Large-scale isolation and fractionation of Tetrahymena histones. Purification of H2B and H4 histones.

Abstract

The histones of the ciliated protozoan Tetrahymena were isolated and fractionated on a large scale by revised procedures, and the H2B and H4 histones were highly purified for sequence determination. The nuclei of an amicronucleate strain, GL, of Tetrahymena pyriformis were isolated, without cell harvesting, from cultures reaching the stationary state, which were continuously centrifuged through 1.0 M sucrose, pH 7.5, containing Nonidet P-40. From the nuclei further purified and washed with 0.15 M NaCl, pH 7.5, histones were extracted with 0.25 M HCl. Thus 1,620 liters of cultures yielded 5.20 g of histone extract. The histones were first separated into a 0.5 M HClO4 soluble fraction (H1) and an insoluble fraction (H2A, H2B, H3, and H4). The insoluble fraction was chromatographed on a large column of Bio-Gel P-60 with 20 mM HCl containing 250 mM NaCL at 15 degrees C, yielding H2A + H3, H2B, and H4 fractions. The H4 fraction was highly pure; the H2B fraction was further purified by the same Bio-Gel P-60 chromatography without NaCl, yielding pure H2B.