Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Dr. John Cairney. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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by Wai-Man Tong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 155-174). / Abstracts in English and Chinese. / ABSTRACT --- p.I-IV / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- General / Chapter 1.1.1. --- Ribosome Inactivating Protein --- p.1 / Chapter 1.1.1.1. --- Ricin --- p.2 / Chapter 1.1.1.2. --- Trichosanthin --- p.5 / Chapter 1.1.2. --- In Vitro Study of RIP --- p.6 / Chapter 1.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 1.2.1. --- Suicide Transport --- p.7 / Chapter 1.2.1.1 . --- Endocytic Uptake of Ricin --- p.8 / Chapter 1.2.1.2. --- Endocytic Uptake of Trichosanthin --- p.11 / Chapter 1.2.2. --- Pervious Studies in This Laboratory --- p.11 / Chapter 1.3. --- Apoptosis And Ribosome Inactivation / Chapter 1.3.1. --- Apoptosis / Chapter 1.3.1.1. --- Morphological Feature of Apoptosis --- p.14 / Chapter 1.3.1.2. --- Molecular Changes of Apoptosis --- p.15 / Chapter 1.3.2. --- Toxicity of Ribosome Inactivating Protein / Chapter 1.3.2.1. --- Toxicity of Ricin --- p.20 / Chapter 1.3.2.2. --- Toxicity of Trichosanthin --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- GENERAL / Chapter 2.1.1. --- Cell Culture / Chapter 2.1.1.1 . --- Schwann Cell Culture --- p.23 / Chapter 2.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.24 / Chapter 2.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.25 / Chapter 2.1.2. --- Labeling of Toxins --- p.26 / Chapter 2.1.3. --- Administration of Toxin --- p.27 / Chapter 2.2. --- UPTAKE OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.2.1. --- Real-Time Observation of Toxin Uptake by Neurons --- p.27 / Chapter 2.3. --- APOPTOSIS STUDY OF RIBOSOME INACTIVATING PROTEINS' TOXICITY / Chapter 2.3.1. --- TUNEL Staining --- p.28 / Chapter 2.3.2. --- Annexin V Staining --- p.30 / Chapter 2.4. --- MOLECULAR STUDY OF THE DEATH MECHANISM OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.4.1. --- NIH/3T3 Cell Line Culture --- p.33 / Chapter 2.4.2. --- Differential Display / Chapter 2.4.2.1. --- Differential Display --- p.34 / Chapter 2.4.2.2. --- Cloning and Sequencing --- p.38 / Chapter 2.4.2.3. --- RT-PCR --- p.42 / Chapter 2.4.3. --- Two Dimension Gel Electrophoresis --- p.43 / Chapter 2.4.4. --- Ribosomal RNA Analysis --- p.48 / Chapter 3. --- RESULTS / Chapter 3.1. --- General / Chapter 3.1.1. --- Cell Culture / Chapter 3.1.1.1 . --- Schwann Cell Culture --- p.50 / Chapter 3.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.51 / Chapter 3.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.51 / Chapter 3.1.2. --- RIPs Labeling --- p.52 / Chapter 3.2. --- Uptake of Ribosome Inactivating Protein / Chapter 3.2.1. --- Real-Time Observation of Toxin Uptake --- p.53 / Chapter 3.3. --- Apoptosis Study of Ribosome Inactivating Proteins' Toxicity / Chapter 3.3.1. --- TUNEL Staining --- p.55 / Chapter 3.3.2. --- Annexin V Assay / Chapter 3.3.2.1. --- Schwann Cell Culture --- p.57 / Chapter 3.3.2.2. --- Dorsal Root Ganglion Neuron Culture --- p.58 / Chapter 3.3.2.3. --- Unique Observable Pattern --- p.60 / Chapter 3.4. --- Molecular Study of the Death Mechanism of Ribosome Inactivating Proteins / Chapter 3.4.1. --- NIH/3T3 Cell Line Culture --- p.60 / Chapter 3.4.1.1. --- TUNEL Staining --- p.61 / Chapter 3.4.1.2. --- Annexin V Staining --- p.61 / Chapter 3.4.2. --- Differential Display / Chapter 3.4.2.1. --- Observation --- p.61 / Chapter 3.4.2.2. --- Primer Combination --- p.62 / Chapter 3.4.2.3. --- Differential Display --- p.62 / Chapter 3.4.3. --- Two-Dimensional Gel Electrophoresis / Chapter 3.4.3.1. --- Observation --- p.63 / Chapter 3.4.3.2. --- Comparison of Gels --- p.63 / Chapter 3.4.4. --- Ribosomal RNA Analysis --- p.63 / Chapter 4. --- DISCUSSION / Chapter 4.1. --- General / Chapter 4.1.1. --- The Selection of In Vitro Model / Chapter 4.1.1.1. --- Schwann Cell Culture --- p.65 / Chapter 4.