by Tam Fung-ping. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 254-264). / Chapter I. --- Title page --- p.I / Chapter II. --- Abstract --- p.II / Chapter III. --- Acknowlegements --- p.III / Chapter IV. --- Table of contents --- p.IV / Chapter V. --- List of tables --- p.V / Chapter VI. --- List of figures --- p.VI / Chapter VII. --- Abbreviations --- p.VII / Chapter Chapter 1. --- Literature Reviews / Chapter 1.1 --- Modes of energy generation in facultative bacteria --- p.1 / Chapter 1.1.1 --- Difference in energy generation mechanism between respiratory and fermentative pathways --- p.2 / Chapter 1.1.2 --- Difference in carbon metabolism during anaerobiosis --- p.6 / Chapter 1.2 --- Repression and derepression of genes during anaerobiosis --- p.8 / Chapter 1.3 --- Global regulatory network for respiratory control --- p.8 / Chapter 1.3.1 --- Fnr-regulated gene expression --- p.10 / Chapter 1.3.2 --- NarL-regulated gene expression --- p.11 / Chapter 1.3.3 --- Crp-regulated gene expression --- p.12 / Chapter 1.3.4 --- ArcA-regulated gene expression --- p.13 / Chapter 1.3.5 --- Overlapping control of gene expression --- p.14 / Chapter 1.3.6 --- Regulatory mechanism of respiratory control --- p.16 / Chapter 1.4 --- Other regulatory systems in respiratory control --- p.19 / Chapter 1.5 --- The puzzle of regulatory network in anaerobiosis --- p.22 / Chapter 1.6 --- ArcA-ArcB system in Escherichia coli --- p.24 / Chapter 1.6.1 --- Arc A and ArcB for aerobic respiratory control --- p.24 / Chapter 1.6.2 --- arcA/dye/msp/fex/sfrA/cpxC gene are on identical genetic locus --- p.26 / Chapter 1.6.3 --- Arc function and Sfr function of Arc A protein are separately regulated --- p.28 / Chapter 1.6.4 --- ArcB-ArcA as sensor regulator in two component system for respiratory control --- p.29 / Chapter 1.7 --- Objectives and strategies of present study --- p.37 / Chapter Chapter 2. --- Materials / Chapter 2.1 --- Bacterial strains --- p.41 / Chapter 2.2 --- Culture mediums --- p.44 / Chapter 2.3 --- "Buffers, chemicals and antibiotics" --- p.46 / Chapter 2.4 --- DNA primers --- p.53 / Chapter Chapter 3. --- Primer extension analysis for locating the transcriptional start point of anaerobic inducible arcA in pFS --- p.34 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Methods --- p.57 / Chapter 3.2.1 --- Preparation of total RNA --- p.59 / Chapter 3.2.2 --- Formaldeyde agarose gel electrophoresis of RNA --- p.60 / Chapter 3.2.3 --- Spectrometric estimation of RNA --- p.61 / Chapter 3.2.4 --- End-labelling of arcAusp primer with 32P --- p.62 / Chapter 3.2.5 --- Precipitation of arcAusp primer with samples RNA --- p.63 / Chapter 3.2.6 --- Primer extension reaction --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Preparation of RNA --- p.67 / Chapter 3.3.2 --- Determination of transcription start site by primer extension --- p.67 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Selective activations of aerobic and anaerobic transcripts in response to oxygen level --- p.76 / Chapter 3.4.2 --- The arcA promoter is a sigma-70 dependent promoter --- p.77 / Chapter 3.4.3 --- Experimental design --- p.77 / Chapter Chapter 4. --- In vitro chemical mutagensis for finding some important regulatory elements of arcA in pFS --- p.34 / Chapter 4.1 --- Introduction / Chapter 4.2 --- Methods --- p.84 / Chapter 4.2.1 --- Large scale preparation of pFS34 plasmid --- p.84 / Chapter 4.2.2 --- PCR-mediated chemical mutagenesis of pFS34 --- p.86 / Chapter 4.2.3 --- Restriction enzyme digestion of PCR-amplified arcA insert after phenol