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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325714 |
| Date | January 2006 |
| Contributors | Lau, Suk Kwan., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xx, 160 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use 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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