DNA methylation, histone modification and the transcription factor dE2F1 in Drosophila [Elektronische Ressource] / Alexandra Weyrich

DNA methylation, histone modification and the transcription factor dE2F1 in Drosophila Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften Eingereicht am Fachbereich Biologie der Johannes Gutenberg Universität Mainz Erarbeitet im DAAD/MPG Labor an der Chinesischen Akademie der Biologischen Wissenschaften, Shanghai/China Alexandra Weyrich geboren am 20. März 1979 in Birkenfeld Mainz, April 2007 Index Index I. DNA methylation in the male germ line of Drosophila 1. Abstract___________________________________________________________ 15 2. Introduction_______________________________________________________ 16 3. Material & Methods________________________________________________ 19 3.1 Subcloning and Microinjection 19 3.1.1 β2t-Dnmt3a/3b constructs 19 3.1.2 UAS-Dnmt3a/3b constructs 22 3.2 GAL4 expression of β2t- and UAS-Dnmt3a/3b 22 3.3 Western Blots 23 3.4 In Vitro Methylation Assay 24 3.5 2D-Thin Layer Chromatography 24 3.6 DAPI- and Immunostaining of Testis 25 3.7 Testis preparation for HPLC and MALDI-TOF 26 3.8 High Performance Liquid Chromatography (HPLC) analysis 26 3.9 Matrix-assisted laser desorption ionization – time of flight mass 27 spectrometry (MALDI-TOF) analysis 4. Results___________________________________________________________ 28 4.1 Does DNA methylation exist in Drosophila? 28 4.2 DNA methylation in Dnmt-transformant flies 31 4.2.1 Dnmt3a 31 4.2.