by Yung Mei Hing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 130-135. / ACKNOWLEDEMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iii / ABBREVIATIONS --- p.viii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Chemistry and Structure of Trichosanthin --- p.1 / Chapter 1.1.1 --- Chemistry --- p.1 / Chapter 1.1.2 --- Primary Structure --- p.2 / Chapter 1.1.3 --- Three-dimensional Structure --- p.4 / Chapter 1.2 --- Biological Activities of Trichosanthin --- p.6 / Chapter 1.2.1 --- Abortifacient Properties --- p.6 / Chapter 1.2.1A --- Termination of Mid-term Gestation --- p.6 / Chapter 1.2.1B --- Inhibition of Early Pregnancy --- p.12 / Chapter 1.2.2 --- Immunological Properties --- p.13 / Chapter 1.2.2A --- Antigenicity and Allergenicity --- p.13 / Chapter 1.2.2B --- Immunosuppressive Effects --- p.14 / Chapter 1.2.3 --- Anti-tumour Activity --- p.15 / Chapter 1.2.4 --- Ribosome-inactivating Activity --- p.16 / Chapter 1.2.5 --- Human Immunodeficiency Virus (HIV) Inhibitory Activity --- p.20 / Chapter 1.3 --- Objectives and Strategy of Cloning the cDNA of Trichosanthin --- p.21 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- General Techniques --- p.24 / Chapter 2.1.1 --- Extraction with Phenol --- p.24 / Chapter 2.1.2 --- Ethanol Precipitation --- p.24 / Chapter 2.1.3 --- Spectrophotometry --- p.25 / Chapter 2.1.4 --- Restriction Digestion of DNA --- p.25 / Chapter 2.1.5 --- End-labelling DNA with Recessed 3'-ends --- p.25 / Chapter 2.1.6 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.1.7 --- Elution of DNA from Agarose Gel --- p.26 / Chapter 2.1.8 --- Minipreparation of Bacteriophage A DNA from Plate Lysates --- p.27 / Chapter 2.1.9 --- Minipreparation of Plasmid DNA --- p.28 / Chapter 2.1.10 --- Large-scale Preparation of Plasmid DNA --- p.29 / Chapter 2.1.10A --- By Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient --- p.29 / Chapter 2.1.10B --- By Using QIAGEN-pack 100 Cartridge --- p.31 / Chapter 2.1.11 --- Preparation and Transformation of Competent Escherichia coli --- p.32 / Chapter 2.1.12 --- Preparation of Nucleic Acid Probes --- p.34 / Chapter 2.1.12A --- By Nick Translation --- p.34 / Chapter 2.1.12B --- By Random-primed Labelling --- p.34 / Chapter 2.1.13 --- Sephadex G-50 Spun-column Chromatography --- p.35 / Chapter 2.1.14 --- Monitoring the Progress of Probe Labelling Reactions --- p.37 / Chapter 2.1.15 --- Liquid Scintillation Counting --- p.37 / Chapter 2.1.16 --- Southern and Northern Hybridizations --- p.38 / Chapter 2.1.17 --- Autoradiography --- p.41 / Chapter 2.2 --- Techniques for Constructing a Complementary DNA (cDNA) Library --- p.42 / Chapter 2.2.1 --- Extraction and Purification of Plant RNA --- p.42 / Chapter 2.2.2 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.44 / Chapter 2.2.3 --- Ohgo(dT)-cellulose Column Chromatography --- p.46 / Chapter 2.2.4 --- In Vitro Translation in Rabbit Reticulocyte Lysate --- p.47 / Chapter 2.2.5 --- SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) of In Vitro Translation Products --- p.48 / Chapter 2.2.6 --- cDNA Synthesis --- p.50 / Chapter 2.2.6A --- First Strand Synthesis --- p.50 / Chapter 2.2.6B --- Second Strand Synthesis --- p.51 / Chapter 2.2.6C --- Purification of the Double-stranded cDNA (ds cDNA) --- p.51 / Chapter 2.2.7 --- Methylation of the cDNA with EcoRI Methylase --- p.52 / Chapter 2.2.8 --- Addition of EcoRl Cohesive Termini onto the cDNA --- p.52 / Chapter 2.2.