Comparative DNA sequence and methylation analyses of orthologous genes in humans and non-human primates [Elektronische Ressource] : genetic and epigenetic footprints of evolution / Ruxandra Denisa Farcas

May0a

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Comparative DNA sequence and methylation
analyses of orthologous genes in humans and
non-human primates:
Genetic and Epigenetic Footprints of
Evolution



Dissertation
Zur Erlangung des Grades
„Doktor der Naturwissenschaften“



am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz





Ruxandra Denisa Farcas
geb. am 24.09.1980 in Klausenburg, Rumänien


Mainz, 2009





































Dekan: _________________

1. Berichterstatter: _________________

2. Berichterstatter:

Tag der mündlichen Prüfung: __________






INDEX
1. INTRODUCTION .................................................................................................................1
1.1 An excursion into genome evolution..........................................................................................1
1.2 Understanding the role of positive and negative selection in evolution.................................. 6
1.3 Gene expression and the importance in primate evolution................................................... 10
1.4 Insight into Epigenetics ............................................................................................................ 13
1.4.1 DNA Methylation............................................................................................................................... 15
1.4.2 DNA Methylation and Evolution........................................................................................................ 19
2. MATERIALS AND METHODS......................................................................................... 21
2.1 MATERIALS ............................................................................................................................ 21
2.1.1 Tissues Samples.................................................................................................................................. 21
2.1.2 Oligonucleotides......................................................................................................... 23
2.2 METHODS ................................................................................................................................ 25
2.2.1 Isolation of nucleic acids .................................................................................................................... 25
2.2.1.1 Isolation of genomic DNA from tissue samples ......................................................................... 25
2.2.1.2 Plasmid DNA isolation ............................................................................................................... 26
2.2.1.3 RNA isolation from tissue sample .............................................................................................. 27
2.2.2 Spectrophotometric quantification of nucleic acid concentration....................................................... 29
2.2.3 Agarose gel electrophoresis................................................................................................................ 29
2.2.4 Gel extraction of PCR fragments........................................................................................................ 30
2.2.5 DNA Cleanup ..................................................................................................................................... 31
2.2.6 Cloning of PCR products.................................................................................................................... 33
2.2.7 Bisulfite conversion and cleanup of DNA for methylation analysis................................................... 37
2.2.8 PCR (Polymerase Chain Reaction)........... 41
2.2.8.1 Standard PCR.............................................................................................................................. 42
2.2.8.2 RT (Reverse Transcription) PCR................................................................................................ 43
2.2.8.3 Quantitative Real-time PCR ....................................................................................................... 44
2.2.9 DNA Sequencing..................... 47

INDEX
2.2.9.1 Bisulfit-sequencing ..................................................................................................................... 49
2.2.9.2 Pyrosequencing........................................................................................................................... 50
2.2.10 Bioinformatic Tools.......................................................................................................................... 53
2.2.10.1 In silico analysis of the coding sequences in the breakpoint regions........................................ 53
2.2.10.2 BioEdit version 5.0.6 ................................................................................................................ 55
2.2.10.3 MEGA 3.1 version (Molecular Evolutionary Genetics Analysis software).............................. 56
2.2.10.4 CpG Island search................ 57
2.2.10.5 Primer Design ........................................................................................................................... 57
2.2.10.6 RepeatMasker (http://www.repeatmasker.org) ......................................................................... 58
2.2.10.7 Giri (http://www.girinst.org)..................................................................................................... 58
2.2.10.8 Transcriptional Regulatory Element Database (http://rulai.cshl.edu) ....................................... 58
2.2.10.9 Panther Database (http://www.pantherdb.org).......................................................................... 58
2.2.10.10 Novartis gene expression Atlas (http://symatlas.gnf.org/SymAtlas) ...................................... 59
3. RESULTS ............................................................................................................................ 60
3.1 Evolution of coding sequences in the evolutionary breakpoint regions ............................... 60
3.1.1 Evolution of the coding sequences in the breakpoint regions of chromosome 1, 4, 5, 9, 12, 17 and 18
..................................................................................................................................................................... 61
3.1.2 Summarized data of the coding sequence comparison between human and chimpanzee................... 69
3.2 Bioinformatical comparison of gene expression in the evolutionary breakpoint regions of
human and chimpanzee.................................................................................................................. 76
3.3 DNA-Methylation analysis in human and non-human primates.......................................... 79
3.3.1 Comparison of CGI promoter methlyation in human and chimpanzee cortex ................................... 80
3.3.2 Variation of CCRK CGI promoter methylation among humans and non-human primates ................ 85
3.4 Expression of CCRK gene in the frontal cortex of human and non-human primates......... 94
3.5 Analysis of DNA methylation patterns using bisulfite Pyrosequencing............................... 97
4. DISCUSSION.................................................................................................................... 102

INDEX
4.1 Natural Selection of genes at evolutionary breakpoint regions .......................................... 102
4.2 Plasticity of CpG islands methylation patterns in human and non-human primates cortices
........................................................................................................................................................ 107
4.3 Comparison of DNA-Methylation patterns in human and chimpanzee cortices by bisulfite
Pyrosequencing ............................................................................................................................. 113
5. SUMMARY........................................................................................................................ 115
6. REFERENCES 118
7 ANNEX ............................................................................................................................... 128
7.1 Abbreviatons ........................................................................................................................... 128
7.2 Figure Index............. 131
7.3 Table Index .............................................................................................................................. 134
7.4 Acknowledgments ................................................................................................................... 136
7.5 Curriculum vitae..................................................................................................................... 137
7.6 Publication............... 138




1. Introduction
1. INTRODUCTION

1.1 An excursion into genome evolution

Hominid evolution and human speciation are the most fascinating topics in evolutionary
biology