The last several years have been a landmark period in the ubiquitin field. The breadth of ubiquitin's roles in cell biology was first sketched, and the importance of ubiquitin-dependent proteolysis as a regulatory mechanism gained general acceptance. The many strands of work that led to this new perception are re counted in this book. A consequence of this progress is that the field has grown dramatically since the first book on ubiquitin was published almost a decade ago [M. Rechsteiner (ed. ), Ubiquitin, Plenum Press, 1988]. In this span, students of the cell cycle, transcription, signal transduction, protein sorting, neuropathology, cancer, virology, and immunology have attempted to chart the role of ubi quit in in their particular experimental systems, and this integration of the field into cell biology as a whole continues at a remarkable pace. We hope that for active researchers in the field as well as for newcomers and those on the fence, this book will prove helpful for its breadth, historical perspective, and practical tips. Structural data are now available on many of the components of the ubiquitin pathway. The structures have provided basic insights into the unusual biochemical mechanisms of ubiquitination and proteasome-mediated proteolysis. Because high-speed computer graphics can convey structures more effectively than print media, we have supplemented the figures of the book with a Worldwide Web site that can display the structures in a flexible, viewer-controlled format.

Substrate selection in ubiquitination reactions is achieved by ubiquitin ligases, which simultaneously bind both the target protein and a ubiquitin conjugating enzyme. We have developed a phosphopeptide based approach to facilitate the identification of ubiquitin ligases (e.g. F-box proteins) that recognize regulatory proteins in a phosphorylation-dependent manner. Thus far, we have used this approach to identify substrates of two F-box proteins, Fbw7 and beta-TRCP. Cyclin E associates with Fbw7 through a phosphodegron near its C-terminus. Experiments in vitro and in vivo have identified critical phosphorylation sites in this degron that are required for interaction with Fbw7. Interestingly, this motif is found in a number of other unstable oncogenic proteins, including c-myc, c-jun, and SREBP. In a second series of studies, we have identified the cell cycle regulatory protein Cdc25A as a target of the beta-TRCP protein. We have found that Chk1 phosphorylates Cdc25A in response to DNA damage to generate a priming event that facilitates phosphorylation of Cdc25A on a motif that then binds to beta-TRCP. Using biochemical and genetic techniques, we demonstrate that this interaction is required for regulated proteolysis of Cdc25A.

The Polymerase Chain Reaction (PCR) technique was invented nearly 20 years ago. Its subsequent variations and applications were many and varied, and today molecular biology, clinical, and forensic laboratories make almost daily use of PCR. This second edition of the much-praised PCR Primer: A Laboratory Manual updates the tried-and-true methods and presents the advances made in the 10 years since the first edition. After introducing the basics for PCR and methods of sample preparation, PCR Primer provides laboratory-tested protocols for RT-PCR methods, detection of PCR products, analysis of differential expression, cloning, and mutagenesis. These step-by-step methods include extensive background information, as well as valuable troubleshooting information provided by the leading experts in this technology. This manual is a comprehensive and reliable source of the full range of PCR methods for novices and experienced investigators alike.

This book covers important topics such as the dynamic structure and function of the 26S proteasome, the DNA replication machine: structure and dynamic function and the structural organization and protein–protein interactions in the human adenovirus capsid, to mention but a few. The 18 chapters included here, written by experts in their specific field, are at the forefront of scientific knowledge. The impressive integration of structural data from X-ray crystallography with that from cryo-electron microscopy is apparent throughout the book. In addition, functional aspects are also given a high priority. Chapter 1 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.