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Includes state-of-the-art procedures for isolating and working with nuclei from a wide range of cell types, and procedures for studying their components including pathology-related structures
Although our understanding of the structure and activities of the cell nucleus and of the nanomachines which it contains is increasing rapidly, much remains to be learned. The application and continuing development of the new, powerful biochemical and biophysical methodologies described here are essential in this quest. In The Nucleus, researchers from more than forty leading international laboratories describe state-of-the-art methods for isolating nuclei and their components and for studying their structure and activities, including some pathology-associated features. Volume I: Nuclei and Subnuclear Structures presents an overview of features of the intranuclear environment, followed by the most recent procedures for isolating nuclei from a wide range of cell types including muscle cells, yeast, oocytes, cells with polytene nuclei, Arabidopsis, trypanosomes, and dinoflagellates. The latest methods are described for isolating and working with nucleoli, constitutive heterochromatin, pathology-associated inclusions, and chromatin and for examining glycosylation, sumoylation, and ADP-ribosylation of nuclear proteins. Written in the highly successful Methods in Molecular Biology™ series format, chapters contain lists of necessary materials and reagents, readily reproducible protocols, and tips for troubleshooting and avoiding known pitfalls.
The Nucleus, Volume I: Nuclei and Subnuclear Structures is an essential reference for scientists who are working on our rapidly growing understanding of the cell nucleus and its activities.
I. THE INTRANUCLEAR ENVIRONMENT 1. The intranuclear environment. Santiago Schnell and Ronald Hancock II. ISOLATION OF NUCLEI 2. Purification of Nuclei and Preparation of Nuclear Envelopes from Skeletal Muscle. Gavin S. Wilkie and Eric C. Schirmer 3. Isolation of Highly Purified Yeast Nuclei for Nuclease Mapping of Chromatin Structure. Joseph C. Reese, Hesheng Zhang and Zhengjian Zhang 4. Working with oocyte nuclei: cytological preparations of active chromatin and nuclear bodies from amphibian germinal vesicles. Garry T. Morgan. 5. Preparation of Arabidopsis Nuclei and Nucleoli. Peter McKeown, Alison F. Pendle and Peter J. Shaw 6. High-Yield Isolation and Subcellular Proteomic Characterization of Nuclear and Subnuclear Structures from Trypanosomes. Jeffrey A. DeGrasse, Brian T. Chait, Mark C. Field and Michael P. Rout 7. Methods for studying the nuclei and chromosomes of dinoflagellates. Marie-Odile Soyer-Gobillard III. THE NUCLEOLUS 8. Isolation of nucleoli from Ehrlich ascites tumor cells and dynamics of nascent RNA within isolated nucleoli. Marc Thiry and Dominique Ploton 9. Time-lapse microscopy and FRET to analyze the dynamics and interactions of nucleolar proteins in living cells. Emilie Louvet, Marc Tramier, Nicole Angelier and Danièle Hernandez-Verdun 10. Three-Dimensional Reconstruction of Nucleolar Components by Electron Microscope Tomography. Pavel Tchelidze, Hervé Kaplan, Adrien Beorchia, Marie-Françoise O’Donohue, Hélène Bobichon, Nathalie Lalun, Laurence Wortham and Dominique Ploton IV : INTRANUCLEAR STRUCTURES 11. The perinucleolar compartment (PNC): detection by immunohistochemistry. Alicja Slusarczyk and Sui Huang 12.Isolation of the Constitutive Heterochromatin from Mouse Liver Nuclei. Olga V. Zatsepina, Oxana O. Zharskaya and Andrei N. Prusov 13. Isolation of pathology-associated intranuclear inclusions. Christine Iwahashi and Paul J Hagerman 14. The nuclear ubiquitin-proteasome system: visualization of proteasomes, protein aggregates and proteolysis in the cell nucleus. Anna von Mikecz, Min Chen, Thomas Rockel and Andrea Scharf V: INTERPHASE CHROMOSOMES 15. Multicolor 3D-FISH for imaging interphase chromosomes. M. Cremer, F. Grasser, C. Lanctot, S. Müller, M. Neusser, I. Solovei, R. Zinner and T. Cremer 16. Fluorescent transgenes to study interphase chromosomes in living plants. Antonius J.M. Matzke, Bruno Huettel, Johannes van der Winden and Marjori Matzke 17. Analysis of telomeres and telomerase . Jirí Fajkus, Martina Dvorácková and Eva Sýkorová 18. Combined immunofluorescence, RNA FISH, and DNA FISH to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation. Julie Chaumeil, Sandrine Augui, Jennifer C. Chow and Edith Heard 19. Analysis of the mobility of DNA double-strand break-containing chromosome domains in living mammalian cells. P.M. Krawczyk, J. Stap, R.A. Hoebe, C.H. van Oven, R. Kanaar, and J.A. Aten