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.66 / Chapter 4.1.2. --- Labeling of Toxins with Fluorochromes --- p.67 / Chapter 4.1.3. --- Dosage Used in In Vitro Study --- p.68 / Chapter 4.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 4.2.1. --- Real-Time Examination of Toxin Uptake --- p.69 / Chapter 4.3. --- Involvement of Apoptosis in Ribosome Inactivating Proteins' Intoxication / Chapter 4.3.1. --- TUNEL Staining --- p.75 / Chapter 4.3.2. --- Annexin V and Propidium Iodide Staining --- p.77 / Chapter 4.3.3. --- Special Pattern of Fluorescence Signal in Neuronal Cell Bodies --- p.82 / Chapter 4.4. --- Molecular Study of Death Mechanism of Ribosome Inactivating Proteins / Chapter 4.4.1. --- NIH/3T3 Cell Line Culture --- p.84 / Chapter 4.4.2. --- Differential Display --- p.84 / Chapter 4.4.3. --- Two Dimensional Polyacrylamide Gel Electrophoresis --- p.86 / Chapter 4.4.4. --- Ribosomal RNA Alternation --- p.88 / Chapter 5. --- CONCLUSIONS --- p.89 / Chapter 6. --- "FIGURES, GRAPHS AND LEGENDS" --- p.91 / Chapter 7. --- REFERENCES --- p.155 / APPENDIX / Appendix A Materials --- p.175 / Appendix B Source of Chemicals and Equipments --- p.184 / ACKNOWLEDGEMENTS --- p.186

Lam Sze Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-200). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Table of contents --- p.ii / Abstract --- p.xii / 撮要 --- p.xv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / TABLE OF CONTENTS / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Overview of Chitinases --- p.3 / Chapter 1.1.1. --- Classification of Chitinases --- p.7 / Chapter 1.1.1.1. --- Family 19 Chitinases --- p.7 / Chapter 1.1.1.1.1. --- Class I Chitinases --- p.9 / Chapter 1.1.1.1.2. --- Class II Chitinases --- p.10 / Chapter 1.1.1.1.3. --- Class IV Chitinases --- p.10 / Chapter 1.1.1.1.4. --- Class V Chitinases --- p.11 / Chapter 1.1.1.1.5. --- Class VI Chitinases --- p.11 / Chapter 1.1.1.2. --- Family 18 Chitinases --- p.12 / Chapter 1.1.1.2.1. --- PR-8/Class III Chitinases --- p.12 / Chapter 1.1.1.2.2. --- PR-11 Chitinases --- p.15 / Chapter 1.1.1.3. --- The PR-4 Family --- p.16 / Chapter 1.1.2. --- Catalytic Mechanism of Chitinases --- p.19 / Chapter 1.1.2.1. --- Catalytic Mechanism of Family 18 Chitinases --- p.20 / Chapter 1.1.2.2. --- Catalytic Mechanism of Family 19 Chitinases --- p.21 / Chapter 1.1.3. --- Biological Properties of Chitinases --- p.22 / Chapter 1.1.3.1. --- Antifungal Activity of Chitinases in vitro --- p.22 / Chapter 1.1.3.2. --- Antifungal Activity of Chitinases in vivo --- p.23 / Chapter 1.1.3.3. --- Other Functions --- p.23 / Chapter 1.2. --- Overview of Ribonucleases --- p.25 / Chapter 1.2.1. --- Classification of Ribonucleases --- p.26 / Chapter 1.2.1.1. --- RNase T1 Family --- p.26 / Chapter 1.2.1.1.1. --- Action Mechanism of RNase T1 Family --- p.32 / Chapter 1.2.1.2. --- RNase T2 Family --- p.34 / Chapter 1.2.1.2.1. --- Action Mechanism of RNase T2 Family --- p.36 / Chapter 1.2.2. --- Biological Activities of Plant Ribonucleases --- p.38 / Chapter 1.2.2.1. --- Phosphate Remobilization --- p.38 / Chapter 1.2.2.2. --- Senescence --- p.39 / Chapter 1.2.2.3. --- Programmed Cell Death --- p.40 / Chapter 1.2.2.4. --- Plant Defense --- p.41 / Chapter 1.2.2.5. --- RNA Processing and Decay --- p.43 / Chapter 1.2.2.6. --- Antitumor Activities --- p.43 / Chapter 1.3. --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.45 / Chapter 1.3.1. --- General properties of RIPs --- p.46 / Chapter 1.3.1.1. --- Classification of RIPs --- p.46 / Chapter 1.3.2. --- Activities of Ribosome-inactivating Proteins --- p.52 / Chapter 1.3.2.1. --- RNA N-glycosidase activity --- p.52 / Chapter 