Regulation of DNA methylation during development [Elektronische Ressource] : alternative isoforms of DNA methyltransferase / von Ana Maria Aguirre-Arteta

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Aus dem Institut für Biologie der Humboldt-Universität Berlin REGULATION OF DNA METHYLATION DURING DEVELOPMENT: ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE DISSERTATION zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat. ) im Fach BIOLOGIE eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Ana Maria Aguirre-Arteta geb. am 02.01.1968 in Bilbao (Spanien) Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. hc. Hans Meyer Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Bernhard Ronacher Gutachter: 1. Prof. Dr. Harald Saumweber 2. PD Dr. Jörn Walter 3. Prof. Dr. Franz Theuring Tag der mündlichen Prüfung: 28.06.00 REGULATION OF DNA METHYLATION DURING DEVELOPMENT: ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE DISSERTATION To obtain the academic degree doctor rerum naturalium (Dr. rer. nat. ) in the field of BIOLOGY submitted at the Faculty of Mathematics and Natural Sciences I Of the Humboldt-University Berlin von Ana Maria Aguirre-Arteta geb. am 02.01.1968 in Bilbao (Spain) Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. hc. Hans Meyer Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Bernhard Ronacher Gutachter: 1. Prof. Dr. Harald Saumweber 2. PD Dr. Jörn Walter 3. Prof. Dr. Franz Theuring Tag der mündlichen Prüfung: 28.06.

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• Biologie

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• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2000
• Nombre de lectures: 46
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem Institut für Biologie der Humboldt-Universität Berlin    

 

REGULATION OF DNA METHYLATION DURING DEVELOPMENT:   ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE  

  DISSERTATION  zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat. ) im Fach BIOLOGIE eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von

Ana Maria Aguirre-Arteta geb. am 02.01.1968 in Bilbao (Spanien)

Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. hc. Hans Meyer

          Gutachter:       Tag der mündlichen Prüfung: 28.06.00

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Bernhard Ronacher

1. Prof. Dr. Harald Saumweber 2. PD Dr. Jörn Walter 3. Prof. Dr. Franz Theuring

         

 

 

REGULATION OF DNA METHYLATION DURING DEVELOPMENT:   ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE  

DISSERTATION  To obtain the academic degree doctor rerum naturalium (Dr. rer. nat. ) in the field of BIOLOGY submitted at the

Faculty of Mathematics and Natural Sciences I Of the Humboldt-University Berlin von

Ana Maria Aguirre-Arteta geb. am 02.01.1968 in Bilbao (Spain)  

Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. hc. Hans Meyer

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Bernhard Ronacher

      Gutachter: 1. Prof. Dr. Harald Saumweber  2. PD Dr. Jörn Walter  3. Prof. Dr. Franz Theuring   Tag der mündlichen Prüfung: 28.06.00

 

 

 

 

           

         

 

THIS WORK IS DEDICATED WITH LOVE TO MY PARENTS, MY BROTHER AND SPECIALLY TO MY SISTER AMAYA

 

 

 

                        DIE WISSENSCHAFT FAENGT EIGENTLICH ERST DA AN INTERESSANT ZU WERDEN, WO SIE AUFHOERT JUSTUS VON LIEBIG (1803-1873)

 

 

Table of contents

  ABBREVIATIONS ................................................................................................. i   SUMMARY ..........................................................................................................v...I   ZUSAMMENFASSUNG  ..................................................................................... vII      1

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INTRODUCTION ................................................................................ 1   1.1 DNA METHYLATION ........................................................... 1 1.1.1 TYPES OF DNA METHYLATION......................................... 1 1.1.2 DNA METHYLATION REAC TION......................................... 2 1.1.3 STRUCTURE OF A C5-CYTOSINE METHYLTRANSFERASE2 1.1.4 DNA METHYLATION AND GENE EXPRESSION.................. 3 1.2 DNA METHYLATION IN NORMAL DEVELOPMENT AND DISEASE ................................... 5 1.2.1 DNA METHYLATION AND DEVELOPMENT......................... 5   1.2.1 DNA METHYLATION AND DISEASE.................................... 6   1.2.3 DNA METHYLATION AND IMPRINTING............................... 8 1.3 REGULATION OF DNA METHYLATION ................... 9 1.4 MAMMALIAN DNA MTases ........................................... 11   MATERIALS AND METHODS .................................................. 13 2.1 RNA ISOLATION ................................................................. 13 2.2 REVERSE TRANSCRIPTION ........................................ 13 2.3 POLYMERASE CHAIN REACTION ............................ 14 2.4 PLASMID DNA TRANSFORMATION INTO ESCHERICHIA COLIAND SELECTION OF RECOMBINANTS ................................................................ 15 2.5 SEQUENCING ....................................................................... 15 2.6 NORTHERN HYBRIDIZATION ...................................... 16 2.6.1 PROBE LABELING............................................................ 16   2.6.2 HYBRIDIZATION................................................................ 16 2.7 IN SITU HYBRIDIZATION ................................................ 17 2.7.1 PROBE LABELING............................................................ 17 2.7.2 SAMPLE PREPARATION.................................................. 18  

 

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Table of contents

2.7.3 HYBRIDIZATION............................................................... 19 2.8 CELL CULTURE AND TRANSFECTION ................. 20 2.9 IMMUNOFLUORESCENCE ANALYSIS ................... 20   2.9.1 TISSUE SECTIONS............................................................ 20   2.9.2 CULTURED CELLS............................................................ 21   2.9.3 IMMUNOSTAINING............................................................ 21   2.10 MICROSCOPY ...................................................................... 21   2.11 CELL EXTRACTS AND WESTERN BLOT ANALYSIS .............................................................................. 22 RESULTS ............................................................................................ 23 3.1 GENOMIC STRUCTURE OFND1TMGENE ..........23 3.2 ALTERNATIVE DNMT1 ISOFORMS DURING GAMETOGENESIS ............................................................. 27   3.2.1 CLONING OF ALTERNATIVE DNMT1 ISOFORMS FROM TESTIS.............................................................................. 27 3.2.2 EXPRESSION OF THE DNMT1 ISOFORMS BY NORTHERN ANALYSIS......................................................................... 29 3.2.3 LOCALIZATION OF THEDNMT1ISOFORMS BY IN SITU HYBRIDIZATION................................................................ 31 3.2.4 LOCALIZATION OF THE DNMT1 PROTEIN IN MALE AND FEMALE GERM CELLS..................................................... 35 3.3 EXPRESSION OF THEDNMT1 ROSMISOF  DURING MYOGENESIS ................................................... 38   3.3.1 CLONING OF THE ALTERNATIVE DNMT1 ISOFORM FROM SKELETAL MUSCLE......................................................... 38 3.3.2 THE ALTERNATIVE MUSCLE ISOFORM IS EXPRESSED SPECIFICALLY IN DIFFERENTIATED MYOTUBES............ 43 3.4 GENOMIC ORGANIZATION OF THE DNMT1 ISOFORMS ............................................................................. 44   3.5 TRANSLATION EFFICIENCY OF THE TESTIS/SKELETAL MUSCLE ISOFORM ................ 45 DISCUSSION .................................................................................... 49 LIST OF REFERENCES .............................................................. 52

 

Table of contents

  APPENDIX  A ACKNOWLEDGEMENTS ...................................................................... I B PERSONAL RECORD ............................................................................ I C STATEMENT ......................I....V ................................................................