Human arsenic methyltransferase pharmacogenetics [Elektronische Ressource] : functional studies of common polymorphisms and its impact on medicine / vorgelegt von Annette Friederike Klumpp
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Human arsenic methyltransferase pharmacogenetics [Elektronische Ressource] : functional studies of common polymorphisms and its impact on medicine / vorgelegt von Annette Friederike Klumpp

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Aus dem Institut für Pharmakologie und Toxikologie Direktor: Prof. Dr. T. Gudermann des Fachbereichs Medizin der Philipps-Universität Marburg Human arsenic methyltransferase pharmacogenetics: functional studies of common polymorphisms and its impact on medicine Inaugural-Dissertation zur Erlangung des Doktorgrades der gesamten Humanmedizin dem Fachbereich Medizin der Philipps-Universität Marburg vorgelegt von Annette Friederike Klumpp aus Herne Marburg, 2008 Angenommen vom Fachbereich Medizin der Philipps-Universität Marburg am: 16.05.2008 Gedruckt mit der Genehmigung des Fachbereichs. Dekan: Prof. Dr. M. Rothmund Referent: Prof. Dr. Dr. J. Krieglstein Korreferent: PD Dr. S. Müller-Brüsselbach “....Si toutes ces excuses ne suffisent pas, je vieux bien dédier ce livre à l’enfant qu’a été autrefois cette grande personne. Toutes les grandes personnes ont d’abord été des enfants. (Mais peu d’entre s’en souviennent)....“ Antoine de Saint-Exupéry Le Petit Prince Table of Contents Table of Contents.......................................................... 5 List of Figures ............................................................... 7 List of Tables................................................................. 7 Abbreviations...............

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Publié le 01 janvier 2008
Nombre de lectures 43

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Aus dem Institut für Pharmakologie und Toxikologie Direktor: Prof. Dr. T. Gudermann des Fachbereichs Medizin der Philipps-Universität Marburg       Human arsenic methyltransferase pharmacogenetics:  functional studies of common polymorphisms  and its impact on medicine
      
 
Inaugural-Dissertation zur Erlangung des Doktorgrades der gesamten Humanmedizin       dem Fachbereich Medizin der Philipps-Universität Marburg vorgelegt von  Annette Friederike Klumpp aus Herne  Marburg, 2008
 
                             Angenommen vom Fachbereich Medizin
der Philipps-Universität Marburg am: 16.05.2008
 
Gedruckt mit der Genehmigung des Fachbereichs.
Dekan: Prof. Dr. M. Rothmund
Referent: Prof. Dr. Dr. J. Krieglstein
Korreferent: PD Dr. S. Müller-Brüsselbach
 
 
 
    
 
  
  
“....Si toutes ces excuses ne suffisent pas, je vieux bien dédier ce livre à l’enfant qu’a été autrefois cette grande personne. Toutes les grandes personnes ont d’abord été des enfants. (Mais peu d’entre s’en souviennent)....“
  
Antoine de Saint-Exupéry Le Petit Prince
 
 
Table of Contents  Table of Contents.......................................................... 5 List of Figures ............................................................... 7 List of Tables................................................................. 7 Abbreviations................................................................ 8 Summary ..................................................................... 11 Zusammenfassung ...................................................... 13 1. Introduction ............................................................ 15 1.1 Pharmacogenetics............................................................................................15 1.2 Polymorphisms.................................................................................................16 1.2.1 Single nucleotide polymorphisms...............................................................17 1.3 Arsenic..............................................................................................................19 1.3.1 Historical remarks.......................................................................................19 1.3.2 Effects and occurrence................................................................................19 1.4 Biotransformation............................................................................................21 1.5 S-Adenosyl-L-Methionine...............................................................................22 1.6 Arsenic methyltransferase..............................................................................23 1.7 Thiopurine methyltransferase........................................................................24 1.8 Goals and research strategy for these studies...............................................25 2. Materials and Methods .......................................... 26 2.1 Materials...........................................................................................................26 2.2 Equipment........................................................................................................27 2.3 Growth of bacterial cultures...........................................................................27 2.4 DNA samples and analysis..............................................................................28 2.5 Synthesis and purification of oligonucleotide primers.................................28 2.5.1 Primer sequences ........................................................................................29 2.6 Quantification of nucleic acids.......................................................................29 2.7 Preparation of plasmid-DNA..........................................................................30 2.7.1 Maxi-purification ........................................................................................30 2.7.2 Mini-purification.........................................................................................30 2.8 Modification of DNA: Adding an “A“ overhang to the end of a DNA fragment..................................................................................................................31 2.9 DNA Preparations...........................................................................................32 2.9.1 Digestion.....................................................................................................32 2.9.2 DNA-sequencing ........................................................................................32 2.9.3 Electrophoresis ...........................................................................................32 2.10 Isolation of DNA fragments..........................................................................33 2.11 Cloning of DNA fragments...........................................................................33 2.11.1 Overview of vectors and expression systems ...........................................33 2.11.2 Ligation and transformation .....................................................................33 2.11.3 Transfection ..............................................................................................34 5
 
