Kazimierz Toczko

Subunit structure of Physarum polycephalum chromatin.

Digestion of animal chromatin [l-4] as well as chromatin from higher plants [S] and yeast [6] by exogenous bacterial nuclease has led to the conclusion that chromatin has a regular subunit structure. It is however still important to establish whether this structure is common especially among organisms in which the histone pattern is not identical with that of higher plants and animals. We report here studies on the chromatin organization in a true slime mold Physarum polycephalum which belongs to a very primitive group of eukaryotes and is widely used as a model organism especially in mitotic studies [7].

HMG-like proteins of Physarum polycephalum: Association with the transcriptionally active chromatin

HMG‐like proteins were isolated from nuclei of the acellular slime mold Physarum polycephalum. These proteins represented 0.6 ± 0.2% of nuclear proteins and approx. 6% of DNA by weight. Polyacrylamide gel electrphoresis demonstrated the presence of 3 major proteins, designated HMG‐1P, HMG‐2P and HMG‐14/17P. None of these proteins comigrated with any of the calf thymus HMGs. Physarum HMG‐like proteins were found to be preferentially associated with transcriptionally active chromatin fraction, as well as being released from nuclei by light DNase I digestion. In contrast, the content of histone H1 was significantly lower in active than in inactive chromatin fraction.

Evidence that neutral protease from calf thymus chromatin is a serine type enzyme

Chromatins from different animal tissues have been shown to contain neutral protease [ 1,2,3]. The enzyme, when being an integral part of nucleohistone or chromatin complex, degrades only the fl and f3 histones. Dissociated from the complex it however degrades equally all five histone fractions. High degree of purification of neutral protease from calf thymus chromatin was achieved by Kurecki and Toczko [4] who also found its mol. wt to be 15 400 f 1000 and optimum pH of 8.5 towards total histone as substrate. The enzyme isolated by these authors was reported not to be of metal-, thio-, or acidic-type protease. In the present paper evidence is given to show that neutral protease from calf thymus chromatin is a serine-type enzyme.

Effect of ethidium bromide on the digestion of chromatin DNA with micrococcal nuclease.

Intercalation of ethidium bromide into DNA influences the rate of its digestion with micrococcal nuclease in opposite directions depending on whether it is free DNA or DNA in chromatin. In the case of free DNA the binding of ethidium bromide, starting from a very low concentration, results in the inhibition of the rate of digestion (increasing constantly with the increase of the ethidium bromide/nucleotide ratio). In contrast to free DNA the digestion rate as well as the overall amount of nuclease susceptible DNA is increased upon ethidium bromide binding to chromatin, with maximum enhancement around the saturation of intercalation sites. The saturation of intercalation sites in chromatin leads also to the disappearance of the typical micrococcal nuclease digestion pattern of DNA upon gel electrophoresis. Instead, a random cleavage pattern is observed. These data indicate that partial unwinding of chromatin DNA by ethidium bromide results in unmasking new sites for nuclease action. Interpretation of this finding in terms of the nucleosomal structure of chromatin and the mode of ethidium bromide binding to chromatin DNA indicates that newly unmasked sites are localized within the core particle DNA.

Identification of a ras gene in the slime mold Physarum polycephalum

A ras homologue was identified in the cDNA library from the slime mold Physarum polycephalum. The cDNA codes for a protein of 189 amino acids, showing high homology to ras genes from other organisms, especially to these from Dictyostelium discoideum. Amino acid sequence at the C-terminus of the putative protein suggests that unlike most other ras proteins, it is not palmitoylated and bears a geranylgeranyl rather than farnesyl chain.

Chromatin condensation Possible dehydrating and stabilizing factors

The effect of Na+, Mg2+, spermidine and spermine on the dehydration of chromatin gel and precipitation of soluble chromatin has been compared. Considerable differences have been found in the relative ratios within the studied group (Na+, Mg2+, spermidine and spermine) between the ability to dehydrate (1 : 32 : 53 : 67) and to precipitate (1 : 53 : 800 : 2000) chromatin. On the basis of the dependence of precipitation on initial chromatin concentration it has been suggested that the observed effect as contributed considerably by interparticle aggregation is a relatively good measure of the ability of cation to stabilize higher order structures of chromatin through direct crosslinking or induction of hydrophobic associations at selected sites. In contrary to that the method estimating the direct dehydration measures the overall dehydrating effect of a cation exerted on the whole chromatin. It has been suggested on the basis of the above comparative data that the in vivo regulation of the degree of overall chromatin hydration should occur through changes in concentration of free small inorganic cations. Larger organic polycations like polyamines should be mainly involved in stabilization of the higher order chromatin structures. The stabilizing role of large polyanions like RNA has been ruled out. It has also been found that the unwinding of chromatin DNA results in considerable chromatin hydration.

Nucleotide and predicted amino acid sequence of a new member of the ras gene family from the slime mold Physarum polycephalum

A second ras homologue, designated Ppras2, has been isolated from Physarum polycephalum mixed amoebae and flagellates cDNA library. Ppras2 encodes a protein of 193 amino acids of a calculated M(r) of 21,633. The deduced amino acid sequence is highly homologous to Ppras1 and other ras genes from slime molds. The amino acid sequence at the C-terminus of the putative protein suggests that like other slime mold ras proteins but not the ones from other organisms, it is modified by geranylgeranylation rather than farnesylation, it is unpalmitoylated and contains a putative lysine-rich domain.

Nonhistone proteins of the transcriptionally active chromatin fraction of Physarum polycephalum, associated with nucleosome linker DNA instead of histone H1

Physarum polycephalum Nonhistone protein Histone H1 Transcriptionally active chromatin

Chromatin reorganization during early differentiation of Physarum polycephalum

Abstract When microplasmodia of Physarum polycephalum are induced to differentiate by starvation, the rate of RNA synthesis measured in vitro with endogenous RNA polymerase B drops rapidly, decreasing after 12 h of starvation to 30% of the initial value. Transcriptional inactivation is accompanied by: (a) 60% decrease in the amount of the chromatin fraction solubilizable by light DNAase I digestion; (b) increase in the content of histone H1 and several nonhistone proteins in the DNAase-I-solubilized chromatin; and (c) formation of a nucleosome-like structure that is absent from this fraction during normal growth. No marked change in the structure and composition of the total chromatin on starvation is evident. These data suggest that the repression of transcription during differentiation in Physarum is correlated with structural reorganization of active chromatin regions.

Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene ☆

We have cloned the genomic copy of the Ppras1 gene, a homologue of the ras proto-oncogene, from the true slime mold Physarum polycephalum. Ppras1 contains five small introns, four of which have a high content of pyrimidines. The (dC)-homopolymers present in introns 4 and 5 may be responsible for the observed recA-independent deletion in Ppras1 upon amplification of the Ppras1-bearing plasmid by choramphenicol. Although Ppras1 exhibits amino acid and nucleotide homologies with the DdrasG gene, a homologue of ras from another slime mold, Distyostelium discoideum, locations and sequences of their introns are quite different. This discordance suggests that introns of the ras genes in these species were acquired independently.