Drosophila Cytogenetics Protocols

Leading drosophilists describe in step-by-step detail all the essential techniques for studying Drosophila chromosomes and suggest new avenues for scientific exploration. The chapters emphasize specimen preparation (from dissection to mounting) and cover both polytene and mitotic/meiotic chromosomes in depth. Each fully tested and readily reproducible protocol offers a background introduction, equipment and reagent lists, and tips on troubleshooting and avoiding pitfalls. A cutting-edge FISH and immunolocalization technique will be important for discovering how DNA sequence influences higher-order chromosome architecture and ultimately gene expression.

This exceptional laboratory manual describes thirty-seven procedures most likely to be used in the next decade for molecular, biochemical, and cellular studies on Drosophila. They were selected after extensive consultation with the research community and rigorously edited for clarity, uniformity, and conciseness.The methods included permit investigation of chromosomes, cell biology, molecular biology, genomes, biochemistry, and development. Each protocol includes the basic information needed by novices, with sufficient detail to be valuable to experienced investigators. Each method is carefully introduced and illustrated with figures, tables, illustrations, and examples of the data obtainable. The book's appendices include key aspects of Drosophila biology, essential solutions, buffers, and recipes.An evolution of Michael Ashburner's 1989 classic Drosophila: A Laboratory Manual, this book is an essential addition to the personal library of Drosophila investigators and an incomparable resource for other research groups with goals likely to require fly-based technical approaches.

This third edition expands on the previous editions by providing updated protocols and current methods that cover recent breakthroughs in Drosophila research. Chapters guide readers through FlyBase, versatile gene expression systems, analysis of microRNA function, single-cell transcriptome data and metabolism, recent applications of CRISPR for precise genome editing, transcriptional activation and cell lineage tracing, protein inhibition and tagging, optogenetic and optochemical control of tissue mechanics, AFM measurements, sample preparation for electron microscopy, live imaging of different tissues and organs, and quantitative image analysis. Written in the format of the highly successful Methods in Molecular Biology series, chapters include an introduction to their topic, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls Authoritative and cutting-edge, Drosophila: Methods and Protocols, Third Edition serves as a useful and practical guide to new researchers and experts using Drosophila as a model system. Chapter 7 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

Leading drosophilists describe in step-by-step detail all the essential techniques for studying Drosophila chromosomes and suggest new avenues for scientific exploration. The chapters emphasize specimen preparation (from dissection to mounting) and cover both polytene and mitotic/meiotic chromosomes in depth. Each fully tested and readily reproducible protocol offers a background introduction, equipment and reagent lists, and tips on troubleshooting and avoiding pitfalls. A cutting-edge FISH and immunolocalization technique will be important for discovering how DNA sequence influences higher-order chromosome architecture and ultimately gene expression.

The theoretical background of cytogenetics as well as the practical methods to investigate chromosomes from Drosophila to mammals and humans are presented in this book. Theory and practical protocols on the following topics are included: Cell culture methods, chromosome preparation and banding techniques, radioactive and fluorescent in situ hybridisation (FISH) methods and their derivates (chromosome painting), meiotic chromosome methods using light and electronic microscopy, the polytenic (Drosophila) and lampbrush (urodeles) chromosomes as well as flow and slit scan cytometry. The editors, all at INRA, played a prominent part in the development of cytogenetics. Paul Popescu, a specialist for domestic animals worked with gene mapping, Helene Hayes, together with Popescu showed the homology of bovine and human chromosome disorders, and Bernard Dutrilaux focused on human cytogenetics.

This detailed volume compiles numerous methods to explore fly oogenesis. Beginning with updated protocols from isolating and staining the ovary to numerous imaging techniques and genetic protocols for cell-specific assessment and CRISPR-mediated mutagenesis, the volume continues by detailing techniques from assessing cytoskeletal structures to uncovering protein-protein interactions, with closing chapters exploring how Drosophila oogenesis can be used in the classroom and in outreach programs to increase interest in biomedical research, STEM education, and STEM careers. 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 and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Drosophila Oogenesis: Methods and Protocols serves as an ideal guide for both new and expert researchers working with this robust and versatile model organism.

Drosophila melanogaster: Practical Uses in Cell and Molecular Biology is a compendium of mostly short technical chapters designed to provide state-of-the art methods to the broad community of cell biologists, and to put molecular and cell biological studies of flies into perspective. The book makes the baroque aspects of genetic nomenclature and procedure accessible to cell biologists. It also contains a wealth of technical information for beginning or advanced Drosophila workers. Chapters, written within a year of publication, make this topical volume a valuable laboratory guide today and an excellent general reference for the future. Key Features* Collection of ready-to-use, state-of-the art methods for modern cell biological and related research using Drosophila melanogaster* Accessible to both experienced Drosophila researchers and to others who wish to join in at the cutting edge of this system * Drosophila offers an easily managed life cycle, inexpensive lifestyle, extraordinarily manipulable molecular and classical genetics, now combined with powerful new cell biology techniques * Introduction and overview sections orient the user to the Drosophila literature and lore * Six full-color plates and over 100 figures and tables enhance the understanding of these cell biology techniques

Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal the unique structural features and biochemical functions of this protein. Several unique properties of p53 posed a challenge to understa- ing its normal function in the initial phase of its research. The low levels of p53 in normal cells, its stabilization under situations of genotoxic stress, induction of growth arrest, and apoptosis with stabilization of the protein, obstructed the visibility of its normal, unmutated function. The property of p53 that can sense a promoter and transactivate or inhibit is still not well understood. It is still not known whether it is the absence of the protein that causes tumorigenesis, or if its mutants have a dominant role in inducing cancer. p53 Protocols comprises eighteen chapters for the study of the diverse properties of p53 and related proteins. The methods included are invaluable for delineating the function of other proteins that may function as tumor suppr- sors or growth suppressors. The chapters are not presented in any schematic order, for the importance and diversity of the functions of p53 make it imp- sible to organize them suitably. We have made a sincere effort to collect the methods most useful to those investigators working on tumor suppressors or growth suppressors. The purpose of p53 Protocols is not only to provide investigators with methods to analyze similar biochemical functions, but also to familiarize them with the associated problems that arose during the course of investigations.

Knowledge of the three-dimensional structure of a protein is absolutely required for the complete understanding of its function. The spatial orientation of amino acids in the active site of an enzyme demonstrates how substrate specificity is defined, and assists the medicinal chemist in the design of s- cific, tight-binding inhibitors. The shape and contour of a protein surface hints at its interaction with other proteins and with its environment. Structural ana- sis of multiprotein complexes helps to define the role and interaction of each individual component, and can predict the consequences of protein mutation or conditions that promote dissociation and rearrangement of the complex. Determining the three-dimensional structure of a protein requires milligram quantities of pure material. Such quantities are required to refine crystallization conditions for X-ray analysis, or to overcome the sensitivity limitations of NMR spectroscopy. Historically, structural determination of proteins was limited to those expressed naturally in large amounts, or derived from a tissue or cell source inexpensive enough to warrant the use of large quantities of cells. H- ever, with the advent of the techniques of modern gene expression, many p- teins that are constitutively expressed in minute amounts can become accessible to large-scale purification and structural analysis.