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA --- p.53 / Chapter 2.2.10 --- Ligation of the cDNA with EcoRI-digested λgt10 --- p.55 / Chapter 2.2.11 --- In Vitro Packaging --- p.56 / Chapter 2.2.12 --- Titration of the λgt10 Library V --- p.56 / Chapter 2.3 --- Screening the cDNA Library in λgt10 with a Nucleic Acid Probe --- p.57 / Chapter 2.4 --- Subcloning DNA Fragments in pUC18 --- p.59 / Chapter 2.4.1 --- Dephosphorylation of Linearized pUC18 with Protruding 5' Termini --- p.59 / Chapter 2.4.2 --- Ligation of Foreign DNA with Linearized pUC18 with Cohesive Termini --- p.60 / Chapter 2.5 --- DNA Sequencing on Double-stranded Templates --- p.60 / Chapter 2.5.1 --- DNA Sequencing Reaction --- p.61 / Chapter 2.5.1A --- Alkaline Denaturation of Double-stranded Plasmid Template --- p.61 / Chapter 2.5.1B --- Labelling Reaction and Termination Reactions --- p.61 / Chapter 2.5.2 --- DNA Sequencing Electrophoresis --- p.62 / Chapter CHAPTER THREE --- CONSTRUCTION AND CLONAL SCREENING OF cDNA LIBRARY FROM ROOT TUBERS OF TRICHOSANTHES KIRILOWII MAXIMOWICZ --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Isolation of Total RNA from Root Tubers --- p.69 / Chapter 3.3 --- Purification of Poly(A)+ RNA from Total RNA --- p.70 / Chapter 3.4 --- Northern Blot Analysis of the Poly(A)+ RNA --- p.72 / Chapter 3.5 --- In Vitro Translation of the Poly(A)+ RNA --- p.75 / Chapter 3.6 --- cDNA Synthesis --- p.78 / Chapter 3.7 --- Southern Blot Analysis of the cDNA --- p.81 / Chapter 3.8 --- Addition of EcoRI Cohesive Termini to the cDNA --- p.81 / Chapter 3.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA vi --- p.84 / Chapter 3.10 --- Ligation of the Size Fractionated cDNA with EcoRI-digested λgt10 and In Vito Packaging of the A Hybrids --- p.85 / Chapter 3.11 --- Analysis of cDNA Inserts --- p.85 / Chapter 3.12 --- Clonal Screening of the cDNA Library --- p.88 / Chapter 3.13 --- Characterization of Positive Clones --- p.88 / Chapter 3.14 --- Subcloning Positive Inserts into pUC18 --- p.92 / Chapter 3.15 --- Discussion --- p.93 / Chapter CHAPTER FOUR --- DNA SEQUENCING OF POSITIVE CLONES --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Sequencing Strategies --- p.104 / Chapter 4.2.1 --- pTCS48210 Sequencing --- p.m / Chapter 4.2.2 --- pTCS5021 Sequencing --- p.107 / Chapter 4.3 --- Results and Discussion --- p.108 / Chapter 4.3.1 --- Nucleotide Sequence and Deduced Amino Acid Sequence --- p.108 / Chapter 4.3.2 --- Secondary Structure Predicted from the Amino Acid Sequence Encoded by the Trichosanthin cDNA --- p.116 / APPENDIX A --- p.124 / APPENDIX B --- p.126 / APPENDIX C --- p.129 / REFERENCES --- p.130
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318561 |
| Date | January 1990 |
| Contributors | Yung, Mei Hing., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Department of Biochemistry, Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, ix, 135 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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