Summary. The histones of the cellular slime mould Dictyostelium discoideum have been separated by electrophoresis using both acid urea and sodium dodecyl sulphate systems, and the gel pattern compared with that of histones from Physarum polycephalum and calf thymus. Dictyostelium is found to possess a full complement of H1, H2A, H2B, H3 and H4.Key words. Dictyostelium; slime mould; histone; electrophoresis.Dictyostelium discoideum, the cellular slime mould, is an extensively studied organism. Its easy growth and culture, the existence of axenic strains of free living amoebae, such as AX-2, which may be cultured indefinitely in liquid suspension cultures, and its small genome size, combine to make the organism interesting and important in the context of biochemistry and genetics. Work on the genome of Dictyostelium has focused attention on its ehromatin, and many laboratories have attempted separation and characterization of its histones ~4.Studies on Dictyostelium histones are of interest since this organism is a primitive eukaryote and there has been some doubt about whether such organisms have the full complement of histones as found in higher eukaryotes. The paths of histone evolution are also interesting, especially since core histone H3 and H4 are the most highly conserved of all proteins whilst H1 is less strongly conserved and, at least in higher organism, is coded for by a small multi gene family. Since the genome of this organism is small, the yield of histones is also small, and identification of histones by comprehensive comparison of the amino acid composition with that of other lower eukaryotes such as Physarum, has not as yet proved possible. We have, therefore, sought to achieve good separation of Dietyostelium histones with both triton/acetic acid urea gels and sodium dodecyl sulphate gels, and also to use a two-dimensional gel electrophoresis separation, somewhat similar to that advocated by Bonner et a12 and thus to proceed to a more accurate identification of these histones than has been previously obtained. In addition, our histones have been extracted from nuclei isolated by the use of digitonin, a natural plant saponin, rather than with nonidet P40. We have already shown that, using a number of criteria, such nuclei are superior to those isolated with NP-40 ~~ and, in particular, that their DNA is not substantially degraded.
Materials and methodsGrowth conditions. Dictyostelium discoideum strain AX-2 cells were grown in suspension culture as previously described ~~ and nuclei were isolated from such cells using the 0.1 mg/ml digitonin method described in the same paper.Isolation ofhistones. Basic nucleoproteins were extracted from isolated nuclei by one of two methods. 1) The acid extraction of Bakke and Bonner 4. 2) The method of Mohberg and Rusch using the modification of Coukell and Walker H the suspension of nuclei being heated in 1 M CaClz at 80~ for 15 min. In both methods basic nucleoproteins were precipitated by adding 4 vol. of ethanol at -20 ~ and allowing precipitation to proceed ...