extraction --- p.90 / Chapter 4.2.4 --- Large scale preparation of vector pFZYl and restriction enzyme digestion --- p.91 / Chapter 4.2.5 --- Ligation of EcoRI-SalI digested pFS34 fragment and vector pFZYl --- p.91 / Chapter 4.2.6 --- Preparation of electrotcompetent cell Salmonella typhymurium JR502 and electro-transformation --- p.92 / Chapter 4.2.7 --- Screening of transformed clones by LB-amp50-xgal plates --- p.93 / Chapter 4.2.8 --- Screening of recombinants colonies by Polymerase chain reaction (PCR) --- p.94 / Chapter 4.2.9 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.96 / Chapter 4.2.10 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.98 / Chapter 4.2.11 --- Sequencing of mutated clones --- p.101 / Chapter 4.2.11.1 --- Recombinant M13 single-stranded sequencing of the mutated clones --- p.101 / Chapter 4.2.11.2 --- pUC18 double-stranded DNA sequencing of mutated clones --- p.105 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3. --- l PCR-mediated chemical mutagenesis of pFS34 --- p.108 / Chapter 4.3.2 --- Screening of transformed clones by LB-amp50-xgal plate --- p.112 / Chapter 4.3.3 --- Screening of recombinants colonies by polymerase chain reaction (PCR) --- p.112 / Chapter 4.3.4 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.114 / Chapter 4.3.5 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.117 / Chapter 4.3.6 --- Sequencing of mutated clones --- p.123 / Chapter 4.4 --- Discussions --- p.135 / Chapter 4.4.1 --- The possible mechanisms in anaerobic transcription --- p.135 / Chapter 4.4.2 --- The possible mechanisms in aerobic transcription --- p.143 / Chapter 4.4.3 --- Experimental design --- p.146 / Chapter Chapter 5 --- Investigation of the effect of integration host factor (IHF) and autoregulation on the expression of pFS34 / Chapter 5.1 --- Introduction --- p.152 / Chapter 5.2 --- Methods --- p.154 / Chapter 5.2.1 --- Construction of Escherichia coli mutant --- p.155 / Chapter 5.2.2 --- PCR check of mutant for the presence of pFS34 and pFZYl plasmid --- p.157 / Chapter 5.2.3 --- β-galactosidase assay of aerobic and anaerobic activities change of pFS34 --- p.157 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Effect of integration factor (IHF) on pFS34 --- p.158 / Chapter 5.3.1.1 --- PCR analysis of E. coli. himA and himD mutant for the presence of pFS34 and pFZYl plasmid --- p.158 / Chapter 5.3.1.2 --- β-galatosidase assay of aerobic and anaerobic activities of pFS34 in E. coli. himA and himD mutant --- p.158 / Chapter 5.3.2 --- Autoregultion on expression of pFS34 --- p.162 / Chapter 5.3.2.1 --- PCR analysis of E. coli. arcA mutant for the presence of pFS34 plasmid --- p.162 / Chapter 5.3.2.2 --- β-galctosidase assay of aerobic and anaerobic activities of pFS34 (arcA-lacZ) in E. coli. arcA mutant --- p.162 / Chapter 5.4 --- Discussions --- p.167 / Chapter 5.4.1 --- Effect of IHF on aerobic and anaerobic expression of arcA --- p.167 / Chapter 5.4.1.1 --- Possible regulatory mechanism of IHF on aerobic transcription --- p.167 / Chapter 5.4.1.2 --- Possible regulatory mechanism of IHF on anaerobic transcription --- p.170 / Chapter 5.4.1.3 --- Affinity binding of IHF depends on topological state of arcA --- p.172 / Chapter 5.4.1.4 --- Possible role of IHF in global regulation of anaerobiosis --- p.173 / Chapter 5.4.1.5 --- Experimental design --- p.174 / Chapter 5.4.2 --- Autoregulatory expression of arcA in pFS34 --- p.176 / Chapter Chapter 6. --- PCR walking of arcA from Salmonella typhimurium LT2 / Chapter 6.1 --- Introduction --- p.177 / Chapter 6.2 --- Methods --- p.186 / Chapter 6.2.