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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322313 |
| Date | January 1998 |
| Contributors | Shiu, Ka Man., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xv, 182 leaves : ill. (some 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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