List of Primers --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- A Brief Introduction of GH --- p.1 / Chapter 1.2 --- Growth Hormone Receptor (GHR) --- p.3 / Chapter 1.2.1 --- Tissue Distribution of GHR --- p.4 / Chapter 1.2.2 --- GHR Biosynthesis and Degradation --- p.7 / Chapter 1.2.3 --- Regulation of GHR level --- p.8 / Chapter 1.2.4 --- Structure of GHR --- p.10 / Chapter 1.2.5 --- Possible Signal Transduction Pathways of GHR --- p.13 / Chapter 1.2.6 --- GHR Related Dwarfism --- p.15 / Chapter 1.2.7 --- Significance of Cloning of GHR cDNA --- p.16 / Chapter 1.3 --- Objectives of the Present Study --- p.17 / Chapter Chapter 2 --- General Materials and Methods / Chapter 2.1 --- Ethanol Precipitation of DNA and RNA --- p.19 / Chapter 2.2 --- Spectrophotometric Determination of DNA and RNA --- p.19 / Chapter 2.3 --- Minipreparation of Plasmid DNA --- p.19 / Chapter 2.4 --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification Kit from Promega --- p.20 / Chapter 2.5 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.21 / Chapter 2.6 --- Preparation and Transformation of Escherichia coli Competent Cell --- p.22 / Chapter 2.7 --- Rapid Screening for the Presence of Desired Plasmid --- p.23 / Chapter 2.8 --- Agarose Gel Electrophoresis --- p.23 / Chapter 2.9 --- Formaldehyde / Agarose Gel Electrophoresis --- p.24 / Chapter 2.10 --- Restriction Digestion of DNA --- p.25 / Chapter 2.11 --- Linearization and Dephosphorylation of Plasmid Vector --- p.25 / Chapter 2.12 --- Purification of DNA form Agarose Gel Using GENECLEAN II® Kit --- p.25 / Chapter 2.13 --- Purification of DNA by Phenol / Chloroform Extraction --- p.26 / Chapter 2.14 --- DNA Radiolabelling --- p.26 / Chapter 2.15 --- Spun-Column Chromatography --- p.27 / Chapter 2.16 --- Capillary Transfer of DNA/RNA to a Nylon Membrane --- p.27 / Chapter 2.16.1 --- DNA Denaturation --- p.27 / Chapter 2.16.2 --- Capillary Transfer --- p.28 / Chapter 2.17 --- Hybridization of DNA/RNA --- p.28 / Chapter 2.18 --- Autoradiography --- p.29 / Chapter 2.19 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.29 / Chapter 2.20 --- Total RNA Isolation --- p.30 / Chapter 2.21 --- mRNA Isolation --- p.31 / Chapter 2.22 --- First Strand cDNA Synthesis --- p.32 / Chapter 2.23 --- Polymerase Chain Reaction --- p.32 / Chapter 2.24 --- 3'End Modification of PCR Amplified DNA --- p.33 / Chapter 2.25 --- Ligation of DNA Fragments --- p.34 / Chapter 2.26 --- DNA Sequencing --- p.34 / Chapter 2.26.1 --- DNA Sequencing Reaction --- p.34 / Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.35 / Chapter 2.27 --- Reagents and Buffers --- p.38 / Chapter 2.27.1 --- Media for Bacterial Culture --- p.38 / Chapter 2.27.2 --- Reagents for Preparation of Plasmid DNA --- p.38 / Chapter 2.27.3 --- Buffers for Agarose Gel Electrophoresis --- p.40 / Chapter 2.27.4 --- Buffers for Formaldehyde Gel Electrophoresis --- p.40 / Chapter 2.27.5 --- Buffers for Preparation Competent Cells --- p.41 / Chapter 2.27.6 --- Buffers for Capillary Transfer and Hybridization --- p.42 / Chapter 2.27.7 --- Buffers for Total RNA Extraction --- p.43 / Chapter 2.27.8 --- 10X CIP Buffers --- p.43 / Chapter 2.28 --- Size of DNA/RNA Molecular Weight Markers --- p.44 / Chapter Chapter 3 --- Molecular Studies on Chicken Growth Hormone Receptor / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Material and Methods --- p.46 / Chapter 3.2.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.46 / Chapter 3.2.1.1 --- Animals and Tissue --- p.46 / Chapter 3.2.1.2 --- Reverse Transcrbed-Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 3.2.1.3 --- Subcloning of PCR Amplified DNA Fragments --- p.47 / Chapter 3.2.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.48 / Chapter 3.2.2.1 --- Animals and Tissues --- p.48 / Chapter 3.2.2.2 --- Northern Analysis --- p.48 / Chapter 3.2.2.3 --- Quantification of GHR mRNA level --- p.49 / Chapter 3.2.