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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_320528 |
| Date | January 1995 |
| Contributors | Cheung, Ka-shing., 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, xiii, 126 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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