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

Lam Kwok Lim. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 107-126). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Abstract --- p.iv / Abbreviations --- p.vii / Abbreviation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Metallothionein (MT) --- p.1 / Chapter 1.1.1 --- Classification of MT --- p.1 / Chapter 1.1.2 --- Structure of MT --- p.2 / Chapter 1.1.3. --- Structure of MT Genes --- p.4 / Chapter 1.1.4 --- Function of MT --- p.5 / Chapter 1.1.5 --- Regulation of MT Expression --- p.7 / Chapter 1.1.6 --- Fish MT --- p.9 / Chapter 1.1.7. --- Aims and Rationale of the Present Study --- p.12 / Chapter 2 --- MT mRNA Induction of Tilapia After Intraperitoneal Injection of Metal --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1. --- Specific Aims of This Chapter --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Regents --- p.20 / Chapter 2.2.1.1 --- Purification of Total RNA --- p.20 / Chapter 2.2.1.2 --- Denaturing Gel and Vacuum Blotting of RNA (Northern Blotting) --- p.20 / Chapter 2.2.1.3 --- Hybridization --- p.21 / Chapter 2.2.2 --- Methods --- p.21 / Chapter 2.2.2.1 --- Purification of Total RNA --- p.21 / Chapter 2.2.2.2 --- Vacuum Blotting of Total RNA (Northern Blotting) --- p.22 / Chapter 2.2.2.3 --- Radioactive Labeling of Nucleic Acid Probes --- p.22 / Chapter 2.2.2.4 --- Hybridization --- p.22 / Chapter 2.2.2.5 --- Densitometric Analysis --- p.23 / Chapter 2.2.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.23 / Chapter 2.2.3 --- Endogenous MT mRNA Expression of Juvenile Tilapia and Carp --- p.23 / Chapter 2.2.4 --- Induction of MT mRNA Juvenile Tilapia and Carp Injected with Metals --- p.24 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Endogenous Levels of MT mRNA in Tilapias in Normal Conditions --- p.25 / Chapter 2.3.2 --- Induction of MT mRNA Levels in Juvenile Tilapia Injected with Metals --- p.25 / Chapter 2.3.1.1 --- Copper Injection --- p.25 / Chapter 2.3.1.2 --- Zinc Injection --- p.25 / Chapter 2.3.1.3 --- Cadmium Injection --- p.26 / Chapter 2.3.3 --- Induction of MT mRNA Levels in Juvenile Carp with Zinc Injection --- p.26 / Chapter 2.4 --- Discussion --- p.26 / Chapter 2.4.1 --- MT mRNA Expression of Tilapia and Carp Injected with Metals --- p.26 / Chapter 2.5 --- Conclusions --- p.29 / Chapter 3 --- Induction Level of MT mRNA in Tilapia After Aqueous Exposure to Metals --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.1.1 --- Specific aims of this chapter --- p.36 / Chapter 3.2 --- Material s and Methods --- p.36 / Chapter 3.2.1 --- 96hours LC-50 values for zinc and copper --- p.36 / Chapter 3.2.2 --- Induction of MT mRNA in Juvenile Tiapias under Metal Aqueous Exposures --- p.37 / Chapter 3.2.3 --- Calculation of Fold Induction of MT mRNA and Analysis of Results --- p.38 / Chapter 3.2.4 --- Metal Analysis --- p.38 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- LC-50 values of metals for Juvenile Tilapia --- p.38 / Chapter 3.3.2 --- Induction of MT mRNA in Juvenile Tilapia under Metal Aqueous Exposures --- p.39 / Chapter 3.3.2.1 --- Aqueous Exposure to Copper --- p.39 / Chapter 3.3.2.2 --- Aqueous Exposure to Zinc --- p.40 / Chapter 3.3.2.3 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.3 --- Induction of MT mRNA in Juvenile Carp after Aqueous Exposures to Metal --- p.41 / Chapter 3.3.3.1 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.4 --- Metal Concentrations of Water Samples