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Publié par | technische_universitat_munchen |
Publié le | 01 janvier 2008 |
Nombre de lectures | 32 |
Langue | English |
Poids de l'ouvrage | 39 Mo |
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TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Biotechnologie
Aggregation propensities of the yeast Sup35p and mouse
prion protein domains in the cytosol of mammalian cells
Dipl.-Biol. (Univ.) Carmen Krammer
Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. Chr. Becker
Prüfer der Dissertation:
1. Univ.-Prof. Dr. J. Buchner
2. Univ.-Prof. H. Schätzl
Die Dissertation wurde am 25.08.2008 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 05.11.2008 angenommen.
Scio me nihil scire
(Sokrates)
Dedicated to Hildegard and Johann Krammer
TABLE OF CONTENTS
TABLE OF CONTENTS .......................................................................................................... I
I. SUMMARY .................................................................................................................. 1
I.A ENGLISH VERSION ............................................................................................................. 1
I.B DEUTSCHE VERSION .......................................................................................................... 3
II. INTRODUCTION .......................................................................................................... 5
II.A PRION DISEASES .............................................................................................................. 5
II.A.1 HUMAN PRION DISEASES ....................................................................................................... 6
II.A.2 ANIMAL PRION DISEASES ....................................................................................................... 8
II.B THE PRION PROTEIN ......................................................................................................... 9
II.B.1 THE PRNP GENE ................................................................................................................... 9
C SCII.B.2 STRUCTURAL AND BIOCHEMICAL CHARACTERISTICS OF PRP AND PRP ...................................... 10
CII.B.3 CELL BIOLOGY OF PRP ....................................................................................................... 12
II.B.4 THE PRION CONVERSION PROCESS ........................................................................................ 13
II.B.5 PRION STRAINS AND THE SPECIES BARRIER .............................................................................. 15
II.C PRP AND ITS ROLE IN NEURODEGENERATION IN PRION DISEASES .............................................. 18
II.D YEAST PRIONS .............................................................................................................. 21
II.D.1 FUNCTION OF SUP35P ....................................................................................................... 23
II.D.2 STRUCTURE AND CHARACTERISTICS OF SUP35P ...................................................................... 25
+II.D.3 GENERATION AND PROPAGATION OF THE [PSI ] PHENOTYPE .................................................... 27
II.D.4 YEAST PRION VARIANTS AND THE SPECIES BARRIER .................................................................. 30
II.E SIMILARITIES AND DIFFERENCES BETWEEN MAMMALIAN PRP AND YEAST SUP35P ........................ 31
II.F YEAST PRIONS AS A MODEL SYSTEM FOR PRION RESEARCH....................................................... 34
II.G OBJECTIVE ................................................................................................................... 34
III. MATERIALS AND METHODS ..................................................................................... 36
III.A MATERIALS ................................................................................................................. 36
III.A.1 CHEMICALS ..................................................................................................................... 36
i
III.A.2 BUFFERS AND SOLUTIONS .................................................................................................. 37
III.A.3 ANTIBIOTICS .................................................................................................................... 38
III.A.4 ENZYMES ........................................................................................................................ 38
III.A.5 ANTIBODIES ..................................................................................................................... 38
III.A.6 PLASMID GENERATION ...................................................................................................... 40
III.A.7 OLIGODEOXYNUCLEOTIDES ................................................................................................. 41
III.A.8 EUCARYOTIC CELL LINES ..................................................................................................... 43
III.A.9 CELL CULTURE MEDIA AND SUPPLEMENTS ............................................................................. 43
III.A.10 KITS ............................................................................................................................. 43
III.A.11 INSTRUMENTS AND ACCESSORIES ...................................................................................... 44
III.B METHODS................................................................................................................... 45
III.B.1 BIOLOGICAL SAFETY ........................................................................................................... 45
III.B.2 MOLECULAR BIOLOGICAL METHODS ..................................................................................... 46
III.B.2.1 Polymerase chain reaction (PCR) .............................................................................. 46
III.B.2.2 Agarose gel electrophoresis (AGE) ........................................................................... 47
III.B.2.3 Elution of DNA fragments from agarose gels ........................................................... 48
III.B.2.4 TOPO cloning ............................................................................................................ 48
III.B.2.5 Enzymatic digestion of plasmid DNA ........................................................................ 48
III.B.2.6 DNA ligation .............................................................................................................. 49
III.B.2.7 Preparation of chemically competent E. coli ............................................................ 50
III.B.2.8 Transformation of E. coli with plasmid DNA ............................................................. 51
III.B.2.9 Isolation of plasmid DNA .......................................................................................... 52
III.B.2.10 Quantification of nucleic acid ................................................................................. 52
III.B.3 PROTEIN BIOCHEMICAL METHODS........................................................................................ 53
III.B.3.1 Preparation of cell lysates from mammalian cells .................................................... 53
III.B.3.2 Determination of protein concentration by Bradford assay .................................... 54
III.B.3.3 Proteinase K (PK) di