Molecular mechanisms controlling the tissue specific activity of the nutritionally regulated promoter I of the acetyl-CoA {carboxylase-α [carboxylase alpha] encoding gene in cattle [Elektronische Ressource] / by Xuanming Shi

Sujets

• Biologie

Informations

• Publié par: universitat_rostock
• Publié le: 01 janvier 2009
• Nombre de lectures: 49
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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 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 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