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.49 / Chapter 3.2.3.1 --- Subcloning of Chicken GHR cDNA into a Prokaryotic Expression Vector --- p.49 / Chapter 3.2.3.2 --- Expression of Chicken GHR cDNAin E.coli --- p.50 / Chapter 3.2.3.3 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 3.2.4 --- Reagents and Buffers / Chapter 3.2.4.1 --- Medium for Bacterial Culture --- p.53 / Chapter 3.2.4.2 --- Reagents for SDS-PAGE --- p.53 / Chapter 3.2.5 --- Size of Protein Molecular Weight Markers --- p.54 / Chapter 3.3 --- Results --- p.55 / Chapter 3.3.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.55 / Chapter 3.3.1.1 --- RT-PCR --- p.55 / Chapter 3.3.1.2 --- Subcloning --- p.56 / Chapter 3.3.1.3 --- Nucleotide Sequence Analysis --- p.57 / Chapter 3.3.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.59 / Chapter 3.3.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.64 / Chapter 3.3.3.1 --- Subcloning --- p.64 / Chapter 3.3.3.2 --- Nucleotide Sequence Analysis --- p.65 / Chapter 3.3.3.3 --- Prokaryotic Expression --- p.66 / Chapter 3.4 --- Discussion --- p.68 / Chapter 3.4.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.68 / Chapter 3.4.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.70 / Chapter 3.4.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.71 / Chapter Chapter 4 --- Molecular Cloning of Pigeon Growth Hormone Receptor Complementary DNA by Polymerase Chain Reaction and Sequence Analysis / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and Methods --- p.75 / Chapter 4.2.1 --- Animals and Tissues --- p.75 / Chapter 4.2.2 --- Cloning of Pigeon GHR cDNA Main Core by PCR --- p.75 / Chapter 4.2.2.1 --- RT-PCR --- p.75 / Chapter 4.2.2.2 --- Southern Analysis of PCR Amplified Product --- p.76 / Chapter 4.2.2.3 --- Subcloning of PCR Amplified DNA Fragment --- p.76 / Chapter 4.2.3 --- Determination of 3' End Coding Sequence of Pigeon GHR cDNA --- p.76 / Chapter 4.2.4 --- Determination of 5' End Coding Sequence of Pigeon GHR cDNA --- p.79 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Cloning of Pigeon GHR cDNA Main Core by PCR --- p.82 / Chapter 4.3.1.1 --- RT-PCR --- p.82 / Chapter 4.3.1.2 --- Southern Analysis --- p.83 / Chapter 4.3.1.3 --- Subcloning of Fragment M --- p.83 / Chapter 4.3.1.4 --- Restriction Digestion of Plasmid --- p.85 / Chapter 4.3.1.5 --- Nucleotide Sequence Analysis --- p.86 / Chapter 4.3.2 --- Determination of 3' End and 5' End coding Sequences of Pigeon GHR cDNA --- p.88 / Chapter 4.3.2.1 --- Random Primer Initiated RNA-PCR --- p.88 / Chapter 4.3.2.2 --- AmpliFINDER RACE --- p.88 / Chapter 4.3.2.3 --- Subcloning of Fragment 3' and Fragment 5' --- p.90 / Chapter 4.3.2.4 --- Nucleotide Sequence Analysis --- p.92 / Chapter 4.3.3 --- Nucleotide Sequence and Predicted Amino Acid Sequence of Pigeon GHR --- p.93 / Chapter 4.4 --- Discussion --- p.100 / Chapter Chapter 5 --- Attempts on Molecular Cloning of Fish Growth Hormone Receptor Complementary DNA / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.107 / Chapter 5.2.1 --- Animals and Tissues --- p.107 / Chapter 5.2.2 --- Design of PCR primers --- p.107 / Chapter 5.2.3 --- RT-PCR and Subcloning of PCR Amplified DNA --- p.108 / Chapter 5.2.4 --- Northern Analysis of Dace Liver RNA --- p.110 / Chapter 5.3 --- Results / Chapter 5.3.1 --- PCR --- p.111 / Chapter 5.3.2 --- Subcloning --- p.112 / Chapter 5.3.3 --- Nucleotide Sequence Analysis --- p.114 / Chapter 5.3.4 --- Northern Analysis --- p.117 / Chapter 5.4 --- Discussion --- p.119 / Chapter Chapter 6 --- General Discussion --- p.123 / References --- p.126 / Appendix --- p.135

