by Leung Sau-mei. / With: Two articles bound together subsequent to publication. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 101-108. / Abstract --- p.i / Acknowledgements --- p.iv / List of Abbreviations --- p.v / Table of content --- p.vi / Chapter Section 1 --- Introduction / Chapter 1.1 --- The phenomenon of host controlled restriction and modification in bacteria --- p.2 / Chapter 1.2 --- Classification of restriction and modification systems --- p.3 / Chapter 1.3 --- The nomenclature system for restriction and modification systems --- p.8 / Chapter 1.4 --- Variety of type II restriction and modification systems --- p.9 / Chapter 1.5 --- Properties of type II restriction endonucleases --- p.11 / Chapter 1.5.1 --- Biological function --- p.11 / Chapter 1.5.2 --- Protein structure --- p.12 / Chapter 1.5.3 --- Genetics --- p.16 / Chapter 1.5.4 --- Cleavage mechanism --- p.18 / Chapter 1.5.5 --- Factors affecting optimal activity --- p.21 / Chapter 1.6 --- Aim of study --- p.27 / Chapter Section 2 --- Materials and methods / Chapter 2.1 --- Screening for type II restriction endonucleases --- p.28 / Chapter 2.1.1 --- Sources --- p.28 / Chapter 2.1.2 --- Preparation of crude enzyme extract --- p.29 / Chapter 2.1.3 --- Assay of restriction enzyme activity --- p.30 / Chapter 2.1.4 --- Characterization of strains --- p.31 / Chapter 2.2 --- Purification of restriction endonucleases --- p.31 / Chapter 2.2.1 --- Preparation of column materials --- p.32 / Chapter 2.2.2 --- Purification of BcoI --- p.33 / Chapter 2.2.3 --- "Purification of Bcol0278I, BspI, Bsu8646I and PvuHKU" --- p.33 / Chapter 2.2.4 --- Purification of Bsu8565I and Pei9403I --- p.33 / Chapter 2.2.5 --- Purification of Sol3335I --- p.34 / Chapter 2.3 --- Characterization of discovered restriction endonucleases --- p.34 / Chapter 2.3.1 --- Determination of recognition specificity --- p.34 / Chapter 2.3.2 --- Determination of cleavage specificity of BcoI --- p.35 / Chapter 2.3.3 --- Unit definition --- p.36 / Chapter 2.3.4 --- Assays for ionic requirement and optimal temperature --- p.37 / Chapter 2.3.5 --- Heat inactivation --- p.37 / Chapter 2.4 --- Construction of Bacillus coagulans SM1 genomic library --- p.38 / Chapter 2.4.1 --- Preparation of chromosomal DNA --- p.38 / Chapter 2.4.1.1 --- Restriction digestion of chromosomal DNA --- p.40 / Chapter 2.4.2 --- Large scale preparation of vector pBR322 DNA --- p.43 / Chapter 2.4.2.1 --- Restriction digestion of vector DNA --- p.44 / Chapter 2.4.2.2 --- Preparation of lambda insert DNA for control tests --- p.45 / Chapter 2.4.3 --- Ligation of insert and vector DNA --- p.46 / Chapter 2.4.4 --- Transformation --- p.46 / Chapter 2.4.4.1 --- Preparation of electro-competent cells --- p.46 / Chapter 2.4.4.2 --- Electro-transformation --- p.47 / Chapter 2.4.5 --- Rapid screening for the presence of plasmid --- p.49 / Chapter Section 3 --- Results / Chapter 3.1 --- Presence of type II restriction endonucleases --- p.50 / Chapter 3.2 --- Strain identification --- p.50 / Chapter 3.3 --- Purification and properties of the discovered restriction endonucleases --- p.52 / Chapter 3.3.1 --- BcoI --- p.55 / Chapter 3.3.2 --- "Bco10278I, BspI, Bsu8646I and PvuHKUI" --- p.59 / Chapter 3.3.3 --- Bsu8565I and Pei9403I --- p.65 / Chapter 3.3.4 --- Sol3335I --- p.70 / Chapter 3.4 --- Construction of Bacillus coagulans SM1 genomic library --- p.73 / Chapter 3.4.1 --- Preparation of chromosomal DNA --- p.73 / Chapter 3.4.1.1 --- Generation of 4-10 kb insert fragments --- p.73 / Chapter 3.4.2 --- Preparation of plasmid DNA --- p.75 / Chapter 3.4.3 --- Ligation of insert and vector DNA --- p.76 / Chapter 3.4.4 --- Rapid screening for the presence of plasmid --- p.77 / Chapter Section 4 --- Discussion / Chapter 4.1 --- Screening of type II restriction endonucleases --- p.79 / Chapter 4.1.1 --- Methods for screening of type II restriction endonucleases --- p.79 / Chapter 4.1.2 --- Factors affecting the detection of restriction endonucleases --- p.83 / Chapter 4.2 --- Purification of the discovered restriction endonucleases --- p.87 / Chapter 4.3 --- Characterization of discovered restriction endonucleases --- p.91 / Chapter 4.3.1 --- Determination of recognition site --- p.91 / Chapter 4.3.2 --- Determination of cleavage specificity --- p.93 / Chapter 4.4 --- Characteristics of the discovered restriction endonucleases --- p.95 / Chapter 4.5 --- Molecular cloning of BcoI --- p.96 / Chapter 4.6 --- Future prospects --- p.99 / References --- p.101 / Appendix I --- p.109
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318585 |
| Date | January 1990 |
| Contributors | Leung, Sau-mei., 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, viii, 109 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/) |
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