1.3.2.2. --- Protein synthesis inhibitory activity --- p.58 / Chapter 1.3.2.3. --- Abortifacient activity --- p.59 / Chapter 1.3.2.4. --- Immunosuppressive activity --- p.60 / Chapter 1.3.2.5. --- Antiviral activity --- p.61 / Chapter 1.3.3. --- Roles of RIPs in plants --- p.63 / Chapter 1.3.3.1. --- Defensive role of RIPs in plants --- p.63 / Chapter 1.3.3.2. --- Role of RIPs in stress adaptation in plants --- p.66 / Chapter 1.3.4. --- Possible application of RIPs --- p.67 / Chapter 1.3.4.1. --- Use of RIPs in therapies --- p.67 / Chapter 1.3.4.1.1. --- Antiviral agents --- p.67 / Chapter 1.3.4.1.2. --- Immunotoxins --- p.68 / Chapter 1.3.4.1.3. --- Anti-HIV drugs --- p.69 / Chapter 1.3.4.2. --- Use of RIPs in agriculture --- p.71 / Chapter 1.4. --- Overview of the PR-5 Family: Thaumatin-Like Proteins (TLPs) --- p.72 / Chapter 1.4.1. --- Occurrence of Thaumatin-Like Proteins --- p.76 / Chapter 1.4.2. --- Biological properties of TLPs --- p.77 / Chapter 1.4.2.1. --- Antifungal Activity --- p.77 / Chapter 1.4.2.2. --- TLPs as Anti-Freeze Protein --- p.78 / Chapter 1.4.3. --- Biotechnological Application ´ؤ Transgenic Plants --- p.79 / Chapter Chapter 2 --- Materials and Methods --- p.81 / Chapter 2.1. --- Materials --- p.81 / Chapter 2.2. --- Preparation of Crude Extract --- p.82 / Chapter 2.3. --- Purification --- p.83 / Chapter 2.4. --- Chromatography --- p.84 / Chapter 2.4.1. --- CM-Cellulose Chromatography --- p.84 / Chapter 2.4.2. --- Mono S® HR 5/5 and Mono Q® HR 5/5 --- p.85 / Chapter 2.4.3. --- Affi-gel Blue gel --- p.86 / Chapter 2.4.4. --- Superdex75 --- p.87 / Chapter 2.5. --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 2.6. --- Protein Concentration Determination --- p.89 / Chapter 2.7. --- Preparation of Rabbit Reticulocyte Lysate --- p.90 / Chapter 2.8. --- Determination of N-terminal Amino Acid Sequence --- p.91 / Chapter 2.9. --- Biological Activity Assays --- p.92 / Chapter 2.9.1. --- Assay for Antifungal Activity --- p.92 / Chapter 2.9.2. --- Assay for Cell-Free Translation Inhibitory Activity --- p.93 / Chapter 2.9.3. --- Assay of Cytotoxic Activity on Cancer Cell Lines --- p.94 / Chapter 2.9.4. --- Assay for HIV-1 Reverse Transcriptase (RT) Inhibitory Activity --- p.95 / Chapter 2.9.5. --- Assay of Mitogenic Activity --- p.97 / Chapter 2.9.6. --- Assay for N-Glycosidase Activity --- p.98 / Chapter 2.9.6.1. --- RNA Extraction --- p.98 / Chapter 2.9.6.2. --- Aniline Treatment --- p.99 / Chapter 2.9.6.3. --- Formaldehyde Gel Electrophoresis --- p.99 / Chapter 2.9.7. --- Assay of Ribonuclease Activity --- p.100 / Chapter 2.9.7.1. --- Assay for Yeast tRNA --- p.100 / Chapter 2.9.7.2. --- Activity toward Polyhomoribonucleotides --- p.100 / Chapter Chapter 3 --- Purification and Characterization of Pathogenesis-Related Proteins from their Respective Sources --- p.101 / Chapter 3.1. --- Purification and Characterization of Chitinase and Ribonuclease from the Roots of Panax notoginseng --- p.102 / Chapter 3.1.1. --- Introduction --- p.102 / Chapter 3.1.2. --- Results --- p.104 / Chapter 3.1.3. --- Purification --- p.107 / Chapter 3.1.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.108 / Chapter 3.1.3.2. --- Affinity Chromatography on Affi-gel Blue gel --- p.111 / Chapter 3.1.3.3. --- Cation-Exchange Chromatography on Mono S Column --- p.114 / Chapter 3.1.3.4. --- Gel Filtration on Superdex 75 Column --- p.115 / Chapter 3.1.4. --- Characterization of Chitinase --- p.117 / Chapter 3.1.4.1. --- N-terminal Amino Acid Sequence --- p.117 / Chapter 3.1.4.2. --- Assay for Antifungal Activity --- p.118 / Chapter 3.1.4.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.120 / Chapter 3.1.4.4. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.120 / Chapter 3.1.5. --- Characterization of Ribonuclease --- p.121 / Chapter 3.1.5.1. --- N-terminal Amino Acid Sequence --- p.121 / Chapter 3.1.5.2. --- Assay for Ribonuclease Activity --- p.122 / Chapter 3.1.5.