Gemin5 是運動神經元綜合體(SMN Complex)的其中一個組件，這綜合體的主要功能是控制小型胞核核糖核蛋白(UsnRNPs)的合成。這些小型胞核核糖核蛋白組合成剪接核糖核酸前體(pre-mRNA)的剪接體(Spliceosome)，使核糖核酸分子可以用來翻譯成蛋白質。失去運動神經元綜合體功能引致脊髓肌肉萎縮症。果蠅是其中一個用作研究人類疾病重要的生物。更重要的是，部分組成運動神經元綜合體的組件也存在於果蠅。是次研究是利用遺傳方式在果蠅內研究Gemin5 的同源基因-- rigor mortis (rig) 的作用。果蠅帶有rig 突變基因表現神經肌肉接頭(neuromuscular junction)上的缺陷和異常的運動行為。這表明，rig 的功能可能與神經退化性疾病有關。為了進一步了解rig 的功能途徑(functional pathway)，已進行了一個利用移除突變體的基因過濾實驗，研究鎖定了 12 個染色體部份可能和rig 有基因上的相互作用，進一步研究與rig 有相互作用的基因有助於了解rig 的功能及研究脊髓肌肉萎縮症的發病機制。 / Gemin5 is a component of the Survival of Motor Neuron (SMN) complex, which is a protein complex regulating biogenesis of various Uridine-enriched small nuclear ribonucleoproteins (UsnRNPs). These UsnRNPs form the molecular machinery spliceosome, which mediates pre-messenger RNA splicing, an important mechanism before an mRNA molecule can be used to translate proteins. Loss-of-function of the SMN complex is now known to cause the neurodegenerative disease, Spinal Muscular Atrophy. Drosophila is one of the well-characterized model organisms for studying human diseases. More importantly, components of the SMN complex are also found in Drosophila. Here, I studied the function of rigor mortis (rig), the Gemin5 orthologue, in Drosophila using a genetic approach. Drosophila carrying mutations in the rig gene show defects in the neuromuscular junction (NMJ) and display abnormal motor behavior. This suggests that the function of rig may link to the neurodegenerative disease. To further characterize the function of rig, a genetic screen was carried out. Twelve chromosomal regions encoding possible rig-interacting genes were identified. Further characterization of these rig-interacting genes may help us better understand the function of rig. / Detailed summary in vernacular field only. / Cheng, Yat Pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 120-125). / Abstracts also in Chinese. / ABSTRACT --- p.i / ABSTRACT IN CHINESE --- p.ii / ACKNOWLEDGEMENT --- p.iii / LIST OF ABBREVIATIONS --- p.iv / LIST OF FIGURES --- p.v / LIST OF TABLES --- p.vi / TABLE OF CONTENTS --- p.vii / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Introduction of rigor mortis / Chapter 1.1.1 --- Orthologue of Gemin5 in Drosophila --- p.1 / Chapter 1.1.2 --- Published Phenotypic Analyses of rig Mutants --- p.1 / Chapter 1.2 --- Introduction of Gemin5 / Chapter 1.2.1 --- Introduction of Gemins --- p.4 / Chapter 1.2.2 --- Structural Properties of Gemin5 --- p.4 / Chapter 1.2.3 --- Gemin5-interacting partners --- p.7 / Chapter 1.2.4 --- Gemin5 as a Component of the Survival of Motor Neuron (SMN) Complex --- p.7 / Chapter 1.2.5 --- Function of the SMN Complex and Spinal Muscular Atrophy --- p.11 / Chapter 1.3 --- Drosophila as a Model Organism / Chapter 1.3.1 --- Advantages of Using Drosophila as a Model Organism --- p.11 / Chapter 1.3.2 --- Neuromuscular Junction of Drosophila --- p.15 / Chapter 1.4 --- Aim of the Present Study --- p.19 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila Culture / Chapter 2.1.1 --- Culture Medium --- p.20 / Chapter 2.1.2 --- Drosophila Stocks and Crosses Maintenance --- p.20 / Chapter 2.1.3 --- Larvae Collection --- p.21 / Chapter 2.1.3.1 --- Reagents --- p.21 / Chapter 2.1.3.2 --- Procedures --- p.21 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Reagents --- p.23 / Chapter 2.2.2 --- Drosophila S2R⁺ Cell Culture --- p.24 / Chapter 2.2.3 --- Establishment of Stable S2R⁺ Cells --- p.24 / Chapter 2.3 --- Genomic Polymerase Chain Reaction (PCR) / Chapter 2.3.1 --- Reagents --- p.25 / Chapter 2.3.2 --- Genomic DNA Extraction from a Single Larva --- p.26 / Chapter 2.3.3 --- Primer Design --- p.26 / Chapter 2.3.4 --- Polymerase Chain Reaction (PCR) --- p.27 / Chapter 2.4 --- Behavioral Assay / Chapter 2.4.1 --- Stable S2R⁺ Cell Staining --- p.29 / Chapter 2.4.1.1 --- Reagents --- p.29 / Chapter 2.4.1.2 --- Procedures --- p.30 / Chapter 2.4.2 --- Larvae Staining --- p.31 / Chapter 2.4.2.1 --- Reagents --- p.31 / Chapter 2.4.2.2 --- Larvae Dissection --- p.32 / Chapter 2.4.2.3 --- Larval Muscle Staining --- p.33 / Chapter 2.4.2.4 --- Larval Neuromuscular Junction Staining --- p.33 / Chapter 2.5 --- Microscopy / Chapter 2.5.1 --- Light Microscopy --- p.34 / Chapter 2.5.1.1 --- Microscopic Observation of Larval Movement --- p.34 / Chapter 2.5.1.2 --- Quantification of Larval Contraction