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Publié par | albert-ludwigs-universitat_freiburg |
Publié le | 01 janvier 2010 |
Nombre de lectures | 22 |
Langue | Deutsch |
Poids de l'ouvrage | 25 Mo |
Extrait
Max-Planck-Institute of Immunobiology
Freiburg im Breisgau
Albert-Ludwigs-Universitat Freiburg
Faculty of Biology
Characterization of neocentromere formation
in Drosophila melanogaster
Agata Olszak
Dissertation submitted to obtain a
Degree of Doctor rerum naturalium by the
Albert-Ludwigs-Universität
Faculty of Biology
Freiburg im Breisgau, Germany
Supervisor
Dr. Patrick Heun
Max-Planck-Institute of Immunobiology
Freiburg im Breisgau 2010
Erklärungen
Die vorliegende Arbeit wurde im Zeitraum von März 2006 bis April 2010 am Max-
Planck-Institut für Immunbiologie in Freiburg im Breisgau unter Anleitung von Herrn Dr.
Patrick Heun angefertigt.
Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und
ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus
anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter
Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche
Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Promotionsberater oder
anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder
mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem
Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch im
Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität
Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion
zur Führung des Doktortitels nicht berechtigt bin.
Dekan der Fakultät: Prof. Dr. Ad Aertsen
Promotionsvorsitzender: Prof. Dr. Eberhard Schäfer
Betreuer der Arbeit: Dr. Patrick Heun
Referent: Prof. Dr. Rudolf Grosschedl
Koreferent: Prof. Dr. Karl-Friedrich Fischbach
3. Prüfer: Dr. Patrick Heun
Tag der mündlichen Prüfung: 29.06.2010
II
Abbreviations
A
Ampere
nd2L
left arm of 2 Drosophila chromosome
nd2R
right arm of 2
3D
three-dimensional
rd3L
left arm of 3 Drosophila chromosome
rd3R
right arm of 3
Ab
antibody
APS
aminopropyltriethoxysilane
ATP
adenosine triphosphate
BAC
bacterial artificial chromosome
bp
base pair
C. albicans
Candida albicans
C. elegans Caenorhabditis elegans
CAD
CENP-A-nucleosome distal
CATD
Centromere protein A targeting domain
CCAN
constitutive centromere-associated network
CEN
centromere
CenH3
centromere specific histone H3 variant
CENP-A-W
centromere protein A-W
chr
chromosome
CID
centromere identifier
Da
Dalton
DAPI
',6-diamidino-2-phenylindole
DMSO
dimethyl sulfoxide
DNA
deoxyribonucleic acid
dNTP
deoxyribonucleotide triphosphate
DSB
double strand break
dsRNA
double stranded RNA
DTT
dithiothreitol
dUTP
deoxyuridine triphosphate
E. coli
Escherichia coli
EDTA
thylenediaminetetraacetic acid
EM
electron microscopy
eu
euchromatin
F
forward
FACS
fluorescence-activated cell sorting
FCS
Fetal Calf Serum
FISH
fluorescence in situ hybridization
FP
fluorescence protein
GFP
green fluorescence protein
H3K9me2
dimethylation on lysine 9 of histone H3
III
H4Ac
acetylation on histone H4
HA
haemagglutinin
HAC
human artificial chromosome
het
heterochromatin
HFD
histone fold domain
HJURP
holliday junction recognition protein
hygro
hygromycin
IF
immunofluorescence
kMT
kinetochore microtubule
LacI
Lac repressor
LacOp
Lac Operator
Mbp
mega base pair
MT
microtubule
mut
mutated
NAC
nucleosome associated complex
NPM1
Nucleophosmin1
PBS
phosphate buffered saline
PCR
polymerase chain reaction
PEV
position effect variegation
PFGE
pulse field gel electrophoresis
Pol
polymerase
prox
proximity
puro
puromycin
rDNA
ribosomal DNA
rev
reverese
RFP
red fluorescence protein
RNAi
RNA interference
rpm
revolutions per minute
RT
room temperature
S. cerevisiae
Saccharomyces cerevisiae
S. pombe
Schizosaccharomyces pombe
SDS
sodium dodecyl sulfate
TdT terminal deoxynucleotidyl transferase
tel
telomere
TEMED
tetramethylethylenediamine
trx
transcript
UV
ultra violet
WT
wild type
IV
V
Erklärungen.................................................................................................................................................II
Abbreviations............III
1
Introduction..........1
1.1
Chromatin.....................................................................................................................................1
1.1.1
Nucleosome..........................1
1.1.2
Higher
order
structure....2
1.2
The
centromere................................................................................................4
1.2.1
Specification
of
centromere..........................7
1.3
Organization
of
centromeric
chromatin..........................................................................9
1.3.1
The
centromere
specific
histone
variant
(CenH3)..............9
1.3.2
Composition
of
CenH3
nucleosomes.....11
1.3.3
Histone
modifications
at
centromeres..................................................................13
1.3.4
CenH3
deposition...........................................13
1.4
The
kinetochore.......................................................16
1.4.1
Constitutive
and
cycle‐dependent
components
of
kinetochore................17
1.5
Neocentromeres......................................................................................20
1.6
Aim
of
the
thesis................................22
2
MATERIALS
&
METHODS............24
2.1
Cloning.........................................................................................................................................24
2.2
Cell
culture.26
2.2.1
Cultivation
of
cells..........26
2.2.2
Stable
transfection.........................................................................................................26
2.2.3
Protein
expression.........28
2.3
Live
imaging..............................................................28
2.4
Image
acquisition
and
analysis.........................................................................................30
2.5
Fluorescent
Activated
Cell
Sorting
(FACS)...................................31
2.6
Cytological
preparations......................................................................31
2.7
Microtubules
fixing................................................32
2.8
Immunofluorescence.............32
2.9
Metamorph®
Analysis..........................................................................35
2.10
Laser‐Microsurgery.............................................35
2.11
HP1
depletion
by
RNA
interference
(RNAi).............................................................35
2.12
Western
analysis..................................................................................36
2.13
Genomiphi
V2
DNA
amplification.................................................37
2.14
Fluorescence
in
situ
Hybridization
(FISH)................................................................38
2.14.1
FISH
probes
preparation..........................................................38
2.14.2
Probes’
purification
and
precipitation...............................40
2.14.3
Hybridization
reaction..............................................................40
2.15
Minichromosome
rescue
with
telomeric
sequences............................................42
3
Results..................................................................................................................43
3.1
Pulse
induction
of
CID
overexpression
leads
to
the
formation
of
ectopic
“CID
islands”
with
kinetochore
function............................................................43
3.2
CID
islands
self‐propagate
for
multiple
cell
generations......................................48
3.3
Overexpression
of
CID
combined
with
the
histone
H3.3
variant
or
bearing
mutations
in
the
CATD
domain
of
CID
interfere
with
CID
islands
formation..........50
3.4
Ectopic
kinetochores
preferentially
form
near
telomeres
and
pericentric
heterochromatin
regions................................................................................................................53
3.5
Formation
of
functional
ectopic
kinetochores
protects
CID
islands
from
clearing...................................................54
3.6
High
levels
of
HP1
expression
interfere
with
CID
islands
formation...............59
3.7
Local
targeting
of
HP1‐LacI
to
Lac
Operator
DNA
sequences
creates
a
new
hotspot
for
CID
deposition.............................................................................................................61