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.186 / Chapter 6.2.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.187 / Chapter 6.2.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R & delC-34R) --- p.188 / Chapter 6.2.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.189 / Chapter 6.2.5 --- High stringency PCR amplification with arcAusp2 and delC-23 primer --- p.190 / Chapter 6.2.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer" --- p.191 / Chapter 6.2.7 --- High stringency PCR amplification with arcAusp2 only and Sau3A restriction enzyme digestion of PCR products --- p.192 / Chapter 6.2.8 --- Cloning of PCR walking products into pUC18 and heat shock transforming into E.coli. JM83 --- p.193 / Chapter 6.2.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.194 / Chapter 6.2.10 --- Dideoxy sequencing of PCR walking arcA fragments in pUC18 --- p.194 / Chapter 6.2.11 --- Subcloning of arcA fragment into pFZYl and PCR analysis for insertion of one insert with proper orientation --- p.195 / Chapter 6.2.12 --- arcA-galactosiadase assay of PCR walking arcA fragment-lacZ fusion --- p.196 / Chapter 6.3 --- Results --- p.198 / Chapter 6.3.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.198 / Chapter 6.3.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.198 / Chapter 6.3.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R and delC-34R) --- p.200 / Chapter 6.3.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.200 / Chapter 6.3.5 --- High stringency PCR amplification with arcAusp2 、 primer and delC-23 prime --- p.203 / Chapter 6.3.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer to check for flanking ends of bands" --- p.205 / Chapter 6.3.7 --- High stringency PCR amplification with arcAusp2 primer and Sau3A restriction enzyme digestion of PCR products --- p.207 / Chapter 6.3.8 --- Cloning of PCR walking products into pUC18 and heat-shock transforming into E. coli. JM83 --- p.210 / Chapter 6.3.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.210 / Chapter 6.3.10 --- Dideoxy sequencing of arc A PCR walking fragment: :pUC18 --- p.210 / Chapter 6.3.11 --- Subcloning of arcA fragment into pFZYl and PCR check for right insertion of single insert with proper orientation --- p.226 / Chapter 6.3.12 --- β-galactosidase assay --- p.232 / Chapter 6.4 --- Discussions --- p.227 / Chapter 6.4.1 --- PCR based gene walking strategy --- p.227 / Chapter 6.4.2 --- Confirmation of cloned arcA gene in pFS34 was a geniune arcA gene of S. typhimurium --- p.240 / Chapter 6.4.3 --- Promoter activity of further upstream arcA clones - AU87::pFZYl --- p.241 / Chapter Chapter 7. --- Overall Discussion --- p.244 / Chapter 7.1 --- Summary --- p.244 / Chapter 7.2 --- Proposed Model of regulation of arcA in Salmonella typhimurium --- p.249 / Chapter 7.3 --- Further Studies --- p.251 / References --- p.254

by Cheung, Man Wai William. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 113-123). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.ii / DEDICATION --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- General Introduction --- p.1 / Chapter 1.2. --- Purpose of Study --- p.3 / Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Central Pathways of Aerobic and Anaerobic Carbon Catabolism --- p.7 / Chapter 2.2. --- Global Regulation of Gene Expression by Oxygen --- p.10 / Chapter 2.2.1. --- Two approaches for the studies --- p.10 / Chapter 2.2.2. --- FNR regulation --- p.12 / Chapter 2.2.3. --- ArcAB regulation --- p.19 / Chapter 2.2.3.1. --- arcA --- p.19 / Chapter 