Molecular analysis of arcA promoter of salmonella typhimurium.

by Cheung, Man Wai William.Thesis (M.Phil.)--Chinese University of Hong Kong, 1992.Includes bibliographical references (leaves 113-123).ABSTRACT --- p.iACKNOWLEDGMENTS --- p.iiDEDICATION --- p.iiiTABLE OF CONTENTS --- p.ivLIST OF FIGURES --- p.viiiLIST OF TABLES --- p.xChapter 1. --- Introduction --- p.1Chapter 1.1. --- General Introduction --- p.1Chapter 1.2. --- Purpose of Study --- p.3Chapter 2. --- Literature Review --- p.7Chapter 2.1. --- Central Pathways of Aerobic and Anaerobic Carbon Catabolism --- p.7Chapter 2.2. --- Global Regulation of Gene Expression by Oxygen --- p.10Chapter 2.2.1. --- Two approaches for the studies --- p.10Chapter 2.2.2. --- FNR regulation --- p.12Chapter 2.2.3. --- ArcAB regulation --- p.19Chapter 2.2.3.1. --- arcA --- p.19Chapter 2.2.3.2. --- arcB --- p.20Chapter 2.2.3.3. --- A member of the Two- Components regulatory systems --- p.21Chapter 2.3. --- Molecular Analysis of Promoters --- p.26Chapter 2.3.1. --- S1 mapping --- p.29Chapter 2.3.2. --- Primer extension --- p.29Chapter 2.3.3. --- DNaseI footprinting --- p.30Chapter 2.3.4. --- Mutational analysis of promoters --- p.32Chapter 3. --- Materials and Methods --- p.35Chapter 3.1. --- Bacterial strains and Plasmids --- p.35Chapter 3.2. --- Media --- p.35Chapter 3.3. --- Solutions --- p.38Chapter 3.4. --- Small Scale Preparation of Plasmid DNA --- p.40Chapter 3.5. --- Large Scale Preparation of Plasmid DNA --- p.41Chapter 3.5.1. --- Growth of bacterial culture --- p.41Chapter 3.5.2. --- Lysis by alkali --- p.43Chapter 3.5.3. --- Purification of closed circular DNA by cesium chloride gradient equilibrium centrifugation --- p.44Chapter 3.5.4. --- Digestion of DNA with restriction endonucleases --- p.45Chapter 3.6. --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.45Chapter 3.7. --- Cloning of DNA Fragments from Nest-deleted M13mpl8 Clones to pFZYl --- p.47Chapter 3.8. --- Introduction of Plasmids into Cells --- p.48Chapter 3.8.1. --- Heat shock transformation --- p.48Chapter 3.8.1.1. --- Preparation of competent cells (I) --- p.48Chapter 3.8.1.2. --- Preparation of competent cells (II) --- p.49Chapter 3.8.2. --- High efficiency transformation by electroporation --- p.50Chapter 3.8.2.1. --- Preparation of electro- competent cells --- p.50Chapter 3.8.2.2. --- Electro-transformation --- p.51Chapter 3.9. --- DNA Sequencing by Chain Termination Method --- p.51Chapter 3.9.1. --- Preparation of single-stranded M13 templates for sequencing reaction --- p.51Chapter 3.9.2. --- Sequencing reactions using single- stranded templates --- p.53Chapter 3.9.3. --- Preparation of polyacrylamide gel for sequencing --- p.54Chapter 3.9.4. --- Electrophoresis of the DNA samples --- p.55Chapter 3.10. --- Construction of Nested Clones by Exonuclease III Unidirectional Deletions --- p.55Chapter 3.10.1. --- Unidirectional nested deletion of M13mpl8 clones --- p.55Chapter 3.10.2. --- Screening of nested clones by Direct gel electrophoresis --- p.56Chapter 3.10.3. --- Screening of nested clones of M13mpl8 and pFZYl by Polymerase Chain Reaction --- p.57Chapter 3.11. --- β-galactosidase Assay --- p.59Chapter 3.12. --- Primer Extension --- p.60Chapter 3.12.1. --- Preparation of total RNA from Gram- negative bacteria --- p.60Chapter 3.12.2. --- Labelling the 5' end of the oligonucleotides --- p.61Chapter 3.12.3. --- Hybridization and primer extension --- p.62Chapter 4. --- Result --- p.63Chapter 4.1. --- Subcloning of arcA promoter into M13mpl8/19 --- p.63Chapter 4.2. --- Sequencing of p34一18i and p3419i using M13 Sequencing primers (-47) and ArcA-cds Primers --- p.63Chapter 4.3. --- Unidirectional Nested Deletion of p3418i using Exonuclease III --- p.65Chapter 4.3.1. --- Large scale preparation of p3A18i DNA for Exonuclease III unidirectional nested deletion --- p.65Chapter 4.3.2. --- Construction of 3' and 5' overhangs --- p.65Chapter 4.3.3. --- Exonuclease III digestion --- p.67Chapter 4.3.4. --- Repairing of the 3' and 5' overhangs to generate blunt ends --- p.67Chapter 4.3.5. --- Blunt-end ligation of the nested deletion M13mpl8 subclones p3418i --- p.67Chapter 4.3.6. --- Transformation --- p.69Chapter 4.3.7. --- Screening of nest-deleted p3418i clones by Direct Gel --- p.71Chapter 4.3.8. --- Screening of nested deletion p3418i clones by PCR Screening --- p.73Chapter 4.3.9. --- Sequencing of the nested deletion p3418i clones --- p.76Chapter 4.4. --- Cloning of Nested Deletion DNA Fragments from M13mpl8 into pFZYl --- p.80Chapter 4.4.1. --- Screening of pFZYl clones using PCR Screening --- p.80Chapter 4.5. --- Expression of Nest-Deleted arcA Promoter Clones in E. coli MC1061-5 --- p.87Chapter 4.6. --- Expression of Nest-Deleted arcA Promoter Clones in S. typhimurium JR501 --- p.89Chapter 4.7. --- Primer Extension --- p.89Chapter 5. --- Discussion --- p.93Chapter 5.1. --- Sequencing of arcA Promoter --- p.93Chapter 5.2. --- Unidirectional Nested Deletion of p3A18i using Exonuclease III --- p.94Chapter 5.3. --- Screening of Nest-deletion p3418i Subclones --- p.95Chapter 5.4 --- Cloning of Nest-deleted DNA Fragments from M13mpl8 Subclones into pFZYl --- p.99Chapter 5.5. --- Screening of Nest-deleted pFZYl Subclones of p3418i --- p.101Chapter 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.102Chapter 5.7. --- Primer Extension --- p.102Chapter 5.8. --- Sequence Analysis of the Cloned arcA Promoter --- p.104Chapter 6. --- Conclusion and Further Studies --- p.111Chapter 7. --- Reference Cited --- p.11