Inhalt

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   1 Introduction
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   1.1 Baker yeast as model organism
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   1.1.1 The yeast cell wall
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   1.1.2 Cell polarity of budding yeast cells
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   1.2 Protein transport in eukaryotic cells
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   1.3 Vesicle transport
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  1.3.1 Analysis of anterograde transport from TGN to vacuole
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   1.3.1.1 CPY transport
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   1.3.1.2 ALP transport
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  1.3.2 Retrograde transport from EE to TGN
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   1.3.2.1 A-ALP transport
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   1.3.2.2 Snc1p transport
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   1.4 SNAREs
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   1.4.1 The SNARE hypothesis
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   1.4.2 Molecular structure
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   1.4.3 SNAREs in membrane fusion
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  1.4.4 Yeast SNAREs
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   1.4.4.1 Pep12p
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   1.4.4.2 Vti1p
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   1.4.4.3 Syn8p
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  1.5 Adaptor proteins
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   1.5.1 ENTH-proteins
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   1.5.2 Structure and function of ENTH domain proteins
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   1.5.3 The yeast ENTH domain protein Ent3p
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   1.5.4 Interaction of Ent3p with endosomal SNAREs
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   1.6 Functions of Tvp23p
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   1.6.1 Yip4p and Yip5p
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   2 Aim of this Work
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  3 Material and Methods
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  3.1 Material
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   3.1.1 Lab Equipment
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   3.1.2 Chemicals
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   3.1.3 Proteaseinhibitors
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   3.1.4 Antibodies
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   3.1.5 Enzymes, Nucleotides and Standards
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   3.1.6 Kits for Isolation and Detection of DNA and Proteins
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   3.1.7 Bacteria Strains and Plasmids
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   3.1.8 Media for S. cerevisiae and E. coli
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   3.1.9 Stock Solutions and Buffers
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   3.1.10 Software
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   3.1.11 Internet services
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  3.2 Methods
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  3.2.1 Molecular Biology
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   3.2.1.1 Preparation of electrocompetent E. coli cells
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   3.2.1.2 Electroporation
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   3.2.1.3 DNA isolation from E. coli
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   3.2.1.4 Determination of DNA concentration
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  3.2.1.5 Cloning techniques
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   3.2.1.5.1 Polymerase chain reaction (PCR)
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   3.2.1.5.2 Site directed mutagenesis by PCR
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   3.2.1.5.3 Colony PCR
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   3.2.1.5.4 Ethanol precipitation
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   3.2.1.5.5 Cloning of PCR-products with pGEM® T-easy
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   3.2.1.5.6 Digestion of DNA with restriction endonucleases
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   3.2.1.5.7 Agarose gel electrophoresis of DNA
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   3.2.1.5.8 Ligation of DNA inserts into plasmid vectors
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   3.2.1.6 Sequencing
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   3.2.1.7 DMSO-Stocks of E. coli and S. cerevisiae
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  3.2.2 Yeast Genetics
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   3.2.2.1 PLATE transformation
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   3.2.2.2 Lithium Acetate (LiAc) transformation
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   3.2.2.3 Isolation of yeast genomic DNA
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   3.2.2.4 Construction of yeast deletion mutants
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   3.2.2.5 Yeast two-hybrid assay
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  3.2.3 Biochemical Methods
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   3.2.3.1 Preparation of protein extract from yeast cells
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   3.2.3.2 Bradford assay for determination of protein concentration
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   3.2.3.3 SDS gel electrophoresis
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  3.2.3.4 Isoelectric focusing (IEF)
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   3.2.3.4.1 Protein extract preparation
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   3.2.3.4.2 IEF-Electrophoresis
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   3.2.3.5 Coomassie blue staining
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   3.2.3.6 Western blot analysis
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   3.2.3.7 Preabsorption of antibodies
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   3.2.3.8 CPY Overlay-Assay
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  3.2.3.9 “Pulse-Chase” Immunoprecipitation (IP)
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   3.2.3.9.1 CPY-IP
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   3.2.3.9.2 ALP-IP
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   3.2.3.9.3 Protein stability
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   3.2.3.10 Test for bacterial expression of recombinant His6 -tagged proteins
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   3.2.3.11 Purification of His6 tagged fusion proteins
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   3.2.3.12 Test for bacterial expression of recombinant Strep-tagged proteins
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   3.2.3.13 Purification of Strep-tag fusion proteins
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   3.2.3.14 Pulldown-Assay
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  3.2.4 Cell Biology
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   3.2.4.1 Growth test with cell wall perturbing agents
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  3.2.4.2 Subcellular fractionation
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   3.2.4.2.1 Differential centrifugation
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   3.2.4.2.2 Sucrose density gradient
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   3.2.4.3 Membrane binding assay
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   3.2.4.4 Indirect immunofluorescence
