Mechanism of metallothionein gene regulation in tilapia.

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.iiiAcknowledgements --- p.vTable of Contents --- p.1List of Tables --- p.4List of Figures --- p.5List of Abbreviations --- p.8Chapter 1. --- Introduction --- p.10Chapter 1.1 --- Biology of metals --- p.10Chapter 1.2 --- Metal detoxification systems --- p.11Chapter 1.3 --- Metallothionein --- p.13Chapter 1.4 --- Classification of MTs --- p.15Chapter 1.5 --- Biological roles of MT --- p.15Chapter 1.5.1 --- Homeostasis of essential transition metal ion --- p.15Chapter 1.5.2 --- Detoxification of non-essential heavy metal ion --- p.17Chapter 1.5.3 --- Protection against oxidative stress --- p.18Chapter 1.5.4 --- Role in neurodegenerative diseases --- p.19Chapter 1.6 --- Molecular biology of MT --- p.19Chapter 1.6.1 --- MT gene structure --- p.19Chapter 1.6.2 --- MT gene regulation --- p.21Chapter 1.7 --- MRE binding transcription factor-1 (MTF-1) --- p.30Chapter 1.8 --- Activation of MTF-1 --- p.31Chapter 1.9 --- Target genes of MTF-1 --- p.32Chapter 1.10 --- Fish MT gene and MTF-1 --- p.33Chapter 1.11 --- Tilapia --- p.39Chapter 1.12 --- Study of tilapia MT --- p.41Chapter 1.13 --- Aims and rationale of study --- p.43Chapter 2. --- Materials and Methods --- p.45Chapter 2.1 --- Cloning of tilapia MT gene 5'-flanking region --- p.45Chapter 2.1.1 --- Animals --- p.45Chapter 2.1.2 --- Preparation of tilapia genomic DNA --- p.45Chapter 2.1.3 --- DNA walking --- p.45Chapter 2.1.4 --- Amplification of whole tiMT gene --- p.50Chapter 2.2 --- Determination of transcription start site --- p.51Chapter 2.2.1 --- Total RNA extraction --- p.51Chapter 2.2.2 --- Rapid amplification of 5，complementary DNA ends (5' RACE) --- p.52Chapter 2.3 --- Transient transfection assay --- p.54Chapter 2.3.1 --- Cell culture --- p.54Chapter 2.3.2 --- Construction of pGL3-tiMT deletion mutants --- p.54Chapter 2.3.3 --- Preparation of heavy metal solutions --- p.56Chapter 2.3.4 --- Determination of heavy metal ion toxicities by alamarBlue´ёØ assay --- p.56Chapter 2.3.5 --- Transient transfection of plasmids to Hepa-T1 cells --- p.56Chapter 2.3.6 --- Metal ions treatment and study of tiMT promoter activities --- p.57Chapter 2.3.7 --- Transient gene expression studies of deletion mutants of tiMT promoter --- p.57Chapter 2.4 --- Site-directed mutagenesis of tiMT promoter --- p.58Chapter 2.4.1 --- Polymerase chain reaction (PCR)-based site-directed mutagenesis --- p.58Chapter 2.4.2 --- Transient transfection of plasmids to Hepa-T1 cells and study of tiMT promoter activities --- p.62Chapter 2.5 --- Electrophoretic mobility shift assay (EMSA) --- p.63Chapter 2.5.1 --- Extract preparation --- p.63Chapter 2.5.2 --- Preparation of radiolabeled tiMRE oligonucleotides --- p.63Chapter 2.5.3 --- Electrophoretic mobility shift assay (EMSA) --- p.64Chapter 3. --- Results --- p.66Chapter 3.1 --- "Cloning of tilapia MT (tiMT) gene 5,-flanking region and amplification of whole tiMT gene" --- p.66Chapter 3.2 --- Determination of transcription start site --- p.69Chapter 3.3 --- Cloning of tiMT promoter fragment into reporter vector --- p.72Chapter 3.4 --- Determination of heavy metal ion toxicities by alamarBlue´ёØ assay --- p.72Chapter 3.5 --- Study of tiMT promoter activities by heavy metal ions exposure..… --- p.72Chapter 3.6 --- Cloning of deletion mutants of tiMT promoter --- p.79Chapter 3.7 --- Transient gene expression studies of deletion mutants of tiMT promoter --- p.80Chapter 3.8 --- Cloning of mutants with site-directed mutagenesis in tiMT promoter --- p.88Chapter 3.9 --- Site-directed mutagenesis of tiMT promoter --- p.92Chapter 3.10 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.97Chapter 4. --- Discussion --- p.102Chapter 4.1 --- Tilapia MT gene --- p.102Chapter 4.2 --- Resistance of tilapia to heavy metal ions --- p.107Chapter 4.3 --- Functional analysis of tiMT gene promoter by transient transfection --- p.111Chapter 4.4 --- DNA binding of metal responsive transcription factor in Hepa-T1 cells --- p.121Chapter 4.5 --- Conclusion --- p.138References --- p.14