Characterization and biosynthesis of histones in cultured cells of Acer pseudoplatanus L.

A new technique has been developed for the isolation of nuclei from suspension culture cells of Acer pseudoplatanus L. This technique involves the use of a glycerol-containing grinding medium at -20°C. The whole isolation process is simple and consistently produces about 20% nuclear yield with reasonable purity. Histone extraction from this nuclear fraction by the H2SO4-EtOH method is superior to other methods examined. The extracted Acer histones exhibit a typical histone pattern on polyacrylamide gels, and the major fractions are identified as H1, H2A, H2B, H3 and H4 (in sequence from the anode to the cathode end on the gel); their molecular weights are respectively 24,500, 13,500, 13,300, 12,800 and 11,000, Both the H3 and H4 histones of Acer cells are identical with those of calf thymus in terms of their mobilities on acid-urea and SDS gels. Identification of the Acer histone fractions has been assisted by a newly developed differential staining method which stains the 5 major histone fractions of calf thymus in 5 different colours. The total extracted Acer histone fraction contains 22% of basic amino acids, and the ratio of lysine to arginine is 2.6 which is higher than that for calf thymus and is probably due to the low content of arginine in Acer H2A. The Acer histones have been shown to be synthesized in the cytoplasm and then transported into the nucleus. The synthesis occurs throughout the cell cycle but reaches its maximum rate while INA is being synthesized. Histone samples obtained from both asynchronous and synchronous cultures at intervals during the progress of their growth show uniform electrophoretic patterns on the gels, suggesting that the Acer histones are generally homogeneous and that any modification of the histones probably affects only a very small proportion of the total histones. The possible existence of such modified histone derivatives and their functions remains to be investigated. Directions along which the present studies could be developed are discussed.