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Inositol Phosphate in the Basidiomycete Fungus Schizophyllum Commune

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Inositol Phosphate in the Basidiomycete Fungus Schizophyllum Commune Inositol phosphate in the basidiomycete fungus Schizophyllum commune Dissertation To fulfill the Requirements for the Degree of „doctor rerum naturalium“ (Dr. rer. nat.) Submitted to the Council of the Faculty of Biological Science of the Friedrich Schiller University Jena by Reyna Carmina Felicia Murry, MSc. born on the 22nd of October 1987 in Bandung, Indonesia First reviewer: Prof. Dr. Erika Kothe, Institut für Mikrobiologie, FSU Jena Second Reviewer: Prof. Dr. Axel A. Brakhage, Institut für Mikrobiologie, FSU Jena Third Reviewer: Prof. Dr. J. Stephen Horton, Department of Biological Sciences, Science and Engineering Center, Union College, Schenectady, New York, USA Date of public defense: 7th May 2019 “Science is the key to our future, and if you don't believe in science, then you're holding everybody back.” Bill Nye Table of Contents 1 Introduction ......................................................................................................................... 1 1.1 Schizophyllum commune .............................................................................................. 1 1.2 Fruiting body development in S. commune ................................................................. 3 1.3 Signal transduction: G-protein-coupled receptors (GPCRs) and Ras signaling .......... 4 1.4 Inositol-based metabolism and signaling: inositol phosphates and inositol lipids ...... 5 1.5 Inositol and inositol monophosphatase ........................................................................ 7 1.6 Inositol hexakisphosphate and the inositol phosphate kinases .................................... 8 1.7 Aim of the study .......................................................................................................... 8 2 Materials and methods ...................................................................................................... 10 2.1 Chemicals, reagents and enzymes ............................................................................. 10 2.2 Cultivation media ...................................................................................................... 10 2.3 Organisms, plasmids, and nucleotides ....................................................................... 11 2.4 Experiments with E. coli DH5α ................................................................................. 13 2.4.1 Growth conditions and production of electrocompetent cells ............................ 13 2.4.2 Transformation of E. coli DH5α ........................................................................ 13 2.5 Experiments with S. commune ................................................................................... 14 2.5.1 Cultivation of S. commune ................................................................................. 14 2.5.2 Growth rate measurement and dry weight determination .................................. 14 2.5.3 Rapid DNA isolation .......................................................................................... 14 2.5.4 DNA isolation using CTAB ............................................................................... 15 2.5.5 RNA isolation ..................................................................................................... 15 2.5.6 Quality, purity, and quantitation for DNA and RNA ......................................... 16 2.6 Inositol monophosphatase activity measurement ...................................................... 16 2.6.1 Crude extraction of S. commune ........................................................................ 16 2.6.2 Protein quantitation ............................................................................................ 16 2.6.3 Malachite Green assay ....................................................................................... 17 2.7 Protoplast preparation and transformation of S. commune an ................................... 17 I 2.8 Construction plasmid: imp overexpression for S. commune ...................................... 18 2.8.1 Yeast Recombination ......................................................................................... 18 2.8.2 Cultivation of S. cerevisiae BY4741, ∆LEU ...................................................... 19 2.8.3 Yeast transformation .......................................................................................... 20 2.8.4 Screening and validation of recombined product ............................................... 20 2.8.5 Cloning of tef1 promoter and imp gene to pTrp ................................................. 21 2.9 Proteome analysis using 2D-PAGE ........................................................................... 21 2.9.1 Cytosolic protein isolation ................................................................................. 21 2.9.2 First and second dimension of 2D gel electrophoresis ....................................... 22 2.9.3 Protein/ peptide analysis using MALDI TOF/MS-MS ...................................... 23 2.10 Signal protein analysis ............................................................................................... 24 2.10.1 Signal protein isolation and purification ............................................................ 24 2.10.2 LC-MS/MS analysis and protein database search .............................................. 25 2.11 Microscopy ................................................................................................................ 26 2.11.1 Stereomicroscopy and fluorescence microscopy ............................................... 26 2.11.2 Confocal Laser Scanning Microscopy ............................................................... 26 2.12 Inositol phosphates profile analysis ........................................................................... 27 2.12.1 Radioactive labelling and extraction of inositol phosphates .............................. 27 2.12.2 HPLC run and detection using scintillation counter .......................................... 27 2.13 Other molecular biology methods ............................................................................. 28 2.13.1 Polymerase chain reaction (PCR) ...................................................................... 28 2.13.2 Real Time-quantitative PCR (RT-qPCR) ........................................................... 29 2.13.3 DNA restriction endonucleases .......................................................................... 29 2.13.4 DNA sequencing ................................................................................................ 29 2.13.5 Plasmid DNA preparation .................................................................................. 30 2.13.6 Determination of Protein Kinase C activity ....................................................... 30 2.14 In silico analysis ........................................................................................................ 30 2.14.1 Inferring phylogeny ............................................................................................ 30 2.14.2 Chromosomal organization of imp surrounding gene ........................................ 31 II 2.14.3 Other protein analysis ......................................................................................... 31 3 Results ............................................................................................................................... 32 3.1 Inositol phosphate in S. commune ............................................................................. 32 3.2 Inositol phosphate phosphatase and inositol phosphate kinases ............................... 34 3.3 Inositol phosphate kinases gene expression .............................................................. 36 3.4 Proteins involved in the development of S. commune ............................................... 37 3.5 Lithium effect on inositol phosphate in S. commune ................................................. 39 3.5.1 Lithium effect on the growth and mating in S. commune .................................. 39 3.5.2 Lithium effect on inositol phosphate and inositol monophosphatase activity ... 41 3.5.3 Lithium effect on S. commune metabolism and cellular signaling..................... 42 3.5.4 Lithium effect on actin dynamics ....................................................................... 44 3.6 Inositol monophosphatase and inositol monophosphatase-like gene family ............. 45 3.7 Inositol monophosphatase ......................................................................................... 46 3.8 Comparative genetic map of imp and surrounding genes .......................................... 48 3.9 Overexpression of inositol monophosphatase (imp) ................................................. 50 3.9.1 Validation of imp overexpression ...................................................................... 50 3.9.2 Characterization of imp overexpressing mutants ............................................... 51 3.9.3 Mating interaction of imp overexpressing mutants ............................................ 53 3.9.4 Inositol phosphates profile in imp overexpressing mutants ............................... 54 3.9.5 Effect of SDS on imp overexpressing mutants ................................................... 55 3.10 Proteome analysis in imp overexpressing mutants .................................................... 56 3.11 Membrane trafficking related proteins are induced in imp overexpressing strains ... 58 3.12 The effect of Brefeldin A on membrane trafficking
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