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.125 / Chapter 3.1.5.4. --- Assay for Antifungal Activity --- p.125 / Chapter 3.1.5.5. --- Assay for Antiproliferative Activity --- p.126 / Chapter 3.1.6. --- Discussion --- p.127 / Chapter 3.2. --- Purification and Characterization of Ribosome-Inactivating Protein and Antifungal Protein from the mushroom Lyophyllum shimeji --- p.131 / Chapter 3.2.1. --- Introduction --- p.131 / Chapter 3.2.2. --- Results --- p.132 / Chapter 3.2.3. --- Purification --- p.134 / Chapter 3.2.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.135 / Chapter 3.2.3.2. --- Affinity Chromatography on Affi-gel Blue Gel --- p.137 / Chapter 3.2.3.3. --- Cation-Exchange Chromatography on Mono S --- p.140 / Chapter 3.2.4. --- Characterization of Ribosome-Inactivating Protein and Antifungal Protein from Lyophyllum shimeji --- p.142 / Chapter 3.2.4.1. --- N-terminal Amino Acid Sequence --- p.142 / Chapter 3.2.4.2. --- Assay for Antifungal Activity --- p.144 / Chapter 3.2.4.3. --- Assay for N-glycosidase Activity --- p.147 / Chapter 3.2.4.4. --- Assay for Mitogenic Activity --- p.147 / Chapter 3.2.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.148 / Chapter 3.2.5. --- Discussion --- p.150 / Chapter 3.3. --- Purification and Characterization of Ribosome-inactivating Protein from the Hypsizigus marmoreus --- p.153 / Chapter 3.3.1. --- Introduction --- p.153 / Chapter 3.3.2. --- Result --- p.154 / Chapter 3.3.3. --- Purification --- p.155 / Chapter 3.3.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.156 / Chapter 3.3.3.2. --- Affinity-Chromatography on Affi-gel Blue Gel --- p.158 / Chapter 3.3.3.3. --- Anion-Exchange Chromatography on Mono Q Column --- p.160 / Chapter 3.3.4. --- Characterization of Ribosome-inactivating Protein from Hypsizigus marmoreus --- p.162 / Chapter 3.3.4.1. --- N-terminal Amino Acid Sequence --- p.162 / Chapter 3.3.4.2. --- Assay for Cell-Free Translation-Inhibiting Activity --- p.163 / Chapter 3.3.4.3. --- Assay for Antifungal Activity --- p.164 / Chapter 3.3.4.4. --- Assay for N-glycosidase Activity --- p.166 / Chapter 3.3.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.166 / Chapter 3.3.4.6. --- Assay for mitogenic Activity --- p.167 / Chapter 3.3.4.7. --- Assay for Antiproliferative Activity --- p.167 / Chapter 3.3.5. --- Discussion --- p.159 / Chapter Chapter 4 --- General Discussion --- p.170 / References --- p.172

植物合成多種發病機理相關蛋白以對抗病原體的侵襲。植物發病機理相關蛋白包括：核糖核酸酶；抗真菌蛋白；凝集素；胰蛋白酶抑制因子等。這些發病機理相關蛋白具有抗病毒，抗細菌，抗真菌，免疫調節及抗腫瘤等活性。從六種植物中提純了七個發病機理相關蛋白，包括三個凝集素，一個核糖核酸酶，兩個種抗真菌蛋白及一個胰蛋白酶抑制因子。 / 從西洋參須中提純了新的核糖核酸酶。核糖核酸酶分子量為26kDa，具有特异N末端氨基酸序列。此核糖核酸酶在 pH5 及 60℃ 條件下活性最高。它能抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 從粉色菜豆及日本大花豆中提純了兩種凝集素。它們由兩個分子量為32kDa的亞基構成雙倍體。他們的活性穩定于0-60℃及3-12 pH。粉色菜豆凝集素的特异性糖基為木糖，日本大花豆凝集素的特异性糖基為半乳糖。從太子參中提純的凝集素分子量為33kDa，其活性穩定于0-60℃及2-5 pH。 這三種凝集素都具有抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 提純的胰蛋白酶抑制因子分子量為21kDa。具有高耐熱及耐酸鹼性并表現出抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。從豇豆中提純的抗真菌肽分子量為5447Da，具有類防御素N末端氨基酸序列。 / Plants produce a diversity of proteins with antipathogenic activities. These proteins comprise among others, (i) ribonucleases, (ii) antifungal proteins, (iii) lectins and (iv) trypsin inhibitor with antiviral, antifungal and anticancer activities. The aim of this project was to isolate antipathogenic plant proteins including a ribonuclease from American ginseng branch roots, a trypsin inhibitor from rambutan seeds, defensin-like antifungal peptides from borlotti beans and king pole beans, and lectins from borlotti beans, Japanese large pinto beans and Pseudostellaria heterophylla. / The isolated 26-kDa ginseng branch root ribonuclease was monomeric with a novel N-terminal amino acid