Search for genes involved in the synthesis of poly(L-malate) in the plasmodium of Physarum polycephalum [Elektronische Ressource] / by Nadthanun Pinchai

Nadthanun Pinchai

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Search for Genes Involved in the Synthesis
of Poly(L-malate) in the Plasmodium of
Physarum polycephalum



Dissertation

zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer.nat.)
der Naturwissenschaftlichen Fakultät III
-Biologie und Vorklinische Medizin-
der Universität Regensburg

by
Nadthanun Pinchai
from Bangkok


Regensburg 2004





















Promotionsgesuch eingereicht am: 20.10.2004

Tag des Kolloquiums: 16.12.2004

Die Arbeit wurde angeleitet von: Prof. Dr. E. Holler

Prüfungsausschuss: Vorsitzender: Prof. Dr. R. Sterner

Erstgutachter: Prof. Dr. E. Holler

Zweigutachter: Prof. Dr. K. Kunzelmann

Drittprüfer: Prof. Dr. Ch. Förster



CONTENTS
Abbreviations...............................................................................................................7
I Introduction ..........................................................................................................9
1 Physarum polycephalum ..................................................................................9
1.1 Taxonomy..................................................................................................9
1.2 Life cycle .................................................................................................10
1.3 Differential gene expression in amoebae and plasmodia ........................12
2 ß-poly(L-malic acid) (PMLA) ...........................................................................14
2.1 Chemical structure and natural sources ..................................................14
2.2 PMLA in Physarum polycephalum...........................................................14
2.3 Biosynthesis of PMLA in Physarum polycephalum..................................15
2.3 Biodegradation ........................................................................................16
3 Goal of the thesis............................................................................................18
II Materials und methods.......................................................................................19
1 Materials.........................................................................................................19
1.1 Apparatus and accessories .....................................................................19
1.2 Consumable goods..................................................................................20
1.3 Chemicals................................................................................................21
1.4 Enzymes, antibodies and vectors............................................................23
1.5 Kits ..........................................................................................................25
1.6 Organisms ...............................................................................................25
1.7 Standard markers....................................................................................26
1.7.1 DNA standard markers.....................................................................26
1.7.2 Protein standard marker...................................................................27
1.9 Solutions and media for cell culture.........................................................28
1.9.1 Solutions and media for plasmodia cultures.....................................28
1.9.2 Solutions and media for amoebae cultures ......................................29
1.10 Solutions for analysis of nucleic acids ....................................................32
®1.10.1 Solutions for mRNA isolation using Dynabeads oligo(dT) ............32 25
1.10.2 Solutions for agarose gel electrophoresis ........................................33

1.10.3 Solutions for suppression subtracted hybridization and knock- down
assays 34
1.10.4 Solutions and media for transformation of DNA ...............................34
1.11 Solutions for SDS-PAGE and Western Blotting.......................................36
1.11.1 Solutions for cell lysis and SDS electrophorese36
1.11.2 Solutions for Western Blotting: .........................................................38
1.12 Solutions for quantitative analysis of PMLA.............................................39
2 Methods..........................................................................................................40
2.1 Cell culture ..............................................................................................40
2.1.1 Cultivation of plasmodia ...................................................................40
2.1.1.1 Cultivation of microplasmodia.......................................................40
2.1.1.2 Induction of Spherules..................................................................41
2.1.1.3 Cultivation of macroplasmodia......................................................41
2.1.2 Cultivation of amoebae.....................................................................41
2.1.2.1 Growth of amoebae on DSDM agar plates...................................41
2.1.2.2 Preparation of amoebal stock culture ...........................................42
2.1.2.3 Growth of amoebae in axenic liquid medium................................42
2.2 Isolation of nucleic acids..........................................................................43
2.2.1 Isolation of total RNA........................................................................43
+ ®2.2.2 Poly A mRNA isolation from total RNA using Dynabeads
Oligo(dT) ......................................................................................................44 25
2.2.2.1 Principle........................................................................................44
2.2.2.2 Procedures ...................................................................................44
2.2.3 Isolation of DNA using QIAquick PCR Purification Kit ......................45
2.2.3 Isolation of DNA from agarose gel using QIAquick...........................45
Gel Extraction Kit............................................................................................45
®2.2.5 Isolation of Plasmid DNA using Nucleospin Plasmid Kit.................46
2.2.6 Isolation of plasmid DNA using QIAGEN Plasmid Maxi Kit ..............47
2.3 Analysis and amplification of nucleic acids..............................................48
2.3.1 Quantification of nucleic acids..........................................................48
2.3.2 Polymerase chain reaction (PCR) ....................................................49
2.3.3 Real time PCR..................................................................................50
2.3.3.1 Principle........................................................................................50

2.3.3.2 Experimental procedure ...............................................................55
2.3.3.2.1 Absolute quantification...........................................................55
2.3.3.2.2 Relative quantification............................................................57
2.3.4 RT-PCR............................................................................................58
2.3.4.2 First-strand cDNA synthesis using oligo(dT) primer .....................59
2.3.4.3 cDNA synthesis using CapFinder.................................................60
2.3.4.3.1 Principle .................................................................................60
2.3.4.3.2 Procedure ..............................................................................62
2.3.4.4 5' RACE........................................................................................63
2.3.4.4.1 Principle63
2.3.4.4.2 Procedures.............................................................................65
2.4 Cloning of DNA fragments.......................................................................66
2.4.1 Principle ...........................................................................................66
2.4.2 Procedures.......................................................................................68
®2.4.2.1 Ligation of DNA fragment with a pGEM -T vector........................68
2.4.2.2 Transformation of ligated DNA .....................................................68
2.4.2.3 Isolation of plasmid DNA ..............................................................69
2.4.2.3 Verification of DNA insertion by restriction enzyme digestion.......69
2.5 Suppression subtractive hybridization70
2.5.1 Principle of suppression subtractive hybridization............................70
2.5.2 Experimental procedure ...................................................................73
+2.5.2.1 Isolation of poly(A) RNA73
2.5.2.2 First-stranded cDNA synthesis .....................................................73
2.5.2.3 Analysis of synthesized cDNA......................................................74
2.5.2.4 Long-distance PCR (LD-PCR)75
2.5.2.5 BstUI digestion .............................................................................78