from the Aquaria in the Metal Exposure Test of Tilapia and Carp --- p.42 / Chapter 3.4 --- Discussion --- p.42 / Chapter 3.4.1 --- LC-50 values of Metals for Tilapia --- p.42 / Chapter 3.4.2 --- MT mRNA Expression of Tilapias under Metal Aqueous Exposure --- p.44 / Chapter 3.4.3 --- Normalization of the Signals of Northern Blot Analysis --- p.47 / Chapter 3.5 --- Conclusions --- p.48 / Chapter 4 --- Field Study --- p.58 / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- Specific Aims of this Chapter --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Sampling Sites --- p.59 / Chapter 4.2.2 --- Data Analysis --- p.60 / Chapter 4.2.3 --- Harvest of Feral Tilapia --- p.60 / Chapter 4.2.4 --- Determination of Metal Concentration of Metal Concentration in the Tissues of Feral Tilapia --- p.60 / Chapter 4.2.5 --- Endogenous MT mRNA Levels Using Northern Blot Analysis --- p.61 / Chapter 4.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Metal Concentrations in the Tissues of Feral Tilapia --- p.62 / Chapter 4.3.2 --- Comparison of Metal Concentrations Among Different Tissues of Feral Tilapia --- p.62 / Chapter 4.3.3 --- MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.3.4 --- Correlation Between Metal Concentrations and Endogenous MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.4 --- Discussion --- p.64 / Chapter 4.4.1 --- Bioaccumulation of Metals --- p.64 / Chapter 4.4.2 --- Endogenous Levels of MT mRNA in the Feral Tilapia --- p.67 / Chapter 4.5 --- Conclusions --- p.68 / Chapter 5 --- Cloning of Tilapia MT Genes --- p.86 / Chapter 5.1 --- Specific Aims of This Chapter 、 --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Regents --- p.87 / Chapter 5.2.1.1 --- Preparation of Plasmid DNA --- p.87 / Chapter 5.2.1.2 --- Preparation of Genomic DNA --- p.87 / Chapter 5.2.1.3 --- Restriction Enzyme Digestion --- p.88 / Chapter 5.2.1.4 --- Vacuum Blotting of DNA (Southern Blotting) --- p.88 / Chapter 5.2.1.5 --- Polymerase Chain Reaction --- p.89 / Chapter 5.2.1.6 --- Transformation of E.coli Competent Cells --- p.89 / Chapter 5.2.1.7 --- Nucleotide Sequence Determination --- p.89 / Chapter 5.2.1.8 --- List of Primers --- p.90 / Chapter 5.2.1.8.1 --- Primers for Nucleotide Sequence Determination --- p.90 / Chapter 5.2.1.8.2 --- Tilapia MT Specific Primers for PCR --- p.90 / Chapter 5.2.2 --- Methods --- p.91 / Chapter 5.2.2.1 --- Preparation of Plasmid --- p.91 / Chapter 5.2.2.2 --- Preparation of Genomic DNA --- p.91 / Chapter 5.2.2.3 --- Preparation of Enzyme Digestion --- p.92 / Chapter 5.2.2.4 --- Vacuum Blotting of Genomic DNA (Southern Blotting) --- p.92 / Chapter 5.2.2.5 --- Radioactive Labeling of Nucleic Acid Probes --- p.92 / Chapter 5.2.2.6 --- Hybridization --- p.93 / Chapter 5.2.2.7 --- Polymerase Chain Reaction --- p.93 / Chapter 5.2.3 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.93 / Chapter 5.2.4 --- Analysis of the Sequences of Tilapia MT Genes --- p.94 / Chapter 5.2.4.1 --- Amplification of MT Genes Using PCR --- p.94 / Chapter 5.2.4.2 --- Cloning of the MT Genes --- p.94 / Chapter 5.2.4.3 --- Transformation of E.coli Competent Cell --- p.94 / Chapter 5.2.4.4 --- Nucleotide Sequence Determination --- p.95 / Chapter 5.3 --- Results --- p.95 / Chapter 5.3.1 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.95 / Chapter 5.3.2 --- Amplification of MT Gene Fragments Using PCR --- p.95 / Chapter 5.3.3 --- Analysis of the Sequences of Tilapia MT Genes --- p.96 / Chapter 5.4 --- Discussion --- p.96 / Chapter 5.4.1 --- Fish MT Genes --- p.96 / Chapter 5.5 --- Conclusions --- p.98 / Chapter 6 --- General Discussion --- p.104 / References --- p.107