2.2.3.2. --- arcB --- p.20 / Chapter 2.2.3.3. --- A member of the Two- Components regulatory systems --- p.21 / Chapter 2.3. --- Molecular Analysis of Promoters --- p.26 / Chapter 2.3.1. --- S1 mapping --- p.29 / Chapter 2.3.2. --- Primer extension --- p.29 / Chapter 2.3.3. --- DNaseI footprinting --- p.30 / Chapter 2.3.4. --- Mutational analysis of promoters --- p.32 / Chapter 3. --- Materials and Methods --- p.35 / Chapter 3.1. --- Bacterial strains and Plasmids --- p.35 / Chapter 3.2. --- Media --- p.35 / Chapter 3.3. --- Solutions --- p.38 / Chapter 3.4. --- Small Scale Preparation of Plasmid DNA --- p.40 / Chapter 3.5. --- Large Scale Preparation of Plasmid DNA --- p.41 / Chapter 3.5.1. --- Growth of bacterial culture --- p.41 / Chapter 3.5.2. --- Lysis by alkali --- p.43 / Chapter 3.5.3. --- Purification of closed circular DNA by cesium chloride gradient equilibrium centrifugation --- p.44 / Chapter 3.5.4. --- Digestion of DNA with restriction endonucleases --- p.45 / Chapter 3.6. --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.45 / Chapter 3.7. --- Cloning of DNA Fragments from Nest-deleted M13mpl8 Clones to pFZYl --- p.47 / Chapter 3.8. --- Introduction of Plasmids into Cells --- p.48 / Chapter 3.8.1. --- Heat shock transformation --- p.48 / Chapter 3.8.1.1. --- Preparation of competent cells (I) --- p.48 / Chapter 3.8.1.2. --- Preparation of competent cells (II) --- p.49 / Chapter 3.8.2. --- High efficiency transformation by electroporation --- p.50 / Chapter 3.8.2.1. --- Preparation of electro- competent cells --- p.50 / Chapter 3.8.2.2. --- Electro-transformation --- p.51 / Chapter 3.9. --- DNA Sequencing by Chain Termination Method --- p.51 / Chapter 3.9.1. --- Preparation of single-stranded M13 templates for sequencing reaction --- p.51 / Chapter 3.9.2. --- Sequencing reactions using single- stranded templates --- p.53 / Chapter 3.9.3. --- Preparation of polyacrylamide gel for sequencing --- p.54 / Chapter 3.9.4. --- Electrophoresis of the DNA samples --- p.55 / Chapter 3.10. --- Construction of Nested Clones by Exonuclease III Unidirectional Deletions --- p.55 / Chapter 3.10.1. --- Unidirectional nested deletion of M13mpl8 clones --- p.55 / Chapter 3.10.2. --- Screening of nested clones by Direct gel electrophoresis --- p.56 / Chapter 3.10.3. --- Screening of nested clones of M13mpl8 and pFZYl by Polymerase Chain Reaction --- p.57 / Chapter 3.11. --- β-galactosidase Assay --- p.59 / Chapter 3.12. --- Primer Extension --- p.60 / Chapter 3.12.1. --- Preparation of total RNA from Gram- negative bacteria --- p.60 / Chapter 3.12.2. --- Labelling the 5' end of the oligonucleotides --- p.61 / Chapter 3.12.3. --- Hybridization and primer extension --- p.62 / Chapter 4. --- Result --- p.63 / Chapter 4.1. --- Subcloning of arcA promoter into M13mpl8/19 --- p.63 / Chapter 4.2. --- Sequencing of p34一18i and p3419i using M13 Sequencing primers (-47) and ArcA-cds Primers --- p.63 / Chapter 4.3. --- Unidirectional Nested Deletion of p3418i using Exonuclease III --- p.65 / Chapter 4.3.1. --- Large scale preparation of p3A18i DNA for Exonuclease III unidirectional nested deletion --- p.65 / Chapter 4.3.2. --- Construction of 3' and 5' overhangs --- p.65 / Chapter 4.3.3. --- Exonuclease III digestion --- p.67 / Chapter 4.3.4. --- Repairing of the 3' and 5' overhangs to generate blunt ends --- p.67 / Chapter 4.3.5. --- Blunt-end ligation of the nested deletion M13mpl8 subclones p3418i --- p.67 / Chapter 4.3.6. --- Transformation --- p.69 / Chapter 4.3.7. --- Screening of nest-deleted p3418i clones by Direct Gel --- p.71 / Chapter 4.3.8. --- Screening of nested deletion p3418i clones by PCR Screening --- p.73 / Chapter 4.3.9. --- Sequencing of the nested deletion p3418i clones --- p.76 / Chapter 4.4. --- Cloning of Nested Deletion DNA Fragments from M13mpl8 into pFZYl --- p.80 / Chapter 4.4.1. --- Screening of pFZYl clones using PCR Screening --- p.80 / Chapter 4.5. --- Expression of Nest-Deleted arcA Promoter Clones in E. coli MC1061-5 --- p.87 / Chapter 4.6. --- Expression of Nest-Deleted arcA Promoter Clones in S. typhimurium JR501 --- p.89 / Chapter 4.7. --- Primer Extension --- p.89 / Chapter 5. --- Discussion --- p.93 / Chapter 5.1. --- Sequencing of arcA Promoter --- p.93 / Chapter 5.2. --- Unidirectional Nested Deletion of p3A18i using Exonuclease III --- p.94 / Chapter 5.3. --- Screening of Nest-deletion p3418i Subclones --- p.95 / Chapter 5.4 --- Cloning of Nest-deleted DNA Fragments from M13mpl8 Subclones into pFZYl --- p.99 / Chapter 5.5. --- Screening of Nest-deleted pFZYl Subclones of p3418i --- p.101 / Chapter 5.6. --- The Effect of 5' Unidirectional Nested Deletion on the Expression of the Cloned arcA promoter in E. coli M1061-5 and S typhimurium JR501 --- p.102 / Chapter 5.7. --- Primer Extension --- p.102 / Chapter 5.8. --- Sequence Analysis of the Cloned arcA Promoter --- p.104 / Chapter 6. --- Conclusion and Further Studies --- p.111 / Chapter 7. --- Reference Cited --- p.113

Briefly, some findings in my research are as follows: (1) The genetic variants of the CA repeats in IGF1 promoter 1 can affect the activity of promoter 1, and the CA repeat showed a suppressive effect on the activity of the promoter 1 of IGF1 gene. EMSA results have shown that the CA repeats could bind to certain nuclear protein. (2) The SNPs T/C (rs5742612) and T/A (rs2288377) can also affect the activity of the promoter 1 in IGF1 gene, and the activity of C-A haplotype is significantly higher than that of T-T haplotype. EMSA results have shown that the SNP T/A (rs2288377) could bind to certain nuclear protein. (3) I developed the new dual reporter assay method to investigate the transactivation interaction between the SNP T/G (rs2071430) and C/A (rs17000900) in the MxA promorer. This new method can not only improve the detection limit for small difference between haplotypes, but also calculate the model of transactivation effect between these two SNPs. The results were better than those of traditional method, and it gave a clear-cut demonstration of the effect of interaction between these two SNPs on the activity of MxA promoter. / In addition, in the IGF1 study, the core promoter region of promoter 2 was identified through 5' deletion mutagenesis methods. Moreover, a cell-type specific mechanism of bidirectional activation of promoter was found. / Recently, more and more studies focus on gene function with the completion of the Human Genome Project. It is well known that polymorphism of human genome sequence is a common phenomenon in the human population. Specially, a lot of genetic polymorphisms, including single nucleotide polymorphisms (SNPs) and microsatellites, have been reported in the regulatory region of many genes. However, the effects of most of these genetic polymorphisms on gene expression are still unknown. The polymorphisms in the promoter can play an important role in the gene regulation. For example, some SNPs located in the transcription factor binding site (TFBS) can affect gene transcription. So, it is very necessary to directly study the effect of genetic variants on promoter transactivation activities. In this study, we