by Agnes Pui-Yee Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 162-177). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.vii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Physiology of growth --- p.3 / Chapter 1.2 --- The anterior pituitary --- p.4 / Chapter 1.3 --- Chemistry of GH and the GH gene family --- p.7 / Chapter 1.4 --- Biochemical effects and mode of action of GH --- p.8 / Chapter 1.5 --- Control of GH at cellular level --- p.10 / Chapter 1.6 --- Control of GH gene expression at molecular level / Chapter 1.6.1 --- Introduction --- p.11 / Chapter 1.6.2 --- Tissue-specific expression of GH gene / Chapter 1.6.2.1 --- Tissue-specific transcription factors of pituitary cells --- p.20 / Chapter 1.6.2.2 --- Non-tissue specific transcription factors of pituitary cells --- p.27 / Chapter 1.6.2.3 --- Negatively-acting transcription factors of non-pituitary cells --- p.34 / Chapter 1.6.2.4 --- Theory for tissue-specific GH gene activation --- p.39 / Chapter 1.7 --- Characteristic of growth in fish --- p.40 / Chapter 1.8 --- Objectives of the present study --- p.42 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Preparation of DNA / Chapter 2.1.1.1 --- Minipreparation of DNA --- p.46 / Chapter 2.1.1.2 --- Preparation of DNA using Qiagen column --- p.47 / Chapter 2.1.1.3 --- Preparation of phage DNA --- p.48 / Chapter 2.1.2 --- Elution of DNA from agarose gel --- p.51 / Chapter 2.1.3 --- Preparation of competence cells and transformation --- p.52 / Chapter 2.1.4 --- Ligation of DNA fragments --- p.53 / Chapter 2.1.5 --- Cell feeding and subculturing --- p.54 / Chapter 2.2 --- Special techniques / Chapter 2.2.1 --- DNA sequencing --- p.56 / Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.67 / Chapter 2.2.3 --- Direct sequencing of PCR products --- p.72 / Chapter 2.2.4 --- Nested-deletion --- p.75 / Chapter 2.2.5 --- DNA transfection --- p.81 / Chapter 2.2.6 --- CAT assay --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequencing of the grass carp GH gene / Chapter 3.1.1 --- Introduction --- p.93 / Chapter 3.1.2 --- Sequencing strategy --- p.94 / Chapter 3.2 --- Sequence analysis of the grass carp GH gene --- p.108 / Chapter 3.3 --- Functional analysis of the grass carp GH gene --- p.115 / Chapter CHAPTER 4 --- DISCUSSIONS / Chapter 4.1 --- DNA sequence comparison between grass carp GH gene and other organisms --- p.137 / Chapter 4.2 --- Amino acid comparisons between grass carp GH and other organisms --- p.143 / Chapter 4.3 --- Tissue-specific expression of GH gene / Activation of transcription --- p.154 / Repression of transcription --- p.155 / Chapter 4.4 --- Electroporation of zebrafish eggs --- p.157 / Chapter 4.5 --- Further studies --- p.160 / REFERENCES --- p.162 / APPENDIX --- p.178

Chan Pat Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 153-177). / Acknowledgments --- p.i / Presentations Derived from the Present thesis Work --- p.ii / Abstract --- p.iii / Abbreviations --- p.v / Abbreviation Table for Amino Acids --- p.viii / List of Figures --- p.ix / List of Tables --- p.xi / Contents --- p.xii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Environmental Pollutants --- p.1 / Chapter 1.2 --- Pollutant Inducible Genes (PIGs) --- p.1 / Chapter 1.2.1 --- Classification of PIGS --- p.2 / Chapter 1.2.1.1 --- Drug Metabolizing Enzymes/Proteins --- p.2 / Chapter 1.2.1.2 --- Stress Proteins --- p.5 / Chapter 1.2.1.3 --- Antioxidant Enzymes --- p.6 / Chapter 1.2.1.4 --- "Hormones, Growth Factors and Their Receptors" --- p.6 / Chapter 1.2.1.5 --- Enzymes/Proteins Involved in Bioenergetics --- p.6 / Chapter 1.2.2 --- PIGs as a Field of Study --- p.8 / Chapter 1.2.2.1 --- Study of the Mechanism of Detoxification and Toxication --- p.8 / Chapter 1.2.2.2 --- Biomarker Study --- p.9 / Chapter 1.2.2.3 --- Study of Regulation of Gene Expression --- p.11 / Chapter 1.2.2.4 --- Study of Evolution --- p.12 / Chapter 1.3 --- Aims and Rational of the Present Study --- p.12 / Chapter 2 --- General Methodology --- p.15 / Chapter 2.1 --- Materials --- p.15 / Chapter 2.2.1 --- Reagents --- p.15 / Chapter 2.1.1.1 --- Preparation of Plasmid DNA --- p.15 / Chapter 2.1.1.2 --- Preparation of Genomic DNA --- p.15 / Chapter 2.1.1.3 --- Purification of Total RNA --- p.16 / Chapter 2.1.1.4 --- Restriction Enzyme Digestion --- p.16 / Chapter 2.1.1.5 --- Capillary Blotting of DNA (Southern Blotting) --- p.16 / Chapter 2.1.1.6 --- Capillary Blotting of Total RNA (Northern Blotting) --- p.17 / Chapter 2.1.1.7 --- Hybridization --- p.17 / Chapter 2.1.1.8 --- Library Screening --- p.18 / Chapter 2.1.1.9 --- Polymerase Chain Reaction --- p.18 / Chapter 2.1.1.10 --- Transformation of E. coli Competent Cells --- p.19 / Chapter 2.1.1.11 --- Nucleotide Sequence Determination --- p.19 / Chapter 2.1.2 --- List of Primers --- p.20 / Chapter 2.1.2.1 --- Primers used for Nucleotide Sequence Determination --- p.20 / Chapter 2.1.2.2 --- Primer Used for First Strand cDNA Synthesis --- p.20 / Chapter 2.1.2.3 --- Primers for Amplifying Actin cDNA Fragment --- p.20 / Chapter 2.1.2.4 --- Common Carp MT Specific Primers --- p.20 / Chapter 2.1.2.5 --- Teleost CYP1A Specific Primers --- p.21 / Chapter 2.1.2.6 --- Common Carp CYP1A Specific Primers --- p.21 / Chapter 2.1.2.7 --- Primers and Cassettes for the Cloning of5' Upstream Regions of MT Genes --- p.21 / Chapter 2.1.3 --- Accession Numbers of Selected P450 and MT Nucleotide and Amino Acid Sequences in the Genebank --- p.21 / Chapter 2.1.3.1 --- MTs of Different Teleost Species --- p.21 / Chapter 2.1.3.2 --- MTs of Other Vertebrate Species' --- p.22 / Chapter 2.1.3.3 --- P450s of Different Teleost Species --- p.22 / Chapter 2.1.3.4 --- CYP1s of Different Mammalian Species --- p.22 / Chapter 2.2 --- Methods --- p.23 / Chapter 2.2.1 --- Preparation of Plasmid --- p.23 / Chapter 2.2.2 --- Preparation of Genomic DNA --- p.23 / Chapter 2.2.3 --- Purification of Total RNA --- p.24 / Chapter 2.2.4 --- Restriction Enzyme Digestion --- p.25 / Chapter 2.2.5 --- Capillary Blotting of DNA (Southern Blotting) --- p.25 / Chapter 2.2.5.1 --- Semi-dry Capillary Blotting --- p.25 / Chapter 2.2.5.2 --- Alkaline Transfer --- p.25 / Chapter 2.2.5.3 --- Transfer of Digested Genomic DNA on to Nylon Membrane --- p.26 / Chapter 2.2.6 --- Capillary Blotting of Total RNA (Northern Blotting) --- p.26 / Chapter 2.2.7 --- Radioactive Labeling of Nucleic Acid Probes --- p.26 / Chapter 2.2.8 --- Hybridization --- p.27 / Chapter 2.2.9 --- Library Screening --- p.27 / Chapter 2.2.9.1 --- Construction of Liver cDNA Library of Adult Common Carp --- p.27 / Chapter 2.2.9.2 --- Preparation of Plating Cells --- p.27 / Chapter 2.2.9.3 --- Phage Tittering --- p.27 / Chapter 2.2.9.4 --- Primary Screening --- p.28 / Chapter 2.2.9.5 --- Secondary Screening / Chapter 2.2.9.6 --- Conversion of Phage DNA to Phagemid by invivo Excision --- p.28 / Chapter 2.2.10 --- First Strand cDNA Synthesis --- p.29 / Chapter 2.2.11 --- Polymerase Chain Reaction --- p.29 / Chapter 2.2.12 --- Ligation of DNA with Linearized Plasmid --- p.30 / Chapter 2.2.13 --- Transformation of E. coli Competent Cell --- p.30 / Chapter 2.2.14 --- Nucleotide Sequence Determination --- p.31 / Chapter 2.2.15 --- Densitometric Analysis --- p.31 / Chapter 3 --- "Cloning of Common Carp MT cDNA and Gene, and Induction of MT mRNA Expression" --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.1.1 --- Metals in Biological System --- p.32 / Chapter 3.1.2 --- Metallothionein --- p.33 / Chapter 3.1.2.1 --- Functions of MT --- p.26 / Chapter 3.1.2.2 --- Regulation of MT Expression --- p.39 / Chapter 3.1.3 --- Fish MTs --- p.44 / Chapter 3.1.3.1 --- Detection of MT in Teleost --- p.46 / Chapter 