Polarized light scattering of nucleosomes and polynucleosomes-in situ and in vitro studies

Nucleosomes, chromatin and nuclei, extracted from rat hepatocytes, were studied by an experimental configuration which measures circular intensity differential scattering (CIDS) and other elements of the polarized light scattering matrix. The Mueller matrix elements, S ₁₄ and S ₃₄, that are related to the geometric parameters of the superhelical arrangement of polynucleosomes point to the existence of a quarternary structure at low ionic strength for chromatin prepared by the cold-water method, which is lost by shearing, and is not found in the soluble chromatin prepared through the nuclease method. Only the addition of salt to produce a final concentration of 5 mM MgCl ₂, 150 mM NaCl and 10 mM Tris HCl (pH 7) yields a sizable (S ₁₄+S ₃₄) signal in the latter chromatin. This signal is however still different from the corresponding signal of native nuclei and of cold-water chromatin. The (S ₁₄+S ₃₄) signal from isolated nucleosomes in consistently very low (nearly zero) as predicted by multiple dipole simulation within the framework of classical electrodynamics. Results are discussed in terms of the topological constraints present in the native long chromatin fiber, which are lost after limited nuclease digestion and after shearing.