Alleles of the Human Adrenergic Receptor Gene and Possible Effects on the Phenotype

Diplomarbeit
zur Erlangung der Magisterwürde
an der Naturwissenschaftlichen Fakultät
der Karl-Franzens-Universität Graz
vorgelegt von
Alexander Binder
Graz, Juni 1996

This book was typeset on an AlphaServer 2000 4/275 system running OpenVMS 6.2-1H2 and an Apple Laserwriter Pro using TeX 3.14159 and LaTeX <1995/12/01> format, including 16 CTAN packages. The running font is Adobe Times Roman. The used TeX implementation is maintained by Alexander Binder <alexander.binder@kfunigraz.ac.at>.

Die vorliegende Arbeit wurde am Institut für Physiologie und am Institut für Biochemie der Universität Graz in der Zeit von November 1992 bis Dezember 1995 unter der Leitung von Ass.-Prof. UD. Dr. Rudolf Jörg Schaur durchgeführt.

• Contents
• List of Figures
• Introduction
  • Adrenergic System, Adrenoceptors - Classes, Functionality, Physiology
    • The Adrenergic System
    • The Adrenergic Receptor
    • Functionality
  • Salt Sensitivity and Adrenergic Receptors
  • Genetics of the Receptor: Receptor Codon, Codon Mutations
  • Objective
• Materials and Methods
  • Materials and Laboratory Apparatus
  • Chemicals
  • Buffers
    • Stock Solutions
    • Other Solutions
  • Culture Media
    • Liquid Media
    • Solid Media
    • Antibiotics
    • Top-Agar
  • General Methods
    • DNA Preparation from Whole Blood
    • Nucleic Acid Quantification
    • Restriction Endonucleases and Their Use
    • Phenol Extraction
    • Ethanol Precipitation
    • Oligonucleotide Purification
    • Heat Denaturation of DNA
  • PCR Techniques
    • Standard PCR Conditions
    • Primer Selection
    • Determination of Melting Temperatures
    • The PCR Buffer
    • Factors Affecting Specifity
    • Fidelity of Amplification
    • Used Materials and Precautions
    • Oligonucleotide Quantification
  • Gel Electrophoresis
    • Agarose Gel Electrophoresis
    • Freeze-Squeeze-Technique
    • Polyacrylamide Gels
• Sequencing of the Coding Region
  • Sequencing Strategy
  • PCR of the Coding Fragment
  • Subcloning the Insert in a pUC18 plasmid
    • Preparation of the Vector
    • Dephosphorylation of the Linearized Vector
    • Preparation of the Insert
    • Ligation of Insert into the Vector
  • Transformation of Bacteria
    • Preparation of Competent Hosts
    • Transformation of Competent DH5 Cells
    • Isolation of the Plasmid after Alkaline Lysis
  • Sequencing
    • Alkali Denaturation of DNA Template
    • Sequencing Protocol
    • Denaturing Polyacrylamide Gels
  • Results
    • Mutations of the -Adrenergic Receptor Gene
• A Screening System for the Human Adrenergic Receptor
  • Methods of Detection of Single Point Mutations
  • Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences
    • Restriction Map of the Adrenergic Receptor Codon
    • Amplified Fragments
    • Selecting the Used Restriction Endonucleases
  • PCR of the RFPL fragments
    • Used Protocol
    • PCR Optimization
  • RFLP Protocol
  • Analysis of RFLPs
  • Silver Staining of DNA in Polyacrylamide Gels
    • Silver Staining Protocol
  • Results
    • Human Adrenergic Receptor Allele Frequencies and Haplotypes
    • Adrenoceptor Alleles in Relation to Adrenoceptor Expression in Cultured Skin Fibroblasts
• Discussion
• References
• Index
• About this document ...