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Identification and Characterization of Carboxylesterase Variants

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Identification and Characterization of Carboxylesterase Variants Identification and characterization of Carboxylesterase variants Master thesis for the degree Master of Pharmacy Kathleen Nanding School of Pharmacy The Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO Department of Pharmacology OSLO UNIVERSITY HOSPITAL, ULLEVÅL April, 2016 I II III IV Copyright © Kathleen Nanding 2016 Identification and characterization of carboxylesterase variants Kathleen Nanding http://www.duo.uio.no Printed Reprosentralen, University of Oslo V VI Preface This thesis is made as a completion of the master education in Pharmacy and it took place at the Department of Pharmacology, Oslo University Hospital, Ullevål during the period of August 2015 to April 2016. First of all, I would like to express my deep gratitude to my external supervisors Marianne K. Kringen and Kari Bente Foss Haug for all the guidance and support through the entire study. I have learnt so much and I am truely insipired. I would also like to thank Hege Gilbø Bakke for helping me with all the practical problems I had, and for her kindness and patience. Furthermore I would like to thank all the colleagues at the department for their help and for a delightful and warm working atomsphere. I am grateful to my internal supervisor Hege Thoresen for following the progress of my study and for her constructive comments on the thesis. My thanks also go to the Class 2016 and my friends for all the support and for joyful memories through the years of my education. Finally I would like to thank my parents for all the love and encouragement, I would have never completed my education without your support. Ullevål, April 2016 Kathleen Nanding VII VIII Abstract Alternative splicing is a naturally occuring process where different protein isoforms could be encoded from the same pre-mRNA. A single pre-mRNA could have a combination of multiple patterns of mechanisms such as exon skipping, intron retention and alternative splice site selection. Alternative splicing has contributed to protein diversity and could influence their biological functions in different aspects. Carboxylesterases 1(CES1) and carboxylesterase 2(CES2) are members of carboxylesterase (CEs) family and are mainly expressed in liver and small intestine tissues. They are involved in drug metabolism, drug activation and other biological processes. Genetic variations of the CES1 and CES2 gene have shown to affect the drug metabolism in different drugs. This study has focused on identification and characterization of carboxylesterase variants and was performed in three parts. In this study, different methods and bioinformatics tools were used for detection, sequencing and analysing of splicing variants in CES1 and CES2. One new alternative splicing variant was found in the CES1 gene with deletion of exon 12, exon 13 and most part of exon 11 (135bp). The deletion of exons has lead to missing of one active site and one mutagenesis site in the CES1 gene. Alternative splicing variant that were known has also been detected, one variant in CES1 with deletion of exon 7 and one variant in CES2 with deletion of exon 2 and 3. The expression level of alternative splicing variants of CES2 was studied with reverse transcriptase-quantitative PCR. The alternative splicing variant has shown highest expression in fetal brain tissue, where the original splicing variant has the highest expression level in liver tissue. A pilot study for enzyme activity of cells with- and without insertion of alternative splicing variant of CES1 was performed where the original plan was to study the enzyme activity of both original- and alternative splicing variant of CES1 and CES2. It is difficult to make valuable conclusion based on the results. IX Abbreviations: 4-MUBA 4-methylumbelliferyl acetate A Adenine A-site Acitivity site AML Acute myeloid leukemia AraC Cytarabine Asn Asparagine Bcl-xl B-cell lymphoma-extra large bp Base pair C Cytosine C-terminus Carboxyl-terminus cDNA Complementary DNA CE Carboxylesterase CES2_EX2_3SPL cDNA fragment with deletion of exon 2 and 3 CES2_EX8_SPL cDNA fragment with deletion of exon 8 COX Cyclooxygenase Ct Threshold cycle Cys Cysteine dCK Deoxycytidine kinase DNA Deoxyribonuclei acid dNTP Nucleoside triphosphate dUTP Deoxyruridine triphosphate EDTA Ethylenediaminetetraacetic acid EMEM Eagle's Minimum Essential Medium ER Endoplasmic reticulum ESE Exonic splicing enhancer ESS Exonic splicing suppressor FAM 6-carboxyfluorescein G Guanine GAPDH Glyceraldehyde 3-phosphate dehydrogenase Glu Glutamic acid HEK Human embryonic kidney X His Histidine ISE Intronic splicing enhancer ISS Intronic splicing suppressor kb Kilo base-pair LacZ β-galactosidase LB Lysogeny broth Leu Leucine mM Milli molar concentration mRNA Mature RNA N-terminus Amino-terminus ng Nanogram NKCC2 Sodium-potassium-chloride transporter 2 NMD Nonsense-mediated decay NSAIDs Nonsteroid anti-inflammatory drug PCR Polymerase chain reaction PGK1 Phosphoglycerate kinase 1 RNA Ribonucleic acid RNase Ribonuclease rpm Revolutions per minute RT-qPCR Reverse transcriptase - quantitative PCR snRNP Small nuclear ribonucleoprotein particle SOC Super optimal broth with Catabolite repression SRE Splicing regulatory elements ss Splice site T Thymidine TBE Tris/Borate/EDTA UV Ultraviolet x-GAL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside µl Microliter µM Micro molar concentration XI XII Table of contents 1 Introduction .............................................................................................................................. 1 1.1 RNA splicing. .................................................................................................................................... 1 1.2 Alternative splicing ....................................................................................................................... 2 1.2.1 Spliceosome and splicing regulatory elements. ......................................................................... 2 1.2.2 Patterns of alternative splicing. ......................................................................................................... 2 1.2.3 Biological influences of alternative splicing. ................................................................................ 3 1.2.4 Alternative splicing in pharmacogenetics. .................................................................................... 5 1.3 Human Carboxylesterase ............................................................................................................ 7 1.3.1 Carboxylesterase family. ...................................................................................................................... 7 1.3.2 CES1 and CES2. ......................................................................................................................................... 7 1.3.3 Biological roles of Carboxylesterase ............................................................................................... 8 1.3.4 The pharmacogenetics of Carboxylesterase ................................................................................ 9 1.4 Aim of the study ........................................................................................................................... 10 2 Materials and Methods ........................................................................................................ 11 2.1 Materials ........................................................................................................................................ 11 2.1.1 Chemicals and Reagents ..................................................................................................................... 11 2.1.2 Tools ............................................................................................................................................................ 13 2.1.3 Instruments .............................................................................................................................................. 14 2.2 Polymerase chain reaction – PCR .......................................................................................... 15 2.2.1 Template RNAs ....................................................................................................................................... 16 2.2.2 cDNA – synthesis .................................................................................................................................... 16 2.2.3 Long-Range PCR (LR-PCR) ................................................................................................................ 17 2.2.4 Hot-Start PCR ........................................................................................................................................... 19 2.2.5 Analyzing PCR products – Agarose gel electrophoresis ....................................................... 21 2.3 DNA extraction – QIAquicK gel extraction .......................................................................... 21 2.4 TOPO Cloning ............................................................................................................................... 22 2.4.1 Vectors ........................................................................................................................................................ 23 2.4.2 PCR products with adenine at the 3’end ..................................................................................... 25 2.4.3 TOPO cloning reaction ........................................................................................................................
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