Shiu Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 176-182). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Chinese Abstract --- p.iii / English Abstract --- p.iv / List of Abbreviations --- p.v / Abbreviation for Amino Acids and Nucleotides --- p.vii / List of Figures --- p.viii / List of Tables --- p.vi / Contents / Chapter Chapter.1 --- Literature Review --- p.1 / Chapter 1.1 --- Transcriptional Regulation of Gene Expression --- p.1 / Chapter 1.2 --- MT: A Brief Review --- p.4 / Chapter 1.3 --- Transcriptional Regulation of MT --- p.15 / Chapter 1.4 --- MT Promoter Organization and Function --- p.18 / Chapter 1.5 --- Fish MT Genes --- p.29 / Chapter 1.6 --- Aim and Rationale of Present Studies --- p.32 / Chapter Chapter 2 --- PCR Cloning of Common Carp MT Gene --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1 --- The Biology of Common Carp --- p.34 / Chapter 2.1.2 --- The Study of Common Carp MT --- p.35 / Chapter 2.2 --- Materials and Methods --- p.39 / Chapter 2.2.1 --- Materials --- p.39 / Chapter 2.2.1.1 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter 2.2.1.2 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.1.3 --- Gene Clean by Sephaglas´ёØ BandPrep Kit (Pharmacia) --- p.40 / Chapter 2.2.1.4 --- TA Cloning --- p.40 / Chapter 2.2.1.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.41 / Chapter 2.2.1.6 --- Preparation of Plasmid DNA --- p.41 / Chapter 2.2.1.7 --- DNA Sequencing --- p.42 / Chapter 2.2.1.7.1 --- Template Denaturation and Primer Annealing --- p.42 / Chapter 2.2.1.7.2 --- Labeling and Termination Reaction --- p.42 / Chapter 2.2.1.7.3 --- DNA Sequencing Electrophoresis --- p.43 / Chapter 2.2.1.8 --- Total RNA Extraction --- p.43 / Chapter 2.2.1.9 --- PolyA RNA Extraction --- p.44 / Chapter 2.2.1.10 --- Micro Bio-Spin Chromatography --- p.44 / Chapter 2.2.1.11 --- Analysis of the Transcription Start Site --- p.45 / Chapter 2.2.2 --- Methods --- p.46 / Chapter 2.2.2.1 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2.2 --- Gene Clean by Sephaglas ´ёØ BandPrep Kit (Pharmacia) --- p.46 / Chapter 2.2.2.3 --- TA Cloning --- p.47 / Chapter 2.2.2.4 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.47 / Chapter 2.2.2.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.48 / Chapter 2.2.2.6 --- Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.1 --- Small Scale Alkali Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.2 --- Large Scale Preparation of Plasmid DNA using Wizard Maxiprep Kit (Promega) --- p.49 / Chapter 2.2.7 --- DNA Sequencing --- p.50 / Chapter 2.2.2.7.1 --- Template Denaturation and Primer Annealing --- p.50 / Chapter 2.2.2.7.2 --- Labeling and Termination Reaction --- p.51 / Chapter 2.2.2.7.3 --- DNA Sequencing Electrophoresis --- p.51 / Chapter 2.2.2.8 --- Total RNA Extraction --- p.52 / Chapter 2.2.2.9 --- PolyA RNA Extraction --- p.53 / Chapter 2.2.2.10 --- Analysis of the Transcription Start Site --- p.55 / Chapter 2.3 --- Results --- p.56 / Chapter 2.3.1 --- PCR Cloning of the MT Gene --- p.56 / Chapter 2.3.2 --- Identification of the Transcriptional Start Site --- p.57 / Chapter 2.4 --- Discussion --- p.60 / Chapter 2.4.1 --- PCR Cloning of the MT Gene --- p.60 / Chapter 2.4.2 --- Comparison of Common Carp MT Promoter with Other --- p.60 / Chapter 2.4.3 --- Identification of the Transcriptional