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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324397 |
| Date | January 2003 |
| Contributors | Cheung, Pok Lap., 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, xvi, 251 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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