i The evolutionary history of the mammalian synaptonemal complex Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius- Maximilians-Universität Würzburg vorgelegt von Johanna Fraune aus Höxter Würzburg, 2014 Eingereicht am: ....................................................................................................................... Mitglieder der Promotionskommission: Vorsitzender: .......................................................................................................................... Gutachter : .............................................................................................................................. Gutachter: ............................................................................................................................... Tag des Promotionskolloquiums: ............................................................................................. Doktorurkunde ausgehändigt am: ............................................................................................ Index 3 Index Index ..................................................................................................................................... 3 1 List of figures ................................................................................................................. 6 2 List of tables ................................................................................................................... 7 3 Summary ....................................................................................................................... 8 4 Zusammenfassung ......................................................................................................... 9 5 General Introduction .................................................................................................... 11 5.1 Meiosis ................................................................................................................. 11 5.1.1 Meiosis I .............................................................................................................. 11 5.1.2 Meiosis II ............................................................................................................. 14 5.2 The synaptonemal complex (SC) ........................................................................... 15 5.2.1 The SC components of metazoan model organisms ........................................... 16 5.2.2 The function of SC components and their assembly to a mature SC .................. 20 6 Aims of this study......................................................................................................... 25 7 Chapter I: The evolution of the two bona fide structural SC components of the mouse 27 Introduction ..................................................................................................................... 27 Results ............................................................................................................................. 28 Sequence and structure of HySYCP1 and HySYCP3............................................................. 29 Expression of HySYCP1 and HySYCP3 in Hydra vulgris AEP ................................................ 32 Discussion ........................................................................................................................ 37 8 Chapter II: The evolution of the central element components of the murine SC ........... 40 Introduction ..................................................................................................................... 40 Results ............................................................................................................................. 40 Phylogeny of SYCE1, SYCE2, SYCE3 and Tex12 .................................................................... 40 Expression of HySYCE2 and HyTex12 in Hydra vulgaris AEP ............................................... 43 Interaction of HySYCE2 and HyTex12 ex vivo ...................................................................... 47 Discussion ........................................................................................................................ 47 Index 4 9 Chapter III: The evolution of the mammalian lateral element protein SYCP2 ................ 50 Introduction ..................................................................................................................... 50 Results ............................................................................................................................. 51 Phylogeny of SYCP2 ............................................................................................................. 51 Sequence and expression of the SYCP2 orthologue in Hydra vulgaris AEP ........................ 53 Discussion ........................................................................................................................ 55 10 General Discussion ................................................................................................... 57 10.1 The dynamic evolution of the SC ........................................................................... 57 10.2 The cnidarian SC: Hydra as model system for meiosis research? ........................... 60 11 Perspectives ............................................................................................................. 67 12 Material ................................................................................................................... 68 12.1 Biological material ................................................................................................ 68 12.1.1 Hydra lineage: Hydra vulgaris AEP ...................................................................... 68 12.1.2 Bacteria ............................................................................................................... 68 12.1.3 Cell lines .............................................................................................................. 69 12.1.4 Antibodies ........................................................................................................... 69 12.2 Molecular material ............................................................................................... 70 12.2.1 Plasmids .............................................................................................................. 70 12.2.2 Oligonucleotides ................................................................................................. 71 12.2.3 Recombinant DNA constructs ............................................................................. 73 12.3 Chemicals ............................................................................................................. 74 12.4 Computer software and Online Tools .................................................................... 75 12.5 Equipment ............................................................................................................ 75 13 Methods ................................................................................................................... 77 13.1 Microbiological methods ...................................................................................... 77 13.1.1 Culturing of bacteria ........................................................................................... 77 13.1.2 Production of competent bacteria ...................................................................... 78 13.1.3 Transformation of E. coli bacteria with plasmid DNA ......................................... 78 13.2 Molecular methods .............................................................................................. 79 13.2.1 RNA extraction .................................................................................................... 79 13.2.2 Purification of plasmid DNA ................................................................................ 79 13.2.3 Polymerase chain reaction (PCR) ........................................................................ 79 13.2.4 DNA sequencing .................................................................................................. 81 13.2.5 DNA gel electrophoresis ...................................................................................... 81 Index 5 13.2.6 Preparative DNA gel electrophoresis and gel extraction .................................... 82 13.2.7 DNA cloning ......................................................................................................... 83 13.3 Biochemical methods ........................................................................................... 84 13.3.1 Protein gel electrophoresis ................................................................................. 84 13.3.2 Coomassie staining .............................................................................................. 86 13.3.3 Western Blot analysis .......................................................................................... 86 13.3.4 Expression and purification of HIS‐tagged fusion proteins ................................. 88 13.3.5 Concentration and purification of proteins from aqueous solutions ................. 90 13.3.6 Synthesis of specific antibodies .......................................................................... 91 13.4 Cell culture ........................................................................................................... 94 13.4.1 Culturing COS‐7 cells ..........................................................................................