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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326527 |
| Date | January 2008 |
| Contributors | Au, Yee Man Candy., 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, xiv, 144 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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