2.11.4 Cell harvesting ..........................................................................................35 2.11.4.1 Cell harvesting for enzyme activity determination............................35 2.11.4.2 Cell harvesting for the luciferase assay .............................................35 2.11.5 Subcloning of the insert from vector pCR2.1 into the vector pGL-3 .......35 2.11.6 Screening the pGL-3 vector for the insert ................................................36 2.12 Preparation of LB-agar with ampicillin and X-gal....................................36 2.13 PCR amplification.........................................................................................36 2.14 Cell culture.....................................................................................................37 2.15 Assays..............................................................................................................38 2.15.1 Arsenic methyltransferase assay...............................................................38 2.15.2 Luciferase assay........................................................................................39 3. Results ..................................................................... 40 3.1 Human AS3MT gene structure......................................................................40 3.1.1 NCBI AS3MT gene annotation ..................................................................41 3.2 Methylation activity.........................................................................................42 3.2.1 Detection of methylation activity ...............................................................42 3.2.2 Optimization of the assay ...........................................................................44 3.2.3 Controls and transfection efficiency...........................................................46 3.2.4 Outcome and further steps ..........................................................................46 3.3 VNTR studies...................................................................................................49 3.3.1 Hardy-Weinberg equilibrium analysis for the VNTR alleles .....................50 3.3.2 Coriell Cell Repository samples analysis ...................................................51 3.3.2.1 Analysis of 120 Coriell Cell Repository samples concerning the SNP in the FR, the number of repeats and the cSNP variant 860 in exon 9 ............52 3.4 Designing constructs........................................................................................54 3.4.1 DNA sequence ............................................................................................54 3.4.2 Designing pCR2.1 constructs .....................................................................56 3.4.3 Designing pGL-3 constructs .......................................................................58 3.5 Luciferase assay...............................................................................................58 3.5.1 Results of the luciferase assay ....................................................................60 4. Discussion ................................................................ 63 4.1 AS3MT and the pioneer of pharmacogenetics..............................................63 4.2 Expression of the cSNPs in the ORF..............................................................63 4.3 Influence of the polymorphisms in the VNTR region on the AS3MT gene activity.....................................................................................................................64 4.4 Linkage between the 2 VNTR repeat and the variant 860...........................65 4.5 Conclusions and future approaches...............................................................66 4.6 Results after my stay.......................................................................................67 4.7 Clinical approach.............................................................................................67 5. References ............................................................... 69 Akademische Lehrer .................................................. 73 Acknowledgements ..................................................... 74 Ehrenwörtliche Erklärung......................................... 75 Veröffentlichung ......................................................... 75 
 
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List of Figures  Fig. 1: Inorganic arsenate, arsenite and methylated metabolites............................................. 20 Fig. 2: Methionine adenosyltransferase catalyzed biosynthesis of S-adenosyl-L-methionine 22 Fig. 3: Human AS3MT gene structure .................................................................................... 40 Fig. 4: AS3MT methylation activity for the expression of recombinant WT and variant 860. .................................................................................................................................................44 Fig. 5: Protein dependence curve for the AS3MT variant 860 ............................................... 45 Fig. 6: Substrate curve for the AS3MT variant 860 ................................................................ 45 Fig. 7: Final results of the AS3MT activity ............................................................................ 48 Fig. 8:Vector pCR2.1 overhang .............................................................................................. 56 Fig. 9: Vector pCR2.1 map ..................................................................................................... 57 Fig. 10: Average levels of Firefly luciferase activity for AS3MT VNTR reporter constructs transfected into HEK cells ...................................................................................................... 60 Fig. 11: Average levels of Firefly luciferase activity for AS3MT VNTR reporter constructs transfected into HepG2 cells ................................................................................................... 61   List of Tables  Table 1: Primer sequences ..................................................................................................... 29 Table 2: Arsenite concentration inµM and activity in cpm.................................................... 46 Table 3: Analysis of 120 samples, for the SNP located 23 nucleotides upstream from the beginning of the VNTR GC, VNTR genotype and the SNP at nucleotide 860 ................. 53 Table 4: Analysis of the AS3MT 5’-FR and UTR .................................................................. 55              
 
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