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Molecular cloning of human glycogen synthase kinase-3α promoter and expression study of the protein.

by Chan Ying Chi, Jessica. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 113-127). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iv / Contents --- p.vi / Abbreviations --- p.xi / Single Letter Amino Acid Code --- p.xvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Glycogen Synthase (EC 2.4.1.11) --- p.1 / Chapter 1.2 --- Glycogen Synthase Kinase-3 --- p.4 / Chapter 1.3 --- Structure of Glycogen Synthase Kinase-3 --- p.5 / Chapter 1.4 --- Functions of Glycogen Synthase Kinase-3 --- p.8 / Chapter 1.4.1 --- Substrate Recognition --- p.8 / Chapter 1.4.2 --- Glycogen Synthase Kinase-3 Homologs --- p.10 / Chapter 1.4.2.1 --- Drosophila --- p.10 / Chapter 1.4.2.2 --- Xenopus --- p.11 / Chapter 1.4.2.3 --- Dictyostelium and Others --- p.12 / Chapter 1.4.3 --- Regulation of Glycogen Synthase-3 in Mammalian Systems --- p.13 / Chapter 1.4.4 --- The role of Glycogen Synthase Kinase-3in Mammalian Brain --- p.16 / Chapter 1.4.4.1 --- Glycogen Synthase Kinase-3β --- p.18 / Chapter 1.4.4.2 --- Glycogen Synthase Kinase-3α --- p.21 / Chapter 1.4.5 --- Glycogen Synthase Kinase-3α in Certain Tumor Cells --- p.23 / Chapter 1.5 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Plasmid Minipreparation --- p.26 / Chapter 2.1.2 --- Large Scale of Plasmid DNA Purification Using QIAGEN-tip500 --- p.28 / Chapter 2.1.3 --- Extraction of Human Blood Genomic DNA --- p.30 / Chapter 2.1.4 --- UV Spectroscopy for determining DNA/RNA Concentration --- p.31 / Chapter 2.1.5 --- Agarose Gel Electrophoresis of DNA --- p.31 / Chapter 2.1.6 --- Purification of DNA Fragment from Agarose Gel using GeneClean III ® (BIO 101 Inc.) Kit --- p.32 / Chapter 2.1.7 --- Restriction Digestion of DNA --- p.32 / Chapter 2.1.8 --- Southern Blot --- p.33 / Chapter 2.1.9 --- Probe Labelling --- p.33 / Chapter 2.1.10 --- Hybridization by Radio-labelling --- p.34 / Chapter 2.1.11 --- DNA Sequencing Reaction --- p.35 / Chapter 2.1.12 --- "Preparation of 6% Polyacrylamide, 8M Urea Denaturing Gel for DNA Sequencing Analysis" --- p.37 / Chapter 2.1.13 --- Preparation of Escherichia coli DH5α Competent Cells --- p.38 / Chapter 2.1.14 --- Modification of 5'Protruding end with T4DNA Polymerase --- p.39 / Chapter 2.1.15 --- Ligation and Transformation of Foregin DNA --- p.39 / Chapter 2.1.16 --- Rapid Screening for the Presence of Plasmid --- p.40 / Chapter 2.2 --- Expression of Glycogen Synthase Kinase-3 / Chapter 2.2.1 --- Preparation of Mammalian cells in Culture --- p.41 / Chapter 2.2.2 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2.3 --- Western Blot Detection of Glycogen Synthase Kianse-3 --- p.43 / Chapter 2.3 --- Assay of Glycogen Synthase Kinase Promoter Activity / Chapter 2.3.1 --- Preparation of SHSY5Y in Culture --- p.45 / Chapter 2.3.2 --- Trypsinization for Removing Adherent Cells --- p.45 / Chapter 2.3.3 --- Transfection of Mammalian Cells by Calcium Phosphate Precipitation --- p.46 / Chapter 2.3.4 --- Stimulation of Transfection Cells by different Chemicals and Preparation of Cell Extract --- p.47 / Chapter 2.3.5 --- CAT-ELISA and β-Gal ELISA Assay --- p.47 / Chapter 2.4 --- Isolation of Glycogen Synthase Kinase-3α 5,Promoter Region / Chapter 2.4.1 --- 5'Rapid Amplification