A Safety Considerations Many techniques described here involve a number of hazards, such as high electrical current and voltage, radioactivity and highly toxic chemicals. It is absolutely essential that the instructions of equipment manufacturers be followed, and that particular attention be paid to the local and federal safety regulations. B Introduction The expression of prokaryotic and eukaryotic genes has been shown most often to be regulated at the level of mRNA synthesis. Thanks to the rapid development of methods for dissecting DNA sequences, cis-acting regulatory elements such as promoters and enhancers have been recognised. More recently, the widely expressed intuition that discrete sequences within these elements constitute binding sites for sequence-specific binding proteins has been confirmed, especially through the use of "footprinting" assays (for examples, Galas and Schmitz, 1978). This and similar assays have already resulted in the recognition, isolation and analysis of DNA-bind­ ing proteins for several genes. Excellent reviews exist of the structural studies on these transcription regulatory proteins and related DNA elements (for example, Glover, 1989 and Johnson and McKnight, 1989), to which the reader is referred for detailed information. To set the scene for applications of the techniques described in this volume, only the barest outline of previous studies is presented here. Protein-DNA interactions are dependent on very specific tertiary configurations of the binding protein which allow the closest contact with the DNA helix.

Inhalt

I Introduction.- The Study of Protein-DNA Interactions by Means of Enzymes.- II The study of Protein-DNA Interactions by Deoxyribonuclease 1 Footprinting.- III Exonuclease III Protection Assay for Specific DNA-Binding Proteins.- IV The Proteolytic Clipping Band-Shift Assay of Protein-DNA Complexes.- The Study of Protein-DNA Interactions by UV Light.- V Visualising Intimate Protein-DNA Contacts and Altered DNA Structures with Ultraviolet Light.- VI UV Cross-Linking of Protein to Bromouridine-Substituted RNA.- VII Molecular Weight Determination of DNA-Binding Proteins by UV Cross-Linking Combined with SDS Polyacrylamide Gel Electrophoresis.- The Study of Protein-DNA Interactions by Means of Chemicals (Interference Assays).- VIII Interaction of Proteins with Partially Depurinated and Depyrimidinated Oligonucleotides (Missing-Contact Probing of Protein-DNA Interactions).- IX The Study of Protein-DNA Contacts by Ethylation Interference.- X The Study of Protein-DNA Interactions by Methylation Interference.- XI Using the Chemistry of the Hydroxyl Radical to Determine Structural Details About DNA and Protein-DNA Complexes.- XII Detection of Unusual DNA Structures and DNA-Protein Interactions by Diethylpyrocarbonate Carbethoxylation.- The Study of Protein-DNA Interactions by Means of Electron Microscopy.- XIII Electron Microscope Visualisation of Protein-DNA Complexes.- The Study of Protein-DNA Interactions by Gel Mobility-shift Assays.- XIV Qualitative and Quantitative Studies of Protein-DNA Interactions by Gel Mobility-Shift Assay.- XV Diagonal Gel Mobility-Shift Assays for the Resolution of Multi-subunit Complexes Binding to Regulatory Elements of Specific Genes.- XVI A Rapid Gel-Shift Technique to Analyze DNA-Protein Interactions Using PhastSystem(TM).- Protein-DNA Interactions on a Solid Support.- XVII Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography.- XVIII Cloning of Sequence-Specific DNA-Binding Proteins by Screening ? cDNA Expression Libraries with Radiolabelled Binding-Site Probes.- The Study of Protein-DNA Interaction Using Special Vectors.- XIX Detection of DNA Bending by Gel Electrophoresis: Use of Plasmid Vectors.- Other Related Techniques.- XX The Use of DNA Amplification for In Vitro Footprinting Experiments.- XXI Non-radioactive Detection Methods for In Vitro Footprinting.- XXII Quantitative Determination of Proteins in Crude Extracts.- Appendices.- Appendix A DNA Isolation from E. coli (J.P. Jost).- Appendix B The Spun Column Procedure.- Appendix C Purification of Oligonucleotides (J. Jiricny).- Appendix D Migration of DNA on Native and Denaturating Gels of Different Polyacrylamide Concentration.- Appendix E Preparation of a Nuclear Lysate from Liver (J.P. Jost).- Appendix F Preparation of a Cell Lysate from Tissue Culture Cells.- Appendix G Preparation of Nuclear Extracts from Cells in Tissue Culture.- Appendix H Inhibitors of Proteases (E. Shaw).- Appendix I The Most Common Protein Kinase and Phosphatase Inhibitors (B. Hemmings).- Appendix J Tables on the Precipitation of Proteins with Ammonium Sulphate.