Start Site --- p.62 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3. --- Functional Assay of Common Carp MT Promoter --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.1.1 --- Fish MT Promoters --- p.64 / Chapter 3.2 --- Materials and Methods --- p.68 / Chapter 3.2.1 --- Materials --- p.68 / Chapter 3.2.1.2 --- Micro Bio-Spin Chromatography --- p.68 / Chapter 3.2.1.3 --- Construction of Deletion Mutants --- p.68 / Chapter 3.2.1.4 --- Isolation of Hepatocytes --- p.69 / Chapter 3.2.1.5 --- Determination of LC50 Values for Common Carp Hepatocytes --- p.69 / Chapter 3.2.1.6 --- Transfection by LipofectAMINE´ёØ (Gibco) --- p.70 / Chapter 3.2.1.9 --- Determination of the Amount of Protein by BCA Protein Assay --- p.70 / Chapter 3.2.1.10 --- β-galactosidase Analysis --- p.71 / Chapter 3.2.2 --- Methods --- p.72 / Chapter 3.2.2.1 --- Subcloning of 5' Flanking Region of Common Carp MT Gene into Reporter Gene --- p.72 / Chapter 3.2.2.2 --- Micro Bio-Spin Chromatography (Bio-rad) --- p.72 / Chapter 3.2.2.3 --- Creating Deletion Mutants --- p.73 / Chapter 3.2.2.4 --- Isolation of Hepatocytes --- p.73 / Chapter 3.2.2.5 --- Determination ofLC50 Values for Common Carp Hepatocytes --- p.74 / Chapter 3.2.2.6 --- Transfection with LipofectAMINE´ёØ (Gibco BRL) --- p.75 / Chapter 3.2.2.7 --- Optimization of Incubation Time of Cells with LipofectAMINE´ёØ --- p.75 / Chapter 3.2.2.8 --- Optimization of Amount of DNA for Transfection --- p.76 / Chapter 3.2.2.9 --- Determination of Protein Concentration by --- p.76 / Chapter 3 2.2.10 --- β-galactosidase Analysis --- p.77 / Chapter 3.2.2.11 --- Fluorescence Measurement --- p.77 / Chapter 3.2.2.12 --- Dose-Response Curve of Different Metals on Transfected Cells --- p.77 / Chapter 3.2.2.13 --- "Fold-Induction of Different Metals, LPS and H202" --- p.78 / Chapter 3.3. --- Result --- p.79 / Chapter 3.3.1 --- Deletion Mutants --- p.79 / Chapter 3.3.2 --- LC50 of Common Carp Hepatocytes --- p.80 / Chapter 3.3.3 --- Optimization of Transfection --- p.81 / Chapter 3.3.4 --- Dose Response Curve --- p.85 / Chapter 3.3.5 --- Deletion Mutants with Different Treatments --- p.95 / Chapter 3.4 --- Discussion --- p.109 / Chapter 3.4.1 --- LC50 Values of Metal Toxicity in Different in vitro Fish Cells Studies --- p.109 / Chapter 3.4.2 --- Dose Response Curve (Figure 3.9 to 3.16) --- p.110 / Chapter 3.4.3 --- Fold Induction in Deletion Mutants --- p.111 / Chapter 3.5 --- Conclusion --- p.128 / Chapter Chapter 4. --- MRE-Binding Proteins --- p.129 / Chapter 4.1 --- Introduction --- p.129 / Chapter 4.1.1 --- MTF-1 --- p.129 / Chapter 4.1.1.1 --- Structure of MTF-1 --- p.129 / Chapter 4.1.1.2 --- MTF-1 is a Zinc Dependent Factor --- p.130 / Chapter 4.1.1.3 --- Band-shift Assay of MTF-1 --- p.132 / Chapter 4.1.1.4 --- MTF-1 is Essential for Both Basal and Metal-Induced MT Transcription --- p.133 / Chapter 4.1.2 --- MBP-l --- p.134 / Chapter 4.1.3 --- MBF-l l --- p.35 / Chapter 4.1.4 --- Rat Zinc Activated Protein --- p.135 / Chapter 4.1.5 --- MREBF-1 and MREBF-2 --- p.136 / Chapter 4.1.6 --- Human Zinc Regulatory Factor --- p.136 / Chapter 4.1.7 --- MREBP --- p.137 / Chapter 4.1.8 --- Aim of This Chapter --- p.138 / Chapter 4.2 --- Materials and Methods --- p.139 / Chapter 4.2.1 --- Materials --- p.139 / Chapter 4.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.139 / Chapter 4.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.139 / Chapter 4.2.1.3 --- Binding Reaction of Protein and DNA --- p.141 / Chapter 4.