Mok Yu-Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (Leaves 195-201). / Abstract --- p.i / Acknowledgements --- p.iii / List of Abbreviations --- p.iv / Table of contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The need to increase the specificity and variety of restriction endonucleases --- p.1 / Chapter 1.2 --- Classification of methods used for increasing the specificity and variety of restriction endonculeases --- p.2 / Chapter 1.3 --- Isolation and characterization of restriction endonucleases from natural sources --- p.3 / Chapter 1.4 --- Modification of DNA substrate to produce new cleavage specificities --- p.6 / Chapter 1.4.1 --- Methylation of the DNA substrate --- p.6 / Chapter 1.4.1.1 --- Achilles' hell cleavage-The use of canonical methylation to produce novel specificities --- p.10 / Chapter 1.4.1.2 --- Cross protection-The use of non-canonical methylation to generate new cleavage specificity --- p.14 / Chapter 1.4.1.2.1 --- Recognition sequence of a restriction endonuclease and a methylase partially overlap --- p.14 / Chapter 1.4.1.2.2 --- Methylase recognizing a subset of the degenerate sequence of the restriction endonuclease --- p.16 / Chapter 1.4.1.2.3 --- Methylase-limited partial digestion --- p.16 / Chapter 1.4.1.3 --- The use of methylation dependent restriction endonucleases and methylases to generate new specificity --- p.17 / Chapter 1.4.1.4 --- Sequential double-methylation-A two step methylation procedure to generate new specificities --- p.20 / Chapter 1.4.2 --- The generation of a universal restriction endonuclease by combining a Type IIS restriction enzyme moiety and an oligonucleotide adaptor --- p.22 / Chapter 1.4.2.1 --- General principle for generating a universal restriction endonuclease --- p.22 / Chapter 1.4.2.2 --- Factors that affect the cleavage efficiency of universal restriction endonuclease --- p.25 / Chapter 1.4.2.3 --- Modifications and potential applications of the universal restriction endonuclease --- p.29 / Chapter 1.4.3 --- DNA triple helix formation-enhance restriction enzyme specificity by site-specific inhibition of restriction/modification enzymes --- p.32 / Chapter 1.5 --- Modification of the cleaving agent to produce new specificities --- p.36 / Chapter 1.5.1 --- Sequence-specific artificial endonucleases --- p.36 / Chapter 1.5.1.1 --- Oligonucleotides as sequence-specific ligand --- p.37 / Chapter 1.5.1.2 --- Protein or peptide as sequence-specific ligand --- p.40 / Chapter 1.5.1.3 --- General limitations and applications of artificial endonucleases --- p.42 / Chapter 1.5.2 --- Molecular cloning and protein engineering of the restriction-modification system of bacteria --- p.43 / Chapter 1.5.2.1 --- Molecular cloning of the bacterial restriction-modification systems --- p.43 / Chapter 1.5.2.1.1 --- The strategies used to clone and screen restriction-modification systems --- p.45 / Chapter 1.5.2.2 --- Protein engineering of the restriction-modification systems of bacteria --- p.50 / Chapter 1.5.2.2.1 --- Pre-requisites for protein engineering on the restriction-modification systems --- p.51 / Chapter 1.5.2.2.2 --- Effects of protein engineering on the activity and specificity of restriction endonuclease and methylase --- p.53 / Chapter 1.6 --- Variation of restriction endonuclease specificity by altering the reaction condition --- p.56 / Chapter 1.6.1 --- Effects of organic solvents --- p.57 / Chapter 1.6.2 --- Effects of pH and ionic environment on restriction endonuclease specificity --- p.58 / Chapter 1.6.3 --- Remarks on the use of star activity to introduce new specificity --- p.59 / Chapter 1.7 --- Aim of study --- p.59 / Chapter Chapter 2 --- Purification and characterization of thermophilic restriction endonucleases from soil Bacillus spp / Chapter 2.1 --- Materials and methods --- p.61 / Chapter 2.1.