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Quantitative Analysis of Chromatin Proteomes in Disease
Published on: December 28, 2012
Chromatin nu bodies: isolation, subfractionation and physical characterization
Nucleic Acids Research
|December 1, 1976
Summary
Researchers isolated monomer chromatin subunits (nu1) from chicken red blood cells. These stable particles were separated into fractions containing specific histones and DNA, aiding structural analysis.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Chromatin, the complex of DNA and proteins that forms chromosomes, is fundamental to eukaryotic genome organization.
- Understanding the structure of chromatin subunits is crucial for elucidating gene regulation and DNA packaging.
- Previous studies have characterized various chromatin components, but isolating stable, large quantities of monomeric subunits presented challenges.
Purpose of the Study:
- To isolate and characterize monomer chromatin subunit particles (nu1) in substantial quantities.
- To investigate the composition and stability of these isolated chromatin monomers.
- To determine the physical parameters of nu1 and its subfractions using multiple biophysical techniques.
Main Methods:
- Large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei.
- Storage of isolated nu1 particles at low temperatures in EDTA.
- Subfractionation of nu1 via dialysis against varying KCl concentrations.
- Analysis using sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy.
Main Results:
- Gram quantities of monomer chromatin subunit particles (nu1) were successfully isolated and found to be stable upon freezing.
- Dialysis against 0.1 M KCl separated nu1 into a soluble fraction (histones H4, H3, H2A, H2B with ~130-140 bp DNA) and a precipitate (all histones including H1, H5 with DNA).
- Comprehensive physical parameters of the total nu1 and the soluble fraction were determined and correlated across multiple biophysical methods.
Conclusions:
- Monomer chromatin subunits (nu1) can be isolated in large, stable quantities, facilitating detailed structural studies.
- The subfractionation method effectively separates core histones and associated DNA from H1/H5-containing fractions.
- The study provides a correlated set of physical parameters for nu1, offering insights into chromatin structure at the nucleosome level.
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