DNA methylation, histone modification and the transcription factor dE2F1 in Drosophila [Elektronische Ressource] / Alexandra Weyrich

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• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

DNA methylation, histone modification
and the transcription factor dE2F1 in Drosophila





Dissertation zur Erlangung des Grades
Doktor der Naturwissenschaften



Eingereicht am Fachbereich Biologie
der Johannes Gutenberg Universität Mainz
Erarbeitet im DAAD/MPG Labor
an der Chinesischen Akademie der Biologischen Wissenschaften,
Shanghai/China




Alexandra Weyrich
geboren am 20. März 1979 in Birkenfeld


Mainz, April 2007 Index
Index
I. DNA methylation in the male germ line of Drosophila
1. Abstract___________________________________________________________ 15
2. Introduction_______________________________________________________ 16
3. Material & Methods________________________________________________ 19
3.1 Subcloning and Microinjection 19
3.1.1 β2t-Dnmt3a/3b constructs 19 3.1.2 UAS-Dnmt3a/3b constructs 22
3.2 GAL4 expression of β2t- and UAS-Dnmt3a/3b 22
3.3 Western Blots 23
3.4 In Vitro Methylation Assay 24
3.5 2D-Thin Layer Chromatography 24
3.6 DAPI- and Immunostaining of Testis 25
3.7 Testis preparation for HPLC and MALDI-TOF 26
3.8 High Performance Liquid Chromatography (HPLC) analysis 26
3.9 Matrix-assisted laser desorption ionization – time of flight mass 27
spectrometry (MALDI-TOF) analysis
4. Results___________________________________________________________ 28
4.1 Does DNA methylation exist in Drosophila? 28
4.2 DNA methylation in Dnmt-transformant flies 31
4.2.1 Dnmt3a 31 4.2.2 Dnmt3b 32
4.2.3 Tissue specific Dnmt expression 37
5. Discussion________________________________________________________ 40
5.1 DNA methylation in Dnmt-transformant flies 40
6. Acknowledgement__________________________________________________ 44
7. References________________________________________________________ 45
8. Appendix_________________________________________________________ 48
8.1 pUAS-Dnmt3 crossed with GAL4 strains 48


Index
II. Histone methylation in the male germ line of Drosophila
1. Abstract__________________________________________________________ 52
2. Introduction_______________________________________________________ 53
3. Material & Methods_______________________________________________ 55
3.1 Drosophila strains 55
3.2 Preparation of slides and Immunocytology 55
4. Results___________________________________________________________ 57
4.1 Germ cells 59
4.2 Somatic cells 61
5. Discussion________________________________________________________ 64
6. Acknowledgement__________________________________________________ 67
7. References________________________________________________________ 68

III. A transcript specific mutation of the dE2F1 transcription
factor in Drosophila melanogaster
1. Abstract__________________________________________________________ 70
2. Introduction_______________________________________________________ 71
2.1 The E2F transcription factor 71
TF 2.2 dE2F1 mutation 73
3. Material & Methods________________________________________________ 75
3.1 Crossings of balanced dE2F1 mutant flies 75
3.2 PCR for dE2F1 homozygosity check 76
3.2.1 DNA-isolation from single flies 76
3.2.2 Polymerase Chain Reaction (PCR) 76
3.3 2-Dimensional Polyacrylamid Gel Electrophoresis (2D-PAGE) 78
3.3.1 Protein sample preparation of Drosophila ovaries 78
st 3.3.2 Passive rehydration and IEF 1 dimension 79
nd 3.3.3 Casting and run of the 2-Dimension 79
3.3.4 Blotting and Silver Staining 81
3.4 Immunoprecipitation of dE2F1 82
3.4.1 Dialysis of the antibody 82
3.4.2 CNBr-Sepharose activation 82
3.4.3 Antibody coupling Index
3.4.4 Sample preparation 83
3.4.5 Antigen binding 83
3.4.6 Elution of the protein complexes 83
3.5 MALDI-Mass Spectrometry (MS) 84
3.6 Real Time PCR 84
3.6.1 Tissue dissection 85
3.6.2 mRNA isolation and cDNA pre-amplification 85
3.6.3 cDNA concentration measured by photometer 88
3.6.4 Standards 89
3.6.5 No template control (NTCs)
3.6.6 SYBR Green reaction mix 90
3.6.7 Plate design 90
3.6.8 Program conditions 91
3.6.9 Final calculation - Relative quantification with external 92
standards
4. Results___________________________________________________________ 93
TF 4.1 Crossings for dE2F1 homozygosity check 93
TF 4.1.1 Single crosses with homozygous dE2F1 mutant flies 93
TF 4.1.2 Crosses with heterozygous dE2F195
TF 4.2 PCR for dE2F1 homozygosity check 96
4.3 dE2F1 protein sequencing by Mass Spectrometry 98
4.3.1 Two Dimensional Poly Acrylamid Gel Electrophoresis 99
(2D-PAGE)
4.3.2 Immunoprecipitation 101
4.4 The expression levels of the dE2F1 transcripts 102
4.4.1 Semi-quantitative RT-PCR 102
4.4.2 Quantitative Real Time PCR 105
4.4.3 Relative quantification 110
5. Discussion________________________________________________________ 113
5.1 The dE2F1 protein(s) 113
5.2 dE2F1 transcripts 114
5.2.1 Improvements of future Real Time PCR 116
6. Acknowledgement__________________________________________________ 118
7. References________________________________________________________ 119
8. Appendix_________________________________________________________ 122Index
8.1 The P-element insertion into the dE2F1 sequence 122
8.1.1 P-element transformation vector 122
8.1.2 BLAST for P-element localization 124
8.1.3 Mutation of dE2F1 gene 126
8.1.4 Blast result of unspecific 500bp fragment 127
8.2 Primers for semi-quantitative RT-PCR 129