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.142 / Chapter 4.2.1.5 --- Screening of Expression Library --- p.142 / Chapter 4.2.1.5.1 --- Preparation of Labeled DNA Probe --- p.142 / Chapter 4.2.1.5.2 --- Plating of the Library --- p.142 / Chapter 4.2.1.6. --- Isolation of Positive Clones In Vivo Excision --- p.143 / Chapter 4.2.2 --- Methods --- p.144 / Chapter 4.2.2.1 --- Gel Mobility-Shift Assays --- p.144 / Chapter 4.2.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.145 / Chapter 4.2.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.145 / Chapter 4.2.2.1.3 --- Binding Reaction of Protein and DNA --- p.146 / Chapter 4.2.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.146 / Chapter 4.2.2.2 --- Screening of Expression Library --- p.146 / Chapter 4.2.2.2.1 --- Preparation of Labeled DNA Probe --- p.147 / Chapter 4.2.2.2.2 --- Plating of the Library --- p.148 / Chapter 4.2.2.2.3 --- Isolation of Positive Clones --- p.150 / Chapter 4.3 --- Results --- p.150 / Chapter 4.3.1 --- Gel Mobility-Shift Assays --- p.150 / Chapter 4.3.2 --- Expression Library Screening --- p.163 / Chapter 4.4 --- Discussion --- p.166 / Chapter 4.4.1 --- Gel Mobility-Shift Assays --- p.166 / Chapter 4.4.2 --- Expression Library Screening --- p.171 / Chapter 4.5 --- Conclusion --- p.172 / Chapter Chapter 5 --- Conclusion --- p.173 / Chapter 5.1 --- Conclusion --- p.173 / Chapter 5 2 --- Model of MT Gene Transcription --- p.174 / Chapter 5.3 --- Future Direction --- p.175 / references --- p.176

Au, Yee Man Candy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 128-144). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter 1. --- Chapter One Introduction / Chapter 1.1 --- Homeostasis and detoxification of metal ions --- p.1 / Chapter 1.2 --- Biochemistry of metallothionein --- p.3 / Chapter 1.2.1 --- Structure of metallothionein --- p.4 / Chapter 1.2.2 --- Isoforms of metallothionein --- p.5 / Chapter 1.2.3 --- Roles of metallothionein --- p.6 / Chapter 1.2.4 --- Structure of metallothionein gene --- p.9 / Chapter 1.2.5 --- Metal responsive element (MRE) --- p.10 / Chapter 1.2.6 --- Regulation of MT gene --- p.11 / Chapter 1.3 --- Metal responsive element-binding transcription factor 1 (MTF-1) --- p.16 / Chapter 1.3.1 --- Structure of MTF-1 --- p.16 / Chapter 1.3.2 --- Target genes of MTF-1 --- p.18 / Chapter 1.4 --- Teleost MT and MTF-1 --- p.20 / Chapter 1.5 --- Tilapia --- p.26 / Chapter 1.6 --- Tilapia MT and MTF-1 --- p.26 / Chapter 1.7 --- Aims of study --- p.30 / Chapter 2. --- Chapter Two Materials and Methods / Chapter 2.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.32 / Chapter 2.1.1 --- Heavy metal exposure on tilapia --- p.32 / Chapter 2.1.1.1 --- Animals --- p.32 / Chapter 2.1.1.2 --- Heavy metal exposure --- p.32 / Chapter 2.1.1.3 --- Total RNA extraction --- p.33 / Chapter 2.1.1.4 --- Reverse Transcription --- p.35 / Chapter 2.1.2 --- Heavy metal exposure on Hepa-Tl cells --- p.36 / Chapter 2.1.2.1 --- Cell Culture --- p.36 / Chapter 2.1.2.2 --- Metal treatment on Hepa-Tl cells --- p.37 / Chapter 2.1.3 --- SYBR green --- p.39 / Chapter 2.1.3.1 --- Primer Design --- p.39 / Chapter 2.1.3.2 --- Validation of cycling condition --- p.41 / Chapter 2.1.3.3 --- Determination of relative amount of target gene present in the samples --- p.43 / Chapter 2.1.3.4 --- Statistical analysis --- p.44 / Chapter 2.1.4 --- TaqMan probes --- p.44 / Chapter 2.1.4.1 --- Primer Design --- p.44 / Chapter 2.1.4.2 --- Validation of cycling condition --- p.45 / Chapter 2.2 --- Localization study of MTF-1 isoforms --- p.46 / Chapter 2.2.1 --- Amplification of the full length tilapia MTF-1 isoforms --- p.46 / Chapter 2.2.2 --- Preparation of Escherichia coli competent cells --- p.48 / Chapter 2.2.3 --- Transformation --- p.49 / Chapter 2.2.4 --- Confirmation of the insert of the ligation products --- p.50 / Chapter 2.2.5 --- Cloning of MTF-1-L and MTF-1-S gene into phrGFPII-1 vector --- p.51 / Chapter 2.2.6 --- Transient transfection of plasmids to Hepa-Tl cells --- p.54 / Chapter 2.2.7 --- Staining of the nucleus by Hoechst 33342 --- p.55 / Chapter 2.2.8 --- Metal treatment on Hepa-Tl cells --- p.55 / Chapter 2.3 --- Electrophoretic mobility shift assay (EMSA) --- p.56 / Chapter 2.3.1 --- Preparation of Hepa-Tl whole-cell protein extract --- p.56 / Chapter 2.3.2 --- In vitro transcription/translation of tilapia MTF-1 isoforms --- p.57 / Chapter 2.3.3 --- Annealing of the tiMREg oligonucleotides --- p.58 / Chapter 2.3.4 --- Labeling of the annealed tiMREg oligonucleotides --- p.58 / Chapter 2.3.5 --- Electrophoretic mobility shift assay --- p.59 / Chapter 3. --- Chapter Three Results / Chapter 3.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.62 / Chapter 3.1.1 --- Validation of primers for real-time PCR --- p.62 / Chapter 3.1.2 --- Tissue distribution of MTF-1 isoforms in tilapia and Hepa-Tl cell-line --- p.63 / Chapter 3.1.3 --- Effect of metal treatment on MTF-1 isoforms and MT gene expression level in different tissues of tilapia and Hepa-Tl cell-line --- p.68 / Chapter 3.2 --- Localization study of MTF-1 isoforms --- p.82 / Chapter 3.2.1 --- Cloning of MTF-1 isoforms into phrGFPII-1 vector --- p.82 / Chapter 3.2.2 --- Transient transfection of phrGFPII-1 plasmids to Hepa-Tl cells --- p.82 / Chapter 3.3 --- Electrophoretic mobility shift assay (EMSA) --- p.96 / Chapter 4. --- Chapter Four Discussion / Chapter 4.1 --- Tissue distribution of MTF-1 isoforms --- p.104 / Chapter 4.2 --- Effect of metal stress on the mRNA expression level of MT and MTF-1 isoforms --- p.106 / Chapter 4.3 --- In vitro study of the localization of the MTF-1 isoforms --- p.114 / Chapter 4.4 --- DNA binding of MTF-1 synthesized by in vitro transcription/translation method --- p.121 / Chapter 4.5 --- Conclusion --- p.125 / Chapter 5. --- REFERENCES --- p.128

Cheuk, Wai Ka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 96-108). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xii / Chapter CHAPTER 1 --- General introduction / Chapter 1.1 --- Metal Contaminations in the environment --- p.1 / Chapter 1.2 --- Biology of Heavy Metal Ions --- p.3 / Chapter 1.2.1 --- Essential and non-essential metal ions --- p.3 / Chapter 1.2.2 --- Toxicities and origins of heavy metal ions --- p.5 / Chapter 1.3 --- Monitoring Of Heavy Metal Contaminations In Aquatic Environment --- p.9 / Chapter 1.3.1 --- Monitoring in chemical approach --- p.9 / Chapter 1.3.2 --- Monitoring in biological approach: biomarkers --- p.11 / Chapter 1.4 --- Metallothionein (MT) --- p.12 / Chapter 1.4.1 --- Biological functions of MT and its regulation --- p.12 / Chapter 1.4.2 --- MT isoforms --- p.14 / Chapter 1.4.3 --- Mechanisms of MT gene regulation --- p.15 / Chapter 1.4.3.1 --- Zinc pool hypothesis --- p.20 / Chapter 1.4.3.2 --- Protein kinase cascade --- p.21 / Chapter 1.5 --- Metal responsive element (MRE) --- p.22 / Chapter 1.6 --- MRE-Binding Transcription Factor-1 (MTF-1) --- p.30 / Chapter 