Molecular mechanisms of circadian photoreception in Neurospora crassa [Elektronische Ressource] / von Zdravko Dragovic

Dragovic Zdravko

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 26
• Poids de l'ouvrage: 9 Mo

Extrait

Molecular Mechanisms of Circadian
Photoreception in Neurospora crassa
Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
von
Zdravko Dragovic
aus
Batajnica in SerbienInaugural dissertation
zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
vorgelegt von
Diplom Biologer Zdravko Dragovic
aus Batajnica in Serbien
Erstgutachter: Herr Prof. Reinhold Herrmann
Zweitgutachter: Herr Prof. Eberhard Gwinner
Sondergutachter: Herr Prof. Till Roenneberg
Mündliche Prüfüng am 16. Februar 2004
21 Introduction.............................................................................................................7
1.1 Circadian rhythms............................................................................................7
1.2 Properties of circadian systems ........................................................................8
1.2.1 Free-running and self-sustainment............................................................8
1.2.2 Period length............................................................................................9
1.2.3 Circadian rhythms are entrainable ............................................................9
1.2.4 Circadian rhythms are compensated .........................................................9
1.2.5 Modules of circadian clock ......................................................................9
1.3 Entrainment of circadian rhythms by light......................................................10
1.3.1 Phase response curves (PRCs)................................................................10
1.3.2 Circadian photosensory system ..............................................................11
1.3.3 The Gonyaulax story ..............................................................................12
1.3.4 Circadian photoreception in Arabidopsis................................................12
1.3.4.1 Cryptochromes...................................................................................12
1.3.4.2 Phytochromes.....................................................................................13
1.3.4.3 Resetting of the Arabidopsis clock with light......................................14
1.3.5 Circadian Photoreception in mice...........................................................15
1.3.5.1 Specialized circadian photoreceptor cells in eye .................................15
1.3.5.2 Resetting of the clock within an SCN cell...........................................16
1.4 Neurospora crassa: A simple model system for clock and photobiology studies
17
1.4.1 The biology of Neurospora crassa .........................................................17
1.4.2 Asexual propagation...............................................................................17
1.4.3 Sexual cycle...........................................................................................18
1.5 Circadian Rhythms in Neurospora crassa ......................................................19
1.5.1 Conidiation is major output of circadian rhythm.....................................19
1.5.2 Genetic analysis of the clock ..................................................................20
1.5.3 Genetic analysis of the light input pathway ............................................21
1.5.4 The Neurospora genome suggests additional photoreceptors..................21
1.6 Action spectra analysis ..................................................................................22
1.7 Molecular studies of the photoreception in Neurospora..................................23
1.7.1 White Collars.........................................................................................23
1.7.2 FRQ protein ...........................................................................................24
1.7.3 Resetting of the Neurospora clock by light.............................................25
1.7.4 Photoadaptation and circadian photoreception........................................26
1.8 Open questions ..............................................................................................27
2 Results...................................................................................................................28
2.1 Molecular and genetic regulation of the light input pathway in Neurospora
crassa 28
2.1.1 Rhythmicity in DD: FRQ and WC-1 ......................................................29
2.1.2 Regulation of frq and wc-1 is interdependent: WC-1 regulates frq and
FRQ regulates wc-1 ...............................................................................................32
2.1.3 frq and the light signaling pathway.........................................................35
2.1.4 Circadian regulation of light signaling....................................................37
2.2 Posttranscriptional control of wc-1 expression ...............................................38
2.2.1 Identification of putative protein binding sites in wc-1 UTRs .................38
32.2.2 Proteins bind to 5’ and 3’ UTRs of wc-1 mRNA ....................................40
2.2.3 Examining specificity of protein-UTR complexes ..................................41
2.2.4 Circadian regulation of complex formation is not observable .................42
2.2.5 Light regulates UTR complex formation ................................................45
2.3 Light-regulated behavior in white collar mutants ...........................................46
2.3.1 white collar mutants synchronize conidiation in response to light...........47
2.3.2 Fluence titration of light induced conidiation in wc mutant strains..........53
2.3.3 A self–sustained circadian rhythm in the wc mutants..............................54
2.4 Molecular characterization of a series of white collar alleles ..........................55
2.4.1 Light induction of FRQ protein in different wc background....................55
2.4.2 Verification of the wc strains..................................................................56
2.4.3 Molecular dissection of light input mechanisms .....................................59
2.4.4 Presence of FRQ but not quantitative or qualitative changes are required
for the regulation of conidiation by light................................................................63
2.4.5 Fluence titration of light induction of FRQ protein .................................64
2.5 Identification of light induced genes in wc-2 mutant.......................................65
2.6 Action spectrum for FRQ protein induction ...................................................68
2.6.1 Equal Intensity action spectrum..............................................................69
2.6.2 Range of reciprocity for FRQ protein light induction..............................71
2.6.3 Dose response curves .............................................................................73
2.6.4 Action spectrum for FRQ protein induction............................................75
2.7 Novel photoreceptor candidates .....................................................................77
3 DISCUSSION .......................................................................................................85
3.1 Regulation of the light input pathway in N. crassa .........................................85
3.1.1 Levels of WC-1 protein are circadianly regulated...................................85
3.1.2 Interdependent regulation of FRQ and WC-1 .........................................86
3.1.3 Circadian regulation of light responses...................................................86
3.2 Novel photoreceptors in Neurospora..............................................................87
3.2.1 wc mutants respond to light....................................................................87
3.2.2 What are the photoreceptors in Neurospora?..........................................89
3.2.3 Dual light input pathway in N. crassa.....................................................91
3.2.4 FRQ role in regulation of conidiation and carotenogenesis .....................92
3.2.5 A self-sustained circadian rhythm in the wc mutants ..............................93
3.2.6 Action spectrum of FRQ protein induction.............................................93
3.3 Posttranscriptional regulation of wc-1 expression...........................................94
3.3.1 Regulation of translation efficiency by binding to the 5’ UTR................95
3.3.2 Adaptation in constant light and regulation of mRNA stability by binding
to the 3’ UTR ........................................................................................................96
4 Materials and Methods ..........................................................................................98
4.1 Physiological Methods...................................................................................98
4.1.1 Strain maintenance.................................................................................98
4.1.2 Media supplements ................................................................................98
4.1.3 Stock management .................................................................................98
4.1.4 Lab practice ...........................................................................................99
4.1.5 Conidial suspensions -inoculum ............