Choi, Ching Gee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 127-132). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ABSTRACT (Chinese version) --- p.iii / ACKNOWLEDGEMENTS --- p.iv / LISTS OF ABBREVIATIONS --- p.vi / LISTS OF TABLES --- p.ix / LISTS OF FIGURES --- p.x / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- The nucleolus ´ؤ a major site for ribosome biogenesis --- p.1 / Chapter 1.1.1 --- Ribosome biogenesis --- p.1 / Chapter 1.1.1.1 --- Eukaryotic ribosomal RNA processing --- p.4 / Chapter 1.1.1.2 --- Ribosome assembly --- p.5 / Chapter 1.1.2 --- Trans-acting proteins in ribosome biogenesis --- p.8 / Chapter 1.1.3 --- rRNA processing and ribosome assembly are two coordinated process --- p.9 / Chapter 1.2 --- Dribble (DBE) ´ؤ an essential nucleolar RNA-binding protein --- p.11 / Chapter 1.2.1 --- DBE carries a RNA-binding KH-domain --- p.12 / Chapter 1.2.2 --- RNA-binding properties of DBE --- p.13 / Chapter 1.2.3 --- DBE is involved in rRNA processing --- p.15 / Chapter 1.2.4 --- Yeast homolog of DBE 一 Krrlp is associated with ribosome biogenesis --- p.19 / Chapter 1.3 --- Aims of Research --- p.22 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Expression and purification of DBE protein in Escherichia coli --- p.24 / Chapter 2.1.1 --- DNA construct --- p.24 / Chapter 2.1.2 --- Bacterial culture --- p.24 / Chapter 2.1.3 --- Purification of DBE protein --- p.25 / Chapter 2.2 --- Drosophila cell culture and preparation of protein lysates --- p.27 / Chapter 2.2.1 --- Drosophila S2 cell culture --- p.27 / Chapter 2.2.2 --- Preparation of protein lysates from Drosophila cultured cells --- p.27 / Chapter 2.2.3 --- Ribonuclease A (RNase A) treatment of protein lysates from Drosophila cultured cells --- p.28 / Chapter 2.3 --- Drosophila culture and genetics --- p.28 / Chapter 2.3.1 --- Drosophila culture --- p.28 / Chapter 2.3.2 --- GAL4/UAS transgene expression in Drosophila --- p.29 / Chapter 2.4 --- Affinity pull-down --- p.29 / Chapter 2.4.1 --- Immobilization of proteins onto AminoLink Plus coupling beads --- p.29 / Chapter 2.4.2 --- Affinity pull-down using protein coupled-beads --- p.32 / Chapter 2.5 --- Trichloroacetic acid protein precipitation --- p.33 / Chapter 2.6 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.7 --- Coomassie blue staining and destaining --- p.35 / Chapter 2.8 --- Protein identification by tandem mass spectrometry --- p.35 / Chapter 2.8.1 --- In-gel trypsin digestion of protein bands --- p.35 / Chapter 2.8.2 --- Peptide extraction --- p.37 / Chapter 2.8.3 --- Desalting of digested peptides --- p.38 / Chapter 2.8.4 --- Mass spectrometric analysis of protein candidates --- p.38 / Chapter 2.9 --- Electrotransfer and Western blotting --- p.39 / Chapter 2.10 --- Sucrose gradient sedimentation --- p.41 / Chapter 2.11 --- Glutathione S-transferase (GST) pull-down assay --- p.42 / Chapter 2.11.1 --- Construction of pRSETA-GST-r̐ư̐ưأ̐ơة plasmid --- p.42 / Chapter 2.11.1.1 --- Total RNA preparation from fly heads --- p.42 / Chapter 2.11.1.2 --- DNase treatment on extracted RNA --- p.43 / Chapter 2.11.1.3 --- Reverse Transcription --- p.44 / Chapter 2.11.1.4 --- PCR amplification of DNA fragment for cloning --- p.45 / Chapter 2.11.1.5 --- Agarose gel electrophoresis --- p.47 / Chapter 2.11.2 --- Overexpression of GST and GST-RpS9 proteins --- p.47 / Chapter 2.11.3 --- GST pull-down assay --- p.48 / Chapter 2.12 --- Immunoprecipitation ofFLAG-DBE in Drosophila --- p.49 / Chapter 2.13 --- Reagents and buffers --- p.50 / Chapter 2.13.1 --- Bacterial culture medium --- p.50 / Chapter 2.13.2 --- Buffers for purification of DBE protein in Escherichia coli --- p.51 / Chapter 2.13.3 --- Reagent for preparing protein lysates from Drosophila cultured cells --- p.53 / Chapter 2.13.4 --- Reagents for Drosophila culture and genetics --- p.53 / Chapter 2.13.5 --- Buffers for immobilization of proteins onto AminoLink Plus coupling gel --- p.55 / Chapter 2.13.6 --- Buffers for affinity pull-down using protein coupled-beads --- p.56 / Chapter 2.13.7 --- Reagents for SDS-PAGE --- p.56 / Chapter 2.13.8 --- Reagent for Coomassie Blue Staining and Destaining --- p.58 / Chapter 2.13.9 --- Reagents for protein identification by tandem mass spectrometry --- p.59 / Chapter 2.13.10 --- Reagents for electrotransfer and Western blotting --- p.62 / Chapter 2.13.11 --- Reagents for sucrose gradient sedimentation --- p.63 / Chapter 2.13.12 --- Reagents for agarose gel electrophoresis --- p.64 / Chapter 2.13.13 --- Reagents for immunoprecipitation --- p.65 / Chapter 2.13.14 --- Other common buffer --- p.66 / Chapter 3. --- RESULTS / Chapter 3.1 --- Identification of DBE protein interactors by affinity pull-down --- p.67 / Chapter 3.1.1 --- Introduction --- p.67 / Chapter 3.1.2 --- Results --- p.68 / Chapter 3.1.2.1 --- Expression and purification of DBE protein in E. coli --- p.68 / Chapter 3.1.2.2 --- Affinity pull-down using DBE-coupled beads and the control using lysozyme-coupled beads --- p.70 / Chapter 3.1.2.3 --- Affinity pull-down using DBE-coupled beads and the control using heat-denatured DBE-coupled beads --- p.73 / Chapter 3.1.2.4 --- Tandem mass spectrometric identification of proteins pulled down by DBE-coupled beads --- p.75 / Chapter 3.1.2.5 --- Study of the RNA-dependence of the interactions between DBE and its interactors --- p.83 / Chapter 3.1.3 --- Discussion --- p.89 / Chapter 3.1.3.1 --- Most DBE-interactors pulled down by DBE-coupled beads were shown to be specific --- p.89 / Chapter 3.1.3.2 --- Most of the potential DBE-interactors were found in the nucleolar proteome --- p.91 / Chapter 3.1.3.3 --- Comparison of protein interactions identified in DBE and Krrlp --- p.92 / Chapter 3.1.3.4 --- The implications of DBE-interacting ribosomal proteins on the role of DBE in ribosome biogenesis --- p.94 / Chapter 3.2 --- Study of the sedimentation behavior of DBE by sucrose gradient sedimentation --- p.97 / Chapter 3.2.1 --- Introduction --- p.97 / Chapter 3.2.2 --- Results --- p.98 / Chapter 3.2.2.1 --- The sedimentation behaviors of endogenous DBE and overexpressed FLAG-DBE --- p.98 / Chapter 3.2.3 --- Discussion --- p.100 / Chapter 3.2.3.1 --- The sedimentation behaviors of endogenous DBE and overexpressed FLAG-DBE was similar --- p.100 / Chapter 3.2.3.2 --- DBE associates with a macromolecular complex --- p.101 / Chapter 3.3 --- Study of the interaction between DBE and RpS9 by GST pull-down assay --- p.103 / Chapter 3.3.1 --- Introduction --- p.103 / Chapter 3.3.2 --- Results --- p.104 / Chapter 3.3.2.1 --- The constructs for GST and GST-RpS9 expression --- p.104 / Chapter 3.3.2.2 --- Purified DBE protein was pulled down by GST-RpS9 --- p.104 / Chapter 3.3.3 --- Discussion --- p.107 / Chapter 3.3.3.1 --- Further investigations on the interaction between DBE and RpS9 --- p.107 / Chapter 3.3.3.2 --- The implications of interaction between DBE and RpS9 on ribosome biogenesis --- p.107 / Chapter 3.3.3.3 --- The RNA dependence of the interaction between RpS9 and DBE --- p.111 / Chapter 3.4 --- Study of the association between DBE and histone proteins by co-immunoprecipitation and sucrose gradient sedimentation --- p.112 / Chapter 3.4.1 --- Introduction --- p.112 / Chapter 3.4.2 --- Result --- p.113 / Chapter 3.4.2.1 --- Histone H3 was not co-immunoprecipitated with FLAG-DBE --- p.113 / Chapter 3.4.2.2 --- Histone H3 did not co-sediment with DBE in sucrose gradient sedimentation --- p.115 / Chapter 3.4.3 --- Discussion --- p.116 / Chapter 3.4.3.1 --- Association between DBE and histone H3 could not be found --- p.116 / Chapter 3.4.3.2 --- Further investigation on the association between DBE and histone proteins --- p.118 / Chapter 4. --- GENERAL DISCUSSION --- p.119 / Chapter 5. --- CONCLUSION --- p.125 / Chapter 6. --- REFERENCES --- p.127
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326088 |
| Date | January 2007 |
| Contributors | Choi, Ching Gee., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvii, 132 leaves : col. 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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