Rate --- p.34 / Chapter 2.5.1.3 --- Quantification of Larval Travelling Distance --- p.34 / Chapter 2.5.2 --- Fluorescence Microscopy --- p.35 / Chapter 2.5.2.1 --- Microscopic Observation of Larval Muscle --- p.35 / Chapter 2.5.2.2 --- Microscopic Observation of Stable S2R⁺ Cells --- p.35 / Chapter 2.5.3 --- Confocal Microscopy --- p.36 / Chapter 2.5.3.1 --- Microscopic Observation of Larval Neuromuscular Junction --- p.36 / Chapter 2.5.3.2 --- Quantification of Larval Neuromuscular Junction --- p.36 / Chapter 2.6 --- Generation of transgenic fly lines expressing rig transgene / Chapter 2.6.1 --- Polymerase Chain Reaction --- p.36 / Chapter 2.6.2 --- Agarose Gel Electrophoresis --- p.38 / Chapter 2.6.2.1 --- Reagents --- p.38 / Chapter 2.6.2.2 --- Procedures --- p.39 / Chapter 2.6.3 --- Restriction Digestion --- p.39 / Chapter 2.6.4 --- Ligation Reaction --- p.39 / Chapter 2.6.5 --- Bacterial Transformation --- p.40 / Chapter 2.6.5.1 --- Reagents --- p.40 / Chapter 2.6.5.2 --- Procedures --- p.40 / Chapter 2.6.6 --- Bacterial Glycerol Stock for Long-term Storage --- p.41 / Chapter 2.7 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting / Chapter 2.7.1 --- Reagents --- p.41 / Chapter 2.7.2 --- Lysate Preparation of Stable S2R⁺ Cells, Adult Fly Heads and Larvae --- p.44 / Chapter 2.7.2.1 --- Stable S2R+ Cells --- p.44 / Chapter 2.7.2.2 --- Adult Fly Heads --- p.44 / Chapter 2.7.2.3 --- Larvae --- p.45 / Chapter 2.7.3 --- SDS-Polyacrylamide Gel Electrophoresis --- p.45 / Chapter 2.7.4 --- Immunoblotting --- p.45 / Chapter CHAPTER 3. --- PHENOTYPIC CHARACTERIZATION OF RIG MUTANT / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Re-balancing of rig Mutant Fly Lines Over the Cy; Tb Compound Balancer for Genotype Identification --- p.48 / Chapter 3.3 --- Verification of Model Genotype --- p.49 / Chapter 3.4 --- rig Mutant Larvae Displayed Abnormal Motor Behavior / Chapter 3.4.1 --- Contraction Rate of rig Mutant Larvae --- p.54 / Chapter 3.4.2 --- Traveling Distance of rig Mutant Larvae --- p.56 / Chapter 3.5 --- rig Mutant Larvae Showed Normal Body Wall Musculature --- p.58 / Chapter 3.6 --- rig Mutant Larvae Displayed Defects in the Neuromuscular Junction / Chapter 3.6.1 --- rig Mutant Larvae Showed Branching Defects --- p.60 / Chapter 3.6.2 --- rig Mutant Larvae Showed Fewer Boutons Number --- p.62 / Chapter 3.7 --- rig Mutant Larvae Showed Normal Active Zone Pattern --- p.64 / Chapter 3.8 --- Discussion --- p.66 / Chapter CHAPTER 4. --- A GENETIC SCREEN TO IDENTIFY GENES THAT INTERACT GENETICALLY WITH RIG / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Candidates and Design of the Screen --- p.72 / Chapter 4.3 --- Re-balancing of Deletion Lines Over the Cy; Tb Compound Balancer --- p.75 / Chapter 4.4 --- Identification of Chromosomal Regions That Genetically Interact With rig --- p.75 / Chapter 4.5 --- Identification of NMJ Genes That Genetically Interact With rig --- p.80 / Chapter 4.6 --- Discussion --- p.83 / Chapter CHAPTER 5. --- ATTEMPTS TO INVESTIGATE RIG FUNCTION IN PRE-AND POST-SYNAPTIC REGIONS OF THE NMJ / Chapter 5.1 --- Introduction --- p.89 / Chapter 5.2 --- Transgenic Rescue Experiment by Transgenic Expression of rig in rig Mutant / Chapter 5.2.1 --- Design of the Rescue Experiment --- p.90 / Chapter 5.2.2 --- Construct of pUAST-rig-FLAG --- p.93 / Chapter 5.2.3 --- Construct of the pUAST-myc-rig --- p.98 / Chapter 5.3 --- Tissue Specific Knockdown of rig expression --- p.102 / Chapter 5.4 --- Discussion --- p.105 / Chapter CHAPTER 6. --- ESTABLISHMENT OF AN INDUCIBLE S2R⁺ CELL MODEL FOR RIG OVEREXPRESSION / Chapter 6.1 --- Introduction --- p.108 / Chapter 6.2 --- Detection of Rig Protein in S2R⁺ Cells by Immunoblotting --- p.111 / Chapter 6.3 --- Detection of Rig Protein in S2R⁺ Cells by Immunostaining --- p.111 / Chapter 6.4 --- Detection of RNA in Immunopurified Rig Protein --- p.113 / Chapter 6.5 --- Discussion --- p.115 / Chapter CHAPTER 7. --- GENERAL DISCUSSION --- p.117 / References --- p.120 / Appendices --- p.126
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328426 |
| Date | January 2013 |
| Contributors | Cheng, Yat Pang., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xii, 132 leaves) : ill. (some col.) |
| 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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