Fractionation of Chromatin by thermal precipitation in phosphate buffer.

The stability of sonicated rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied as a function of buffer concentration (0.012 to 0.16 m) and temperature (20 to 98 °C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 50 °C, 70 to 75 °C and above 90 °C) and two transitional zones (55 to 70 °C and 75 to 90 °C) on the A₃₂₀ curves and on the percentage precipitated nucleoprotein versus temperature curves as well.This permitted the fractionation of chromatin in 0.08 m-phosphate buffer into three fractions by a stepwise heating at 50 °C (50 °C-pellet) and 98 °C (50–98 °C-pellet and post 98 °C-supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with heterogeneous nuclear RNA. The hybridization studies showed a different ability of these three DNA preparations in binding nuclear heterogeneous RNA: 16%, 8% and 30% for DNA isolated from 50 °C-pellet, 50–98 °C-pellet and post 98 °C-supernatant, respectively. The results are discussed in terms of chromatin structure and function.