Chan chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 89-95). / Abstracts in English and Chinese. / Dedication --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Abbreviations --- p.vi / List of Figures --- p.ix / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1. --- Thesis synopsis --- p.1 / Chapter 1.2. --- hNP220 protein --- p.1 / Chapter 1.2.1. --- Domain organization --- p.1 / Chapter 1.2.2. --- Known splice variants --- p.5 / Chapter 1.2.3. --- Subcellular localization --- p.7 / Chapter 1.2.4. --- Proposed roles in transcriptional activation and RNA processing --- p.7 / Chapter 1.2.5. --- Interaction between C-terminal of hNP220 and FHL2 --- p.9 / Chapter 1.3. --- Hypothesis --- p.12 / Chapter 1.4. --- Principles of key methods --- p.14 / Chapter 1.4.1. --- RLM-RACE --- p.14 / Chapter 1.4.2. --- CytoTrap® two-hybrid system --- p.15 / Chapter CHAPTER 2 --- Materials and Methods --- p.18 / Chapter 2.1. --- Cloning protocol --- p.18 / Chapter 2.1.1. --- Amplification of DNA fragment --- p.18 / Chapter 2.1.2. --- Purification of PCR product --- p.19 / Chapter 2.1.3. --- Restriction endonuclease digestion --- p.20 / Chapter 2.1.4. --- Dephosphorylation of cloning vector 5'-termini --- p.20 / Chapter 2.1.5. --- Insert/vector ligation --- p.20 / Chapter 2.1.6. --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.21 / Chapter 2.1.7. --- Transformation of ligation product into chemically competent bacterial cells --- p.22 / Chapter 2.1.8. --- Small-scale preparation of bacterial plasmid DNA --- p.22 / Chapter 2.1.9. --- Screening for recombinant clone --- p.24 / Chapter 2.1.10. --- Dideoxy DNA sequencing --- p.24 / Chapter 2.1.11. --- Midi-scale preparation of recombinant plasmid DNA --- p.25 / Chapter 2.2. --- Determination of the transcription start site (TSS) of hNP220 gene --- p.27 / Chapter 2.2.1. --- RNA ligase-mediated rapid amplification of cDNA 5'-end (5-RLM-RACE) --- p.27 / Chapter 2.3. --- Isolation and identification of the third splice variant of HNP220 (hNP220ε) --- p.29 / Chapter 2.3.1. --- PCR from human heart/testis cDNAs pool --- p.29 / Chapter 2.3.2. --- RT-PCR --- p.29 / Chapter 2.3.3. --- Northern hybridization --- p.30 / Chapter 2.4. --- Human tissue distribution of hNP220 --- p.31 / Chapter 2.4.1. --- RT-PCR --- p.31 / Chapter 2.4.2. --- Northern hybridization --- p.31 / Chapter 2.5. --- Visualization of the subcellular localization patterns of GFP-tagged hNP220ε in HepG2 cell line --- p.32 / Chapter 2.5.1. --- Cloning of hNP220a and hNP220s into vector pEGFP-Cl --- p.32 / Chapter 2.5.2. --- Transfection of GFP fusion constructs into HepG2 cell line --- p.32 / Chapter 2.5.3. --- Epi-fluorescence microscopy --- p.33 / Chapter 2.6. --- Identification of the protein-protein interaction between hNP220ε and MAPRE1 --- p.34 / Chapter 2.6.1. --- CytoTrap® XR HeLa Cell cDNA Library screening --- p.34 / Chapter 2.6.1.1. --- Cloning of hNP220ε into yeast two-hybrid bait vector pSos --- p.34 / Chapter 2.6.1.2. --- Preparation of cdc25Ha & cdc25Hα yeast competent cells --- p.34 / Chapter 2.6.1.3. --- Autonomous activation study of bait fusion construct pSos-hNP220ε --- p.36 / Chapter 2.6.1.4. --- Cotransformation of pSos-hNP220ε and CytoTrap® XR HeLa Cell cDNA Library --- p.36 / Chapter 2.6.1.5. --- Verification of interaction by yeast mating --- p.38 / Chapter 2.6.1.5.1. --- Generation of yeast plasmid segregant for mating --- p.38 / Chapter 2.6.1.5.2. --- Yeast mating in 96-well plate --- p.39 / Chapter 2.6.1.6. --- Identification of putative interaction partner --- p.39 / Chapter CHAPTER 3 --- Results --- p.42 / Chapter 3.1. --- Transcription start site of the HNP220 gene is located 312 nucleotides upstream the initiation codon --- p.42 / Chapter 3.2. --- Third splice variant of hNP220 gene hNP220s) is identified --- p.44 / Chapter 3.3. --- In silico analysis of hNP220ε --- p.54 / Chapter 3.4. --- hNP220a and hNP220s are ubiquitously expressed in human fetal and adult tissues --- p.65 / Chapter 3.5. --- hNP220ε shows a punctate subnuclear localization pattern in HepG2 cell line --- p.67 / Chapter 3.6. --- hNP220ε interacts with MAPRE1 --- p.69 / Chapter CHAPTER 4 --- Discussion --- p.71 / Chapter 4.1. --- "Identification of hNP220s, the third splice variant of hNP220 gene" --- p.71 / Chapter 4.2. --- Biological resemblance between hNP220α (hNP220) and hNP220ε --- p.73 / Chapter 4.3. --- Protein-protein interaction between hNP220ε and MAPRE1 --- p.74 / Chapter 4.3.1. --- MAPRE1 protein --- p.77 / Chapter 4.3.2. --- Wnt signaling pathway --- p.78 / Chapter 4.4. --- Potential roles of hNP220 in the regulation of chromosome stability and oncogenesis --- p.82 / Chapter 4.5. --- Summary --- p.85 / Chapter 4.6. --- Concluding questions --- p.86 / Chapter 4.7. --- Future work --- p.87 / References --- p.89 / Appendix --- p.96
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324423 |
| Date | January 2003 |
| Contributors | Chan, Chi-wai., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvi, 98 leaves : ill. (some col.) ; 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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