Tang Wai-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-147). / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / Lists of Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Isolation of Mas Oncogene --- p.1 / Chapter 1.2 --- Distribution of Mas Oncogene..........…… --- p.3 / Chapter 1.3 --- Developmental Expression of Mas Oncogene --- p.5 / Chapter 1.4 --- Study of Mas-deficient Mice --- p.7 / Chapter 1.5 --- Signal Transduction of Mas Oncogene --- p.8 / Chapter 1.6 --- Other Family Member of Mas Oncogene --- p.9 / Chapter 1.7 --- Mas and Angiotensin Receptor --- p.11 / Chapter 1.8 --- Angiotensin Receptors / Chapter 1.8.1 --- Classification of Angiotensin AT1 Receptor --- p.14 / Chapter 1.8.2 --- Cloning of Angiotensin Receptor --- p.15 / Chapter 1.9 --- Expression of Angiotensin Receptor / Chapter 1.9.1 --- Physiological Factors --- p.17 / Chapter 1.9.2 --- Cis-regulatory Elements / Chapter 1.9.2.1 --- Organization and Regulatory Elements of AT1 Receptor --- p.19 / Chapter 1.9.2.2 --- Expression of AT1a Receptor Promoter was Induced by AP-1 and GATA-4 in Pressure Overload Model --- p.20 / Chapter 1.9.2.3 --- AT1a Receptor Reveals Three Glucocorticoid Responsive Elements --- p.22 / Chapter 1.10 --- Signal Transduction of Angiotensin Receptor --- p.22 / Chapter 1.11 --- Aim of Project --- p.25 / Chapter Chapter 2: --- Mas Oncogene in AR4-2J cells / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Reagents --- p.27 / Chapter 2.2.1.2 --- Enzymes --- p.27 / Chapter 2.2.1.3 --- DNA Purification Kits --- p.28 / Chapter 2.2.1.4 --- Materials and Antibodies for Western Blot --- p.28 / Chapter 2.2.1.5 --- Others --- p.28 / Chapter 2.2.2 --- Restriction Enzyme Digestion --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis --- p.29 / Chapter 2.2.4 --- DNA Extraction and Purification --- p.29 / Chapter 2.2.5 --- Plasmid Vector Modification and DNA Ligation --- p.30 / Chapter 2.2.6 --- Bacterial Transformation --- p.31 / Chapter 2.2.7 --- Preparation of Plasmid DNA / Chapter 2.2.7.1 --- Minipreps --- p.32 / Chapter 2.2.7.2 --- Midipreps and Maxipreps --- p.33 / Chapter 2.2.8 --- Genomic DNA Extraction From Tissue and Cell Culture --- p.34 / Chapter 2.2.9 --- RT-PCR Cloning of Mas Oncogene --- p.35 / Chapter 2.2.10 --- Construction of Full Length Mas cDNA into pBluescript® II SK Vector --- p.38 / Chapter 2.2.11 --- Southern Blot Analysis / Chapter 2.2.11.1 --- Preparation of DIG-labeled Mas Probe --- p.38 / Chapter 2.2.11.2 --- Enzyme Restriction of Genomic DNA --- p.39 / Chapter 2.2.11.3 --- Transferring DNA to Nylon Membrane --- p.40 / Chapter 2.2.11.4 --- Prehybridization and Hybridization --- p.40 / Chapter 2.2.11.5 --- Post-hybridization Washes and Blocking --- p.41 / Chapter 2.2.11.6 --- Detection --- p.41 / Chapter 2.2.12 --- DNA Sequencing / Chapter 2.2.12.1 --- Manual Sequencing --- p.42 / Chapter 2.2.12.2 --- Autosequencing --- p.43 / Chapter 2.2.12.3 --- Sequencing Primers --- p.44 / Chapter 2.2.13 --- Cell Culture --- p.45 / Chapter 2.2.14 --- Protein Assay by Modified Lowery --- p.46 / Chapter 2.2.15 --- SDS-PAGE and Western Blot Analysis --- p.47 / Chapter 2.3 --- Results --- p.49 / Chapter 2.4 --- Discussion --- p.60 / Chapter Chapter 3: --- Analysis of Transfected Mas Cell Lines / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials --- p.62 / Chapter 3.2.2 --- Cell Culture and Transfection / Chapter 3.2.2.1 --- Cell Culture --- p.62 / Chapter 3.2.2.2 --- Transfection Optimization --- p.62 / Chapter 3.2.2.3 --- Fluorescent SEAP Assay --- p.63 / Chapter 3.2.2.4 --- Transient Transfection --- p.64 / Chapter 3.2.2.5 --- Stable Cell Line Construction --- p.64 / Chapter 3.2.3 --- Protein Assay ESL --- p.65 / Chapter 3.2.4 --- SDS-PAGE and Western Blot Analysis --- p.65 / Chapter 3.2.5 --- Preparation of an AT1a Receptor Internal Standard for Quantitative RT-PCR Analysis / Chapter 3.2.5.1 --- Preparation of an AT1a Receptor cDNA by RT-PC --- p.66 / Chapter 3.2.5.2 --- Cloning of AT1A Receptor cDNA into pBluescript® II SK Vector --- p.67 / Chapter 3.2.5.3 --- Autosequence of pBluescript® II SK Vector/AT1AR --- p.68 / Chapter 3.2.5.4 --- Preparation of 100 bp Deleted AT1a Receptor cDNA by RT- PCR --- p.68 / Chapter 3.2.5.5 --- Cloning of Deleted AT1a R cDNA into pCAPs Vector --- p.71 / Chapter 3.2.6 --- Construction of Full Length Mas cDNA into pOPRSVI/MCS Operator Vector --- p.71 / Chapter 3.2.7 --- Preparation of an Mas Internal Standard for Quantitative RT-PCR Analysis / Chapter 3.2.7.1 --- Preparation of 100 bp Deleted Mas cDNA by RT- PCR --- p.72 / Chapter 3.2.7.2 --- Cloning of 100 bp Deleted Mas cDNA into pCAPs Vector (Mas/pCAPs) --- p.73 / Chapter 3.2.8 --- Quantitative RT-PCR Analysis of AT1A R Expression --- p.74 / Chapter 3.2.9 --- Quantitative RT-PCR Analysis for the Expression of Mas --- p.74 / Chapter 3.3 --- Results --- p.76 / Chapter 3.4 --- Discussions --- p.100 / Chapter Chapter 4: --- Cloning of AT1A Receptor Promoter / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials --- p.105 / Chapter 4.2.2 --- Genomic DNA Extraction From Rat Pancreas --- p.105 / Chapter 4.2.3 --- "Nest PCR Amplification of 3.2, 2.8 and 1.4kb AT1a Receptor Promoter" --- p.105 / Chapter 4.2.4 --- PCR Amplification of 2.2 kb Aproximal Portion of AT1a Receptor Promoter --- p.107 / Chapter 4.2.5 --- Construction of PCR Fragment of Angiotensin Receptor Promoter into Various Vector --- p.108 / Chapter 4.2.5.1 --- pSEAP2-Basic --- p.108 / Chapter 4.2.5.2 --- pBluescript® II SK Vector --- p.109 / Chapter 4.2.5.3 --- PCR Cloning Kit (pCAPs vector) --- p.109 / Chapter 4.2.5.4 --- PCR-TRAP Cloning System --- p.109 / Chapter 4.2.6 --- Direct PCR Analysis --- p.110 / Chapter 4.2.7 --- Autosequencing of PCR Fragment of AT1A Receptor Promoter --- p.111 / Chapter 4.3 --- Results --- p.114 / Chapter 4.4 --- Discussions --- p.130 / Chapter Chapter 5: --- General Discussion --- p.131 / Chapter Appendix 1 --- Composition of Solutions --- p.133 / Chapter Appendix 2 --- Published Abstract --- p.141 / References --- p.142