New insights into circadian photoreception and the molecular regulation of the resetting of Drosophila's circadian clock [Elektronische Ressource] / vorgelegt von Nicolai Peschel

Niki Delaunay

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

New Insights into Circadian Photoreception
and the Molecular Regulation of the Resetting of
Drosophila’s Circadian Clock







DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER
NATURWISSENSCHAFTLICHEN FAKULTÄT III - BIOLOGIE
UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT
REGENSBURG

vorgelegt von
Nicolai Peschel
aus Nürnberg
im Juli des Jahres 2008





























Promotionsgesuch eingereicht am: 16.7.2008
Die mündl. Prüfung (Kolloquium) wurde am 16.09.2008 abgelegt.
Die Arbeit wurde angeleitet von Prof. Dr. Ralf Stanewsky
Queen Mary University of London






Prüfungsausschuss:
Vorsitzender: Prof. Dr. Stephan Schneuwly
Erster Gutachter: Prof. Dr. Ralf Stanewsky
Zweiter Gutachter: Prof. Dr. Charlotte Förster
Dritter GuProf. Dr. Gernot Längst

































Teile dieser Arbeit wurden bereits veröffentlicht:

N. Peschel, S. Veleri, and R. Stanewsky
Veela defines a molecular link between Cryptochrome and Timeless in the light-input
pathway to Drosophila's circadian clock
PNAS, November 14, 2006; 103(46): 17313 - 17318.

Nicolai Peschel and Ralf Stanewsky
Light-dependent interactions between Cryptochrome and Jetlag regulate circadian
clock resetting in Drosophila.
(Under Review in Current Biology)



Table Of Contents

1. Introduction………......................…………………………………….…....1
1.1 General Introduction and History…………………..…..…....………1
1.2 The Basics and Characteristics of Circadian Clocks….....….………3
1.3 The Circadian Clock of Drosophila melanogaster…….....….……….5
1.4 Input into Drosophilas Clock………………………..………………..8
1.5 Circadian Photoreception in Drosophila…........……………………...9
1.6 Cryptochrome…………………………………………………………...9
1.7 The Molecular Pathway of Drosophilas Circadian................
Photoreception……………………………..…………..………..........10
1.8 Anatomy of Drosophilas Circadian System……….....…...…..…......11
1.9 Evening and Morning Oscillators…..………………………….…….13
1.10 Neurochemistry of the Drosophila Circadian Clock…………….14
1.11 Electrical Activity in Clock Neurons……………………………….15
1.12 Aim of this Work……………………………...............………….16

2. Materials and Methods……………………………….….17
2.1 Materials…………………………………………………...………….…...17
2.1.1 Chemicals, enzymes and consumables…………………………..17
2.1.2 Fly, Bacteria and Yeast Strains...............................................17
2.1.3 Oligonucleotides and Vectors......................................................19
2.1.4 Solutions and Buffers....................................................................21
2.2 Methods................................................................................................23
Genomic DNA.................................................................................23
RNA.......................................................................................
cDNA...............................................................................................23
PCR..............................................23
qPCR...............................................................................................23
Sequencing...........................................................
Cloning...........................................................24
Packard .......................................................................................29
Behavior........................................................................................29
Antibodies....................................................................................30
Westernblot and Initial S2 Cell Transfection Experiments......30
CoIP...............................................................................................31
Immunocytochemistry (ICC) ................................................................32
P-element Transformation..........................................................32
Yeast-Two-Hybrid........................................................................33

3. Quasimodo.............................................................................34
3.1 Background..................................................................................................34 3.2 Introduction..................................................................................................35
3.2.1 Quasimodo is a Zona Pellucida Protein..............................................35
3.2.2 The quasimodo gene locus....................................................................38
3.2.3 MicroRNA Cluster 310-313 and a General Insight into........................
miRNAs..................................................................................................39
3.3 Quasimodo Results.......................................................................................40
3.3.1 Rhythmic Expression of quasimodo...........................................40
3.3.2 Behavior Analysis.......................................................................41
3.3.3 Period and Timeless Expression in the Adult Brain................43
3.3.4 QsmRNAi(16) in Constant Light Conditions...........................47
3.3.5 Localization of Quasimodo......................................................48
3.3.5.1 Qsm Antibody...................................................................48
05510 3.3.5.2 Reporter Gene Expression in P(PZ)l(2)05510 .........
Animals.................................................................................51
01 3.3.6 per and Quasimodo..............................................................53
3.3.6.1 Behavior........................................53
01 3.3.6.2 Tim Amount in per ............................................................55
3.3.7 PDF and Quasimodo................................................................58
3.3.8 miRNA Involvement in Circadian Rhythm.........................64
3.3.8.1 Function and Interaction....................................................68
3.3.9 Cg31547....................................................................................70
3.4 Discussion.....................................................................................72
3.4.1 Rhythmic Expression.............................................................72
3.4.2 Behavior Analysis...................................................................72
3.4.3 Localization of Quasimodo.....................................................75
01 3.4.4 per and quasimodo..................................................................77
3.4.5 PDF and quasimodo.................................................................80
3.4.6 The EP2586 Insertion..............................................................81
3.5 Outlook...............................................................................................83

4. Veela and Jetlag.............................................................85
4.1 Background......................................................................................85
4.2 Veela Results....................................................................................86

Veela defines a molecular link between Cryptochrome and Timeless in the light-
input pathway to Drosophila's circadian clock.....................................87

4.3 jetlag regulates circadian clock resetting in Drosophila..............87

Light-dependent interactions between Cryptochrome and Jetlag regulate
circadian clock resetting in Drosophila..........................................................88

4.4 Influence of Shaggy on Cryptochromes stability..........................89
4.4.1 Introduction Shaggy.................................................................89
4.4.2 Shaggy Results.........................................................................90
4.4.3 Discussion Shaggy.................................................................94
4.4.4 Outlook Shaggy........................................................................98

5. The roundabout Gene’s Function in Circadian Rhythm..99
5.1 Introduction.......................................................................................99
5.2 Results.............................................................................................101
5.2.1 Adult Flies.................................................................................101
5.2.2 Semi Cultures...........................................................................102
5.3 Discussion......................................................