Neurospora crassa [Elektronische Ressource] : a model system for photoperiodism and circadian rhythm research / vorgelegt von Ying Tan

Ying Tan

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 35
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Institut für Medizinische Psychologie der Universität München
Vorstand: Prof. Dr. E. Pöppel
Neurospora crassa –
A Model System for Photoperiodism and Circadian Rhythm Research
Dissertation
zum Erwerb des Doktorgrades der Medizin
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München
vorgelegt von
Ying Tan
aus P.R.China
2003
1Mit Genehmigung der Medizinischen Fakultät
der University München
Berichterstatter: Prof. Dr. Till Roenneberg
Mitberichterstatter: Prof. Dr. Thomas Cremer
Prof. Dr. Walter Neupert
Mitbetreuung durch den
promovierten Mitarbeiter: Priv. Doz. Martha Merrow
Dekan: Prof. Dr. med. Dr.h.c. K. Peter
Tag der mündlichen Prüfung:____ 26.06, 2003_____
21. Introduction................................................................................................................6
1.1 Photoperiodism.................................................................................................... 6
1.1.1 Specific examples of photoperiodism........................................................... 7
1.2 Circadian Rhythm.............................................................................................. 10
1.2.1 Circadian rhythm is common in nature ...................................................... 10
1.2.2 Circadian rhythms of human ...................................................................... 11
1.2.3 Properties of circadian rhythms.................................................................. 11
1.2.4 General description of circadian system..................................................... 12
1.3 Mechanism of Photoperiodism.......................................................................... 13
1.1.3 Neurospora crassa – a potential model to attack questions of
photoperiodism at molecular level.......................................................................15
1.1.3.1 The life cycles of Neurospora crassa.................................................. 15
1.1.3.2 The conditions of in nature allow a photoperiodic
response ........................................................................................................... 16
1.4 Circadian Rhythms of Neurospora crassa ........................................................ 17
1.4.1 Circadian rhythm of conidiation of Neurospora crassa............................. 17
1.4.2 The molecular mechanism of circadian rhythms in Neurospora crassa ....18
1.4.2.1 The “central” components of the oscillation ....................................... 18
1.4.2.2 The molecular hallmarks of the Neurospora clock cycle.................... 19
1.4.3 Circadian gated light signaling in Neurospora crassa ............................... 20
2. Materials and Methods ............................................................................................ 22
2.1 Neurospora crassa Strains................................................................................. 22
2.2 Physiological Methods.......................................................................................25
2.2.1 Strain maintenance......................................................................................25
2.2.2 Growth conditions 25
2.2.3 Neurospora cultures....................................................................................26
2.2.4 Conidia counting......................................................................................... 26
2.2.4.1 Preparation of slants ............................................................................ 26
2.2.4.2 Conidia harvesting and counting ......................................................... 27
2.2.5 Race tubes................................................................................................... 27
2.2.6 Sexual crosses............................................................................................. 27
2.2.7 Protoperithecia counting............................................................................. 28
2.2.7.1 Preparation of conidia for inoculation ................................................. 28
2.2.7.2 Processing and counting of protoperithecia from conidial strains.......28
2.2.7.3 Preparation and counting of protoperithecia from aconidial strains....29
2.2.8 Spore counting............................................................................................ 29
2.2.9 Carotenoid assay......................................................................................... 30
2.2.9.1 Media for carotenoid assay.................................................................. 30
2.2.9.2 Inoculation and harvesting................................................................... 30
2.2.9.3 Carotenoid extraction...........................................................................31
2.2.10 Light-dark cycles ...................................................................................... 31
2.2.11 Temperature cycles................................................................................... 31
2.3 Molecular Methods 32
2.3.1 DNA preparation ........................................................................................ 32
2.3.1.1 Genomic DNA preparations ................................................................ 32
2.3.1.2 Amplification of DNA with Polymerase Chain Reaction (PCR) ........32
2.3.1.3 Prepare plasmid DNA from E.coli.......................................................33
2.3.2 Quantitation of DNA concentration............................................................34
2.3.2.1 Quantitation of DNA with spectrophotometer .................................... 34
2.3.2.2 Quantitation of DNA with gel electrophoresis 34
32.3.3 Plasmid DNA cloning................................................................................. 34
2.3.3.1 Cloning pKSbar2cpc1frq..................................................................... 34
2.3.3.2 Cloning the 5’ end of the wc-1 ORF.................................................... 35
2.3.4 Transformation of E.coli by electroporation .............................................. 36
2.3.5 Protein analysis........................................................................................... 37
2.3.5.1 Neurospora protein extraction............................................................. 37
2.3.5.2 Establishing a standard curve for quantitation of protein extracts ......37
2.3.5.3 Quantitation of protein concentration.................................................. 37
2.3.5.4 SDS-PAGE .......................................................................................... 38
2.3.5.5 Protein transfer from gel to nitrocellulose membrane ......................... 39
2.3.5.6 Probing the membrane......................................................................... 39
2.3.5.7 Developing the membrane................................................................... 40
2.3.5.8 Analysis of the blots ............................................................................ 40
2.3.6 RNA analysis.............................................................................................. 41
2.3.6.1 Neurospora RNA extraction................................................................ 41
2.3.6.2 Quantitation of RNA concentration..................................................... 41
2.3.6.3 RNA gel electrophoresis...................................................................... 41
2.3.6.4 Northern blot: Transfer of RNA from the gel to a nylon membrane...42
2.3.6.5 RNA hybridization...............................................................................42
2.3.7 RNA analysis with TaqMan RT-PCR ........................................................ 43
2.3.7.1 RNA digestion with DNase ................................................................. 43
2.3.7.2 Reverse transcription ........................................................................... 43
2.3.8 Primers used in this study 45
2.4 Genetic Methods................................................................................................ 45
2.4.1 Spheroplast Preparation.............................................................................. 45
2.4.2 Transformation of DNA into Spheroplasts................................................. 46
2.5 Time Scales........................................................................................................ 47
3. Results......................................................................................................................48
3.1 Photoperiodic Responses in a 24-hour Day....................................................... 48
3.1.1 Phase of conidiation in different photoperiods........................................... 48
3.1.2 Photoperiodic response of the formation of asexual spores – conidia........51
3.1.3 Photoperiodic response of light induced mycelial carotenoids .................. 53
3.1.4 Photoperiodic response of protoperithecia development............................ 55
3.1.3 Photoperiodic response of ascospore-shooting 59
3.2 Physiological responses in a non-24-hour photoperiod (T=18h) ...................... 60
3.2.1 Influence of photoperiod on carotenoid production ................................... 60
3.2.2 Photoperiodic response of protoperithecia development............................ 61
3.3 Physiological Responses in Night-break Experiments...................