sequence. It exhibited maximal robonucleolytic activity toward yeast tRNA at pH 5 and 60℃. It inhibited proliferation of MCF7 human breast cancer cells and HepG2 human hepatoma cells. It also inhibited the activity of HIV-1 reverse transcriptase. / Both borlotti bean lectin and Japanese large pinto bean lectin were dimeric with a subunit molecular mass of 32-kDa. They were stable from 0℃ to 60℃ and from pH 3 to pH 12. Borlotti bean lectin was xylose-specific whereas Japanse large pinto bean lectin was galactose-specific. The 33-kDa Pseudostellaria heterophylla lectin could not be inhibited by the simple sugars tested. It was stable from 0℃ to 60℃ and from pH 2 to 5. All three isolated lectins suppressed proliferation of MCF7 and HepG2 cells and inhibited HIV-1 reverse transcriptase. / The isolated 21-kDa rambutan trypsin inhibitor has relatively high pH stability and thermostability, and exhibited HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity toward a variety of tumor cells. The isolated 5447-Da king pole bean defensin-like peptide inhibited fungal growth. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xii / Chapter Chapter 1 --- Overview of Plant Defense-related Protein --- p.1 / Chapter 1.1 --- Overview of Lectins and hemagglutinins --- p.4 / Chapter 1.1.1 --- History and definition of lectins and hemagglutinins --- p.4 / Chapter 1.1.2 --- Occurrence and distribution of plant lectins --- p.6 / Chapter 1.1.3 --- Classification of lectins --- p.7 / Chapter 1.1.3.1 --- Classification of lectins on the basis of overall structure of lectin subunits --- p.7 / Chapter 1.1.3.2 --- Classification of lectins based on binding specificty to carbohydrates --- p.11 / Chapter 1.1.3.3 --- Classification of lectins according to families --- p.12 / Chapter 1.1.3.3.1 --- Legume lectins --- p.12 / Chapter 1.1.3.3.2 --- Monocot mannose-binding lectins --- p.13 / Chapter 1.1.3.3.3 --- Other lectins --- p.14 / Chapter 1.1.4 --- Defensive role of plant lectins --- p.15 / Chapter 1.1.5 --- Applications of plant lectins --- p.18 / Chapter 1.1.5.1 --- The antibacterial activity --- p.18 / Chapter 1.1.5.2 --- Anti-insect activity --- p.19 / Chapter 1.1.5.3 --- Antifungal activity --- p.21 / Chapter 1.1.5.4 --- The antiviral activity --- p.22 / Chapter 1.1.5.5 --- Lectin affinity chromatography --- p.23 / Chapter 1.1.5.6 --- Lectin microarray --- p.23 / Chapter 1.2 --- Overview of Ribonucleases --- p.26 / Chapter 1.2.1 --- History and definition of Ribonucleases --- p.26 / Chapter 1.2.2 --- Classification of Ribonucleases --- p.27 / Chapter 1.2.2.1 --- T1 Ribonucleases family --- p.27 / Chapter 1.2.2.2 --- RNase T2 family --- p.28 / Chapter 1.2.3 --- Biological activities of plant ribonucleases --- p.28 / Chapter 1.2.3.1 --- Phosphate remobilization --- p.28 / Chapter 1.2.3.2 --- Senescence --- p.29 / Chapter 1.2.3.3 --- Programmed cell death --- p.30 / Chapter 1.2.3.4 --- Plant defense --- p.31 / Chapter 1.2.3.5 --- RNA processing and decay --- p.32 / Chapter 1.2.3.6 --- Antitumor activities --- p.33 / Chapter 1.3 --- Other plant pathogen-related proteins --- p.34 / Chapter 1.3.1 --- Overview of chitinase --- p.34 / Chapter 1.3.1.1 --- Classification of chitinases --- p.35 / Chapter 1.3.1.2 --- Biological properties of chitinases --- p.38 / Chapter 1.3.2 --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.41 / Chapter 1.3.2.1 --- Classification of RIPs --- p.42 / Chapter 1.3.2.2 --- Roles of RIPs in plants --- p.44 / Chapter 1.3.2.3 --- Possible application of RIPs --- p.46 / Chapter 1.3.3 --- Overview of thaumatin-like proteins (TLPs) --- p.50 / Chapter 1.3.3.1 --- Occurrence of TLPs --- p.51 / Chapter 1.3.3.2 --- Biological properties of TLPs --- p.52 / Chapter 1.4 --- Aim of this study --- p.54 / Chapter Chapter 2 --- Isolation of a lectin and an antifungal protein from Phaseolus