Studies on the Growth in Culture of Plant Cells: XXIII ISOLATION OF NUCLEI AND HISTONES FROM CULTURED CELLS OF ACER PSEUDOPLATANUS L.

A technique is described for the isolation of purified nucleifrom suspension culture cells of Acer pseudoplatanus. This involvesa grinding medium containing 70% (v/v) glycerol, 1 mM Mg²⁺,2 mM Ca²⁺, and Tris buffer at pH 7.8, prestorage and disruptionof the cells at –20 °C in a Potter-Elvehjem homogenizer,and purification by filtration and centrifugation in the presenceof Triton X-100. The nuclear yield is c. 25% as assessed bynuclear count or DNA estimation and the nuclei are active inthe RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H₂SO₄ andprecipitated by ethanol, 113 µg histone is obtained perµg nuclear DNA and the histone fraction contains 22% basicamino acids and has a lysine: arginine ratio of 2.6. Acid-ureagel electrophoresis shows the presence of five major histones(H1, H2A, H2B, H3, and H4 in sequence from anode to cathode)having respectively molecular weights of 24 500, 13 500, 13300, 12 800, and 11 000. There is very good correspondence betweencalf thymus histones H3 (reduced form) and H4 and two of theseAcer histones. The other Acer histones differ from the calfthymus histones H1, H2A, H2B in molecular weight but can beprovisionally equated with these by a newly developed differentialstaining reaction. Calf thymus histone H2A appears to be lessrich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (¹⁴C)lysine and[³H]tryptophan is in favour of the cytoplasmic synthesis ofthe histones.