List of Abbreviations
A adenosine
Å angstrom
ABE aminobenzyl-EDTA
Amp ampicilin
APB (p)-azidophenacylbromide
APS ammoniumperoxidisulphate
AS anchoring site (sequence)
ATP adenosine-5'-triphosphate
BABE bromoacetylbromide-EDTA
BPS branchpoint sequence
BSA bovine serum albumin
bp base pair
C cytidine
°C degree Celsius
Ci Curie
CE cacodylic acid-EDTA
cm centimeter
cpm counts per minute
CTP cytidine-5'-triphosphate
Da Dalton
DE53 diethyl amino ethyl cellulose 53
DNA deoxynucleic acid
dNTPs deoxynucleotide-5'-triphosphates
DMSO dimethylsulfoxide
dpm desintegrations per minute DTT 1,4-dithiothreitol
DTE 1,4-dithioerythrol
E.coli Escherischia coli
EDTA ethylendiamine-N,N,N',N'-tetraacetic acid
EM electron microscopy
EtOH ethanol
fmol femptomole
G guanosine
GpppG diguanosine triphosphate GTP guanosine-5'-triphosphate
h hour
HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
kDa kilodalton
l liter
# micro
m methyl
M molar
m3G cap 2,2,7-trimethyl-diguanosine triphosphate 5' cap m7G cap 7-monomethyl-diguanosine triphosphate 5' cap
mAb monoclonal antibody
Mg magnesium
min minute
ml mililiter
mRNA messenger RNA
N any nucleotide
nM nanomolar
NMR nuclear magnetic resonance
NP-40 Nonidet P-40
nt nucleotide
nxt nuclear extract
OH hydroxy
O/N overnight
PAGE polyacrylamide gel electrophoresis
PAS Protein A-sepharose
PCR polymerase chain reaction
pCp [5'-32P] 5',3'-cytidine biphosphate
pmole picomole
ppt polypyrimidine tract pre-mRNA pre-messenger RNA
R purin
RNA ribonucleic acid
RNase ribonuclease
RNasin ribonuclease inhibitor
RNP ribonucleoprotein
RT room temperature
S.cerevisiae Saccharomyces cerevisiae SDS sodium dodecyl sulfate
snRNA small nuclear ribonucleic acid snRNP small nuclear ribonucleoprotein snoRNP small nucleolar ribonucleoprotein
SR serine-arginine
ss splice site
3D three-dimensional
T thymidin
TBE tris-borate-EDTA buffer TCEP tris(2-carboxyethyl) phosphine
TEMED N,N,N',N'-tetramethylethylenediamine TPs total proteins from HeLa snRNP particles
tRNA transfer RNA
# pseudouridine
U uridine
U snRNP uridine-rich small nuclear ribonucleoprotein particle UTP uridine-5'-triphosphate
UV ultraviolet
V volume
v/v volume per volume
w weight
w/v weight per volume
xc xylene cyanol
Y pyrimidine
Acknowledgements
Firstly, I would like to thank Prof. Dr. Reinhard Lührmann for this interesting topic, for his intellectual stimulation, his support, his advices, fruitful discussions and his interest in this project. I am also grateful to him for giving me the chance to attend international conferences and present my results.
I would like to thank Prof. Dr. Ralf Ficner, Dr. Winfried Kramer and Dr. Markus Hauck for being the members of my examination committee. I am also thankful to Prof. Dr.
Reinhard Jahn, Prof. Dr. R. Hardeland and Prof. Dr. Jörg Stühlke for being the members of my external PhD thesis committee.
I thank Dr. Klaus Hartmuth for his support and help during the course of this work. I also thank him for the paper discussions and the critical reading of my thesis.
Many special thanks to Dr. Berthold Kastner for preparing nice figures for my U1 data, always providing us with schemes, all the U1 discussions and for his continuous help.
I am very much grateful to Dr. Patrizia Fabrizio for all her support and friendship, scientific and non-scientific discussions and helping me whenever I need. I thank her very much for her advices about my future and being with me in my good and bad days.
I am grateful to Dr. Reinhard Rauhut for all his help, support and advices all the time. I thank him for scientific and non-scientific discussions and helping me with the procedure to send my paper.
I would like to thank Dr. Cindy Will for scientific discussions, help with my paper and help for the talks and abstracts of the RNA meetings. I also thank her for the critical reading of my thesis abstract.
I am very much grateful to Abdullah Yalcin for helping me doing my clonings and showing me the best ways and strategies.
I am grateful to Rebecca Mathew for her close friendship, support, for our coffee breaks and sharing my happiness and also my problems.