vulgaris cv. Borlotti beans / Chapter 2.1 --- Introduction --- p.55 / Chapter 2.2 --- Materials and Methods --- p.55 / Chapter 2.3 --- Results --- p.64 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Isolation of a lectin from Pinto beans (Phaseolus vulgaris pinto bean) / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.3 --- Results --- p.87 / Chapter 3.4 --- Discussion --- p.103 / Chapter Chapter 4 --- Isolation of a lectin from Pseudostellaria hetorophylla roots / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Materials and Methods --- p.107 / Chapter 4.3 --- Results --- p.110 / Chapter 4.4 --- Discussion --- p.122 / Chapter Chapter 5 --- Isolation of a ribonuclease from branch roots of American ginseng (Panax quinquefolium) / Chapter 5.1 --- Introduction --- p.124 / Chapter 5.2 --- Materials and Methods --- p.126 / Chapter 5.3 --- Results --- p.129 / Chapter 5.4 --- Discussion --- p.142 / Chapter Chapter 6 --- Isolation of a trypsin inhibitor in rambutan (Nephelium lappaceum L) seeds / Chapter 6.1 --- Introduction --- p.144 / Chapter 6.2 --- Materials and Methods --- p.147 / Chapter 6.3 --- Results --- p.152 / Chapter 6.4 --- Discussion --- p.163 / Chapter Chapter 7 --- Isoation of a defensin-like antifungal peptide from Phaseolus vulgaris cv. 'King Pole Bean' / Chapter 7.1 --- Introduction --- p.168 / Chapter 7.2 --- Materials and Methods --- p.170 / Chapter 7.3 --- Results --- p.173 / Chapter 7.4 --- Discussion --- p.181 / Chapter Chapter 8 --- Overall discussion --- p.183 / References --- p.186

Polyribosome aggregation state in growing tissues of barley and wheat leaf or stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf or stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of ³²PO₄³⁻ into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) ⁺RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) ⁺RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system; activity of nonpolysomal mRNP was variable, but usually less than that of polysomal mRNP. Deproteinization of mRNP failed to improve its activity. No inhibition of translation of poly(A) ⁺RNA by nonpolysomal mRNP was observed in mixing experiments with the wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described.

Whole safflower seeds of the Mexican variety Kino'76 with a protein content of 17.30% (dwb) were subjected to the processes of dehulling, defatting (n-hexane extraction) and debittering (70% methanol extraction) to produce four types of meals preparations: whole safflower meal, dehulled safflower meal, debittered, whole meal and debittered, dehulled meal with protein contents of 26.90, 66.93, 26.70 and 69.92%, respectively. The proteins of each meal were studied in detail by means of protein fractionation, gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Osborne solubility fractionation of the protein of whole safflower meal showed that the amount of protein in the alkali soluble fraction was approximately 71% of the total and the alcohol soluble fraction did not contain any protein. After dehulling and debittering, the amount of protein in the alkali soluble fraction decreased by 30%, whereas the amount of protein in the insoluble residue increased by 12%. SDS-PAGE of the proteins of the water-, salt- and alkali soluble fractions revealed that they consisted of 8, 13 and 13 distinct subunits, respectively, with apparent molecular weights ranging from 14.7 to 88.0 kDa. The number of subunits and molecular weight distribution decreased as a result of debittering. Fractionation of the proteins of each meal by gel filtration chromatography followed by SDS-PAGE demonstrated that proteins of safflower seed are highly heterogeneous. The process of debittering caused major alteration of the molecular weight profile and subunit composition of the gel filtration protein fractions.