3.1.3.2 --- MT Studies in Common Carp --- p.47 / Chapter 3.1.4 --- Specific Aims of This Chapter --- p.49 / Chapter 3.2 --- Strategies --- p.50 / Chapter 3.3 --- Specific Methods --- p.50 / Chapter 3.3.1 --- Cloning of MT cDNAs of Common Carp --- p.50 / Chapter 3.3.2 --- Analysis of MT cDNA Sequences --- p.51 / Chapter 3.3.3 --- Southern Blot Analysis of Common Carp Genomic DNA --- p.52 / Chapter 3.3.4 --- Amplification of MT Gene Fragments Using PCR --- p.52 / Chapter 3.3.5 --- Amplification of the 5' Upstream Regions of MT Genes Using PCR --- p.52 / Chapter 3.3.6 --- Endogenous MT mRNA Expression of Juvenile and Adult Common Carp --- p.54 / Chapter 3.3.7 --- Induction of MT mRNA of Juvenile Common Carp Injected with Cadmium --- p.55 / Chapter 3.4 --- Results --- p.56 / Chapter 3.4.1 --- Cloning of Common Carp MT cDNAs --- p.56 / Chapter 3.4.2 --- Analysis of the MT cDNA Sequences --- p.57 / Chapter 3.4.3 --- Southern Blot Analysis of the Common Carp Genomic DNA --- p.59 / Chapter 3.4.4 --- Amplification of the MT Gene Fragments of Common Carp Using PCR --- p.62 / Chapter 3.4.5 --- Amplification of the 5' Upstream Regions of MT Genes using PCR --- p.65 / Chapter 3.4.6 --- Endogenous MT mRNA Expression of Juvenile and Adult Common Carp --- p.67 / Chapter 3.4.7 --- Induction of MT mRNA of Juvenile Common Carp Injected with Cadmium --- p.68 / Chapter 3.5 --- Discussion --- p.72 / Chapter 3.5.1 --- MT cDNAs of Common Carp --- p.72 / Chapter 3.5.1.1 --- Coding Region --- p.72 / Chapter 3.5.1.2 --- The 3' Untranslated Region --- p.75 / Chapter 3.5.1.3 --- The 5' Untranslated Region --- p.76 / Chapter 3.5.2 --- MT Genes of Common Carp --- p.77 / Chapter 3.5.3 --- MT mRNA Expression of Common Carp --- p.82 / Chapter 3.5.4 --- Normalization of the Signals of Northern Blot Analysis --- p.85 / Chapter 3.5.5 --- Common Carp MT mRNA as Biomarker of Heavy Metal Exposure? --- p.87 / Chapter 3.6 --- Conclusion --- p.89 / Chapter 4 --- Cloning of Common Carp CYP1A cDNAs and Induction of CYP1A mRNA Expression --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.1.1 --- Cytochrome P450s --- p.90 / Chapter 4.1.2 --- Cytochrome P450 1 (CYP1) --- p.93 / Chapter 4.1.3 --- AhR Mediated CYP1A1 Gene Induction --- p.94 / Chapter 4.1.3.1 --- Anthropogenic Sources of AhR Ligands --- p.95 / Chapter 4.1.3.2 --- Natural Sources of AhR Ligands --- p.97 / Chapter 4.1.3.3 --- Potency of Inducibility --- p.97 / Chapter 4.1.3.4 --- Induction of CYP1A1 Gene Transcription by AhR --- p.98 / Chapter 4.1.3.5 --- Non-AhR Mediated CYP1A1 Gene Transcription? --- p.105 / Chapter 4.1.4 --- CYP1A Studies in Teleost Species --- p.107 / Chapter 4.1.4.1 --- Regulation of CYP1A in Teleost --- p.109 / Chapter 4.1.4.2 --- Detection of CYP1A in Teleost --- p.111 / Chapter 4.1.4.3 --- CYP1A Studies of Common Carp --- p.113 / Chapter 4.1.5 --- Specific Aims of This Chapter --- p.114 / Chapter 4.2 --- Strategies --- p.115 / Chapter 4.3 --- Specific Methods --- p.119 / Chapter 4.3.1 --- RT-PCR of CYP1A cDNAs of Common Carp --- p.119 / Chapter 4.3.2 --- Determination of the Nucleotide Sequences of the CYP1A cDNAs of Common Carp --- p.119 / Chapter 4.3.3 --- Library Screening --- p.119 / Chapter 4.3.4 --- Analysis of the CYP1A Genes of Common Carp --- p.121 / Chapter 4.3.5 --- Induction of CYP1A mRNA of Common Carp Injected with 3-MC --- p.122 / Chapter 4.4 --- Results --- p.123 / Chapter 4.4.1 --- RT-PCR of CYP1A cDNAs of Common Carp --- p.123 / Chapter 4.4.2 --- Determination of the Nucleotide Sequences of the CYP1A cDNAs of Common Carp --- p.124 / Chapter 4.4.3 --- Library Screening --- p.124 / Chapter 4.4.4 --- Analysis of the CYP1A Genes of Common Carp --- p.128 / Chapter 4.4.5 --- Induction of CYP1A mRNA of Common Carp Injected with 3-MC --- p.131 / Chapter 4.5 --- Discussion --- p.134 / Chapter 4.5.1 --- On the Use of Rainbow Trout CYP1A1 cDNA Probe --- p.134 / Chapter 4.5.2 --- CYP1A cDNAs of Common Carp --- p.134 / Chapter 4.5.3 --- CYP1A Genes of Common Carp --- p.138 / Chapter 4.5.4 --- CYP1A Expression in Uninduced and Induced Tissues --- p.142 / Chapter 4.5.5 --- The Use of CYP1A cDNAs As Biomarkers --- p.146 / Chapter 4.6 --- Conclusion --- p.148 / Chapter 5 --- General Conclusion --- p.149 / Chapter 6 --- References --- p.153

