A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X-100.Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum.The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F I . After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.The cellular slime mold Dictyostelium discoideum undergoes a period of morphogenesis during its life cycle which can be clearly distinguished from the phase of vegetative growth. Following a period of starvation the amoebae aggregate and differentiate to form only two major cell types, spore cells and stalk cells [l].This relatively uncomplicated system is a useful model for the study of the biochemical changes associated with cell differentiation and has been extensively investigated [2-51. The role of the nucleus in the differentiation of D. discoideum has, however, only recently received attention [6] due to the difficulties involved in isolating useful quantities of clean, intact nuclei and to the low DNA content of the organism [7,8]. The recent development of axenic strains [9,10] of D. discoideum has permitted the production of large quantities of amoebae in pure culture and facilitated the isolation of cellular components.The work described in this paper was undertaken firstly to develop a method for the isolation of clean, intact nuclei from D. discoideum; secondly, it was undertaken to establish whether the nuclei contained basic proteins and if so to what extent they resembled the histones isolated from other organisms. . The first of these methods gave variable results with rather low yields and nuclei of doubtful quality. Neither method was really suitable for dealing with the large quantities of cells required to obtain useful amounts of nuclear material (see Discussion).A paper by Coukell and Walker [6] appeared during the course of this work describing the isolation of nuclei using Triton X-100 and of basic nuclear proteins from D. discoideum. We also used their method but with varying success. As a result of work on the isolation of intact mitochondria and peroxisomes from D. discoideum using sorbitol/Ficoll medium [15,16] andTritonX-100, wemodifiedCoukel1 and Walker's method and developed the one described below. The method is suitable for the isolation of nuclei and other organelles from small or large quantities of cells and from non-axenic as well as from axenic strains of D . discoideum.Subsequent isolation and partial characterisation by polyacrylamide gel electrophoresis of basic nucleoproteins produced consistent results which differed somewhat from those of Coukell and Walker. Althoug...

The nuclei from Dictyostelium discoideum were isolated using a number of detergents. Microscopically clean and intact nuclei were obtained using Kyro EOB, Cemulsol NPT 12 and Triton X-100. While density gradient purification of Kyro-EOB-isolated nuclei led to some loss of basic nucleoproteins, Triton and Cemulsol-isolated nuclei gave relatively consistent patterns of basic nucleoproteins. The variabilities are discussed.No reproducible differences were found between basic nucleoproteins of vegetative and differentiating cells.The basic nucleoproteins were separated on sodium dodecylsulfate as well as acid/urea gels and compared with calf thymus histones. The perchloric-acid-soluble protein (fraction 1) which comigrates with histone H1 on acid/urea gels migrates faster than H1 on dodecylsulfate gels ( M , 19000-20000). The ethanol/HCl fraction ('arginine-rich') contained most of bands 3 and 4 and all of band 5 ; the latter comigrating with H4 on acid/urea gels.The amino acid compositions of whole basic nucleoprotein, fraction 1 protein and the ethanol/ HC1-soluble fraction were determined. The whole basic nucleoprotein contained a high proportion of arginine and lysine, although like many lower eukaryotes the basic/acidic amino acid ratio was relatively low (1.18). The ethanoliHC1-soluble fraction contained about 13.7 mol lysine and 9 mol arginine per 100 mol total residues compared 9.6 and 12 mo1/100 mol respectively in a similar fraction from calf thymus histones.The fraction 1 protein is a lysine-rich protein, but contains more arginine and less lysine than calf thymus H1. Furthermore, it contains a much higher content of acidic (or amide) residues than H1. Hence, it resembles the two lysine-rich basic nucleoproteins of Physarum polycephalum.Tryptic peptide maps of fraction 1 protein and H1 were compared. Some 14 spots were superimposable and one group of five small, highly charged peptides showed a striking correspondence. The possibility that they occur in a conserved region of the protein involved in DNA binding is considered. The similarity between fraction 1 protein and the calf thymus highmobility group proteins is discussed.We have previously described the isolation of nuclei and basic nucleoproteins from the cellular slime mould Dictyostelium discoideum [l]. In this paper we examine more closely the effects of nuclei isolation procedures on the pattern of basic proteins. In particular, since there are conflicting reports concerning the effects of Triton X-100 on nuclei [2,3], we decided to see whether somewhat milder detergents offered any advantages. Modifications of our original method for the isolation of nuclei are also described. In addition we have separated the basic nucleoproteins on sodium dodecylsulfate as well as acid/urea gels.Recently, Osborn and Ashworth [4] reported that there was a difference between the basic nucleoproteins of growing and differentiating cells and we have checked this possibility.We have as yet been unable to separate the individual basic nucleoproteins using methods ...