Poon Yin-tat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 101-111). / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter CHAPTER 2: --- PURIFICATION OF α- AND β-MOMORCHARINS --- p.26 / Chapter CHAPTER 3: --- N-GLYCOSIDASE ACTIVITY OF α- AND β-MOMORCHARINS --- p.45 / REFERENCES --- p.101

Leung Chun-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 113-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgment --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of abbreviations --- p.xiv / List of figures --- p.xvii / List of tables --- p.xix / Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Artificial sweeteners --- p.3 / Chapter 1.2.1 --- SACCHARIN --- p.3 / Chapter 1.2.2 --- cyclamate --- p.4 / Chapter 1.2.3 --- Aspartame --- p.4 / Chapter 1.2.4 --- acesulfame-k --- p.5 / Chapter 1.2.5 --- SUCRALOSE --- p.5 / Chapter 1.3 --- natural sweet plant proteins --- p.7 / Chapter 1.3.1 --- THAUMATIN --- p.7 / Chapter 1.3.2 --- MONELLIN --- p.10 / Chapter 1.3.3 --- CURCULIN --- p.11 / Chapter 1.3.4 --- PENTADIN AND BRAZZEIN --- p.11 / Chapter 1.3.5 --- MIRACULIN --- p.12 / Chapter 1.3.6 --- MABINLIN --- p.12 / Chapter 1.4 --- Genetic Engineering of Sweet Plant Protein --- p.19 / Chapter 1.4.1 --- biotechnological studies on thaumatin --- p.20 / Chapter 1.4.1.1 --- Protein modification and sweetness --- p.20 / Chapter 1.4.1.2 --- Transgenic expression in microbes --- p.21 / Chapter 1.4.1.3 --- Transgenic expression in higher plants --- p.23 / Chapter 1.4.2 --- BIOTECHNOLOGICAL STUDIES ON MONELLIN --- p.24 / Chapter 1.4.2.1 --- Gene modification and transgenic expression in microbes --- p.24 / Chapter 1.4.2.2 --- Transgenic expression in plants --- p.25 / Chapter 1.4.3 --- TRANSGENIC EXPRESSION OF MABINLIN IN PLANTS --- p.26 / Chapter 1.5 --- phaseolin and its regulatory sequences --- p.27 / Chapter 1.6 --- ARABIDOPSIS --- p.29 / Chapter 1.6.1 --- ARABIDOPSIS THALIANA as a model plant --- p.29 / Chapter 1.6.2 --- Transformation methods --- p.29 / Chapter 1.6.2.1 --- Direct DNA uptake --- p.30 / Chapter 1.6.2.2 --- Agrobacterium-mediated transformation --- p.31 / Chapter 1.6.2.3 --- In planta transformation --- p.31 / Chapter 2 --- GENKRAL INTRODUTION AND HYPOTHESIS --- p.22 / Chapter 2.1 --- General Introduction --- p.33 / Chapter 2.2 --- Hypothesis --- p.34 / Chapter 3 --- MOLECULAR STUDIES ON SWEET PROTEIN MARINLIN : THERMAL STABILITY --- p.28 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Materials --- p.40 / Chapter 3.2.1 --- laboratory wares --- p.40 / Chapter 3.2.2 --- Equipments --- p.40 / Chapter 3.2.3 --- Chemicals --- p.40 / Chapter 3.2.4 --- commerical kits --- p.41 / Chapter 3.2.5 --- DNA primers --- p.42 / Chapter 3.2.6 --- DNA plasmids --- p.43 / Chapter 3.2.7 --- bacterial strains --- p.43 / Chapter 3.2.8 --- Plant materials --- p.44 / Chapter 3.2.9 --- Protein and Antibody --- p.44 / Chapter 3.3 --- Methods --- p.45 / Chapter 3.3.1 --- Transformation of Arabidopsis with mbliii and mbli genes --- p.45 / Chapter 3.3.1.1 --- Construction of mutant MBLIII and MBLI genes containing single codon mutation by megaprimer PCR --- p.45 / Chapter 3.3.1.2 --- Cloning of PCR-amplified MBLIII and MBLI cDNAs into vector pD3-8 --- p.48 / Chapter 3.3.1.3 --- In vitro site-directed mutagensis (for the construction of MBLIII and MBLI cDNAs containing single codon mutation) --- p.49 / Chapter 3.3.1.4 --- Cloning of the wild-type and mutated MBLIII and MBLI cDNA into vector pTZ / phas --- p.53 / Chapter 3.3.1.5 --- Confirmation of sequence fidelity and mutated codon in MBLIII and MBLI cDNA by DNA sequencing --- p.53 / Chapter 3.3.1.6 --- Transfer of wild-type MBLIII and MBLI cDNA flanked by phaseolin regulatory sequence into Agrobacterium binary vector --- p.55 / Chapter 3.3.1.7 --- Transformation of Agrobacterium with pBI / phas / MBLIII and pBI / phas / MBLI chimeric gene constructs --- p.57 / Chapter 3.3.1.8 --- Vacuum infiltration transformation of A rabidopsis --- p.58 / Chapter 3.3.1.9 --- Screening of homozygous transgenic Arabidopsis --- p.59 / Chapter 3.3.2 --- Expression analysis of MBLIII transgene --- p.61 / Chapter 3.3.2.1 --- GUS assay of transgenic plants --- p.61 / Chapter 3.3.2.2 --- Genomic DNA isolation from transgenic plants --- p.61 / Chapter 3.3.2.3 --- PCR amplification of transgene --- p.62 / Chapter 3.3.2.4 --- Total RNA isolation from transgenic Arabidopsis --- p.63 / Chapter 3.3.2.5 --- RT-PCR of total RNA from transgenic Arabidopsis --- p.64 / Chapter 3.3.2.6 --- Verification of the presence of mutagenic site and the fidelity of RNA transcript from transgenic Arabidopsis --- p.65 / Chapter 3.3.2.7 --- Protein extraction and tricine SDS-PAGE of putative transgenic protein from Arabidopsis --- p.65 / Chapter 3.3.2.8 --- N-terminal amino acid sequencing --- p.66 / Chapter 3.3.2.9 --- Isoelectric precipitation of MBL --- p.67 / Chapter 3.3.2.10 --- Production of polyclonal antibody against purified MBL --- p.67 / Chapter 3.3.2.11 --- Western-blotting and immunodectection of Arabidopsis protein by anti-MBL polyclonal antibody --- p.69 / Chapter 3.4 --- results & discussion --- p.71 / Chapter 3.4.1 --- Site-specific mutations of Arginine residue in mbliii cdna and glutamine in mbli cdna --- p.71 / Chapter 3.4.1.1 --- Megaprimer PCR --- p.71 / Chapter 3.4.1.2 --- Cloning into the seed-specific expression vector pD38 --- p.74 / Chapter 3.4.1.3 --- In vitro site-directed mutagenesis --- p.76 / Chapter 3.4.2 --- Construction of plant expression vectors containing chimeric MBLIII and MBLI --- p.80 / Chapter 3.4.2.1 --- Cloning of MBLIII and MBLI cDNAs into the seed-specific expression vector pTZ / phas --- p.80 / Chapter 3.4.2.2 --- Cloning into the plant expression vector pBI121 --- p.83 / Chapter 3.4.3 --- Generation of homozygous transgenic Arabidopsis --- p.84 / Chapter 3.4.3.1 --- Screening of transgenic R1 Arabidopsis --- p.84 / Chapter 3.4.3.2 --- Screening of transgenic R2 plants --- p.86 / Chapter 3.4.3.3 --- Screening of homozygous R3 transgenic plants --- p.88 / Chapter 3.4.4 --- Detection of MBLIII transgene in Arabidopsis --- p.89 / Chapter 3.4.4.1 --- Gus Assay --- p.89 / Chapter 3.4.4.2 --- Detection of transgene integration --- p.90 / Chapter 3.4.5 --- DETECTION of MBLIII TRANSCRIPT IN TRANSGENIC arabidopsis --- p.92 / Chapter 3.4.5.1 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.92 / Chapter 3.4.5.2 --- Verification of the presence of the mutant codon and sequence fidelity of the RT-PCR product --- p.94 / Chapter 3.4.6 --- DETECTION OF MBL III PROTEIN IN TRANSGENIC arabidopsis --- p.97 / Chapter 3.4.6.1 --- Expression of MBL protein --- p.97 / Chapter 3.4.6.2 --- Isoelectric precipitation --- p.101 / Chapter 3.4.6.3 --- Assay of titers and quality of primary polyclonal antibody against purified MBL protein --- p.103 / Chapter 3.4.6.4 --- Western blot / Immunodetection --- p.106 / Chapter 4 --- GENERAL DISCUSSION --- p.109 / Conclusion --- p.112 / References --- p.113