Two Hybrid Systems

The yeast two-hybrid system is one of the most widely used and productive techniques available for investigating the macromolecular interactions that affect virtually all biological processes. In Two-Hybrid Systems: Methods and Protocols, Paul N. MacDonald has assembled a collection of these powerful molecular tools for examining and characterizing protein-protein, protein-DNA, and protein-RNA interactions. The techniques range from the most basic (introducing plasmids into yeasts, interaction assays, and recovering the plasmids from yeast) to the most advanced alternative strategies (involving one-hybrid, split two-hybrid, three-hybrid, membrane recruitment systems, and mammalian systems). Methods are also provided for dealing with the well-known problems of artifacts and false positives and for identifying the interacting partners in important biological systems, including the Smad and nuclear receptor pathways. To ensure ready reproducibility and robust results, each technique is described in step-by-step detail by researchers who employ it regularly. Comprehensive and highly practical, Two-Hybrid Systems: Methods and Protocols not only reveals how the great variety of plasmid vectors and approaches may be optimally deployed, but also quickly empowers novices to establish two-hybrid systems in their laboratories, and experienced researchers to expand their repertoire of techniques.

Gabriel Waksman Institute of Structural Molecular Biology, Birkbeck and University College London, Malet Street, London WC1E 7HX, United Kingdom Address for correspondence: Professor Gabriel Waksman Institute of Structural Molecular Biology Birkbeck and University College London Malet Street London WC1E 7H United Kingdom Email: g. waksman@bbk. ac. uk and g. waksman@ucl. ac. uk Phone: (+44) (0) 207 631 6833 Fax: (+44) (0) 207 631 6833 URL: http://people. cryst. bbk. ac. uk/?ubcg54a Gabriel Waksman is Professor of Structural Molecular Biology at the Institute of Structural Molecular Biology at UCL/Birkbeck, of which he is also the director. Before joining the faculty of UCL and Birkbeck, he was the Roy and Diana Vagelos Professor of Biochemistry and Molecular Biophysics at the Washington University School of Medicine in St Louis (USA). The rapidly evolving ?eld of protein science has now come to realize the ubiquity and importance of protein–protein interactions. It had been known for some time that proteins may interact with each other to form functional complexes, but it was thought to be the property of only a handful of key proteins. However, with the advent of hi- throughput proteomics to monitor protein–protein interactions at an organism level, we can now safely state that protein–protein interactions are the norm and not the exception.

This volume, part of the Advances in Molecular Biology series, presents work by pioneers in the field and is the first publication devoted solely to the yeast two-hybrid system. It includes detailed protocols, practical advice on troubleshooting, and suggestions for future development. In addition, it illustrates how to construct an activation domain hybrid library, how to identify mutations that disrupt an interaction, and how to use the system in mammalian cells. Many of the contributors have developed new applications and variations of the technique.

Proteins are indispensable players in virtually all biological events. The functions of proteins are coordinated through intricate regulatory networks of transient protein-protein interactions (PPIs). To predict and/or study PPIs, a wide variety of techniques have been developed over the last several decades. Many in vitro and in vivo assays have been implemented to explore the mechanism of these ubiquitous interactions. However, despite significant advances in these experimental approaches, many limitations exist such as false-positives/false-negatives, difficulty in obtaining crystal structures of proteins, challenges in the detection of transient PPI, among others. To overcome these limitations, many computational approaches have been developed which are becoming increasingly widely used to facilitate the investigation of PPIs. This book has gathered an ensemble of experts in the field, in 22 chapters, which have been broadly categorized into Computational Approaches, Experimental Approaches, and Others.

Experimental techniques are the life blood of science. The better the methodology is, the more reliable and accurate the results will be. Ultimately, this will lead to a clearer interpretation of those results and firmer conclusions from any set of experiments. Experimental methodology in the area of cardiovascular biochemistry and molecular biology has advanced considerably in the last decade. Because of these factors, it was thought that a focused issue of Molecular and Cellular Biochemistry dedicated to the novel, latest technological advances in the field was warranted. We must thank Dr Naranjan S. Dhalla, Editor-in-Chief of Molecular and Cellular Biochemistry, for his willingness to publish an issue with such a focus. We have attracted some of the leaders in the field of cardiovascular biology to submit articles describing some of the most novel, significant techniques currently in use in their laboratories. The purpose of the manuscripts was not to describe the recent experimental findings from each laboratory as is done in most conventional manuscripts. Instead, the purpose of the articles found within this focused volume of Molecular and Cellular Biochemistry was to describe how the technique is performed on the laboratory bench so that others less familiar with the technique may be able to use it in their own labs. The subjects described in this volume can be generally subdivided into three categories: molecular biology, cell biology and basic biochemistry. The methods cover wide areas including various DNA and RNA expression technologies, transfection techniques, quantification of ion flux movement, measurements of lipid metabolism, advances in the culture of specific cardiovascular cell populations, and the use of confocal microscopy to examine cell structure and function. We thank all of the authors who have contributed so much of their time and efforts and, most importantly, shared the `secrets' of these valuable techniques with the rest of the cardiovascular research community.

The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.

This book is about dynamical systems that are "hybrid" in the sense that they contain both continuous and discrete state variables. Recently there has been increased research interest in the study of the interaction between discrete and continuous dynamics. The present volume provides a first attempt in book form to bring together concepts and methods dealing with hybrid systems from various areas, and to look at these from a unified perspective. The authors have chosen a mode of exposition that is largely based on illustrative examples rather than on the abstract theorem-proof format because the systematic study of hybrid systems is still in its infancy. The examples are taken from many different application areas, ranging from power converters to communication protocols and from chaos to mathematical finance. Subjects covered include the following: definition of hybrid systems; description formats; existence and uniqueness of solutions; special subclasses (variable-structure systems, complementarity systems); reachability and verification; stability and stabilizability; control design methods. The book will be of interest to scientists from a wide range of disciplines including: computer science, control theory, dynamical system theory, systems modeling and simulation, and operations research.

This book brings together recent methods and theoretical approaches developed to dissect the activity and function of bacterial and eukaryotic Structural Maintenance of Chromosome (SMC) proteins. The protocols explore numerous subjects such as depletion systems to assess SMC function, genetic manipulation of SMC proteins, chromosomal, biochemical, and microscopy-based assays of SMC activity, as well as theoretical modeling of SMC activity. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, SMC Complexes: Methods and Protocols will greatly aid researchers looking into the mechanism of action of SMC complexes on DNA. Chapters 10 and 19 are available Open Access under a CC BY 4.0 license via link.springer.com.