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Characterization of acetylcholinesterase and its promoter region in Tetraodon nigroviridis. / Characterization of acetylcholinesterase & its promoter region in Tetraodon nigroviridis

Lau Suk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 128-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Table of content --- p.ii / List of Figures --- p.x / List of Tables --- p.xiv / Abbreviation --- p.xv / Abstract --- p.xviii / 論文摘要 --- p.xx / Chapter 1 --- Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Tetraodon nigroviridis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Genomic Sequencing Project --- p.3 / Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4 / Chapter 1.1.3.1 --- Genomic Comparison --- p.4 / Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5 / Chapter 1.1.3.3 --- Genomic Evolution --- p.6 / Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7 / Chapter 1.2.1 --- Transcriptional Regulation --- p.7 / Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7 / Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8 / Chapter 1.2.1.3 --- Promoter Structure --- p.9 / Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10 / Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11 / Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12 / Chapter 1.3 --- Acetylcholinesterase --- p.15 / Chapter 1.3.1 --- Background --- p.15 / Chapter 1.3.2 --- Regulation ofAChE --- p.17 / Chapter 1.3.2.1 --- Transcriptional Level --- p.17 / Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19 / Chapter 1.3.2.3 --- Post-translational Level --- p.20 / Chapter 1.3.2.3.1 --- Oligomerization --- p.20 / Chapter 1.3.2.3.2 --- Glycosylation --- p.21 / Chapter 1.3.2.3.3 --- Phosphroylation --- p.22 / Chapter 1.3.3 --- Functions of AChE --- p.23 / Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23 / Chapter 1.3.3.2 --- Embryonic Development --- p.23 / Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24 / Chapter 1.3.3.4 --- Neuritogensis --- p.24 / Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24 / Chapter 1.3.3.6 --- Apoptosis --- p.25 / Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25 / Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27 / Chapter 1.4 --- Inducible Cell Expression Systems --- p.28 / Chapter 1.5 --- Objectives --- p.32 / Chapter 2 --- Chapter 2 Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Primer Design --- p.34 / Chapter 2.2.2 --- Cell Culture --- p.34 / Chapter 2.2.3 --- Transformation --- p.35 / Chapter 2.2.4 --- Plasmids Preparation --- p.35 / Chapter 2.2.5 --- Plasmids Screening --- p.36 / Chapter 2.2.6 --- RNA Extraction --- p.36 / Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37 / Chapter 2.2.8 --- Genomic Analysis --- p.37 / Chapter 2.2.9 --- Protein Sequence Analysis --- p.38 / Chapter 2.2.10 --- Genomic DNA Extraction --- p.39 / Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39 / Chapter 2.2.12 --- Luciferase Assay --- p.40 / Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40 / Chapter 2.2.14 --- Protein Assay --- p.41 / Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41 / Chapter 2.2.16 --- Histochemistry --- p.42 / Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42 / Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43 / Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43 / Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44 / Chapter 2.2.21 --- SDS Electrophoresis --- p.44 / Chapter 2.2.22 --- Western Blotting --- p.45 / Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45 / Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46 / Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47 / Chapter 2.2.26 --- MTT Assay --- p.47 / Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48 / Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49 / Chapter 3.1 --- Results --- p.49 / Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49 / Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49 / Chapter 3.1.3 --- Primary Sequence Analysis --- p.52 / Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60 / Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60 / Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76 / Chapter 3.2 --- Discussion --- p.78 / Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91 / Chapter 4.1 --- Results --- p.91 / Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91 / Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94 / Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94 / Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94 / Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99 / Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99 / Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99 / Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101 / Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103 / Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103 / Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107 / Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112 / Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112 / Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112 / Chapter 4.2 --- Discussion --- p.116 / Chapter 4.2.1 --- Bacterial Expression System --- p.116 / Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119 / Chapter 5 --- General Discussion --- p.124 / Chapter 5.1 --- Summaries --- p.124 / Chapter 5.2 --- Further works --- p.126 / Chapter 6 --- References --- p.128 / Appendix 1 internet software and database used in this project --- p.151 / Appendix 2 tnAChE mRNA sequence --- p.152 / Appendix 3 ptnAChE-1143 sequence --- p.154 / Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.156

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325714
Date January 2006
ContributorsLau, Suk Kwan., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xx, 160 leaves : ill. (some col.) ; 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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