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

Parkash Amarender. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.xi / 撮要 --- p.xiv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.2 --- General Properties of RIPs --- p.5 / Chapter 1.2.1 --- Structure --- p.5 / Chapter 1.2.1.1 --- Type I and Type II RIPs --- p.5 / Chapter 1.2.1.2 --- Small RIPs --- p.10 / Chapter 1.2.2 --- Distribution --- p.12 / Chapter 1.2.3 --- Physicochemical properties --- p.15 / Chapter 1.3 --- Enzymatic activities of RIPs --- p.17 / Chapter 1.3.1 --- N-glycosidase activity --- p.17 / Chapter 1.3.2 --- Polynucleotide:adenosine glycosidase activity --- p.21 / Chapter 1.3.3 --- Ribonuclease (RNase) activity --- p.24 / Chapter 1.3.4 --- Deoxyribonucleolytic (DNase) activity --- p.25 / Chapter 1.3.5 --- Multiple depurination --- p.26 / Chapter 1.3.6 --- Inhibition of protein synthesis --- p.27 / Chapter 1.4 --- Biological activities of RIPs --- p.29 / Chapter 1.4.1 --- Interaction of ribosome-inactivating proteins with cells --- p.29 / Chapter 1.4.1.1 --- Internalization of type 1 ribosome-inactivating proteins --- p.29 / Chapter 1.4.1.2 --- Internalization of type 2 ribosome-inactivating proteins --- p.32 / Chapter 1.4.2 --- Effects on laboratory animals --- p.33 / Chapter 1.4.3 --- Immunosuppressive activity --- p.33 / Chapter 1.4.4 --- Abortifacient activity --- p.34 / Chapter 1.4.5 --- Antiviral activity --- p.35 / Chapter 1.5 --- Physiological roles of RIPs --- p.37 / Chapter 1.6 --- Applications of RIPs --- p.39 / Chapter 1.6.1 --- Possible uses in experimental and clinical medicine --- p.39 / Chapter 1.6.1.1 --- Anti-tumor therapy --- p.40 / Chapter 1.6.1.2 --- Immune disorders --- p.42 / Chapter 1.6.1.3 --- Neuroscience research --- p.43 / Chapter 1.6.2 --- Applications in agriculture --- p.44 / Chapter 1.7 --- Arginine/Glutamate Rich Polypeptides (AGRPs) --- p.46 / Chapter 1.8 --- Objectives of the present study --- p.48 / Chapter 1.8.1 --- Rationale of the study --- p.48 / Chapter 1.8.2 --- Outline of the thesis --- p.50 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Introduction --- p.52 / Chapter 2.2 --- Materials and methods --- p.54 / Chapter 2.2.1 --- Materials --- p.54 / Chapter 2.2.2 --- Preparation of crude extract --- p.55 / Chapter 2.2.3 --- Purification of proteins --- p.55 / Chapter 2.2.4 --- Molecular weight determination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.61 / Chapter 2.2.5 --- Protein determination --- p.64 / Chapter 2.2.6 --- N-terminal amino acid sequence --- p.64 / Chapter 2.2.7 --- Preparation of rabbit reticulocyte lysate --- p.65 / Chapter 2.2.8 --- Assay for cell-free protein synthesis- inhibiting activity --- p.65 / Chapter 2.2.9 --- Assay for N-glycosidase activity --- p.66 / Chapter 2.2.10 --- Assay for ribonuclease activity --- p.70 / Chapter 2.2.11 --- Assay for antifungal activity --- p.71 / Chapter 2.2.12 --- Assay for dehydrogenase activity --- p.71 / Chapter Chapter 3 --- Purification and characterization of proteins from their respective sources. / Chapter 3.1. --- Purification and Characterization of Hispidin from Hairy melon (Benincasa hispida var. chieh-qua) / Chapter 3.1.1. --- Introduction --- p.73 / Chapter 3.1.2. --- Results --- p.76 / Chapter 3.1.2.1. --- Purification --- p.78 / Chapter 3.1.2.2. --- Molecular weight determination --- p.84 / Chapter 3.1.2.3. --- N-terminal amino acid sequence --- p.85 / Chapter 3.1.2.4. --- Assay for cell-free protein synthesis-inhibiting activity --- p.86 / Chapter 3.1.2.5. --- Assay for N-glycosidase activity --- p.87 / Chapter 3.1.2.6. --- Assay for ribonuclease activity --- p.88 / Chapter 3.1.2.7. --- Assay for dihydrodiol dehydrogenase activity --- p.88 / Chapter 3.1.2.8. --- Assay for antifungal activity --- p.89 / Chapter 3.1.2.9. --- "Assessment of purity, yield and activity" --- p.91 / Chapter 3.1.3. --- Discussion --- p.92 / Chapter 3.2. --- Purification and Characterization of Momorchin from Dried Bitter Gourd (Momordica charantia) Seeds / Chapter 3.2.1. --- Introduction --- p.95 / Chapter 3.2.2. --- Results --- p.99 / Chapter 3.2.2.1. --- Purification --- p.100 / Chapter 3.2.2.2. --- Molecular weight determination --- p.103 / Chapter 3.2.2.3. --- N-terminal amino acid sequence --- p.104 / Chapter 3.2.