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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326040 |
| Date | January 2007 |
| Contributors | Chan, Wai Lun., 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, v, 157 leaves : ill. ; 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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