studied the effect of genetic polymorphisms on gene expression through reporter gene assay, electrophoretic mobility shift assay (EMSA), and so on. And the candidate genes include insulin-like growth factor 1 (IGF1) and myxovirus resistence 1 (MxA). Some SNPs and microsatellites have been reported in the promoters of these genes. In our previous researches, we focused on the study of the association between these polymorphisms and some diseases, and it was found that a few SNPs significantly associated with relevant diseases. Based on the previous results, in my project, I developed new functional assays and also improved existing methods to analyse the functional effect of these genetic variants of promoters on transactivation activities. / by Huang, Wei. / "March 2008." / Adviser: Nelson Leung Sang Tang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 139-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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

Ho, Sui Fan Tong. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 68-71). / LIST OF TABLES / LIST OF FIGURES / ACKNOWLEDGEMENTS / ABSTRACT / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 2. --- OBJECTIVES --- p.12 / Chapter 3. --- MATERIALS AND METHODS --- p.13 / Chapter 3.1 --- Materials --- p.13 / Chapter 3.1.1 --- Enzymes --- p.13 / Chapter 3.1.2 --- DNA Markers --- p.13 / Chapter 3.1.3 --- General Reagents --- p.13 / Chapter 3.2 --- General Methods --- p.15 / Chapter 3.2.1 --- Phenol and Phenol/Chloroform (1:1) Preparation --- p.15 / Chapter 3.2.2 --- Buffer Preparation --- p.15 / Chapter 3.2.3 --- Agarose Gel Electrophoresis --- p.18 / Chapter 3.2.4 --- Polyacrylamide Gel Electrophoresis --- p.18 / Chapter 3.3 --- DNA Study --- p.19 / Chapter 3.3.1 --- Haemophilia B Patient --- p.19 / Chapter 3.3.2 --- Blood Collection --- p.20 / Chapter 3.3.3 --- DNA Extraction --- p.20 / Chapter 3.3.4 --- DNA Quantitation --- p.21 / Chapter 3.3.5 --- Polymerase Chain Reaction --- p.22 / Chapter 3.3.6 --- Purification of PCR Products --- p.28 / Chapter 3.3.7 --- Sequencing --- p.32 / Chapter 3.3.8 --- Cloning --- p.37 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- DNA Extraction --- p.40 / Chapter 4.2 --- Calibration of the Coy TempCycler --- p.42 / Chapter 4.3 --- Optimization of PCR --- p.44 / Chapter 4.3.1 --- PCR-1 --- p.44 / Chapter 4.3.2 --- PCR-2 --- p.46 / Chapter 4.3.3 --- PCR-3 --- p.46 / Chapter 4.3.4 --- PCR-4 --- p.48 / Chapter 4.3.5 --- PCR-5 --- p.49 / Chapter 4.3.6 --- PCR-6 --- p.50 / Chapter 4.3.7 --- PCR-7 --- p.51 / Chapter 4.4 --- Purification of PCR Product --- p.52 / Chapter 4.4.1 --- GC-1 --- p.52 / Chapter 4.4.2 --- GC-2 --- p.52 / Chapter 4.4.3 --- GC-3 --- p.53 / Chapter 4.4.4 --- PAGE-1 --- p.54 / Chapter 4.4.5 --- PAGE-2 --- p.54 / Chapter 4.4.6 --- Agarose Gel Extraction with Glasswool Exclusion --- p.55 / Chapter 4.5 --- Direct Sequencing of PCR Products --- p.55 / Chapter 4.6 --- Cloning --- p.55 / Chapter 5. --- DISCUSSION --- p.57 / Chapter 5.1 --- DNA Extraction --- p.57 / Chapter 5.2 --- Polymerase Chain Reaction --- p.57 / Chapter 5.3 --- Purification of PCR Products --- p.58 / Chapter 5.4 --- Sequencing --- p.61 / Chapter 5.5 --- Cloning --- p.61 / Chapter 6. --- CONCLUSION --- p.67 / Chapter 7. --- PHOTOGRAPHS --- p.64 / Chapter 8. --- REFERENCES --- p.68

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 84-91).

Thesis (Ph. D)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-137).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 72-93).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [131]-144).