The function of the RNA-Binding protein CHLAMY1 in the circadian clock and its temperature integration process [Elektronische Ressource] / von Olga Voytsekh

Olga Voytsekh

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2008
• Nombre de lectures: 3
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

The Function of the RNA-Binding Protein CHLAMY1
in the Circadian Clock and its Temperature
Integration Process



Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium
(Dr. rer. nat.)



vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena









von Diplom-Pflanzenphysiologin
Olga Voytsekh
geboren am 23. Juli 1979 in Moskau, Russland























1. Gutachter: Prof. Dr. Maria Mittag
2. Gutachter: Prof. Dr. Ralf Oelmüller
3. Gutachter: Prof. Dr. Birgit Piechulla

Datum der öffentlichen Verteidigung: 26.06.2008
Content 3
Content
1. Introduction..........................................................................................................10
1.1 Physiological properties of circadian rhythms............................................................12
1.2 Principles of clock architecture...................................................................................13
1.3 The main components of the circadian clock in different organisms........................ 14
1.3.1 The central oscillator...................................................................................................14
1.3.2 The input pathway.......................................................................................................16
1.3.2.1 Temperature................................................................................................................16
1.3.2.2 Light............................................................................................................................17
1.3.3. The output pathway.....................................................................................................17
1.4 The circadian system of Chlamydomonas reinhardtii................................................18
1.4.1 C. reinhardtii, a eukaryotic model organism..............................................................18
1.4.2 The circadian clock of C. reinhardtii.........................................................................19
1.4.3 Molecular components of C. reinhardtii circadian system........................................20
1.4.4 The circadian RNA-binding protein CHLAMY1 of C. reinhardtii............................21
1.5 Main aims of the PhD work........................................................................................23
24 2. Materials and Methods......................................................................................
2.1 Materials.....................................................................................................................24
2.1.1 Devices.......................................................................................................................24
2.1.2 Chemicals....................................................................................................................25
2.1.3 Enzymes......................................................................................................................25
2.1.4 Kits and consumables.................................................................................................25
2.1.5 C. reinhardtii strains...................................................................................................26
2.1.6 Escherichia coli strains...............................................................................................27
2.1.7 Vectors........................................................................................................................27
2.1.8 Recombined plasmids.................................................................................................27
2.1.9 Oligonucleotides.........................................................................................................29
2.1.10 Molecular standards....................................................................................................29
2.1.10.1 DNA standards............................................................................................................29
2.1.10.2 Protein standards.........................................................................................................29
2.1.11 Antibodies...................................................................................................................29
2.1.11.1 Primary antibodies......................................................................................................29
2.1.11.2 Secondary antibodies..................................................................................................30
2.1.12 Cultivation media........................................................................................................30
2.1.12.1 E. coli cultivation media................................30
2.1.12.2 C. reinhardtii cultivation media..................................................................................30
2.1.13 Buffers and solutions..................................................................................................31
2.1.13.1 Solutions for C. reinhardtii cultivation media............................................................31 Content 4
2.1.13.2 Solutions for gel electrophoresis and standard molecular
biological methods......................................................................................................32
2.1.13.3 Buffers and solutions for protein biochemical experiments.......................................32
2.1.13.4 Buffers for immunochemical experiments..................................................................34
2.2 Methods......................................................................................................................34
2.2.1 Methods for a vector construction in E. coli...............................................................34
2.2.1.1 DNA amplification.....................................................................................................34
2.2.1.1.1 Design of oligonucleotides („primers“)......................................................................34
2.2.1.1.2 Polymerase chain reaction (PCR)...............................................................................34
2.2.1.1.3 Purification of a DNA product of the PCR.................................................................35
®2.2.1.1.3.1 Purification of DNA fragments using the „QIAquick
PCR Purification Kit“ (Qiagen, Hilden).....................................................................35
2.2.1.1.3.2 Separation of DNA fragments in agarose gel.............................................................35
2.2.1.1.3.3 Elution of DNA fragments from agarose gel..............................................................36
2.2.1.2 Determination of DNA concentration........................................................................36
2.2.1.2.1 Photometric analysis...................................................................................................36
2.2.1.2.2 "Dot"-test....................................................................................................................36
2.2.1.3 Transformation of E. coli by heat shock method........................................................37
2.2.1.4 Preparation of competent E. coli cells........................................................................37
2.2.1.5 Methods of DNA isolation..........................................................................................37
2.2.1.5.1 Isolation of plasmid DNA from E. coli in small scale................................................37
2.2.1.5.2 High pure plasmid isolation........................................................................................38
2.2.1.5.3 Large scale isolation of plasmid DNA from E. coli...................................................38
2.2.1.5.4 Phenol-chloroform extraction.....................................................................................39
2.2.1.5.5 Ethanol DNA precipitation.........................................................................................
2.2.1.5.6 Isolation of genomic DNA from C. reinhardtii..........................................................39
2.2.1.6 DNA restriction..........................................................................................................40
2.2.1.7 Polishing of sticky ends using Klenow enzyme.........................................................40
2.2.1.8 Ligation.......................................................................................................................40
2.2.2 Work with C. reinhardtii cultures..............................................................................41
2.2.2.1 Cultivation of C. reinhardtii.......................................................................................41
2.2.2.2 Harvest ofC. reinhardtii cells....................................................................................41
2.2.2.3 Cell concentration determination................................................................................41
2.2.2.4 Autolysin preparation....................................................................