I thank to Elif Karagöz very much for her friendship and support and for our cheese-sandwich lunches together.
Many thanks to Christian Merz for sharing our small bench together for all these years and being very nice to me when I occupy more space with my huge primer extension gels.
I thank Juliane Moses very much for helping us all the time with our administrative or visa problems and doing her best.
I would like to thank Uschi Drössler and Gertrud Nowak for being so kind, cheerful and helpful all the time. I also thank Peter Kempkes, Thomas Conrad, Kami and Belinda for preparing the nuclear extracts.
I finally would like to thank everybody, all my past and present collegues for the nice working atmosphere in this lab.
Very very special thanks to my parents, my brother and my sister for their continiuos support and help. They always had to miss me because I am far away but they always believed in me and were proud of me.
Most of all, I am very much thankful to my beloved husband Abdullah Yalcin for all his help, interest, trust and ever-lasting love. He always helped me, believed in me and stood beside me.
Cirriculum Vitae
PERSONAL INFORMATION
Name Gizem
Surname Dönmez
Date of birth 17.04.1978 Place of birth Nazilli, Turkey
Natonality Turkish
Gender Female
EDUCATION
2002-present graduate study in Department of Cellular Biochemistry Max Planck Institute of Biophysical Chemistry, Göttingen Superviser: Prof. Dr. Reinhard Lührmann
2000-2002 MSc International MSc/PhD program of Molecular Biology and Max Planck Institute of Biophysical Chemistry, Göttingen Department of Cellular Biochemistry
Superviser: Prof. Dr. Reinhard Lührmann
1996-2000 B.Sc Department of Molecular Biology and Genetics (high honor) Middle East Technical University, Ankara, Turkey
1989-1996 Nazilli Anatolian High School
1984-1989 Beseylul Primary School
SCHOLARSHIPS
Max Planck Scholarship (2001-present)
International Msc/PhD program of Molecular Biology scholarship (2000-2001)
Sabanci Group Holding scholarship (1996-2000) due to very high ranking in nationwide university entrance examinations
Turkey Scientific and Technical Research Corporation Scholarship (1996-2000) due to very high ranking in nationwide university entrance examinations
CONFERENCES
28th FEBS Meeting
October 2002, Istanbul Turkey
Poster presentation: “Investigation of the modified nucleotides in the 5' end of human U2 snRNA in pre-mRNA splicing”
International symposium: Structure, function and dynamics of RNA-protein complexes September 2003, Göttingen, Germany
Poster presentation: “Investigation of the modified nucleotides in the 5' end of human U2 snRNA in pre-mRNA splicing”
9th annual meeting of the RNA Society June 2004, Madison USA
Oral presentation: “Modified nucleotides in the 5' end of the human U2 snRNA are required for early spliceosome (E complex) formation in vitro”
11th annual meeting of the RNA Society June 2006, Seattle, Washington, USA
Oral presentation: “U1 and U2 snRNPs are in close proximity in early spliceosomal complexes as revealed by U2-tethered hydroxyl radical probing”
PUBLICATIONS
Toyran N, Zorlu F, Dönmez G, Oge K, Severcan F
Chronic hypoperfusion alters the content and structure of proteins and lipids of rat brain homogenates: a Fourier transform infrared spectroscopy study. Eur Biophys J. 2004 Oct;33(6):549-54
Dönmez G, Hartmuth K, Lührmann R
Modified nucleotides at the 5' end of human U2 snRNA are required for spliceosomal E-complex formation RNA. 2004 Dec;10(12):1925-33
Dönmez G, Hartmuth K, Kastner B, Will CL, Lührmann R
The 5' end of U2 snRNA is in close proximity to reactive groups of the pre-mRNA and to U1 in early spliceosomal complexes (manuscript submitted).
Declaration
I herewith declare that I prepared the PhD thesis "Investigation of the higher order structure of the spliceosomal RNA network" on my own and with no other sources and aids than those quoted.
Ich versichere, daß ich die vorliegende Arbeit selbständig verfaßt und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe.
Die Arbeit hat in gleicher oder ähnlicher Form noch keiner Prüfungsbehörde vorgelegen.
Parts of this thesis were published in:
Dönmez G., Hartmuth K., and Lührmann R. (2004) Modified nucleotides at the 5' end of human U2 snRNA are required for spliceosomal E-complex formation. RNA 10, 1925-33
Dönmez G., Hartmuth K., Kastner B., Will CL., and Lührmann R (2006) The 5' end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes. (manuscript submitted)
Göttingen, December 2006