Characterization of the enzymes involved in the glutathione synthesis and the redox enzyme glutathione reductase in Caenorhabditis elegans (Maupas, 1900) Dissertation Submitted for the attainment of a doctoral degree from the Department of Biology, Faculty of Mathematics, Informatics and Natural Sciences of the University of Hamburg, Germany by Caroline Enjuakwei Ajonina Buzie (Bamenda, Cameroon) Hamburg 2006 Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes (DAAD) Table of contents Table of contents CHAPTER 1 INTRODUCTION ................................................................................... 1 1.1 Nematodes ............................................................................................................................... 1 1.1.1 Pathogenesis and host- parasite relationship ................................................................. 1 1.1.2 Caenorhabditis elegans ...................................................................................................... 2 1.1.2.1 Classification .................................................................................................................. 3 1.1.2.2 Life cycle of C. elegans ................................................................................................. 3 1.1.2.3 C. elegans as a model system .................................................................................... 4 1.2 Microinjection in C. elegans ................................................................................................... 5 1.3 RNA interference in C. elegans ............................................................................................. 5 1.3.1 Mechanism of RNA interference ....................................................................................... 6 1.4 Oxidative stress and its effect to the cell .............................................................................. 7 1.4.1 Sources of reactive oxygen species. ................................................................................ 9 1.4.2 The effect of reactive oxygen species in biological systems........................................ 9 1.5 Glutathione ............................................................................................................................. 10 1.5.1 Glutathione biosynthesis, metabolism, and utilization ................................................. 11 1.6 Enzymes of the GSH synthesis ........................................................................................... 17 1.6.1 γ-Glutamylcysteine synthetase ........................................................................................ 17 1.6.2 Glutathione synthetase ..................................................................................................... 20 1.6.3 Glutathione reductase ...................................................................................................... 22 1.7 Research objectives .............................................................................................................. 24 CHAPTER 2 MATERIALS AND METHODS .............................................................. 26 2.1 Chemicals and Bioreagents ................................................................................................. 26 2.2 Standard Solutions and Buffers ........................................................................................... 29 2.2.1 Media .............................................................................................................................. 31 2.3 Vectors and bacterial cells ................................................................................................... 31 2.3.1 Plasmid vectors ................................................................................................................. 31 Table of contents 2.3.2 Escherichia coli strains ..................................................................................................... 32 2.3.3 C. elegans strains .............................................................................................................. 32 2.4 Culture of C. elegans ............................................................................................................ 32 2.4.1 Growth conditions .............................................................................................................. 32 2.4.2 Separation of C. elegans from bacteria ......................................................................... 33 2.4.3 Axenic C. elegans culture ................................................................................................ 33 2.4.4 Freezing of worms ............................................................................................................. 33 2.5 Photometric determination of GSH concentration in C. elegans .................................... 34 2.6 Preparation of nucleic acids ................................................................................................. 35 2.6.1 Preparation of genomic DNA ........................................................................................... 35 2.6.2 Preparation of RNA from C. elegans .............................................................................. 36 2.6.3 Ethanol precipitation ......................................................................................................... 36 2.6.5 Phenol-chloroform extraction .......................................................................................... 36 2.7 Polymerase chain reaction (PCR) ....................................................................................... 37 2.7.1 Single worm PCR .............................................................................................................. 37 2.8 Reverse transcription ............................................................................................................ 37 2.9 Preparations of plasmid DNA .............................................................................................. 38 2.9.1 Mini preparation of plasmid DNA .................................................................................... 38 2.9.2 Midi and Maxi preparation of plasmid DNA ................................................................... 38 2.11 Agarose gel electrophoresis of DNA .................................................................................. 39 2.12 Recovery of DNA from agarose gels .................................................................................. 39 2.13 Ligation of DNA fragments sub-cloning in plasmid vectors ............................................. 39 2.14 Transformation ....................................................................................................................... 40 2.14.1 Competent bacteria production ....................................................................................... 40 2.14.2 Transformation of competent cells ................................................................................. 40 2.15 DNA sequencing and in silico analyses ............................................................................. 41 2.16 Recombinant expression of C. elegans GS ( Ce GS) ...................................................... 41 2.16.1 Cloning of Ce GS ............................................................................................................... 41 2.16.2 Induction of protein expression ....................................................................................... 42 2.16.3 Purification of re Ce GS ...................................................................................................... 42 Table of contents 2.16.4 Enzyme assay and kinetic analyses of recombinant Ce GS ....................................... 43 2.17 Recombinant expression of C. elegans GR ( Ce GR) ....................................................... 43 2.17.1 Cloning of Ce GR ............................................................................................................... 43 2.17.2 Induction of protein expression. ...................................................................................... 44 2.17.3 Purification of r Ce GR ........................................................................................................ 44 2.17.4 Kinetic analysis of Ce GR protein .................................................................................... 45 2.18 Recombinant expression of C. elegans γ-GCS ( Ce GCS) ................................................. 45 2.18.1 Cloning of Ce GCS ............................................................................................................ 45 2.18.2 Recombinant expression of Ce GCS .............................................................................. 45 2.19 Protein determination methods ............................................................................................ 47 2.19.1 Determination of protein concentration .......................................................................... 47 2.19.2 SDS-PAGE under denaturing conditions ...................................................................... 47 2.19.3 Protein staining gels ......................................................................................................... 48 2.19.3.1 Coomassie blue staining ........................................................................................... 48 2.19.3.2 Silver staining .............................................................................................................