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Determination of phosphorylation sites of Drosophila melanogaster exuperantia protein by site-directed mutagenesis.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322811
Date January 1999
ContributorsChan, Kam Leung., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvi, 187 leaves : ill. (some col., some mounted) ; 30 cm.
RightsUse 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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