of cDNA End (5'RACE) --- p.48 / Chapter 2.4.2 --- PromoterFinder DNA Walking --- p.49 / Chapter 2.4.3 --- YAC Clone Genomic Construction --- p.50 / Chapter 2.5 --- Construction of Plasmid for Assay of Glycogen Synthase Kinase-3α Promoter Activity --- p.53 / Chapter 2.6 --- Genomic Organization of Glycogen Synthase Kinase-3α --- p.53 / Chapter 2.7 --- Primer Extension Assay / Chapter 2.7.1 --- Isolation of Total RNA by TRIZOL Reagent --- p.57 / Chapter 2.7.2 --- Primer Extension by SuperScript II --- p.57 / Chapter 2.8 --- Reagents and Buffers / Chapter 2.8.1 --- Nucleic Acid Electrophoresis Buffers --- p.59 / Chapter 2.8.2 --- Reagents for Preparation of Plasmid DNA --- p.59 / Chapter 2.8.3 --- Media for Bacterial Culture --- p.60 / Chapter 2.8.4 --- Reagents for Southern Blot --- p.60 / Chapter 2.8.5 --- Reagents for SDS-PAGE --- p.61 / Chapter 2.8.6 --- Reagents for Western Blot --- p.62 / Chapter 2.8.7 --- Reagents for DNA Sequencing --- p.62 / Chapter Chapter 3 --- Isolation of 5´ة Glycogen Synthase Kinase-3α Promoter Region / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.66 / Chapter 3.2.1 --- 5' Rapid Amplification of cDNA End (5'RACE) --- p.66 / Chapter 3.2.2 --- PromoterFinder DNA Walking --- p.68 / Chapter 3.2.3 --- YAC Clone Library Construction --- p.71 / Chapter 3.2.3.1 --- Southern Blotting --- p.71 / Chapter 3.2.3.2 --- Isolation of Sequence Upstream of Glycogen Synthase Kinase-3α region from YAC Clone Using PromoterFider DNA Walking --- p.71 / Chapter 3.2.3.3 --- Sequences of 5,Glycogen Synthase Kinase -3α Promoter --- p.73 / Chapter 3.2.4 --- Primer Extension Assay --- p.78 / Chapter 3.2.5 --- Assay of Glycogen Synthase Kinase-3α Promoter Activity using CAT-ELISA --- p.78 / Chapter 3.2.6 --- Genomic Structure of Glycogen Synthase Kinase-3α --- p.84 / Chapter 3.3 --- Discussion --- p.90 / Chapter 3.3.1 --- Glycogen Synthase Kinase-3a Promoter --- p.90 / Chapter 3.3.2 --- Glycogen Synthase Kianse-3a Promoter Activity --- p.92 / Chapter 3.3.3 --- Prospective and Future Studies --- p.94 / Chapter Chapter 4 --- Expression of Glycogen Synthase Kinase-3 / Chapter 4.1 --- Introduction --- p.96 / Chapter 4.2 --- Results Expression of GSK-3 under Stresses --- p.97 / Chapter 4.3 --- Discussion --- p.105 / Chapter 4.3.1 --- Post-translation regulation of Glycogen Synthase Kinase-3 --- p.105 / Chapter 4.3.2 --- Prospective and Future Studies --- p.107 / Chapter Chapter 5 --- Conclusion --- p.109 / Chapter 5.1 --- Promoter study --- p.110 / Chapter 5.2 --- Genomic organization study --- p.111 / Chapter 5.3 --- Expression study --- p.112 / Reference --- p.113 / Appendices / Appendix I G/C contents of GSK-3α Promoter Region --- p.128 / Appendix II Restriction sites of GSK-3α Promoter Region --- p.134 / Appendix III Primers designed on GSK-3α Promoter Region --- p.139 / Appendix IV Restriction sites of GSK-3α cDNA --- p.142 / Appendix V Vectors --- p.150 / Appendix VI Adaptors Sequences --- p.152 / Appendix VII Anti-GSK-3 Antibody --- p.153 / Appendix VIII Raw data of GSK-3α promoter activity assay --- p.154

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322413
Date January 1998
ContributorsChan, Ying Chi Jessica., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xiv, 155 leaves : ill. ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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