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Tsang Sup-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 176-185). / Abstracts in English and Chinese. / Acknowledgments --- p.1 / Abstract --- p.2 / 摘要 --- p.4 / List of Abbreviation --- p.6 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Isolation and activation of mas oncogene --- p.11 / Chapter 1.2 --- Amino acid sequence of mas oncogene --- p.14 / Chapter 1.3 --- Expression of mas oncogene --- p.18 / Chapter 1.4 --- Possible physiological role of mas oncogene --- p.20 / Chapter 1.5 --- Gene related to mas family --- p.23 / Chapter 1.6 --- Aims of study --- p.26 / Chapter Chapter 2 --- Over-expression of mas oncogene / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.1.1 --- Chemicals --- p.30 / Chapter 2.2.1.2 --- Enzyme --- p.30 / Chapter 2.2.1.3 --- DNA Purification Kit --- p.31 / Chapter 2.2.1.4 --- Others --- p.31 / Chapter 2.2.2 --- Methods --- p.31 / Chapter 2.2.2.1 --- Strategy of construct preparation --- p.31 / Chapter 2.2.2.2 --- "Preparation of linearized vector, pFRSV" --- p.32 / Chapter 2.2.2.2.1 --- Cloning of vectors --- p.32 / Chapter 2.2.2.2.2 --- Restriction enzyme digestion and DNA dephosphorylation --- p.34 / Chapter 2.2.2.2.3 --- DNA purification by agarose gel electro-elution --- p.34 / Chapter 2.2.2.3 --- Preparation of pFRSV/mas construct --- p.35 / Chapter 2.2.2.3.1 --- PCR amplification --- p.35 / Chapter 2.2.2.3.2 --- Restriction enzyme digestion --- p.36 / Chapter 2.2.2.4 --- Ligation and analysis --- p.37 / Chapter 2.2.2.5 --- Purification of DNA by cesium chloride --- p.38 / Chapter 2.2.2.5.1 --- Large-scale bacterial culturing --- p.38 / Chapter 2.2.2.5.2 --- Ethanol precipitation --- p.39 / Chapter 2.2.2.5.3 --- Cesium chloride purification --- p.39 / Chapter 2.2.2.5.4 --- Removal of DNA dye by dialysis and ethanol precipitation --- p.40 / Chapter 2.2.2.6 --- Transfection by electroporation --- p.41 / Chapter 2.2.2.7 --- Screening for the stably transfected cells --- p.42 / Chapter 2.2.2.8 --- RT-PCR analysis of the mas transfectant --- p.43 / Chapter 2.2.2.8.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.43 / Chapter 2.2.2.8.2 --- Reverse transcription of the total RNA into cDNA --- p.44 / Chapter 2.2.2.8.3 --- Analysis of the transfected mas expression by PCR --- p.44 / Chapter 2.2.2.8.4 --- Analysis of the transfected DHFR expression by PCR --- p.45 / Chapter 2.2.2.8.5 --- Analysis of endogenous GAPDH expression by PCR --- p.46 / Chapter 2.2.2.9 --- Amplification of mas transgene by using methotrexate --- p.47 / Chapter 2.2.2.9.1 --- Amplification by low dosage MTX treatment --- p.47 / Chapter 2.2.2.9.2 --- Amplification by high dosage MTX treatment --- p.49 / Chapter 2.2.2.10 --- Southern blot analysis --- p.50 / Chapter 2.2.2.10.1 --- Preparation of DIG-labelled mas probe --- p.51 / Chapter 2.2.2.10.2 --- Preparation of DIG-labelled DHFR probe --- p.51 / Chapter 2.2.2.10.3 --- Preparation of DIG-labelled GAPDH probe --- p.52 / Chapter 2.2.2.10.4 --- Isolation of Genomic DNA from the mas transfectants by DNAzol® Reagent / Chapter 2.2.2.10.5 --- Enzymatic restriction of genomic DNA and Gel electrophoresis --- p.54 / Chapter 2.2.2.10.6 --- DNA transferring to positive charged Nylon membrane --- p.54 / Chapter 2.2.2.10.7 --- Pre-hybridization and hybridization --- p.56 / Chapter 2.2.2.10.8 --- Post-hybridization washing and blocking --- p.56 / Chapter 2.2.2.10.9 --- Detection --- p.57 / Chapter 2.2.2.11 --- Northern blot analysis --- p.57 / Chapter 2.2.2.11.1 --- Preparation of the agarose gel containing formaldehyde --- p.58 / Chapter 2.2.2.11.2 --- Preparation of the RNA sample --- p.58 / Chapter 2.2.2.11.3 --- Gel electrophoresis and transferring --- p.59 / Chapter 2.2.2.11.5 --- Pre-hybridization and hybridization --- p.60 / Chapter 2.2.2.11.4 --- Post-hybridization washing and blocking --- p.60 / Chapter 2.2.2.11.6 --- Detection --- p.61 / Chapter 2.2.2.11.7 --- Stripping and rehybridization --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- RT-PCR analysis of gene expression in the stably transfectant --- p.62 / Chapter 2.3.2 --- Morphology of the mas trasnfectant --- p.64 / Chapter 2.3.3 --- Determination of mas gene copy number by Southern blot analysis in the mas transfectants --- p.66 / Chapter 2.3.4 --- Northern blot analysis of the transcriptional level of mas transcriptsin mas transfectants --- p.76 / Chapter 2.4 --- Discussion --- p.87 / Chapter Chapter 3 --- In vivo study of physiological effect of over-expression of mas / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Materials --- p.93 / Chapter 3.2.2 --- Methods --- p.93 / Chapter 3.2.2.1 --- Cell culture --- p.93 / Chapter 3.2.2.2 --- Subcutaneous injection of nude mice --- p.94 / Chapter 3.2.2.3 --- Isolation of the total RNA from the tumor tissues --- p.95 / Chapter 3.2.2.4 --- Northern blot analysis --- p.96 / Chapter 3.3 --- Results --- p.96 / Chapter 3.3.1 --- Tumorgenicity assay of mas oncogene in nude mice --- p.96 / Chapter 3.3.2 --- Northern blot analysis of mas expression in the tumor tissues --- p.103 / Chapter 3.4 --- Discussion --- p.109 / Chapter Chapter 4 --- Fluorescent differential display analysis of mas transfectants / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and Methods --- p.112 / Chapter 4.2.1 --- Materials --- p.112 / Chapter 4.2.1.1 --- Chemicals --- p.112 / Chapter 4.2.1.2 --- Enzyme --- p.113 / Chapter 4.2.1.3 --- Kits --- p.113 / Chapter 4.2.1.4 --- Others --- p.114 / Chapter 4.2.2 --- Methods --- p.114 / Chapter 4.2.2.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.114 / Chapter 4.2.2.2 --- DNase I treatment --- p.115 / Chapter 4.2.2.3 --- Reverse transcription (RT) and non-fluorescent PCR --- p.116 / Chapter 4.2.2.4 --- Reverse transcription and fluorescent differential display-PCR --- p.118 / Chapter 4.2.2.5 --- High resolution fluorescent differential display (Fluoro DD) gel --- p.118 / Chapter 4.2.2.6 --- Gel band excision of differentially expressed cDNA fragments --- p.120 / Chapter 4.2.2.7 --- Gel band reamplification --- p.120 / Chapter 4.2.2.8 --- Subcloning of reamplified cDNA fragments --- p.121 / Chapter 4.2.2.9 --- Purification of plasmid DNA from recombinant clones for reverse dot blot analysis --- p.122 / Chapter 4.2.2.10 --- Reverse dot blot analysis --- p.123 / Chapter 4.2.2.10.1 --- Preparation of cDNA dot blot --- p.123 / Chapter 4.2.2.10.2 --- Preparation of DIG-labeled cDNA library probes --- p.124 / Chapter 4.2.2.10.3 --- Hybridization --- p.126 / Chapter 4.2.2.11 --- Northern blot analysis --- p.127 / Chapter 4.3 --- Results --- p.128 / Chapter 4.3.1 --- Fluorescent differential display (FluoroDD) --- p.128 / Chapter 4.3.2 --- Reverse dot blot analysis --- p.135 / Chapter 4.3.3 --- DNA sequencing analysis of the clones --- p.141 / Chapter 4.3.4 --- Confirmation of differential display pattern of the subclones by Northern blot analysis --- p.160 / Chapter 4.4 --- Discussion --- p.166 / Chapter Chapter 5 --- General Discussion / Chapter 5.1 --- General model for mos-induced tumor formation --- p.169 / Chapter 5.2 --- Future aspect --- p.174 / References --- p.176 / Appendix I Buffer composition --- p.186 / Appendix II Sequences of fluoroDD TMR-Anchored primers and arbitrary primers --- p.190

Thesis (Ph. D.)--Cornell University, January, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Biochemistry. Bibliography: leaves 83-87. Also available online.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [74]-91).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-109).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

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