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.105 / Chapter 3.2.2.5. --- Assay for ribonuclease activity --- p.105 / Chapter 3.2.2.6. --- Assay for N-glycosidase activity --- p.106 / Chapter 3.2.2.7. --- "Assessment of purity, yield and activity" --- p.107 / Chapter 3.2.3. --- Discussion --- p.108 / Chapter 3.3.3. --- Purification and Characterization of Luffacylin from Sponge Gourd (Luffa cylindrica) / Chapter 3.3.1. --- Introduction --- p.110 / Chapter 3.3.2. --- Results --- p.113 / Chapter 3.3.2.1. --- Purification --- p.115 / Chapter 3.3.2.2. --- Molecular weight determination --- p.119 / Chapter 3.3.2.3. --- N-terminal amino acid sequencing --- p.120 / Chapter 3.3.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.121 / Chapter 3.3.2.5. --- Assay for ribonuclease activity --- p.121 / Chapter 3.3.2.6. --- Assay for N-glycosidase activity --- p.122 / Chapter 3.3.2.7. --- Assay for antifungal activity --- p.123 / Chapter 3.3.2.8. --- "Assessment of purity, activity and yield" --- p.124 / Chapter 3.3.3. --- Discussion --- p.125 / Chapter 3.4. --- Purification and Characterization of α and β Benincasin from fresh Winter Melon {Benincasa hispida var. dong-gua) Seeds / Chapter 3.4.1. --- Introduction --- p.127 / Chapter 3.4.2. --- Results --- p.129 / Chapter 3.4.2.1. --- Purification --- p.130 / Chapter 3.4.2.2. --- Molecular weight determination --- p.135 / Chapter 3.4.2.3. --- N-terminal amino acid sequence --- p.136 / Chapter 3.4.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.137 / Chapter 3.4.2.5. --- Assay for ribonuclease activity --- p.137 / Chapter 3.4.2.6. --- Assay for antifungal activity --- p.138 / Chapter 3.4.2.7. --- "Assessment of purity, activity and yield" --- p.140 / Chapter 3.4.3. --- Discussion --- p.141 / Chapter 3.5. --- Purification and characterization of Moschins from Pumpkin (Cucurbita moschata) Seeds / Chapter 3.5.1. --- Introduction --- p.143 / Chapter 3.5.2. --- Results --- p.145 / Chapter 3.5.2.1. --- Purification --- p.146 / Chapter 3.5.2.2. --- Molecular weight determination --- p.149 / Chapter 3.5.2.3. --- N-terminal amino acid sequence --- p.150 / Chapter 3.5.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.151 / Chapter 3.5.2.5. --- Assay for ribonuclease activity --- p.151 / Chapter 3.5.2.6. --- "Assessment of purity, activity and yield" --- p.152 / Chapter 3.5.3. --- Discussion --- p.153 / Chapter Chapter 4 --- General Discussion and Conclusion --- p.154 / References --- p.158

RNA interference (RNAi) is a conserved mechanism in a wide range of eukaryotes. Introduction of synthetic dsRNA could specifically target suppression of a gene or could result in off-target silencing of another gene due to sequence similarity. To verify if the observed phenotype in an RNAi transgenic line is due to silencing of a specific gene or if it is due to another nontarget gene, a synthetic gene complementation approach could be used. Synthetic gene complementation described in this method uses the technology of synthesizing a variant of a native gene (used in RNAi silencing) to maximize the difference in DNA sequences while coding for the exact same amino acids as the original native gene. This is achieved through the use of alternate codons. The new variant gene is expressed in the original RNAi transgenic lines and analyzed for complementation of the RNAi phenotype. Complementation of the RNAi-induced phenotype will indicate gene-specific silencing and not off-target silencing.