130Zusammenfassung

131Curriculum Vitae

Index of Figures
Index of Figures
I. DNA methylation in the male germ line of Drosophila
Fig. 1 Murine DNA-methyltransferases 17 2 Subcloning of Dnmt3a 20
Fig. 3 Dnmt3b 21 4 100bp DNA Ladder Plus 23
5mC-Immunocytology of Drosophila melanogaster germ cells Fig. 5 29
5mC-Immu testis envelope 6 29
cells
Fig. 7 Immunocytology of primary spermatocytes in Drosophila hydei 30 8 Immunocytology of young round spermatids in Drosophila hydei 30
spermatocytes
Fig. 9 RT-PCR of β2t-Dnmt3a/3b 31 10 Western blot of testis protein from β2t-Dnmt3b transformant strains 32
Fig. 11 Schemata of 5mC in 2D- thin layer chromatography 33 12 2D-thin layer chromatography 34
Fig. 13 HPLC analysis 35 14 RT-PCR products of pUAS-Dnmt3a/3b 39


II. Histone modification in the male germ line of Drosophila
Fig. 1 Illustration of histone 3 and 4 modifications 54
Antibody labelling in D. melanogaster spermatocytes 2 59
Antibody labelling in spermatocytes of D. hydei Fig. 3 60 4 Antibody labelling in somatic cells of D. melanogaster 62


III. A transcript specific mutation of the dE2F1 transcription
factor in Drosophila melanogaster
Fig. 1 E2F/Rb pathway 71 2 dE2F1 gene and its three transcripts E2F-RA, -RB and -RC 73
Fig. 3 The balancer system 75 4 PageRuler™ Unstained Protein Ladder 80
Loading of IPG-strip Index of Figures
P-element insertion into the dE2F1 gene and primer positions Fig. 5 96 6 Separation of PCR products by agarose gel electrophoresis 97
TF Fig. 7 2D- gel electrophoresis of white and dE2F1 ovaries 1008 Silver stained gel after IP 102
Fig. 9 Map of the three dE2F1 transcripts and primer locations 10310 Semi-quantitative RT-PCR 104
Fig. 11 Amplification curve of Rp49 standards 10512 Standard curve of Rp49 105
Fig. 13 Melting peaks of the four primer pairs in real time PCR reactions 10614 Rp49 product after real time PCR 107
Diagram of the reaction mean concentrations Fig. 15 10816 dE2F1-RB products after real time PCR 109
TF Fig. 17 Diagram fold changes of the three transcripts of dE2F to white 11118 pBac vector construct including the P-element 123
Index of Tables
Index of Tables
I. DNA methylation in the male germ line of Drosophila
Table 1 Primer for RT-PCR of Dnmt3a and Dnmt3b transcripts 23 2 In vitro methylation analysis measurement 34 3 MALDI-TOF analysis 36
Table 4 GAL4 strains 38
Crossing data of parent (P) generation: pUAS-Dnmt3a/3b strain 1 5 49
Crossing data of first offspring (F1) generation: pUAS-Dnmt3a/3b 6 50
strain 1
Table 7 Crossing data of P and F1 generation of pUAS-Dnmt3a/3b strain 2 51


II. Histone modification in the male germ line of Drosophila
Table 1 Antibodies and their reaction in immunostaining experiments 58
targeting histone modifications in Drosophila melanogaster


III. A transcript specific mutation of the dE2F1 transcription
factor in Drosophila melanogaster
Table 1 PCR primer for homozygous check 77 2 Running conditions for IEF 79
nd3 Running conditions for 2 dimension 81
Table 4 Silver staining procedure 81 5 Tissues dissected for real time PCR 85 6 Primers for real time PCR 87
Photometric measuring of the amplified cDNA samples Table 7 88
Photometric mtandards 8 89 9 96 well plate design 91
Table 10 Program settings for PCR run 91
TF11 Data of homozygous dE2F1 single crosses 94
TF12 Crossings of homozygous with heterozygous dE2F1