Chan Kam Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 175-182). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Drosophila as a model for studying development --- p.1 / Chapter 1.2 --- The formation of the body axis in Drosophila --- p.2 / Chapter 1.3 --- The maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.11 / Chapter 1.5 --- The formation of the biocid protein gradient from anterior pole to posterior pole of the embryo --- p.13 / Chapter 1.6 --- The bed protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.15 / Chapter 1.7 --- The formation of the bed protein gradient in embryo --- p.17 / Chapter 1.8 --- Components required for bcd mRNA localization at anterior pole of oocyte --- p.21 / Chapter 1.8.1 --- Cis-acting elements --- p.21 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.9 --- The properties of exuperantia protein --- p.25 / Chapter 1.9.1 --- The function of exu protein --- p.25 / Chapter 1.9.2 --- Exuperantia is a phosphoprotein --- p.26 / Chapter 1.9.3 --- Phosphorylation pattern of exuperantia protein is stage-specific --- p.28 / Chapter 1.9.4 --- Reversible phosphorylation is one of the major mechanisms to control protein activity in all eukaryotic cells --- p.29 / Chapter 1.9.5 --- The relationship between the exu protein phosphorylation and the bcd mRNA localization --- p.30 / Chapter 1.10 --- Aim of project --- p.31 / Chapter CHAPTER 2 --- Preparation of the exuperantia genomic DNA and complement DNA (cDNA) mutant Constructs / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- DNA preparation methods --- p.35 / Chapter 2.2.1.1 --- Preparation of double-stranded DNA by polyethylene glycol6000 --- p.35 / Chapter 2.2.1.2 --- Preparation of M13mp8 single-stranded DNA --- p.37 / Chapter 2.2.1.3 --- "Preparation of double-stranded DNA by Biol prep (Modified from Maniatis et al.,1989)" --- p.38 / Chapter 2.2.2 --- "Preparation of DH5α，JM109, TG1 competent cells" --- p.39 / Chapter 2.2.3 --- Bacteria transformation --- p.40 / Chapter 2.2.4 --- Restriction enzyme digestion --- p.40 / Chapter 2.2.5 --- Phenol/chloroform extraction --- p.41 / Chapter 2.2.6 --- Purification of DNA fragment by electro-elution --- p.42 / Chapter 2.2.7 --- DNA ligation --- p.43 / Chapter 2.2.8 --- DNA dephosphorylation --- p.43 / Chapter 2.2.9 --- In vitro site-directed mutagenesis --- p.44 / Chapter 2.2.9.1 --- The Sculptor´ёØ in vitro mutagenesis --- p.44 / Chapter 2.2.9.2 --- The GeneEditor´ёØ in vitro site-directed mutagenesis --- p.47 / Chapter 2.2.10 --- The double-stranded or single-stranded DNA sequencing by T7 DNA polymerase sequencing system --- p.50 / Chapter 2.2.11 --- Denatured polyacrylamide gel electorphoresis --- p.51 / Chapter 2.2.11 --- Nucleotide sequence of the sequencing primers and the mutageneic oligonucleotides --- p.54 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Design exuperantia mutant constructs --- p.55 / Chapter 2.3.1.1 --- Comparison of exu protein amino acids sequence with different Drosophila species --- p.56 / Chapter 2.3.2 --- The exu genomic mutant constructs --- p.63 / Chapter 2.3.3 --- The exu cDNA mutant constructs --- p.63 / Chapter 2.4 --- Discussion --- p.76 / Chapter CHAPTER 3 --- Epitope tagging of exuperantia protein with c-myc eptiope / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Materials and methods --- p.84 / Chapter 3.2.1 --- Preparation of the c-myc eptiope DNA fragment --- p.84 / Chapter 3.2.2 --- End-filling of 5'overhang DNA fragment by Klenow fragment --- p.86 / Chapter 3.2.3 --- In vitro translation of protein by TNT® Quick coupled transcription and translation system --- p.86 / Chapter 3.2.4 --- Immunoprecipitation of recombinant exu protein --- p.87 / Chapter 3.2.5 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 