Siu Shu Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 100-106). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Contents --- p.ii / Abstract / 摘要 --- p.iv / Abbreviations --- p.vi / List of figures --- p.vii / List of tables --- p.x / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- "Human EST sequencing project, the role and goal" --- p.1 / Chapter 1.2 --- Human liver cDNA sequencing --- p.2 / Chapter 1.3 --- The role of membrane-associated proteins in hepatocellular functions --- p.3 / Chapter 1.3.1 --- Outline of the liver function --- p.3 / Chapter 1.3.2 --- Basic structure of hepatocyte --- p.4 / Chapter 1.3.3 --- Category of membrane associated proteins --- p.5 / Chapter 1.4 --- Identification of human OAT2 gene --- p.7 / Chapter 1.5 --- The multispecific transporter family --- p.8 / Chapter 1.5.1 --- Classification --- p.8 / Chapter 1.5.2 --- The human OAT family --- p.9 / Chapter 1.6 --- The characteristics of rat multispecific OAT2 --- p.11 / Chapter 1.7 --- Clinical significance of organic anion transport proteins --- p.14 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Human liver EST sequencing project --- p.16 / Chapter 2.1.1 --- Plating out the adult human liver phage library --- p.16 / Chapter 2.1.2 --- PCR detection and amplification of the cDNA clone --- p.17 / Chapter 2.1.3 --- Automatic cDNA sequencing --- p.18 / Chapter 2.2 --- Cloning of hOAT2 gene into TA cloning vector pT-Adv --- p.19 / Chapter 2.2.1 --- Amplification of hOAT2 by PCR --- p.19 / Chapter 2.2.2 --- Ligation reaction --- p.19 / Chapter 2.2.3 --- Transformation of recombinant plasmid into competent cells --- p.20 / Chapter 2.3 --- Sequence analysis and structural prediction --- p.20 / Chapter 2.4 --- Cloning of the hOAT2 gene into the pQE30 expression vector --- p.21 / Chapter 2.4.1 --- PCR amplification and restriction endonuclease cutting --- p.21 / Chapter 2.4.2 --- Gene clean --- p.22 / Chapter 2.4.3 --- Preparation of bacterial competent cells --- p.23 / Chapter 2.5 --- Small scale synthesis of plasmid DNA --- p.24 / Chapter 2.6 --- Large scale synthesis of plasmid DNA --- p.25 / Chapter 2.7 --- Cloning of the hOAT2 gene into the pSecTag2B mammalian expression vector --- p.26 / Chapter 2.8 --- Cloning of the hOAT2 gene into the pEGFP-C2 fluorescent vector --- p.27 / Chapter 2.8.1 --- Tissue culture and transfection --- p.27 / Chapter 2.8.2 --- Fluorescence microscopy examination --- p.28 / Chapter 2.9 --- Chromosomal mapping of the hOAT2 gene --- p.29 / Chapter 2.9.1 --- Somatic cell hybrids mapping --- p.29 / Chapter 2.9.2 --- Radiation hybrids mapping --- p.29 / Chapter 2.10 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.30 / Chapter 2.11 --- Western hybridization --- p.32 / Chapter 2.11.1 --- Preparation of anti-hOAT2 antibodies --- p.32 / Chapter 2.11.1.1 --- Synthetic peptide conjugation --- p.32 / Chapter 2.11.1.2 --- Immunizing rabbit polyclonal antibodies for human OAT2 --- p.32 / Chapter 2.11.1.3 --- Purification of the rabbit polyclonal IgG antibodies --- p.33 / Chapter 2.11.2 --- Western blot analysis --- p.33 / Chapter 2.11.2.1 --- Protein isolation from rat liver --- p.33 / Chapter 2.11.2.2 --- Prote in preparation from cell lysate --- p.34 / Chapter 2.11.2.3 --- Quantitation of total proteins by Bradford protein assay --- p.35 / Chapter 2.11.2.4 --- Blotting and