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by Ka-shing Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 112-114). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.v / Abbreviations --- p.x / List of figures --- p.xi / List of tables --- p.xiii / Chapter 1. --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Purpose of study --- p.3 / Chapter 2. --- Literature review / Chapter 2.1 --- Cellulose: properties and degradation --- p.4 / Chapter 2.2 --- Cellulase system / Chapter 2.2.1 --- Definition and substrate specificity --- p.5 / Chapter 2.2.2 --- Co-operation of cellulases --- p.5 / Chapter 2.2.3 --- Multiplicity of cellulases --- p.6 / Chapter 2.2.4 --- Regulation of cellulase synthesis --- p.6 / Chapter 2.2.5 --- Architecture of cellulase protein --- p.8 / Chapter 2.3 --- Molecular biology of fungal cellulase genes / Chapter 2.3.1 --- Structural organization of fungal cellulase genes --- p.15 / Chapter 2.3.1.1 --- Promoter and regulatory sequence --- p.15 / Chapter 2.3.1.2 --- Sequence at transcriptional start point (tsp) --- p.16 / Chapter 2.3.1.3 --- Signal peptide --- p.18 / Chapter 2.3.1.4 --- Intron --- p.18 / Chapter 2.3.1.5 --- General sequence homology --- p.21 / Chapter 2.3.2 --- Regulation of cellulase production at molecular level --- p.23 / Chapter 2.3.3 --- Multiplicity of cellulase gene --- p.24 / Chapter 2.3.4 --- Tactics to clone fungal cellulase genes --- p.25 / Chapter 2.3.4.1 --- Past experience --- p.25 / Chapter 2.3.4.2 --- Present approach --- p.28 / Chapter 2.3.5 --- The importance of cellulase gene cloning --- p.29 / Chapter 2.4 --- Cellulolytic microorganisms / Chapter 2.4.1 --- Ecological roles and diversity --- p.31 / Chapter 2.4.2 --- "Biology of the straw mushroom, Volvariella volvacea" --- p.31 / Chapter 3. --- Materials and methods / Chapter 3.1 --- Recipes of media and solutions / Chapter 3.1.1 --- Culture media and microbial-growth related chemicals --- p.34 / Chapter 3.1.2 --- Solutions --- p.36 / Chapter 3.2 --- Bacterial and fungal strains and the growth and storage of mycelium / Chapter 3.2.1 --- Bacterial and fungal strains --- p.42 / Chapter 3.2.2 --- Growth and storage of mycelium --- p.42 / Chapter 3.3 --- Extraction of DNA from mycelium --- p.43 / Chapter 3.4 --- Degenerate polymerase chain reaction (PCR) / Chapter 3.4.1 --- Primers --- p.45 / Chapter 3.4.2 --- Amplification conditions of degenerate PCR --- p.46 / Chapter 3.5 --- Cloning of PCR products / Chapter 3.5.1 --- Ligation --- p.47 / Chapter 3.5.2 --- Transformation --- p.47 / Chapter 3.5.3 --- Screening by blue/white selection --- p.47 / Chapter 3.5.4 --- Screening by PCR --- p.48 / Chapter 3.6 --- Plasmid extraction by alkaline lysis / Chapter 3.6.1 --- Midi-preparation of plasmid by Qiagen column --- p.51 / Chapter 3.6.2 --- Preparation of plasmid using Promega's Wizard minipreps DNA purification system --- p.51 / Chapter 3.7 --- Sequencing analysis of cloned PCR products / Chapter 3.7.1 --- Growth and titering of helper phage R408 --- p.53 / Chapter 3.7.1.1 --- Plate elution method --- p.53 / Chapter 3.7.1.2 --- Liquid culture method --- p.53 / Chapter 3.7.1.3 --- Titering of R408 --- p.53 / Chapter 3.7.2 --- Rescue of single-stranded DNA from pCR-Script phagemid --- p.54 / Chapter 3.7.3 --- Sequencing by chain-termination reaction --- p.54 / Chapter 3.7.4 --- Preparation of polyacrylamide gel for DNA sequencing --- p.56 / Chapter 3.7.5 --- Running a sequencing gel --- p.57 / Chapter 3.7.6 --- "Fixation, exposure and development of sequencing gel and X-ray film" --- p.57 / Chapter 3.7.7 --- Sequence analysis --- p.58 / Chapter 3.8 --- Digestion of DNA with restriction enzymes --- p.59 / Chapter 3.9 --- Agarose gel electrophoresis --- p.60 / Chapter 3.10 --- Purification of DNA from agarose gel by Qiaex --- p.61 / Chapter 3.11 --- Southern hybridization / Chapter 3.11.1 --- Southern blotting and DNA immobilization --- p.62 / Chapter 3.11.2 --- Random-labelling of DNA probe and removal of unincorporated nucleotides --- p.63 / Chapter 3.11.3 --- Pre-hybridization and hybridization --- p.63 / Chapter 3.11.4 --- Exposure and development --- p.64 / Chapter 3.11.5 --- Determination of molecular weight of hybridization signals --- p.65 / Chapter 4. --- Results / Chapter 4.1 --- Extraction of DNA from the straw mushroom mycelium --- p.66 / Chapter 4.2 --- Amplification of V. volvacea genomic DNA using degenerate primers --- p.70 / Chapter 4.3 --- Cloning of PCR products using pCR-Script SK (+) cloning kit / Chapter 4.3.1 --- Screening by blue/white selection --- p.77 / Chapter 4.3.2 --- Screening by PCR --- p.77 / Chapter 4.4 --- Plasmid extraction by alkaline lysis --- p.80 / Chapter 4.5 --- Preparation of single-stranded DNA template for sequencing / Chapter 4.5.1 --- Growth and titering of helper phage R408 --- p.82 / Chapter 4.5.2 --- Rescue of single-stranded DNA from pCR-Script phagemid --- p.82 / Chapter 4.6 --- Sequencing of cloned PCR products / Chapter 4.6.1 --- The choice of template --- p.84 / Chapter 4.6.2 --- DNA and translated amino acid sequence of PCR clones --- p.84 / Chapter 4.6.3 --- Alignment of DNA sequences against other fungal cellulase genes --- p.93 / Chapter 4.6.4 --- Alignment of translated amino acid sequences against other fungal cellulase --- p.96 / Chapter 4.7 --- Purification of DNA from agarose gel by Qiaex --- p.98 / Chapter 4.8 --- Southern hybridization / Chapter 4.8.1 --- Restriction digestion of genomic DNA --- p.101 / Chapter 4.8.2 --- Hybridization --- p.104 / Chapter 5. --- Discussion / Chapter 5.1 --- Extraction of DNA from V. volvacea mycelium --- p.107 / Chapter 5.2 --- Rationales of designing degenerate primers from heterologous amino acid sequence --- p.107 / Chapter 5.3 --- Amplification of V. volvacea DNA using degenerate primers --- p.110 / Chapter 5.4 --- Cloning of PCR products using pCR-Script system --- p.111 / Chapter 5.5 --- The precaution of using Qiaex-purified DNA --- p.112 / Chapter 5.6 --- Sequencing analysis / Chapter 5.6.1 --- DNA sequence analysis --- p.113 / Chapter 5.6.2 --- Protein sequence analysis --- p.114 / Chapter 5.7 --- Southern hybridization --- p.116 / Chapter 6. --- Conclusion and further analysis --- p.117 / Chapter 7. --- References --- p.119

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

by Wong Mee-wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 100-104. / Contents / Abstract / Abbreviations / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Biological Functions and Structure of GH --- p.1 / Chapter 1.3 --- Molecular Cloning of GH cDNA and its Expression --- p.3 / Chapter 1.4 --- Molecular Cloning of the Genomic Sequence of Growth Hormone --- p.5 / Chapter 1.4.1 --- The Isolation of Mammalian GH gene --- p.5 / Chapter 1.4.2 --- Molecular Cloning of Rainbow Trout Growth Hormone Gene --- p.6 / Chapter 1.4.3 --- Molecular Cloning of Atlantic Salmon Growth Hormone Gene --- p.9 / Chapter 1.4.4 --- Molecular Evolution of GH gene --- p.9 / Chapter 1.4.5 --- Control Elements on GH gene --- p.12 / Chapter 1.4.6 --- Production of Transgenic Fish --- p.14 / Chapter 1.5 --- Purpose of present Study --- p.15 / Chapter Chapter 2 --- Construction of a Genomic Library --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Strategy --- p.19 / Chapter 2.3 --- Materials and Methods --- p.20 / Chapter 2.3.1 --- Materials --- p.20 / Chapter 2.3.2 --- Procedure --- p.24 / Chapter 2.3.2.1 --- Extraction of Total Genomic DMA --- p.24 / Chapter 2.3.2.2 --- Southern Blotting and Hybridization --- p.25 / Chapter 2.3.2.3 --- Extraction of the GH Enriched DNA Fraction --- p.28 / Chapter 2.3.2.4 --- Cloning into Phage Vector Lambda GT11 --- p.29 / Chapter 2.3.2.5 --- Studies on the Enriched Genomic Library --- p.31 / Chapter 2.4 --- Result --- p.33 / Chapter 2.5 --- Discussion --- p.43 / Chapter Chapter 3 --- Screening --- p.46 / Chapter 3.1 --- Strategy --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Materials --- p.47 / Chapter 3.2.2 --- Procedure --- p.48 / Chapter 3.2.2.1 --- Primary Screening --- p.48 / Chapter 3.2.2.2 --- Purificaiton of Positive Signal --- p.49 / Chapter 3.2.2.3 --- DNA Extraction from Positive Clone --- p.49 / Chapter 3.3 --- Result --- p.50 / Chapter 3.4 --- Discussion --- p.58 / Chapter Chapter 4 --- Studies on the Positive Clone --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.2 --- Materials and Methods --- p.60 / Chapter 4.2.1 --- Materials --- p.60 / Chapter 4.2.2 --- Procedure --- p.62 / Chapter 4.2.2.1 --- Insert DNA Preparation --- p.62 / Chapter 4.2.2.2 --- Single Enzyme Digestion --- p.62 / Chapter 4.2.2.3 --- Double Enzyme Digestion --- p.62 / Chapter 4.2.2.4 --- Preparation of Position Specific Probes --- p.62 / Chapter 4.2.2.5 --- Hybridization with Position Specific Probes --- p.64 / Chapter 4.2.2.6 --- Preparation of Competent Cells --- p.65 / Chapter 4.2.2.7 --- Subcloning into pUC18 --- p.65 / Chapter 4.2.2.8 --- Plasmid Preparation --- p.66 / Chapter 4.2.2.9 --- Sequencing --- p.67 / Chapter 4.3 --- Result --- p.69 / Chapter 4.4 --- Discussion --- p.78 / Chapter Chapter 5 --- General Discussion --- p.80 / Appendix A --- p.83 / Appendix B --- p.93 / References --- p.100

Chan chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 89-95). / Abstracts in English and Chinese. / Dedication --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Abbreviations --- p.vi / List of Figures --- p.ix / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1. --- Thesis synopsis --- p.1 / Chapter 1.2. --- hNP220 protein --- p.1 / Chapter 1.2.1. --- Domain organization --- p.1 / Chapter 1.2.2. --- Known splice variants --- p.5 / Chapter 1.2.3. --- Subcellular localization --- p.7 / Chapter 1.2.4. --- Proposed roles in transcriptional activation and RNA processing --- p.7 / Chapter 1.2.5. --- Interaction between C-terminal of hNP220 and FHL2 --- p.9 / Chapter 1.3. --- Hypothesis --- p.12 / Chapter 1.4. --- Principles of key methods --- p.14 / Chapter 1.4.1. --- RLM-RACE --- p.14 / Chapter 1.4.2. --- CytoTrap® two-hybrid system --- p.15 / Chapter CHAPTER 2 --- Materials and Methods --- p.18 / Chapter 2.1. --- Cloning protocol --- p.18 / Chapter 2.1.1. --- Amplification of DNA fragment --- p.18 / Chapter 2.1.2. --- Purification of PCR product --- p.19 / Chapter 2.1.3. --- Restriction endonuclease digestion --- p.20 / Chapter 2.1.4. --- Dephosphorylation of cloning vector 5'-termini --- p.20 / Chapter 2.1.5. --- Insert/vector ligation --- p.20 / Chapter 2.1.6. --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.21 / Chapter 2.1.7. --- Transformation of ligation product into chemically competent bacterial cells --- p.22 / Chapter 2.1.8. --- Small-scale preparation of bacterial plasmid DNA --- p.22 / Chapter 2.1.9. --- Screening for recombinant clone --- p.24 / Chapter 2.1.10. --- Dideoxy DNA sequencing --- p.24 / Chapter 