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.61 / Chapter 2.1.1.1 --- Preparation of crude enzyme extract --- p.61 / Chapter 2.1.1.2 --- Purification of BsiB I and BsiE 1 --- p.63 / Chapter 2.1.1.3 --- Purification of BsiY I --- p.63 / Chapter 2.1.1.4 --- Preparation of BsiG I and BsiU I --- p.64 / Chapter 2.1.1.5 --- Concentration and storage of the purified restriction endonucleases --- p.64 / Chapter 2.1.1.6 --- Regeneration of the columns --- p.64 / Chapter 2.1.2 --- Characterization of restriction endonucleases --- p.65 / Chapter 2.1.2.1 --- Assay for the working temperature and ionic requirement for the restriction enzymes --- p.65 / Chapter 2.1.2.2 --- Unit determination of the restriction endonucleases --- p.66 / Chapter 2.1.2.3 --- Assay for the purities of restriction endonucleases --- p.66 / Chapter 2.1.2.4 --- Determination of recognition specificity --- p.67 / Chapter 2.1.2.5 --- Determination of the restriction endonuclease's sensitivity to dam and dcm methylation --- p.68 / Chapter 2.1.2.6 --- Determination of the cleavage specificities of restriction endonucleases --- p.70 / Chapter 2.1.2.7 --- Sequencing using Deaza dGTP --- p.73 / Chapter 2.2 --- Results --- p.73 / Chapter 2.2.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.73 / Chapter 2.2.1.1 --- Strain identification --- p.74 / Chapter 2.2.1.2 --- Elution properties of the restriction endonucleases from columns --- p.74 / Chapter 2.2.1.2.1 --- BsiB I --- p.74 / Chapter 2.2.1.2.2 --- BsiE I --- p.77 / Chapter 2.2.1.2.3 --- BsiY 1 --- p.78 / Chapter 2.2.1.3 --- The working digestion temperature and ionic strength requirement --- p.81 / Chapter 2.2.1.4 --- Unit determination --- p.82 / Chapter 2.2.1.5 --- Purities of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6 --- Recognition sites of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6.1 --- BsiB I --- p.83 / Chapter 2.2.1.6.2 --- BsiE I --- p.85 / Chapter 2.2.1.6.3 --- BsiY 1 --- p.87 / Chapter 2.2.1.6.4 --- BsiU I and BsiG I --- p.88 / Chapter 2.2.1.7 --- Sensitivity of restriction endonucleases to dam and dcm methylation --- p.90 / Chapter 2.2.1.8 --- Cleavage specificities of the purified restriction endonucleases --- p.91 / Chapter 2.2.1.8.1 --- BsiB I --- p.91 / Chapter 2.2.1.8.2 --- BsiE I --- p.92 / Chapter 2.2.1.8.3 --- BsiY I --- p.93 / Chapter 2.2.1.9 --- Sequencing of a wrongly sequenced site in pACYC177 using Deaza-dGTP --- p.94 / Chapter Chapter 3 --- The use of Xcm I and BsiY I as an universal restriction endonuclease / Chapter 3.1 --- Materials and methods --- p.98 / Chapter 3.1.1 --- Assay of universal restriction endonuclease using ss DNAs --- p.98 / Chapter 3.1.1.1 --- Annealing reaction between adaptors and ss DNAs --- p.99 / Chapter 3.1.1.2 --- Digestion of the annealed DNA complex --- p.100 / Chapter 3.1.1.3 --- Assay of the digested ss DNA on alkaline denaturing agarose gel --- p.100 / Chapter 3.1.2 --- Assay system involving 5' end-labelled oligonucleotide --- p.101 / Chapter 3.1.2.1 --- Purification of oligonucleotides using preparative polyacrylamide gel electrophoresis --- p.102 / Chapter 3.1.2.2 --- 5'end-labelling of the oligonucleotide DNA substrate --- p.104 / Chapter 3.1.2.3 --- The annealing between adaptors and oligonucleotide DNA substrate and the digestion condition --- p.104 / Chapter 3.1.2.4 --- Assay of the labelled oligonucleotides in polyacrylamide gel after digestion --- p.105 / Chapter 3.2 --- Results --- p.106 / Chapter 3.2.1 --- Xcm I adaptors #2 and #4 --- p.106 / Chapter 3.2.1.1 --- Assay conditions used for the universal restriction endonucleases --- p.107 / Chapter 3.2.1.1.1 --- Conditions used for hybridization --- p.107 / Chapter 3.2.1.1.2 --- Conditions used for digestion --- p.108 / Chapter 3.2.1.2 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #4 --- p.110 / Chapter 3.2.1.2.1 --- Methods used to optimize the hybridization process --- p.110 / Chapter 3.2.1.2.2 --- Methods used to relax