Role of Mas oncogene on angiotensin receptor expression.

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