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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_319192 |
| Date | January 1993 |
| Contributors | Tam, Fung-ping., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, vii, 264 leaves : ill. (chiefly mounted) ; 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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