The role of cofactors, replication, and intranucleosomal UASp elements in chromatin remodeling at yeast PHO promoters [Elektronische Ressource] / Franziska Ertel

Franziska Ertel

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 19 Mo

Extrait

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





The Role of Cofactors, Replication, and
Intranucleosomal UASp Elements
in Chromatin Remodeling
at Yeast PHO Promoters

Franziska Ertel
München


Juni 2010



































Eingereicht am 02.06.2010

Mündliche Prüfung am 16.07.2010




1. Gutachter: Prof. Peter Becker
2. Gutachter: Prof. Dirk Eick
3. Gutachter: Prof. Charles David
4. Gutachter: Prof. Kirsten Jung
5. Gutachter: Prof. Böttger
6. Gutachter: Prof. Leonhardt





































Ehrenwörtliche Versicherung


Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir
selbständig und ohne unerlaubte Hilfe angefertigt wurde.


München, den .................................... ............................................................
(Franziska Ertel)





































Erklärung


Hiermit erkläre ich, dass ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg
unterzogen habe.


München, den .................................... ............................................................
(Franziska Ertel)
I
Acknowledgements

First of all I want to thank Dr. Philipp Korber. I am grateful for the opportunity to work on this
exciting project. Your support, advice and the stimulating discussions throughout my studies
strengthened my interest in science and encourage me to further my scientific development.
I am especially grateful to Prof. Dr. Peter Becker who despite having a busy time schedule al-
ways showed interest in my topic and found time and the right words for motivation and sup-
port. Thank you for providing such a stimulating scientific environment and a particularly good
lab atmosphere.
I thank Prof. Dr. Dirk Eick for being my second reviewer.
Prof. Dr. Ralph Rupp and PD Dr. Anton Eberharter I want to thank for their time and helpful
comments as members of my thesis advisory committee.
My heartfelt thanks go to Christian Wippo and Alexandra Lantermann. Lab life and beyond will
never be the same without you.
I owe gratitude to all members of the molecular biology department for their helpful discussion
in scientific questions and the fun during and after lab hours. I want to thank Dorle Blaschke
for her scientific support and humorous remarks and Gözde Güclüler for her help on my
project.
I want to thank our secretaries Edith Müller and Carolin Brieger for all the help they provided.
I thank the Elite Network of Bavaria for financial support of conference visits and the members
of the PhD program “Protein Dynamics in Health and Disease” for their continuing effort in
organizing seminars, soft skill courses, retreats and the fun we had.
My special thanks go to Sandra Vengadasalam and Annette Scharf for their friendship.
Ich danke meinen Eltern, Renate und Wolfgang Ertel, für ihre andauernde Unterstützung und
das Vertrauen, das sie in mich setzen.
Most of all I thank Severin.
II
Table of contents
Acknowledg ements ....................................................................................................................... I ........................................................................................................................ II 
Summary ....................................................................................................................................... 1 
Zusammenfassung ........................................................................................................................ 2 
1  Introduction ...................................................................................................................... 4 
1.1  Chromatin .......................................................................................................................... 4 
1.1.1  Organization ...................................................................................................................... 4 
1.1.2  Posttranslational modifications and histone variants ......................................................... 6 
1.1.3  Chromatin remodeling machines ....................................................................................... 7 
1.1.4  Replication ....................................................................................................................... 10 
1.1.5  What defines a promoter .................................................................................................. 12 
1.2  The PHO system in Saccharomyces cerevisiae ............................................................... 12 
1.2.1  Signal transduction .......................................................................................................... 13 
1.2.2  Chromatin structure ......................................................................................................... 14 
1.2.3  Cofactor requirements for promoter chromatin remodeling at the PHO5 and PHO8
promoters ......................................................................................................................... 16 
1.2.4  Replication and influence of the cell cycle ...................................................................... 19 
1.2.5  In vitro chromatin assemblies .......................................................................................... 19 
1.3  Goals of this study ........................................................................................................... 21 
1.3.1  In vitro reconstitution of promoter chromatin remodeling at the PHO promoters .......... 21 
1.3.2  The role of replication during PHO5 and PHO8 promoter remodeling .......................... 21 
2  Materials and Methods .................................................................................................. 23 
2.1  Materials .......................................................................................................................... 23 
2.1.1  Chemicals ........................................................................................................................ 23 
2.1.2  Enzymes ........................................................................................................................... 24 
2.1.3  Others ............................................................................................................................... 24 
2.2  Standard methods ............................................................................................................. 25 
2.3  Saccharomyces cerevisiae strains .................................................................................... 25 
2.4  Media for growing S. cerevisiae ...................................................................................... 27 
2.4.1  YPDA medium ................................................................................................................ 27 
2.4.2  YNB minimal medium .................................................................................................... 27 
2.4.3  Phosphate-free minimal medium ..................................................................................... 27 
2.5  Induction of PHO genes .................................................................................................. 28 
2.5.1  Phosphate starvation ........................................................................................................ 28 
2.5.2  Growth of replicating and non-replicating cells .............................................................. 28 
2.6  General yeast methods ..................................................................................................... 28  III
2.6.1  Transformation of S. cerevisiae cells ............................................................................... 28 
2.6.2  DNA isolation from S. cerevisiae .................................................................................... 28 
2.7  Extract and protein preparation ........................................................................................ 29 
2.7.1  Yeast whole cell extract .......