3.2.5.1 --- SDS-PAGE preparation --- p.88 / Chapter 3.2.5.2 --- SDS-PAGE electrophoresis --- p.90 / Chapter 3.2.6 --- Western blot analysis --- p.90 / Chapter 3.2.6.1 --- Transfer the protein to a nitro-cellulose membrane by semi-dried blotting --- p.90 / Chapter 3.2.6.2 --- Western blot blocking and antibody recognition --- p.91 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Construction of the plasmid containing exu cDNA tagging with a c-myc epitope --- p.92 / Chapter 3.3.2 --- In vitro translation of c-myc epitope tagged exu protein --- p.102 / Chapter 3.3.3 --- Immunoprecipitation of c-myc labeled exu protein by a polyclonal rabbit anti-exu antibody and monoclonal mouse anti-myc antibody --- p.104 / Chapter 3.4 --- Discussion --- p.109 / Chapter CHAPTER 4 --- In vitro phosphorylation of exuperantia Protein / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and methods --- p.113 / Chapter 4.2.1 --- Exogenous kinase phsophorylation reactions --- p.113 / Chapter 4.2.2 --- Separation of the phosphorylated exu protein variants by SDS- PAGE --- p.114 / Chapter 4.3 --- Results --- p.115 / Chapter 4.3.1 --- Western blot analysis of in vitro translated exu protein variants --- p.115 / Chapter 4.3.2 --- Phosphorylation of in vitro translated exu protein variants by exogenous cAMP-dependent protein kinase --- p.118 / Chapter 4.3.3 --- Phosphorylation of in vitro translated exu protein variants by exogenous cGMP-dependent protein kinase --- p.123 / Chapter 4.3.4 --- Phosphorylation of in vitro translated exu protein variants by exogenous protein kinase C --- p.128 / Chapter 4.4 --- Discussion --- p.133 / Chapter CHAPTER 5 --- Introduction of the exuperantia genomic constrcuts into the germline of Drosophila by P element-mediated transformation / Chapter 5.1 --- Introduction --- p.136 / Chapter 5.2 --- Materials and methods --- p.138 / Chapter 5.2.1 --- Construction of a genomic construct for production of transgenic flies --- p.138 / Chapter 5.2.2 --- Preparation of double-stranded DNA by ultra-centrifugation --- p.142 / Chapter 5.2.3 --- P-element mediated transformation --- p.143 / Chapter 5.2.3.1 --- Eggs collection --- p.143 / Chapter 5.2.3.2 --- Dechorionating the eggs --- p.143 / Chapter 5.2.3.3 --- Orientating the eggs --- p.144 / Chapter 5.2.3.4 --- Microinjection --- p.145 / Chapter 5.2.4 --- Collecting virgin female Drosophila --- p.146 / Chapter 5.2.5 --- Setup a crossing experiment --- p.146 / Chapter 5.2.6 --- Preparation of total ovaries and testes extracts exu protein from Female and male Drosophila --- p.147 / Chapter 5.2.7 --- Immunohistochemical distribution of exuperantia protein --- p.147 / Chapter 5.3 --- Results --- p.150 / Chapter 5.3.1 --- Insertion of the mutated exu fragments into the Drosophila Transformation vector (pCaSpeR) --- p.150 / Chapter 5.3.2 --- Introduction of the mutated exu gene into the genome of Drosophila by P-element mediated transformation --- p.153 / Chapter 5.3.3 --- Western blot analysis of the exu protein in the exu (ES2.1) transgenic fly --- p.160 / Chapter 5.3.4 --- Immunohistochemical distribution of exu protein in exuES21 mutants --- p.162 / Chapter 5.3.5 --- Rescue test of exuES2.1 trangenic flies --- p.165 / Chapter 5.4 --- Discussion --- p.168 / Chapter CHAPTER 6 --- General Discussion --- p.171 / References --- p.173 / Chapter Appendix I: --- List of reagents --- p.183 / Chapter Appendix II: --- Publication --- p.187
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322811 |
| Date | January 1999 |
| Contributors | Chan, Kam Leung., 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, 187 leaves : ill. (some col., some mounted) ; 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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