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Regulation of tilapia metallothionein (MT) gene expression.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324397
Date January 2003
ContributorsCheung, Pok Lap., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvi, 251 leaves : ill. (some col.) ; 30 cm.
RightsUse 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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