hybridization --- p.35 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Catalogue of the 500 liver ESTs --- p.37 / Chapter 3.2 --- Nomenclature of human NLT gene --- p.47 / Chapter 3.3 --- Cloning and characterization of the hOAT2 sequence --- p.48 / Chapter 3.3.1 --- Isolation of hOAT2 cDNA from human liver cDNA library --- p.48 / Chapter 3.3.2 --- The primary and secondary structural analysis of hOAT2 --- p.53 / Chapter 3.3.3 --- Motif search and prediction --- p.61 / Chapter 3.3.4 --- Homology alignment --- p.64 / Chapter 3.4 --- Chromosomal mapping of hOAT2 gene --- p.67 / Chapter 3.4.1 --- Somatic cell hybrid mapping of hOA T2 gene --- p.67 / Chapter 3.4.2 --- Radiation hybrid mapping of hOA T2 gene --- p.69 / Chapter 3.4.3 --- Identification of partial human genomic sequence --- p.73 / Chapter 3.5 --- Detection of the hOAT2 gene expression in human tissues by RT- PCR assay --- p.76 / Chapter 3.6 --- Detection of subcellular localization of hOAT2 protein by conjugating fluorescence protein --- p.81 / Chapter 3.7 --- Immunodetection of protein extracts from cultured cells --- p.83 / Chapter Chapter 4: --- Discussion / Chapter 4.1 --- Characterization of the hepatocellular ESTs --- p.85 / Chapter 4.1.1 --- Classification and frequency distribution of the 500 ESTs --- p.85 / Chapter 4.1.2 --- The expression pattern of membrane associated proteins --- p.87 / Chapter 4.2 --- Tissue distribution and expression profiles of hOAT2 --- p.88 / Chapter 4.3 --- HOAT2 in fetal development --- p.89 / Chapter 4.4 --- Predicting the topology of membrane proteins --- p.90 / Chapter 4.5 --- Chromosomal mapping of human OAT2 --- p.91 / Chapter 4.6 --- Possible functions of hOAT2 --- p.93 / Chapter 4.6.1 --- Hepato-renal relation --- p.93 / Chapter 4.6.2 --- Substrate diversity --- p.95 / Chapter 4.7 --- Fluorescence detection for subcellular localization --- p.96 / Chapter 4.8 --- Conclusion --- p.97 / Chapter 4.9 --- Further aspects --- p.99 / References --- p.100 / Appendix --- p.107

by Sharon, Chui-Wah Luk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 128-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

by Cheung Pok Lap. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 225-251). / Abstracts in English and Chinese. / Abstract --- p.i / 摘錄 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xiv / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Biology of Metals --- p.1 / Chapter 1.1.1 --- Mechanism for Monitoring and Controlling Intracellular Metal Ions --- p.2 / Chapter 1.1.2 --- Metal Ions Homeostasis --- p.5 / Chapter 1.2 --- Metallothionein (MT) --- p.8 / Chapter 1.2.1 --- Classification of MT --- p.8 / Chapter 1.2.2 --- Structure of MT --- p.9 / Chapter 1.2.3 --- Organization of MT Genes --- p.11 / Chapter 1.2.4 --- Biological Functions of MT --- p.13 / Chapter 1.2.5 --- MT as a model of Transcriptional Regulation of Gene Expression --- p.19 / Chapter 1.3 --- Fish MT Genes --- p.24 / Chapter 1.4 --- Aims and Rationale of Present Study --- p.28 / Chapter CHAPTER 2 --- CLONING AND CHARACTERIZATION OF TILAPIA MT (tiMT) GENE PROMOTERS --- p.30 / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- The Biology of Tilapia --- p.30 / Chapter 2.1.2 --- The Study of Tilapia MT --- p.31 / Chapter 2.1.3 --- Fish MT Promoters --- p.35 / Chapter 2.1.4 --- Specific Aims of This Chapter --- p.36 / Chapter 2.2 --- Materials and methods --- p.37 / Chapter 2.2.1 --- Animals --- p.37 / Chapter 2.2.2 --- General Molecular Biology Technique --- p.37 / Chapter 2.2.3 --- PCR Primers Used --- p.40 / Chapter 2.2.4 --- Cloning of tilapia MT Gene 5'-flanking Region Using Inverse PCR --- p.41 / Chapter 2.2.5 --- Cloning of full length of tilapia MT Genes / Chapter 2.2.6 --- Transient Transfection Assay --- p.43 / Chapter 2.3 --- Results --- p.47 / Chapter 2.3.1 --- Tilapia MT Genes --- p.47 / Chapter 2.3.2 --- Functional Analysis of tiMT Gene Promoter by Transient Transfection --- p.49 / Chapter 2.4 --- Discussions --- p.58 / Chapter 2.4.1 --- Tilapia MT Genes --- p.58 / Chapter 2.4.2 --- Functional Analysis of tiMT Gene Promoter by Transient Transfection --- p.63 / Chapter 2.5 --- Conclusion --- p.67 / Chapter CHAPTER 3 --- DETECTION OF MT mRNA LEVELS BY QUANTITATIVE RT-COMPETITIVE PCR --- p.68 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.1.1 --- Quantitative RT-competitive PCR --- p.69 / Chapter 3.1.2 --- Specific Aims of This Chapter --- p.71 / Chapter 3.2 --- Materials and methods --- p.72 / Chapter 3.2.1 --- Animals --- p.72 / Chapter 3.2.2 --- Isolation of total RNA and preparation of first strand cDNA --- p.72 / Chapter 3.2.3 --- Design of primers for preparation of MT mimic and competitive PCR --- p.73 / Chapter 3.2.4 --- Preparation of MT mimic cDNA --- p.74 / Chapter 3.2.5 --- Optimization PCR cycle number for β-actin and MT amplification --- p.77 / Chapter 3.2.6 --- Quantitative competitive PCR --- p.78 / Chapter 3.2.7 --- Quantitative analysis --- p.81 / Chapter 3.2.8 --- Statistical analysis --- p.82 / Chapter 3.3 --- Results --- p.83 / Chapter 3.3.1 --- Preparation of MT mimic cDNA --- p.83 / Chapter 3.3.2 --- Optimization PCR cycle number for MT and β-actin amplification --- p.84 / Chapter 3.3.3 --- Quantification of the MT cDNA levels by competitive PCR --- p.86 / Chapter 3.4 --- Discussions --- p.154 / Chapter 3.4.1 --- Comparison of MT Gene Expression in both in vivo tilapia liver andin vitro PLHC-1 fish cell line --- p.154 / Chapter 3.4.2 --- Absolute Quantification of mRNA using Real Time RT-PCR --- p.159 / Chapter 3.5 --- Conclusion --- p.160 / Chapter CHAPTER 4 --- TILAPIA MTF-1: PCR-CLONING AND GENE EXPRESSION STUDIES --- p.161 / Chapter 4.1 --- Introduction --- p.161 / Chapter 4.1.1 --- General Features of MTF-1 --- p.164 / Chapter 4.1.2 --- Activation MRE-binding of the MTF-1 --- p.166 / Chapter 4.1.3 --- Possible Models for Heavy-metal Regulated MT Genes Transcription --- p.169 / Chapter 4.1.4 --- Genes Under the Regulation of MTF-1 --- p.175 / Chapter 4.1.5 --- Specific Aims of This Chapter --- p.176 / Chapter 4.2 --- Materials and methods --- p.177 / Chapter 4.2.1 --- Cloning of a partial fragment of MTF-1 in tilapia --- p.177 / Chapter 4.2.2 --- Rapid Amplification of cDNA 5'ends --- p.180 / Chapter 4.2.3 --- Rapid Amplification of cDNA 3'ends --- p.184 / Chapter 4.2.4 --- Cloning of The Full-Length Tilapia MTF-1 cDNA Isoforms --- p.185 / Chapter 4.2.5 --- Northern blot analysis of MTF-1 transcripts in tilapia tissues --- p.186 / Chapter 4.2.6 --- Differential Expression of MTF-1 Isoforms in Tilapia Tissues --- p.187 / Chapter 4.2.7 --- Cotransfection Study on Tilapia MTF-1 cDNAs --- p.189 / Chapter 4.3 --- Results --- p.191 / Chapter 4.3.1 --- PCR Amplification of the Partial Sequence of tiMTF-1 --- p.191 / Chapter 4.3.2 --- Rapid Amplification of cDNA 5' and 3' ends of tiMTF-1 --- p.193 / Chapter 4.3.3 --- Cloning of The Full-Length Tilapia MTF-1 cDNA Isoforms --- p.196 / Chapter 4.3.4 --- Northern Blot Analysis of tilapia MTF-1 isoforms --- p.205 / Chapter 4.3.5 --- Differential Expression of MTF-1 Isoforms in Tilapia Tissues --- p.206 / Chapter 4.3.6 --- Cotransfection Study on Tilapia MTF-1 cDNAs --- p.207 / Chapter 4.4 --- Discussions --- p.212 / Chapter 4.4.1 --- Tilapia MTF-1 --- p.212 / Chapter 4.4.2 --- Biological Activity of MTF-1 --- p.216 / Chapter 4.5 --- Conclusion --- p.218 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.220 / REFERENCES --- p.225

Shaojun Ding. / "October 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, / Includes bibliographical references (p. 173-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.