Mechanism and control of nuclear cytoplasmic translocation of the transporter regulator RS1 [Elektronische Ressource] = Mechanismus und Kontrolle der Translokation der Transporterregulator RS1 zwischen Kern und Zytoplasma / submitted by Alina Filatova

Alina Filatova

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• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 55
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Mechanism and Control of Nuclear-Cytoplasmic Translocation of the
Transporter Regulator RS1

Mechanismus und Kontrolle der Translokation der Transporterregulator RS1
zwischen Kern und Zytoplasma


Doctoral thesis for a doctoral degree
at the Graduate School of Life Sciences,
Julius-Maximilians-Universität Würzburg,
Section Biomedicine

submitted by

Alina Filatova


from

Khimki, Russia


Würzburg, 2009














Submitted on:
…………………………………………………………..……………
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Members of the Promotionskomitee:

Chairperson: …….………Prof. Dr. Thomas Hünig……………......

Primary Supervisor: ……Prof. Dr. Hermann Koepsell…..………

Supervisor (Second): ……Prof. Dr. Roland Benz.….…………......

Supervisor (Third): ……...Dr. Heike Hermanns………….....….....


Date of Public Defence: ……………………………………………..

Date of receipt of Certificates: ……………………………………...
Table of contents .
Table of contents
1. INTRODUCTION ........................................................................................................ 1
1.1. The RS1 protein ...................................................................................................... 1
1.2. Transport of proteins in and out of the nucleus................................................... 5
1.2.1. Nuclear protein import pathways ......................................................................... 6
1.2.2. Nuclear export pathways ..................................................................................... 10
1.2.3. Regulation of nuclear transport .......................................................................... 11
1.2.3.1. Regulation of the karyopherin-cargo interaction ....................................... 11
1.2.3.2. Regulation of transport by the NPC ............................................................ 14
2. THE AIM OF THIS STUDY..................................................................................... 16
3. MATERIALS AND METHODS............................................................................... 18
3.1. Materials................................................................................................................ 18
3.1.1. Chemicals............................................................................................................... 18
3.1.2. Antibodies.............................................................................................................. 18
3.1.3. Affinity matrices ................................................................................................... 19
3.1.4. DNA and Protein Markers................................................................................... 19
3.1.5. Enzymes ................................................................................................................. 19
3.1.6. Inhibitors and activators...................................................................................... 19
3.1.7. Reaction kits.......................................................................................................... 20
3.1.8. Peptides.................................................................................................................. 20
3.1.9. Synthetic Oligonucleotides................................................................................... 20
3.1.10. Plasmids and constructs..................................................................................... 21
3.1.11. Bacteria................................................................................................................ 24
3.1.12. Cell lines............................................................................................................... 24
3.1.13. Buffers and solutions .......................................................................................... 24
3.1.14. Software............................................................................................................... 25
3.2. Methods ................................................................................................................. 26
3.2.1. Molecular biology ................................................................................................. 26
3.2.1.1. Mutagenesis .................................................................................................... 26
3.2.1.2. Generation of the GFP-TEV-S-Tag vector.................................................. 26
Table of contents .
3.2.1.3. Generation of the GFP-TEV-S-Tag-CK2-NS-PKC-PKC vector .............. 26
3.2.1.4. Annealing of oligonucleotides ....................................................................... 27
3.2.1.5. Polymerase chain reaction (PCR) ................................................................ 27
3.2.1.6. DNA isolation by phenol/chloroform extraction......................................... 28
3.2.1.7. Digestion of DNA with restriction endonucleases....................................... 28
3.2.1.8. Analytical agarose gel electrophoresis of DNA ........................................... 28
3.2.1.9. Preparative agarose gel electrophoresis....................................................... 29
3.2.1.10. Ligation........................................................................................................... 29
3.2.1.11. Analytical agarose gel electrophoresis of RNA ........................................... 29
3.2.1.12. Desalting of DNA samples............................................................................. 30
3.2.1.13. Transformation of bacteria and clone selection.......................................... 30
3.2.1.14. Isolation of plasmid DNA from E.coli.......................................................... 31
3.2.1.15. Determination of DNA and RNA concentration by spectrophotometry .. 32
3.2.2. Protein analysis methods...................................................................................... 32
3.2.2.1. Preparation of the whole-cell extracts ......................................................... 32
3.2.2.2. Purification of GFP fusion proteins ............................................................. 32
3.2.2.3. Immunoprecipitation of GFP fusion proteins and associated proteins .... 33
3.2.2.4. Determination of protein concentration ...................................................... 34
3.2.2.5. SDS-polyacrylamide gel electrophoresis...................................................... 34
3.2.2.6. Western blot and immunodetection ............................................................. 35
3.2.2.7. Staining of protein polyacrylamide gels with Coomassie blue .................. 37
3.2.2.8. Silver staining of protein polyacrylamide gels ............................................ 37
3.2.2.9. Gel drying ....................................................................................................... 38
3.2.3. Generation and testing of phosphospecific antibodies ...................................... 38
3.2.3.1. Immunization of rabbits................................................................................ 39
3.2.3.2. Identification of the antibody titer. Enzyme-linked Immunosorbent Assay
(ELISA). 39
3.2.3.3. Affinity purification of antibodies................................................................ 40
3.2.4. Cell Culture ........................................................................................................... 41
3.2.4.1. Cultivation of mammalian cells .................................................................... 41
3.2.4.2. Passage ............................................................................................................ 41
3.2.4.3. Cryoculture..................................................................................................... 41
3.2.4.4. Transient transfection of mammalian cells ................................................. 42
Table of contents .
3.2.4.5. Generation of stable cell lines ....................................................................... 42
3.2.4.6. Inhibitor treatment of cells ........................................................................... 43
3.2.5. Analysis of gene expression in mouse embryonic fibroblasts (MEFs) ............. 43
3.2.5.1. Isolation and cultivation of MEFs ................................................................ 43
3.2.5.2. Cryoculture..................................................................................................... 44
3.2.5.3. Synchronization of MEFs.............................................................................. 44
3.2.5.4. Isolation of Total RNA .................................................................................. 45
3.2.5.5. Gene expression microarray analysis .......................................................... 45
3.2.6. Fluorescence an