A Laboratory Guide To In Vitro Studies Of Protein Dna Interactions

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.

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.

This laboratory guide comes at a time when several other method books have already been published in this field. Is this one different from the others? Yes and no. There was no attempt made to be comprehensive. Rather, data were brought to bear on areas where enough competence has been gathered in our laboratories and to complement recent method books (many of which cover extensively various aspects of molecular biology) in those matters which appeared to us somewhat neglected. There was a constant preoccupation and effort to provide miniaturized proce dures that are both simple and time-saving. Interest was devoted to standardized procedures and culture conditions, avoiding dogmas such as those giving excessive importance to sophisticated culture media with endless adjustments for local or personal considerations. The key to success is the quality of the plant material serving as a source of cells. Consequently, isolation. extraction or culture techniques can be simplified and standardized. This is symptomatic for our times as it marks the end of a period when methodological matters were frequently above the biological problems. The times of "methods above all" is basically over, despite the fact that many of us still believe that, say, tissue culture is a "science" per se. By presenting a few original techniques we believe that one seriously reduces the empiricism still prevailing in this area of research.

Gene expression can mean the difference between a functional and non-functional genome, between health and disease, and with the development of transgenic crops, the difference between survival and starvation. In DNA-Protein Interactions: Principles and Protocols, Third Edition, this vital subject is brought up to date with protocols exploring the most cutting-edge developments in the field, including in vivo and genome-wide interaction techniques. Addressing topics such as chromatin immunoprecipitation, topological studies, photocrosslinking, FRET and imaging techniques, the volume fully updates and expands upon the successful previous editions. Written in the convenient and informative Methods in Molecular BiologyTM series format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and authoritative, DNA-Protein Interactions: Principles and Protocols, Third Edition serves as an ideal guide for all those exploring this dynamic, essential, and increasingly affordable area of research.