the secondary DNA structures --- p.112 / Chapter 3.2.1.2.2.1 --- Linearization of M13mp7 with BamH I befor annealing the adaptor --- p.113 / Chapter 3.2.1.2.2.2 --- Relaxation of secondary structure using boiling and NaOH denaturation --- p.114 / Chapter 3.2.1.2.3 --- Methods used to optimize the digestion process --- p.115 / Chapter 3.2.1.2.3.1 --- Addition of BSA --- p.115 / Chapter 3.2.1.2.3.2 --- Addition of the restriction endonuclease in separate batches --- p.115 / Chapter 3.2.1.3 --- Digestion of ss M13mpl8 and ssM13mpl9 DNA using Xcm I adaptor #2 and adaptor #4 --- p.116 / Chapter 3.2.2 --- Xcm I adaptor #1 and #3 --- p.118 / Chapter 3.2.2.1 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #1 and adaptor #3 --- p.119 / Chapter 3.2.2.1.1 --- Methods used to relax the secondary structure --- p.119 / Chapter 3.2.2.1.1.1 --- Linearization of M13mp7 with BamH I before the annealing reaction --- p.120 / Chapter 3.2.2.1.1.2 --- Relaxation of secondary structure by NaOH denaturation --- p.121 / Chapter 3.2.2.1.1.3 --- Relaxation of secondary structure by adding DMSO and urea --- p.122 / Chapter 3.2.2.1.2 --- Methods used to optimize the digestion and hybridization processes --- p.123 / Chapter 3.2.2.1.2.1 --- Annealing of M13mp7 with a different amount of adaptor #3 and digesting the DNA complex with Xcm I at different temperatures --- p.123 / Chapter 3.2.2.1.2.2 --- Optimization of digestion by adding Xcm I in separate batches --- p.124 / Chapter 3.2.3 --- BsiY I adaptor --- p.124 / Chapter 3.2.3.1 --- Methods used to optimize the cleavage of M13mp7-BsiY I adaptor complex with BsiY I --- p.126 / Chapter 3.2.3.1.1 --- Optimization of hybridization using various concentrations of NaCl during the annealing reaction --- p.126 / Chapter 3.2.3.1.2 --- Optimization of digestion by binding BsiY I to the BsiY I adaptor before annealing --- p.127 / Chapter 3.2.4 --- The use of 5' end-labelled oligonucleotide DNA substrates for digestion with universal restriction endonuclease --- p.128 / Chapter Chapter 4 --- Molecular cloning of the BsiY I restriction-modification system / Chapter 4.1 --- Materials and methods --- p.132 / Chapter 4.1.1 --- Preparation of chromosomal DNA from BsiY I producing Bacillus stearothermophilus --- p.132 / Chapter 4.1.1.1 --- Restriction digestion of the chromosomal DNA --- p.134 / Chapter 4.1.1.2 --- Southern hybridization to locate the position of the DNA fragment coding for the restriction-modification system --- p.135 / Chapter 4.1.1.2.1 --- Southern transfer of DNA fragments onto nitro-cellulose paper --- p.135 / Chapter 4.1.1.2.2 --- Labelling of the probes by Nick-translation --- p.136 / Chapter 4.1.1.2.3 --- Hybridization of the nick-translated probes onto the DNA fragments fixed on NC paper --- p.137 / Chapter 4.1.2 --- Large-scale preparation of the cloning vector --- p.137 / Chapter 4.1.2.1 --- Restriction endonuclease digestion and dephosphorylation of the vector ´Ø.… --- p.139 / Chapter 4.1.3 --- Ligation between vector and DNA inserts --- p.139 / Chapter 4.1.4 --- Transformation of the ligated DNA into competent cells --- p.140 / Chapter 4.1.4.1 --- Preparation of competent cells --- p.140 / Chapter 4.1.4.2 --- Transformation of the ligated vector and insert DNA into competent cells --- p.142 / Chapter 4.1.5 --- Rapid alkaline lysis method for screening transformants that contains an insert --- p.143 / Chapter 4.1.6 --- Preparation of the genomic library and its plasmid DNA --- p.144 / Chapter 4.1.7 --- Screening procedures used to clone the BsiY I restriction-modification system --- p.144 / Chapter 4.1.7.1 --- In vitro selection using Hungarian Trick --- p.145 / Chapter 4.1.7.2 --- In vivo selection using the host strain AP1-200 and AP1-200-9 --- p.145 / Chapter 4.1.7.2.1 --- Preparation of competent AP1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.7.2.2 --- Transformation of the genomic library