1.6.1 --- Structure of MTF-1 --- p.30 / Chapter 1.6.2 --- Physiological functions of MTF-1 --- p.32 / Chapter 1.6.3 --- The role of MTF-1 in MT gene regulation --- p.33 / Chapter 1.6.4 --- Regulation of MTF-1 by various heavy metals --- p.34 / Chapter 1.7 --- Zebrafish (Daino reio) --- p.36 / Chapter 1.8 --- Project aim --- p.37 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Cell Culture --- p.40 / Chapter 2.1.1 --- ZFL cell line --- p.40 / Chapter 2.1.2 --- SJD cell line --- p.41 / Chapter 2.2 --- Alarmar blue̐ưؤ M assay --- p.41 / Chapter 2.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.1 --- Metal treatment of the SJD and ZFL cell lines --- p.42 / Chapter 2.3.2 --- Isolation of total RNA --- p.43 / Chapter 2.3.3 --- Quantification of mRNA by spectrophotometer --- p.43 / Chapter 2.3.4 --- Reverse Transcription --- p.44 / Chapter 2.4 --- Quantifications of mRNA levels by using real-time PCR technique --- p.44 / Chapter 2.4.1 --- Primer design --- p.44 / Chapter 2.4.2 --- PCR components and cycling condition --- p.45 / Chapter 2.4.3 --- Determination of relative amount of target gene present in the samples --- p.49 / Chapter 2.5 --- Cloning of zMT-II gene promoter and its transient expression studies --- p.50 / Chapter 2.5.1 --- Purification of genomic DNA --- p.50 / Chapter 2.5.2 --- Preparation of Escherichia coli competent cell --- p.51 / Chapter 2.5.3 --- PCR-Cloning of a 1.4 kb zMT-II gene promoter --- p.51 / Chapter 2.5.4 --- Purification of plasmid DNA --- p.53 / Chapter 2.5.5 --- Transient transfection of plasmid into SJD and ZFL cells --- p.54 / Chapter 2.5.6 --- Heavy metal treatments and measurement of luciferase activities --- p.54 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Toxicities of various heavy metal ions --- p.56 / Chapter 3.2 --- Relative mRNA fold induction of zMT in SJD and ZFL cell lines --- p.59 / Chapter 3.3 --- The zMT-II gene and its induction by metal ions in zebrafish cell-lines --- p.63 / Chapter 3.4 --- MTF-1 mRNA levels in SJD and ZFL cell lines exposed to heavy metal ions --- p.74 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Comparison of metal toxicities in the two cell lines studied --- p.78 / Chapter 4.2 --- zMT gene expression study --- p.80 / Chapter 4.2.1 --- zMT mRNA regulation by heavy metal ions in the two cell lines --- p.80 / Chapter 4.2.2 --- The potential use of MT regulation as exposure biomarker --- p.82 / Chapter 4.3 --- Structure of the zMT-II gene promoter region --- p.82 / Chapter 4.4 --- Metal responsiveness of zMT-II promoter --- p.84 / Chapter 4.5 --- Mechanism of MT gene expression and the MTF-1 mRNA inductions in SJD and ZFL cell lines --- p.86 / Chapter 4.6 --- Concluding Remarks --- p.93 / References --- p.96

by Yan Hiu Mei, Carol. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 114-126). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.xii / Chapter CHAPTER 1 --- TRANSGENIC TECHNOLOGY --- p.1 / Chapter 1.1 --- Transgenesis in animals --- p.1 / Chapter 1.2 --- Transgenic fish in toxicology --- p.4 / Chapter 1.2.1 --- Aquatic metal toxicity --- p.4 / Chapter 1.2.2 --- Environmental monitoring of aquatic metal toxicity --- p.5 / Chapter 1.2.3 --- Biomarkers --- p.6 / Chapter 1.3 --- Transgenics in aquaculture --- p.9 / Chapter 1.3.1 --- Revolution is needed in aquaculture --- p.9 / Chapter 1.3.2 --- Aquaculture potential of tilapia in China --- p.10 / Chapter 1.3.3 --- Endocrinology for fish growth --- p.12 / Chapter 1.3.4 --- Growth