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Doktorarbeit-Endfassung 5 Das BTB-Protein Psg1 als Suppressor von O-Glykosylierungsdefekten in Candida albicans Inaugural-Dissertation Zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Steffen Ullmann aus Lutherstadt Eisleben Düsseldorf 2013 I Aus dem Department Biologie; Institut für molekulare Mykologie, Heinrich-Heine- Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. J.F. Ernst Koreferent: Prof. Dr. R. Wagner Tag der mündlichen Prüfung: II Inhaltsverzeichnis Inhaltsverzeichnis 1. Einleitung ......................................................................................................... 1 1.1 Candida albicans als humanpathogener Pilz ....................................................................... 1 1.2 Glykosylierung als Form der Proteinmodifikation ............................................................... 2 1.2.1 N-Glykosylierung .......................................................................................................... 2 1.2.2 O-Glykosylierung .......................................................................................................... 3 1.3 Protein O-Mannosyltransferasen und die Phänotypen von pmt-Mutanten in C. albicans....5 1.3.1 Phänotypen von pmt-Mutanten in C. albicans.................................... .............................. 7 1.4 Andere Mutationen, welche die Pmt-Mannosylierung beeinflussen.................................... 9 1.5 Das Ubiquitin-Proteasom-System ...................................................................................... 10 1.5.1 E3-Ubiquitinligasen ..................................................................................................... 11 1.5.2 Die BTB-Domäne ........................................................................................................ 14 1.6 Zielsetzung ......................................................................................................................... 14 2. Material und Methoden ................................................................................ 16 2.1 Chemikalien und Enzyme .................................................................................................. 16 2.2 Stämme und Medien ........................................................................................................... 17 2.2.1 E. coli Stämme ......................................................................................................... 17 2.2.2 S. cerevisiae Stämme ............................................................................................... 17 2.2.3 C. albicans Stämme ................................................................................................. 17 2.2.4 C. albicans Stammkonstruktion ............................................................................... 19 2.2.5 Medien zur Anzucht von E. coli .............................................................................. 21 2.2.6 Medien zur Anzucht von Hefen ............................................................................... 21 2.2.7 Medien zur Anzucht von C. albicans....................................................................... 21 2.2.8 Sensitivitätstest ........................................................................................................ 22 2.2.9 Medien mit verschiedenen Substanzen .................................................................... 22 2.2.10 Hypheninduktion von C. albicans ......................................................................... 22 2.3 Primer und Plasmide .......................................................................................................... 23 2.3.1 Plasmide ................................................................................................................... 23 2.3.2 Primer ....................................................................................................................... 26 2.3.2 Plasmidkonstruktion ................................................................................................ 28 III Inhaltsverzeichnis 2.4 Präparation, Konstruktion und Analyse von Nukleinsäuren .............................................. 29 2.4.1 Isolierung von Plasmid-DNA aus E. coli ................................................................. 29 2.4.2 Isolierung von Gesamt-DNA aus Hefen .................................................................. 29 2.4.3 DNA-Restriktion ...................................................................................................... 29 2.4.4 Entfernen von 3´-überhängenden Enden.................................................................. 29 2.4.5 Auffüllen von 5´-überhängenden Enden .................................................................. 30 2.4.6 Dephosphorylierung von DNA ................................................................................ 30 2.4.7 Ligation .................................................................................................................... 30 2.4.8 Isolierung von DNA-Fragmenten aus Agarosegelen ............................................... 30 2.4.9 DNA-Größenstandards ............................................................................................ 30 2.4.10 Konzentrationsabschätzung von Nukleinsäuren .................................................... 30 2.4.11 Nachweis spezifischer Sequenzen über Southernblot-Analyse ............................. 30 2.4.11.1 Sondenmarkierung .............................................................................................. 31 2.4.11.2 Transfer auf eine Nylonmembran durch Vacuum-Blot ...................................... 31 2.4.11.3 Hybridisierung und Detektion ............................................................................. 31 2.4.12 Polymerase-Kettenreaktion (PCR) ......................................................................... 31 2.4.12.1 Kolonie-PCR ....................................................................................................... 31 2.4.12.2 Quantitative RT-PCR/ Real Time-PCR .............................................................. 32 2.5 Transformation ................................................................................................................... 35 2.5.1 Transformation von E. coli ...................................................................................... 35 2.5.2 Transformation von C. albicans .............................................................................. 35 2.6 Proteinbiochemische Methoden ......................................................................................... 36 2.6.2 Präparation von Hefe-Rohextrakten ........................................................................ 36 2.6.3 Bestimmung der Proteinkonzentration nach Bradford ............................................ 36 2.6.3.1 Proteinbestimmung nach Lowry ........................................................................... 36 2.6.4 PNGase F-Behandlung ............................................................................................. 37 2.6.5 SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) ............................................ 37 2.6.6 Nachweis spezifischer Proteine durch Immunoblot-Analyse .................................. 37 2.7 Zellbiologische Methoden .................................................................................................. 38 2.7.1 Immunfluoreszenz .................................................................................................... 38 2.7.1.1 Fixieren von Zellen ............................................................................................... 38 2.7.1.2 Herstellen der Präparate ........................................................................................ 38 2.8 Transkriptomanalyse .......................................................................................................... 39 2.8.1 DNA-Microarrays .................................................................................................... 39 IV Inhaltsverzeichnis 2.8.2 Anzucht der Zellen für Microarrays......................................................................... 39 2.8.3 Synthese der Komplementär-DNA .......................................................................... 39 2.8.4 Hybridisieren, Waschen und Scannen der DNA Microarrays ................................. 40 2.8.5 Normalisierung und statistische Auswertung .......................................................... 40 2.9 Proteinanalysen .................................................................................................................. 41 2.9.1 Qualitative Bestimmung der !-Galaktosidase-Aktivität („X-Gal Overlay-Assay“) 41 2.9.2 Quantitative Bestimmung der !-Galaktosidase-Aktivität („LacZ-Assay“) ............. 41 3. Ergebnisse ....................................................................................................................... 42 3.1 Analyse der Nukleotid- und Aminosäuresequenz von PSG1 ............................................. 43 3.1.1 Sequenz- und genomische Analyse von PSG1 in C. albicans .................................... 43 3.1.2 Aminosäuresequenz von Psg1 ..................................................................................... 45 3.1.2.1 Proteinmotive ...........................................................................................................
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