Molecular cloning and functional studies of Neurospora crassa KIF1, a new member of the UNC-104, KIF1 family of kinesin-like proteins [Elektronische Ressource] / vorgelegt von Michaela Hartel

Hartel, Michaela

Description

Molecular Cloning and Functional Studies of Neurospora crassa KIF1,a New Member of the UNC-104/KIF1 Familyof Kinesin-Like ProteinsMichaela Hartel2004Aus dem Adolf-Butenandt-Institutder Ludwig-Maximilians-Universität MünchenVorstand: Prof. Dr. rer. nat. Dr. med. Walter NeupertMolecular Cloning and Functional Studies of Neurospora crassa KIF1,a New Member of the UNC-104/KIF1 Familyof Kinesin-Like ProteinsDissertationzum Erwerb des Doktorgrades der Medizinan der Medizinischen Fakultät derLudwig-Maximilians-Universität zu Münchenvorgelegt vonMichaela HartelausBerlinJahr2004Mit Genehmigung der Medizinischen Fakultätder Universität MünchenBerichterstatter: Prof. Dr. Manfred SchliwaMitberichterstatter: Prof. Dr. R. Mocikat Priv. Doz. Dr. St. LinderMitbetreuung durch denpromovierten Mitarbeiter: Dr. Günther WoehlkeDekan: Prof. Dr. med. Dr. h. c. K. PeterTag der mündlichen Prüfung: 29. 04. 2004ndParts of this work were presented in a poster at the 2 Munich Symposium on Cell Dynamics,Munich, Germany: Hartel, M., Schliwa, M. and Woehlke G. (2002). Neurospora crassaKIF1, a new short member of the UNC104/KIF1-Family of Kinesins.CONTENTS 1CONTENTSABBREVIATIONS 41.

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Molecular Cloning and Functional Studies of

Neurospora crassaa New Member of the UNC-104/KIF1 Family

of Kinesin-Like Proteins

Michaela Hartel

2004

KIF1,

Aus dem Adolf-Butenandt-Institut

der Ludwig-Maximilians-Universität München

Vorstand: Prof. Dr. rer. nat. Dr. med. Walter Neupert

Molecular Cloning and Functional Studies of

Neurospora crassaa New Member of the UNC-104/KIF1 Family

of Kinesin-Like Proteins

Dissertation

zum Erwerb des Doktorgrades der Medizin

an der Medizinischen Fakultät der

Ludwig-Maximilians-Universität zu München

vorgelegt von

Michaela Hartel

aus

Berlin

Jahr

2004

KIF1,

Mit Genehmigung der Medizinischen Fakultät

Berichterstatter:

Mitberichterstatter:

Mitbetreuung durch den

promovierten Mitarbeiter:

Dekan:

Tag der mündlichen Prüfung:

der Universität München

Prof. Dr. Manfred Schliwa

Prof. Dr. R. Mocikat

Priv. Doz. Dr. St. Linder

Dr. Günther Woehlke

Prof. Dr. med. Dr. h. c. K. Peter

29. 04. 2004

nd Munich Symposium on Cell Dynamics,Parts of this work were presented in a poster at the 2

Neurospora crassaMunich, Germany: Hartel, M., Schliwa, M. and Woehlke G. (2002).

KIF1, a new short member of the UNC104/KIF1-Family of Kinesins.

CONTENTS

CONTENTS

ABBREVIATIONS

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Materials

2.2. Vectors and strains

2.2.1.Vectors

2.2.2. Bacterial strains

strains N. crassa 2.2.3.

E. coli 2.3. Cultivation of

Neurospora crassa 2.4. Cultivation of

2.4.1. Growing and storage of conidia

2.4.2. Culture and media

2.5. Molecular biology methods

2.5.1. Agarose gel electrophoresis

2.5.2. DNA extraction from agarose gels

2.5.3. Determination of DNA and RNA concentration

2.5.4. Preparation of plasmid DNA

2.5.4.1. Analytical preparation of plasmid DNA

2.5.5. DNA cleavage with restriction enzymes

2.5.6. Ligation of DNA into a plasmid vector

cells E. coli 2.5.7. Preparation of electrocompetent

cells E. coli 2.5.8. Electrotransformation of

cells E. coli 2.5.9. Preparation of SEM-competent

cells E. coli 2.5.10. Transformation of SEM-competent

E. coli 2.5.11. Identification of transformed clones in

2.5.12. Filling in DNA 5' overhangs

2.5.13. Phosphorylation and dephosphorylation of DNA

2.5.14. Polymerase chain reaction (PCR)

2.5.15. Oligonucleotides

2.5.15.1. Vector primers

2.5.15.2. Sequencing primers

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CONTENTS

2.5.15.3. NcKin primers

N. crassa 2.5.16. Isolation of RNA from

2.5.17. Electrophoresis of RNA and Northern blotting

2.5.17.1. Construction of the probes for Northern blotting

2.5.17.2. Radioactive labelling of the probes, hybridization and detection

2.5.18. Screening of cDNA

2.5.18.1. Digoxygenin labelling of the probes, hybridization and detection

2.5.19. Construction of the vectors

2.6. Biochemical methods

2.6.1. SDS-Polyacrylamide gel electrophoresis (PAGE)

2.6.2. Coomassie staining

2.6.3. Determination of protein concentration

2.6.4. Purification of tubulin

2.6.5. Polymerisation of microtubules

2.6.6. Determination of microtubule concentration

E. coli 2.6.7. Expression and purification of recombinant NcKIF1 from

2.6.7.1. Expression studies

2.6.7.2. Testing of the purification conditions of recombinant NcKIF1

2.6.8. Density gradient centrifugation and gel filtration

2.6.9. NEM-inhibition test

2.6.10. Cy3-labelling of cys-tagged NcKIF1 constructs

2.6.11. Biotinylation of cys-tagged NcKIF1 constructs

2.6.12. Multiple motor gliding assay

2.6.13. ATPase assay

2.6.13.1. Basal activity of kinesin

2.6.13.2. Coupled ATPase assay

2.6.13.3. Calculations for the ATPase assay

2.6.14. Antibodies

2.6.14.1. Immunization and preparation of antiserum

2.6.14.2. Detection of proteins by Western blotting

2.6.14.3. Affinity purification with nitrocellulose strips

2.6.14.4. Dot blot test for determination of antibody sensitivity

crude extract N. crassa 2.6.15. Preparation of

2.6.16. Microtubule affinity enrichment from N. crassa cude extract

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CONTENTS

3. RESULTS

3.1. Sequence of NcKIF1 3.1.1. Sequence of the NcKIF1 gene