2.1.11. --- Midi-scale preparation of recombinant plasmid DNA --- p.25 / Chapter 2.2. --- Determination of the transcription start site (TSS) of hNP220 gene --- p.27 / Chapter 2.2.1. --- RNA ligase-mediated rapid amplification of cDNA 5'-end (5-RLM-RACE) --- p.27 / Chapter 2.3. --- Isolation and identification of the third splice variant of HNP220 (hNP220ε) --- p.29 / Chapter 2.3.1. --- PCR from human heart/testis cDNAs pool --- p.29 / Chapter 2.3.2. --- RT-PCR --- p.29 / Chapter 2.3.3. --- Northern hybridization --- p.30 / Chapter 2.4. --- Human tissue distribution of hNP220 --- p.31 / Chapter 2.4.1. --- RT-PCR --- p.31 / Chapter 2.4.2. --- Northern hybridization --- p.31 / Chapter 2.5. --- Visualization of the subcellular localization patterns of GFP-tagged hNP220ε in HepG2 cell line --- p.32 / Chapter 2.5.1. --- Cloning of hNP220a and hNP220s into vector pEGFP-Cl --- p.32 / Chapter 2.5.2. --- Transfection of GFP fusion constructs into HepG2 cell line --- p.32 / Chapter 2.5.3. --- Epi-fluorescence microscopy --- p.33 / Chapter 2.6. --- Identification of the protein-protein interaction between hNP220ε and MAPRE1 --- p.34 / Chapter 2.6.1. --- CytoTrap® XR HeLa Cell cDNA Library screening --- p.34 / Chapter 2.6.1.1. --- Cloning of hNP220ε into yeast two-hybrid bait vector pSos --- p.34 / Chapter 2.6.1.2. --- Preparation of cdc25Ha & cdc25Hα yeast competent cells --- p.34 / Chapter 2.6.1.3. --- Autonomous activation study of bait fusion construct pSos-hNP220ε --- p.36 / Chapter 2.6.1.4. --- Cotransformation of pSos-hNP220ε and CytoTrap® XR HeLa Cell cDNA Library --- p.36 / Chapter 2.6.1.5. --- Verification of interaction by yeast mating --- p.38 / Chapter 2.6.1.5.1. --- Generation of yeast plasmid segregant for mating --- p.38 / Chapter 2.6.1.5.2. --- Yeast mating in 96-well plate --- p.39 / Chapter 2.6.1.6. --- Identification of putative interaction partner --- p.39 / Chapter CHAPTER 3 --- Results --- p.42 / Chapter 3.1. --- Transcription start site of the HNP220 gene is located 312 nucleotides upstream the initiation codon --- p.42 / Chapter 3.2. --- Third splice variant of hNP220 gene hNP220s) is identified --- p.44 / Chapter 3.3. --- In silico analysis of hNP220ε --- p.54 / Chapter 3.4. --- hNP220a and hNP220s are ubiquitously expressed in human fetal and adult tissues --- p.65 / Chapter 3.5. --- hNP220ε shows a punctate subnuclear localization pattern in HepG2 cell line --- p.67 / Chapter 3.6. --- hNP220ε interacts with MAPRE1 --- p.69 / Chapter CHAPTER 4 --- Discussion --- p.71 / Chapter 4.1. --- "Identification of hNP220s, the third splice variant of hNP220 gene" --- p.71 / Chapter 4.2. --- Biological resemblance between hNP220α (hNP220) and hNP220ε --- p.73 / Chapter 4.3. --- Protein-protein interaction between hNP220ε and MAPRE1 --- p.74 / Chapter 4.3.1. --- MAPRE1 protein --- p.77 / Chapter 4.3.2. --- Wnt signaling pathway --- p.78 / Chapter 4.4. --- Potential roles of hNP220 in the regulation of chromosome stability and oncogenesis --- p.82 / Chapter 4.5. --- Summary --- p.85 / Chapter 4.6. --- Concluding questions --- p.86 / Chapter 4.7. --- Future work --- p.87 / References --- p.89 / Appendix --- p.96

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 92-101).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (M. Sc.)--York University, 2002. Graduate Programme in Biology. / Title on thesis acceptance page : Molecular cloning of an [alpha]-tubulin from the Cestode, Hymenolepis, diminuta. Includes bibliographical references (leaves 110-125). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ71611.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy