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Identification and Characterization Of IDENTIFICATION AND CHARACTERIZATION OF DEAFNESS GENES IN DROSOPHILA MELANOGASTER Dissertation for the award of the degree “Doctor rerum naturalium” Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Pingkalai Senthilan from Inuvil, Sri Lanka Göttingen 2010 Page 1 Members of the Thesis Committee Prof. Dr. Martin Göpfert (Supervisor) Georg-August-University Göttingen, Cellular Neurobiology Prof. Dr. André Fiala (Reviewer) Georg-August-University Göttingen, Molecular Neurobiology of Behavior Prof. Dr. Ralf Heinrich (Reviewer) Georg-August-University, Neurobiology Oral examination: 24.01.2011 Page 2 I herewith declare that the Ph.D. thesis entitled “Identification and Characterization of Deafness Genes in Drosophila melanogaster” has been written independently and with no other sources and aids than quoted. Pingkalai Senthilan Göttingen, December 2010 Page 3 Page 4 Table of Contents 1 Summary ......................................................................................................................................... 9 2 Introduction ................................................................................................................................... 11 2.1 Hearing impairment .............................................................................................................. 11 2.2 Genes involved in human hearing impairment ..................................................................... 12 2.3 Drosophila as a model organism for the study of hearing .................................................... 12 2.4 Drosophila sound communication ........................................................................................ 13 2.5 The Drosophila hearing organ ............................................................................................... 13 2.6 atonal .................................................................................................................................... 15 2.7 Similar molecular machineries .............................................................................................. 16 2.8 Genetic Screens ..................................................................................................................... 16 2.9 Reverse genetics .................................................................................................................... 17 3 Material and Methods ................................................................................................................... 19 3.1 Microarray Assay ................................................................................................................... 19 3.1.1 Total RNA Preparation ................................................................................................... 19 3.1.2 Two-cycle amplification and hybridization.................................................................... 20 3.1.3 Data analysis .................................................................................................................. 20 3.2 Microarray Validation ............................................................................................................ 21 3.2.1 RNA extraction and cDNA amplification........................................................................ 21 3.2.2 Quantitative real-time PCR (qPCR) ................................................................................ 21 3.2.3 Comparing qPCR Data with Microarray Data ................................................................ 22 3.3 In situ hybridization ............................................................................................................... 22 3.3.1 cDNA synthesis .............................................................................................................. 22 3.3.2 PCR using BioThermD-™ Taq DNA Polymerase ............................................................. 23 3.3.3 PCR using illustraTM PuReTaqTM READY-TO-GOTM PCR beads ......................................... 23 3.3.4 Gel Electrophoresis ........................................................................................................ 24 3.3.5 TOPO cloning ................................................................................................................. 24 3.3.6 Chemical Competent Cells ............................................................................................. 24 3.3.7 Transformation .............................................................................................................. 25 3.3.8 Colony PCR..................................................................................................................... 25 Page 5 3.3.9 Mini Preparation............................................................................................................ 26 3.3.10 Midi Preparation............................................................................................................ 26 3.3.11 Sequencing .................................................................................................................... 27 3.3.12 Restriction digestion ...................................................................................................... 27 3.3.13 IVT (in vitro transcription) ............................................................................................. 28 3.3.14 Fixation of Antennae ..................................................................................................... 28 3.3.15 Vibratome Sections ....................................................................................................... 29 3.3.16 In situ hybridization in antennal sections ...................................................................... 29 3.3.17 Collection and Fixation of Drosophila embryos ............................................................ 30 3.3.18 In situ hybridization in Drosophila embryos .................................................................. 31 3.3.19 Light Microscopy ........................................................................................................... 31 3.4 GAL4-lines .............................................................................................................................. 32 3.4.1 Long Range PCR ............................................................................................................. 32 3.4.2 Molecular Cloning ......................................................................................................... 32 3.4.3 Mini Prep & Restriction Digestion ................................................................................. 33 3.4.4 Sequencing .................................................................................................................... 33 3.4.5 TheBestGene ................................................................................................................. 33 3.4.6 Crossing ......................................................................................................................... 34 3.4.7 Confocal Microscopy ..................................................................................................... 34 3.5 Fly pushing ............................................................................................................................. 34 3.5.1 Fly Food ......................................................................................................................... 34 3.6 Mechanical measurements ................................................................................................... 35 3.7 Mutant qPCR ......................................................................................................................... 35 3.7.1 RNA extraction & cDNA synthesis ................................................................................. 35 3.7.2 qPCR .............................................................................................................................. 35 3.8 Antibody staining................................................................................................................... 36 3.8.1 Single staining ................................................................................................................ 36 3.8.2 Double staining .............................................................................................................. 36 3.8.3 Propium idodide to stain the nucleus ........................................................................... 36 Page 6 3.9 Materials ................................................................................................................................ 37 3.9.1 Chemicals ....................................................................................................................... 37 3.9.2 Enzymes, Kits and Substrates ........................................................................................ 39 3.9.3 Other Reagents .............................................................................................................. 42 3.9.4 Buffers and Solutions .................................................................................................... 43 3.9.5 Antibodies...................................................................................................................... 46 3.9.6 DNA Ladder...................................................................................................................
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