Molecular mechanisms controlling the tissue-specific activity of the nutritionally regulated promoter I of the acetyl-CoA carboxylase-encoding gene in cattle Inaugural dissertation for the academic degree Doctor rerum naturalium of Mathematisch-Naturwissenschaftlichen Fakultät Universität Rostock By Xuanming Shi (M. Sc.), born on 17-09-1975, in Anhui, China From Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere in Dummerstorf Rostock (2009) urn:nbn:de:gbv:28-diss2009-0184-9 Dean: Prof. Dr. Hendrik Schubert 1. Reviewer: Prof. Dr. Hans-Martin Seyfert Molecular Biology Research Unit, Research Institute for the Biology of Farm Animals; Wilhelm-Stahl-Allee 2; D-18196 Dummerstorf; Germany. 2. Reviewer: Prof. Dr. Martin Hagemann Department of Plant Physiology, Institute of Life Sciences, University of Rostock; Albert-Einstein-Strasse 3, 18051 Rostock, Germany; 3. Reviewer: PD. Dr. Monika Schweigel Research Institute for the Biology of Farm Animals (FBN), Department of Nutritional Physiology “Oskar Kellner,” Wilhelm-Stahl-Alee 2, D-18196 Dummerstorf, Germany. Date of defense: 19-Oct-2009 Dedicated to my family Table of contents TABLE OF CONTENTS 1 Introduction ..................................................................................................................................... 1 1.1 Fat metabolism and acetyl-CoA carboxylase (ACC) ............................................................... 1 1.1.1 Fat deposition and mobilization ....................................................................................... 1 1.1.2 Biosynthesis of fatty acids and ACC................................................................................ 1 1.1.3 Roles of ACC in fatty acid synthesis and oxidation......................................................... 2 1.1.4 Functional domains of the ACC- ................................................................................... 3 1.2 Regulation of ACC-............................................................................................................... 4 1.2.1 Long-term regulation........................................................................................................ 4 1.2.1.1 Multi-promoters regulate ACC- transcription initiation......................................... 5 1.2.1.2 Pre-mRNA splicing................................................................................................... 7 1.2.1.3 Stability of mRNA.................................................................................................... 7 1.2.2 Short-term regulation ....................................................................................................... 7 1.2.2.1 Allosteric regulation ................................................................................................. 8 1.2.2.2 Reversible phosphorylation ...................................................................................... 9 1.3 PI is regulated by a bipartite repressor................................................................................... 10 1.4 Transcription factors relevant to regulating ACC- PI activity ..............................................11 1.4.1 Roles of CCAAT/Enhancer Binding Protein (C/EBP) ....................................................11 1.4.2 Roles of Nuclear Factor-Y (NF-Y)................................................................................. 14 1.5 Goals of this study ................................................................................................................. 14 2 Materials and Methods .................................................................................................................. 17 2.1 Materials ................................................................................................................................ 17 2.1.1 Bacterial strains .............................................................................................................. 17 2.1.2 Reagents and antibodies ................................................................................................. 17 2.1.3 Plasmid vectors............................................................................................................... 18 2.2 Preparation of DNA ............................................................................................................... 19 2.2.1 Mini-preparation of plasmid DNA ................................................................................. 19 2.2.2 Midi-preparation of plasmid DNA ................................................................................. 20 2.2.3 Preparation of genomic DNA......................................................................................... 21 2.2.4 Preparation of BAC DNA .............................................................................................. 21 2.3 Preparation of RNA ............................................................................................................... 22 2.3.1 Preparation of RNA from cells and tissues..................................................................... 22 2.3.2 Electrophoresis of RNA through agarose gels containing formaldehyde....................... 23 2.4 Cloning of DNA..................................................................................................................... 23 2.4.1 Digestion by restriction enzyme..................................................................................... 23 2.4.2 Detection of DNA in Agarose Gels ................................................................................ 24 2.4.3 Purification of DNA fragment from gel ......................................................................... 24 2.4.4 Blunting with Klenow enzyme....................................................................................... 24 2.4.5 Ligation .......................................................................................................................... 25 2.4.6 Transformation of DNA into E. coli............................................................................... 25 2.4.7 Screening of the recombinant clones.............................................................................. 26 2.5 Polymerase Chain Reaction (PCR) ........................................................................................ 27 2.5.1 Primer design.................................................................................................................. 28 2.5.2 Standard PCR ................................................................................................................. 28 2.5.3 High-fidelity PCR........................................................................................................... 28 2.5.4 GC-rich PCR .................................................................................................................. 29 I Table of contents 2.5.5 Reverse transcription PCR (RT-PCR) ............................................................................ 29 2.5.6 Rapid amplification of cDNA ends (RACE) .................................................................. 30 2.5.7 Genomic Walking........................................................................................................... 31 2.5.8 PCR- mediated site-directed mutagenesis...................................................................... 32 2.5.9 Quantitative Real-Time PCR.......................................................................................... 33 2.6 Cell culture and transfection .................................................................................................. 34 2.6.1 Cell line .......................................................................................................................... 34 2.6.2 Transient transfection ..................................................................................................... 34 2.6.3 Luciferase activity assays............................................................................................... 35 2.7 Preparation and purification of antigen and antibodies.......................................................... 35 2.7.1 Protein expression and preparation (for inclusion bodies) ............................................. 35 2.7.2 Affinity purification via Ni-NTA sepharose................................................................... 36 2.7.3 Measurement of protein concentration........................................................................... 37 2.7.4 SDS-PAGE electrophoresis............................................................................................ 37 2.7.5 Antigen preparation........................................................................................................ 37 2.7.6 Immunization ................................................................................................................. 38 2.7.7 Antibody purification ..................................................................................................... 39 2.8 Immunological methods......................................................................................................... 40 2.8.1 Western blot....................................................................................................................40 2.8.2 Protein-protein interaction by immunoprecipitation ...................................................... 41 2.8.3 Indirect sandwich ELISA for bovine ACC- ................................................................