Mechanism of metallothionein gene regulation involving metal responsive element binding transcription factor-1 and its short-form variant in tilapia.

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.iiiAcknowledgements --- p.vList of Tables --- p.xList of Figures --- p.xiList of Abbreviations --- p.xiiiChapter 1. --- Chapter One IntroductionChapter 1.1 --- Homeostasis and detoxification of metal ions --- p.1Chapter 1.2 --- Biochemistry of metallothionein --- p.3Chapter 1.2.1 --- Structure of metallothionein --- p.4Chapter 1.2.2 --- Isoforms of metallothionein --- p.5Chapter 1.2.3 --- Roles of metallothionein --- p.6Chapter 1.2.4 --- Structure of metallothionein gene --- p.9Chapter 1.2.5 --- Metal responsive element (MRE) --- p.10Chapter 1.2.6 --- Regulation of MT gene --- p.11Chapter 1.3 --- Metal responsive element-binding transcription factor 1 (MTF-1) --- p.16Chapter 1.3.1 --- Structure of MTF-1 --- p.16Chapter 1.3.2 --- Target genes of MTF-1 --- p.18Chapter 1.4 --- Teleost MT and MTF-1 --- p.20Chapter 1.5 --- Tilapia --- p.26Chapter 1.6 --- Tilapia MT and MTF-1 --- p.26Chapter 1.7 --- Aims of study --- p.30Chapter 2. --- Chapter Two Materials and MethodsChapter 2.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.32Chapter 2.1.1 --- Heavy metal exposure on tilapia --- p.32Chapter 2.1.1.1 --- Animals --- p.32Chapter 2.1.1.2 --- Heavy metal exposure --- p.32Chapter 2.1.1.3 --- Total RNA extraction --- p.33Chapter 2.1.1.4 --- Reverse Transcription --- p.35Chapter 2.1.2 --- Heavy metal exposure on Hepa-Tl cells --- p.36Chapter 2.1.2.1 --- Cell Culture --- p.36Chapter 2.1.2.2 --- Metal treatment on Hepa-Tl cells --- p.37Chapter 2.1.3 --- SYBR green --- p.39Chapter 2.1.3.1 --- Primer Design --- p.39Chapter 2.1.3.2 --- Validation of cycling condition --- p.41Chapter 2.1.3.3 --- Determination of relative amount of target gene present in the samples --- p.43Chapter 2.1.3.4 --- Statistical analysis --- p.44Chapter 2.1.4 --- TaqMan probes --- p.44Chapter 2.1.4.1 --- Primer Design --- p.44Chapter 2.1.4.2 --- Validation of cycling condition --- p.45Chapter 2.2 --- Localization study of MTF-1 isoforms --- p.46Chapter 2.2.1 --- Amplification of the full length tilapia MTF-1 isoforms --- p.46Chapter 2.2.2 --- Preparation of Escherichia coli competent cells --- p.48Chapter 2.2.3 --- Transformation --- p.49Chapter 2.2.4 --- Confirmation of the insert of the ligation products --- p.50Chapter 2.2.5 --- Cloning of MTF-1-L and MTF-1-S gene into phrGFPII-1 vector --- p.51Chapter 2.2.6 --- Transient transfection of plasmids to Hepa-Tl cells --- p.54Chapter 2.2.7 --- Staining of the nucleus by Hoechst 33342 --- p.55Chapter 2.2.8 --- Metal treatment on Hepa-Tl cells --- p.55Chapter 2.3 --- Electrophoretic mobility shift assay (EMSA) --- p.56Chapter 2.3.1 --- Preparation of Hepa-Tl whole-cell protein extract --- p.56Chapter 2.3.2 --- In vitro transcription/translation of tilapia MTF-1 isoforms --- p.57Chapter 2.3.3 --- Annealing of the tiMREg oligonucleotides --- p.58Chapter 2.3.4 --- Labeling of the annealed tiMREg oligonucleotides --- p.58Chapter 2.3.5 --- Electrophoretic mobility shift assay --- p.59Chapter 3. --- Chapter Three ResultsChapter 3.1 --- Quantification of MTF-1 isoforms and MT mRNA levels in tilapia and Hepa-Tl cells by real-time PCR --- p.62Chapter 3.1.1 --- Validation of primers for real-time PCR --- p.62Chapter 3.1.2 --- Tissue distribution of MTF-1 isoforms in tilapia and Hepa-Tl cell-line --- p.63Chapter 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.68Chapter 3.2 --- Localization study of MTF-1 isoforms --- p.82Chapter 3.2.1 --- Cloning of MTF-1 isoforms into phrGFPII-1 vector --- p.82Chapter 3.2.2 --- Transient transfection of phrGFPII-1 plasmids to Hepa-Tl cells --- p.82Chapter 3.3 --- Electrophoretic mobility shift assay (EMSA) --- p.96Chapter 4. --- Chapter Four DiscussionChapter 4.1 --- Tissue distribution of MTF-1 isoforms --- p.104Chapter 4.2 --- Effect of metal stress on the mRNA expression level of MT and MTF-1 isoforms --- p.106Chapter 4.3 --- In vitro study of the localization of the MTF-1 isoforms --- p.114Chapter 4.4 --- DNA binding of MTF-1 synthesized by in vitro transcription/translation method --- p.121Chapter 4.5 --- Conclusion --- p.125Chapter 5. --- REFERENCES --- p.12