Purification and characterization of retinoblastoma like factor-containing protein complexes from Drosophila melanogaster [Elektronische Ressource] / Michael Korenjak

Michael Korenjak

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München





Purification and Characterization of
Retinoblastoma like Factor-containing
Protein Complexes from Drosophila melanogaster









Michael Korenjak
aus
Lienz

2006

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. Dr. Peter Becker betreut und von Herrn Prof. Dr. Dirk Eick vor
der Fakultät für Biologie vertreten.









Ehrenwörtliche Versicherung

Ich versichere, dass ich die vorliegende Arbeit selbständig durchgeführt und keine anderen
als die angegebenen Hilfsmittel und Quellen benutzt habe.


München, am 23. Oktober 2006






………………………………………..
Michael Korenjak







Dissertation eingereicht am 30.10.2006
1. Gutachter Prof. Dr. Peter Becker
2. Gutachter Prof. Dr. Dirk Eick
Mündliche Prüfung am 19.12.2006








Im Gedenken an Walter Korenjak.





















Acknowledgements

Here, I would like to thank the people who have supported me during this PhD thesis and
participated, directly or indirectly, in its realization.

First of all, I am deeply grateful to Alexander Brehm for giving me the possibility to perform
this thesis in his group and his continous support from the first TRAX to the last paragraph of
the work. Thank you for always being available for questions and discussions, for the
motivation and expertise and providing a great environment for this work, scientifically and
socially.

I would like to thank Peter Becker for providing an excellent infrastructure for this work, for
critical and helpful comments on the project and creating a great working atmosphere.

I am greatly thankful to Nick Dyson, Barbie Taylor-Harding, Uli Binne, John Satterlee, Helen
White-Cooper and Rein Aasland for excellent collaborations on the dissection of the function
of the complexes and the help with the bioinformatics. Moreover, I would like to thank Nick
Dyson for helpful discussions and the providing of material, crucial for this work.

I am grateful to Alexander Brehm and Cristina Chioda for proof-reading and helpful
suggestions on this manuscript, Ina Dahlsveen for the help with polytene stainings and Stefan
Gaubatz, Mike Botchan, Helen White-Cooper, Alain Verreault, Bryan Turner and Renato Paro
for antibodies and communicating results prior to publication.

I would like to thank all people from the BREHM GROUP for their help, support and creating
greating working conditions.
Angie Mitterweger, who, as my bench neighbour, had to cope with the full chaos. Thanks for
always being open for a PhD students´ complaints and questions and for the many
“recreational” activities.
Jo Sutcliffe and Karim Bouazoune for the scientific and “less-scientific” discussions, which
made working a pleasure. (Thank you Karim for giving me the feeling to start early. It was
never the same again!)
Yvonne Berghöfer-Hochheimer for the many discussions on complex purification and the
“New York-experience” and Natascha Kuhnert for always being helpful and open for
questions.

Moreover, I would like to thank all the people working at the ABI department of molecular
biology for their permanent help and advice in the lab. Thanks to all for creating a great
working atmosphere!

I thank Axel Imhof and Gernot Längst for providing material, and helpful comments on this
work. A special thanks to the beer club members and football players for “hard fights” at Café
Schiller and Theresienwiese and Andreas “King Nothing” Hochheimer for the ultimative MP3-
collection. Not to forget Felix Hartlepp as my standard “meeting roommate” and short-term
bench neighbour.

Mein ganz besonderer Dank gilt meiner Mutter Theresia Korenjak, und Hans-Jürgen Caroli für
Ihre tolle Unterstützung während des gesamten Studiums in Innsbruck und München!
Weiters möchte ich Doris Caroli, Andreas Müller, sowie Andrea und Markus Meßner für den
manchmal nötigen Abstand von der Arbeit danken. (Auch wenn ab und zu ein regelrechtes
“wegzerren” nötig war)

thFinally, a special thanks goes to Italy for the 4 of July, 2006! ;-)

TABLE OF CONTENTS
Table of contents

1 Summary ...................................................................................................1
2 Introduction...............................................................................................2
2.1 The pRb-E2F network in mammals .............................................................................. 2
2.1.1 The E2F transcription factor family............................................................................ 3
2.1.2 E2F target genes ........................................................................................................ 5
2.1.3 The pocket protein family........................................................................................... 5
2.1.4 Mechanisms of pocket protein mediated transcriptional repression....................... 7
2.1.5 The role of pocket proteins and E2F proteins in development................................ 9
2.2 The pRb-E2F network in flies and worms ................................................................. 10
2.2.1 The Drosophila melanogaster pRb-E2F network................................................... 11
2.2.1.1 Transcriptional regulation by dE2F and RBF proteins .................................... 11
2.2.1.2 RBF and chromatin regulation.......................................................................... 12
2.2.1.3 Cell cycle control by dE2F and RBF proteins.................................................. 13
2.2.1.4 dE2F target genes ............................................................................................. 13
2.2.2 The Caenorhabditis elegans pRb-E2F network ..................................................... 15
2.2.2.1 EFL-1, DPL-1, LIN-35 and cell cycle regulation .............................................. 15
2.2.2.2 EFL-1, DPL-1, LIN-35 and development ......................................................... 16
2.2.2.3 LIN-35 and chromatin regulation ...................................................................... 18
2.3 Objectives....................................................................................................................... 19
2.3.1 Pocket protein containing complexes in mammals ................................................ 19
2.3.2 Drosophila melanogaster as a model system to analyze pocket protein-containing
complexes .............................................................................................................................. 19

3 Material and methods.............................................................................21
3.1 Material............................................................................................................................ 21
3.1.1 Chemicals, enzymes, chromatographic and radioactive material......................... 21
3.1.1.1 Enzymes............................................................................................................. 21
- I - TABLE OF CONTENTS
3.1.1.2 Chromatographic material................................................................................. 21
3.1.1.3 Affinity purification material............................................................................... 21
3.1.1.4 Blotting material ................................................................................................. 22
3.1.1.5 Dialysis and filtration material........................................................................... 22
3.1.2 Standard solutions.................................................................................................... 22
3.1.3 Antibodies ................................................................................................................. 23
3.1.4 Plasmids.................................................................................................................... 26
3.1.5 Oligonucleotides ....................................................................................................... 28
3.1.6 Bacteria, flies and cells ............................................................................................ 29
3.1.6.1 Bacteria .............................................................................................................. 29
3.1.6.2 Flies .................................................................................................................... 29
3.1.6.3 Cell lines and tissue culture media................................................................... 29
3.2 Methods.....