plasmid into competent AP 1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.8 --- Assay of BsiY I restriction endonuclease and methylase activities in the suspecting clones --- p.147 / Chapter 4.1.8.1 --- Assay to BsiY I methylase activity - resistance of the plasmid to BsiY I digestion --- p.147 / Chapter 4.1.8.2 --- Assay of BsiY I methylase activity - ability to incorporate H3-methyl group from H3-SAM into DNA substrate molecules --- p.148 / Chapter 4.1.8.3 --- Assay of BsiY I restriction endonuclease activity - ability of crude enzyme extract to cleave DNA --- p.149 / Chapter 4.2 --- Results --- p.150 / Chapter 4.2.1 --- Construction of the BamH I genomic library --- p.150 / Chapter 4.2.1.1 --- Vector and insert used --- p.150 / Chapter 4.2.1.2 --- Optimization of the ligation and transformation process --- p.151 / Chapter 4.2.1.3 --- Preparation of the BamH I library --- p.153 / Chapter 4.2.1.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.155 / Chapter 4.2.1.4.1 --- The Hungarian Trick --- p.155 / Chapter 4.2.1.4.2 --- Screening of the restriction-modification system using the strains API-200 and AP1-200-9 --- p.159 / Chapter 4.2.2 --- Construction of the Hind III library --- p.161 / Chapter 4.2.2.1 --- Vector and insert used --- p.161 / Chapter 4.2.2.2 --- Optimization of the ligation and transformation process --- p.162 / Chapter 4.2.2.3 --- Preparation of the Hind III library --- p.164 / Chapter 4.2.2.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.165 / Chapter 4.2.2.4.1 --- The Hungarian Trick --- p.165 / Chapter 4.2.2.4.2 --- Screening of the restriction-modification system using the strain AP1-200 and AP1-200-9 --- p.168 / Chapter 4.2.2.5 --- Assay of methylase activity using H3-SAM --- p.170 / Chapter 4.2.3 --- The use of Southern blotting and hybridization to find if two available probes have homology to the BsiY I restriction-modification system --- p.173 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Purification and characterization of restriction endonucleases from Bacillus spp --- p.176 / Chapter 5.1.1 --- Methods used to purify the restriction endonuclease --- p.177 / Chapter 5.1.2 --- Characterization of the restriction endonucleases --- p.179 / Chapter 5.1.2.1 --- Determination of the purities of the purified restriction endonucleases --- p.179 / Chapter 5.1.2.2 --- Determination of the recognition site --- p.179 / Chapter 5.1.2.3 --- Determination of the cleavage site --- p.180 / Chapter 5.1.2.4 --- Sequencing using Deaza-dGTP --- p.181 / Chapter 5.2 --- The use of Xcm I and BsiY I as universal restriction endonucleases --- p.182 / Chapter 5.2.1 --- The adverse effects of hair-pin loop on the cleavage with universal restriction enzymes --- p.183 / Chapter 5.3 --- Molecular cloning of the BsiY I restriction-modification system --- p.187 / Chapter 5.3.1 --- Construction of the genomic library --- p.187 / Chapter 5.3.1.1 --- Preparation of the insert and vector --- p.188 / Chapter 5.3.1.2 --- Optimization of the ligation and transformation processes --- p.188 / Chapter 5.3.2 --- Screening strategies used to clone the BsiY I restriction-modification system --- p.189 / Chapter 5.3.2.1 --- The Hungarian Trick --- p.189 / Chapter 5.3.2.2 --- Screening using the strains AP1-200 and AP1-200-9 cells --- p.191 / Chapter 5.3.3 --- Assay of the gene products from the cloned restriction-modification system --- p.192 / Chapter 5.3.3.1 --- Methylase activity --- p.192 / Chapter 5.3.3.2 --- Restriction endonuclease activity --- p.193 / Chapter 5.4 --- Future prospects --- p.193 / References --- p.195 / Appendix --- p.201
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318939 |
| Date | January 1992 |
| Contributors | Mok, Yu-Keung., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiii, 201 leaves : ill. ; 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/) |
Page generated in 0.0034 seconds