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   3.2.4.5 GFP fluorescence microscopy
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   3.2.4.6 Calcofluor staining
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   3.2.4.7 FM4-64 staining
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   3.2.4.8 Sedimentation-Assay
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  4 Results
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  4.1 Characterization of the interaction of Ent3p with Pep12p, Vti1p and Syn8p
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  4.1.1 Pep12p interacts with Ent3p via its FSD motif
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   4.1.1.1 Yeast-Two-Hybrid and Pull-down interactions
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  4.1.1.2 In vivo interactions of Ent3p with Pep12p FSD mutants
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   4.1.1.2.1 Localization of Pep12 mutants in immunofluorescence microscopy
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   4.1.1.2.2 Localization by sucrose density gradient
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   4.1.1.2.3 Pep12 stability is enhanced in Pep12p F20L
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   4.1.1.3 CPY transport
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  4.1.2 Characterization of the interaction surface of Ent3p with the endosomal SNAREs
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   4.1.2.1 Ent3p interaction with SNAREs is specific for the ENTH domain
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   4.1.2.2 Vti1p, Syn8p and Pep12p bind the same interaction surface of Ent3p but different amino acids
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   4.1.2.3 Interacting amino acids on Vti1p and Pep12p
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   4.1.2.4 Vti1p, Pep12p and Syn8p were sorted together
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   4.1.2.5 Location of Pep12p, Vti1p and Syn8p on vesicles or endosomes
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   4.1.2.6 Functions of Ent3p point mutants in vivo
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   4.1.3 The A-ALP transport is defective in ent3Dent5D double mutants
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   4.1.4 Recycling of GFP-Snc1p is blocked in ent3Dent5D cells
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   4.1.5 Yck2p is mislocalized in ent3Dent5D cells
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  4.2 Functions of the Ent3p domains
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   4.2.1 Functions of the Ent3p domains in the retrograde EE to TGN transport
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   4.2.2 For CPY transport the Ent3p C-terminus and the ENTH domain were necessary
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   4.2.3 Localization of the Ent3p domains via immunofluorescence microscopy
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   4.2.4 Function of Ent3p domains in cell wall assembly and budding and cell separation
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   4.2.4.1 The Ent3p domain mutants could not rescue the defect of the double mutant in a sedimentation assay
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   4.2.4.2 Budding patterns and cell separation
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   4.2.4.3 Cell wall assembly
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   4.2.5 Interactions of the Ent3p ENTH domain with its own C-terminus and the Ent5 ANTH domain
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   4.2.6 Ent3p phosphorylation assay by isoelectric focusing
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   4.3 Tvp23p has a role in retrograde transport from EE to TGN and interacts genetically with VTI1
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   4.3.1 Genetic interaction between TVP23 and VTI1
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  4.4 Yip4p has a function in CPY and retrograde EE to TGN transport
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   4.4.1 Function of Yip4p and Yip5p in a CPY overlay assay
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   4.4.2 GFP-Snc1p transport in yip4D and yip5D deletion mutants
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  5 Discussion
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   5.1 Characterization of the Ent3p-SNARE interaction
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   5.1.1 Pep12p interacts with Ent3p via its FSD motif
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  5.1.2 Structural characterization of the Ent3p interaction surface
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   5.1.2.1 Both halves of the ENTH domain are specifically involved in the SNARE binding
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   5.1.2.2 Vti1p, Pep12p and Syn8p bind to the same surface on Ent3p but to different amino acids
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  5.1.3 Proof of interacting amino acids by charge-swap experiments
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   5.1.3.1 Interacting amino acids on Vti1p
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   5.1.3.2 Interacting amino acids on Pep12p
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   5.1.4 Vti1p, Pep12p and Syn8p were sorted as complex
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   5.1.5 Vti1p and Pep12p are t-SNAREs and Syn8p is a v-SNARE
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  5.1.6 In vivo functions of the Ent3p point mutants
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   5.1.6.1 The Y60D mutant has a severe growth defect
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   5.1.6.2 Rescuing abilities of the Ent3p F62D, E103W and R154E mutants
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  5.1.7 Ent3p and Ent5p have a function in retrograde transport to the TGN independently from SNARE binding.
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   5.1.7.1 A-ALP transport from LE to the TGN
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   5.1.7.2 GFP-Snc1p
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   5.1.7.3 GFP-Yck2p
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  5.2 Functions of the Ent3p domains
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   5.2.1 The Ent3p C-terminus is sufficient for GFP-Snc1p and GFP-Yck2p recycling
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   5.2.1.1 Vacuole morphology of the Ent3p/Ent5p mutants
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   5.2.2 Both Ent3p domains were necessary for CPY sorting
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   5.2.3 Cell wall defects
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   5.2.4 Interactions of the ENTH domain with ANTH and itself
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   5.2.5 Ent3p phosphorylation
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  5.3 Tvp23p and Yip4p have a function in the retrograde EE to TGN transport
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   5.3.1 Vti1p and Tvp23p show genetic interactions in retrograde transport.
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   5.3.2 Yip4p has a function in retrograde EE to TGN and CPY transport
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   5.4 Outlook
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   6 Summary
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   7 Bibliography
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    I Oligonucleotides
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    II Plasmids
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    III Yeast Strains
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    IV Curriculum Vitae
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    V Publications
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   Danksagung
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   Erklärung