promotion by exogenous growth hormone in tilapia --- p.14 / Chapter 1.3.5 --- Accelerated growth in transgenic fish --- p.15 / Chapter 1.4 --- General principle in transgenic fish production --- p.16 / Chapter 1.5 --- Project aim --- p.22 / Chapter CHAPTER 2 --- ISOLATION AND CHARACTERIZATION OF ZEBRAFISH METALLOTHIONEIN GENE PROMOTER --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.1.1 --- Metallothionein --- p.23 / Chapter 2.1.2 --- Biological functions --- p.24 / Chapter 2.1.3 --- Metallothionein gene regulations --- p.25 / Chapter 2.1.4 --- Metallothionein as biomarker for metal pollution --- p.26 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- General molecular biology techniques --- p.28 / Chapter 2.2.2 --- Sequences of PCR primers used --- p.31 / Chapter 2.2.3 --- Cloning zebrafish MT gene 5-flanking region --- p.31 / Chapter 2.2.4 --- Cloning zebrafish MT gene --- p.32 / Chapter 2.2.5 --- Cloning full length zMT gene --- p.33 / Chapter 2.2.6 --- Cell culture --- p.35 / Chapter 2.2.7 --- Transient transfection assay --- p.37 / Chapter 2.2.8 --- Electrophoretic mobility shift assay --- p.39 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Zebrafish metallothionein gene --- p.42 / Chapter 2.3.2 --- Deletion analysis of zMT promoter by transient transfection assay --- p.48 / Chapter 2.3.3 --- Functional characterization of zebrafish metallothionein promoter --- p.57 / Chapter 2.4 --- Discussions --- p.61 / Chapter 2.4.1 --- Zebrafish MT gene --- p.61 / Chapter 2.4.2 --- Functional characterization of zebrafish MT promoter --- p.61 / Chapter CHAPTER 3 --- PREPARATION OF GENE CONSTRUCTS FOR TRANSFER IN ZEBRAFISH --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.1.1 --- Zebrafish as model in toxicological studies --- p.65 / Chapter 3.1.2 --- Reporter gene system --- p.66 / Chapter 3.1.3 --- Transgenic reporter fish --- p.68 / Chapter 3.1.4 --- Gene transfer by electroporation in zebrafish --- p.68 / Chapter 3.1.5 --- Objective --- p.69 / Chapter 3.2 --- Materials and methods --- p.70 / Chapter 3.2.1 --- Design of gene constructs for ectopic expression in zebrafish --- p.70 / Chapter 3.2.2 --- Testing electroporation conditions for zebrafish --- p.72 / Chapter 3.3 --- Results --- p.73 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Engineering gene constructs --- p.76 / Chapter 3.4.2 --- Applications of transgenic zebrafish --- p.79 / Chapter CHAPTER 4 --- GENE TRANSFER EXPERIMENTS ON TILAPIA --- p.82 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and methods --- p.85 / Chapter 4.2.1 --- Isolation of O. aureus growth hormone --- p.85 / Chapter 4.2.2 --- Engineering gene constructs for ectopic expression in tilapia --- p.86 / Chapter 4.2.3 --- Gene transfer in tilapia --- p.87 / Chapter 4.2.4 --- Screening transgenic tilapia --- p.89 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Tilapia growth hormone --- p.91 / Chapter 4.3.2 --- Gene constructs for ectopic expression in tilapia --- p.94 / Chapter 4.3.3 --- Testing electroporation conditions --- p.96 / Chapter 4.3.4 --- PCR screening for transgenic fish --- p.97 / Chapter 4.4 --- Discussions --- p.101 / Chapter 4.4.1 --- Tilapia growth hormone --- p.101 / Chapter 4.4.2 --- Electroporation experiments on of tilapia eggs --- p.101 / Chapter 4.4.3 --- Improvements on gene construct design for tilapia --- p.104 / Chapter 4.4.4 --- Ethical and safety considerations --- p.106 / Chapter CHAPTER 5 --- REFERENCES --- p.114 / APPENDIX --- p.127