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Full Description
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 2: Chromatin, Transcription, Envelope, Proteins, Dynamics, and Imaging presents biophysical approaches to study the mechanical properties of nuclei, together with a comprehensive range of imaging methods. These include FISH, FRAP, FRET, molecular beacons, fluorescence correlation spectroscopy, single molecule tracking, and combing DNA for optical microscopy, recognition imaging for atomic force microscopy, and hybridisation, tomography, and spectroscopic imaging for electron microscopy. 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 2: Chromatin, Transcription, Envelope, Proteins, Dynamics, and Imaging provides a comprehensive collection of the cutting-edge methods making a major contribution to understanding the nucleus and its nanostructure today.
Contents
Physical and Mechanical Properties.- Physical Properties of the Nucleus Studied by Micropipette Aspiration.- Mechanical Properties of Interphase Nuclei Probed by Cellular Strain Application.- Chromatin and Transcription.- Gene Expression in Polytene Nuclei.- Electron Microscope Visualization of RNA Transcription and Processing in Saccharomyces cerevisiae by Miller Chromatin Spreading.- Combing Genomic DNA for Structural and Functional Studies.- Using Molecular Beacons to Study Dispersal of mRNPs from the Gene Locus.- Mapping Cis- and Trans- Chromatin Interaction Networks Using Chromosome Conformation Capture (3C).- Recognition Imaging of Chromatin and Chromatin-Remodeling Complexes in the Atomic Force Microscope.- Using Cells Encapsulated in Agarose Microbeads to Analyse Nuclear Structure and Functions.- The Nuclear Envelope.- Investigation of Nuclear Envelope Structure and Passive Permeability.- Reconstitution of Nuclear Import in Permeabilized Cells.- Nuclear Envelope Formation In Vitro: A Sea Urchin Egg Cell-Free System.- Modifications of Nuclear Proteins.- Detection and Analysis of (O-linked ?-N-Acetylglucosamine)-Modified Proteins.- Detection of Sumoylated Proteins.- Detection of the Nuclear Poly(ADP-ribose)-Metabolizing Enzymes and Activities in Response to DNA Damage.- Purification and Analysis of Variant and Modified Histones Using 2D PAGE.- Quantification of Redox Conditions in the Nucleus.- Protein Dynamics in the Nucleus.- Fluorescence Correlation Spectroscopy to Assess the Mobility of Nuclear Proteins.- Single Molecule Tracking for Studying Nucleocytoplasmic Transport and Intranuclear Dynamics.- Fluorescence Recovery After Photobleaching (FRAP) to Study Nuclear Protein Dynamics in Living Cells.- Imaging Methods.- Nanosizing by Spatially ModulatedIllumination (SMI) Microscopy and Applications to the Nucleus.- Visualisation of RNA by Electron Microscopic In Situ Hybridisation.- Electron Spectroscopic Imaging of the Nuclear Landscape.- Cryoelectron Microscopy of Vitreous Sections: A Step Further Towards the Native State.
