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.

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.

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 and sequence analysis of a rap gene from the true slime mold Physarum polycephalum.

A member of the ras gene superfamily, belonging to the rap family and designated Pprap1, was isolated from a cDNA library from the true slime mold Physarum polycephalum by plaque hybridization in combination with 5'-RACE. The assembled nucleotide sequence of Pprap1 (1062 bp) has an open reading frame coding for a protein of 188 amino acids of a calculated M(r) of 21035. This protein exhibits: (i) a highly conserved GTP binding domain containing a putative effector domain, with the threonine-for-glutamine substitution characteristic of rap proteins, (ii) a hypervariable domain, and (iii) the CAAX motif. Analysis of the C-terminal amino acid sequence of Pprap1 shows that it presumably undergoes geranylgeranylation but is not palmitoylated; however, it contains a lysine-rich domain which might serve as the second membrane localization signal. Pprap1 exhibits significantly high amino acid homology within the GTP binding domain with its homologues: Ddrap1 from Dictyostelium discoideum (92%) and human Rap1A (83%), and relatively low homology (59%) with the Saccharomyces cerevisiae homologue, RSR1. It has also 59% and 61% homology with the P. polycephalum Ppras1 and Ppras2 proteins, respectively. This gene is the third member of the ras gene superfamily identified in P. polycephalum so far.

Nucleoprotein chromatin subunit from Physarum polycephalum.

The nucleoproteins resulting from digestion of the nuclei of the true slime mold Pysarum polycephalum with micrococcal nuclease have been resolved according to the size classes in linear sucrose gradients containg 0.5 M NaCl, and analysed for DNA, RNA and protein content. The basic nucleoprotein subunit has been found to contain a DNA fragment of about 150--170 base pairs complexed with an approximately equal amount, on a weight basis, of basic proteins and a relatively small amount of non-histone proteins (about 35% of the amount of DNA). Higher nucleoprotein oligomers were shown to contain spacer DNA fragments between adjacent subunits and a considerably higher ratio of non-histone proteins to DNA than the basic subunit. Both the basic subunit and higher nucleoprotein oligomers of Physarum chromatin contain some amount of tightly bound RNA. However, in contrast to the distribution of the non-histone proteins, the ratio of RNA to RNA is similar in both fractions.

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.

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.

Changes in size and shape of chromatin particles after successive removal of histones.

The preparations of whole chromatin, chromatin selectively depleted of histone f1, depleted of all lysine-rich histones (f1, f2b, f2a2), and DNA was studied by viscosimetric and light scattering methods. The obtained results were used for calculation of the dimensions and packing ratios of DNA for the preparations studied. The packing ratio in whole chromatin is 7.2 and is almost unaffected by selective removal of histone f1 (6.9), but decreases on successive removal of the remaining four histones, the decrease being dependent more on the quantity than the kind of the dissociated histones.

Isolation and susceptibility to nucleases of transcriptionally active and inactive chromatin fractions from Physarum polycephalum.

Transcriptionally active and inactive chromatin fractions were isolated from Physarum polycephalum after depolymerization of chromatin with DNAase II or micrococcal nuclease, followed by fractionation in 5 mM-MgCl2. The active fraction of chromatin comprised up to 21% of nuclear DNA and was enriched 22-fold in the labelled nascent RNA. Both chromatin fractions were shown to have the nucleosomal structure. DNA of the active fraction of chromatin was degraded much faster with DNAase I and micrococcal nuclease than the DNA of the inactive fraction.

The structure of chromatin synthesized in the presence of cycloheximide in Physarum polycephalum.

The effect of cycloheximide on protein and DNA synthesis and on the structure of chromatin was studied. Changes in the rate and extent of DNA synthesis in response to cycloheximide were highly variable in contrast to the extremely rapid and reproducible inhibition of protein synthesis. No differences in the rate of the release of acid-soluble products by nucleases and in the nature of the nucleoprotein particles were found in chromatin from plasmodia treated and non-treated with cycloheximide. It is concluded that, in Physarum polycephalum, unlike in higher animals, chromatin from the antibiotic-treated plasmodia is structurally indiscernible by the methods applied from normal chromatin.

On the arrangement of histones in chromatin.

It was found that nucleoprotein particles formed after DNase I action on calf thymus chromatin contain single-stranded DNA fragments, associated with histones only by ionic linkages. These results suggest that histones in chromatin are bound ionically only to one polynucleotide strand of double-helical DNA, protecting it against nucleolytic attack.

Comparison of susceptibility to staphylococcal nuclease and behaviour in metrizamide gradients of normal and 5-bromodeoxyuridine-substituted chromatin from Physarum polycephalum.

Replacement of 20--30% of thymine by 5-bromodeoxyuridine in chromatin DNA of Physarum polycephalum does not cause any visible change in a typical, regular pattern of DNA products obtained upon digestion of chromatin with staphylococcal nuclease. The time course of digestion is similar for normal and substituted chromatin even under conditions when the nuclease cleaves preferentially the dAT regions in DNA. 5-Bromodeoxyuridine label does not significantly affect the DNA/protein ratio in chromatin; this is reflected by similar behaviour of normal and substituted chromatin in metrizamide-density gradients.

Some unusual features of Physarum polycephalum chromatin are due to the presence of slime.

Chromatin of lower eukaryote Physarum polycephalum, while showing typical nucleosomal organization, reveals upon digestion with micrococcal nuclease certain features not found in chromatins of higher eukaryotes, the most pronounced of which is the unusual pattern of degradation of core-size DNA, without accumulation of subcore fragments. It has been shown that these peculiarities are not due to intrinsic features of Physarum nucleohistone complex but to the presence of a specific polysaccharide, the main component of Physarum slime, contaminating chromatin preparations.

Isolation of chromatin subcore particles by chromatography on Biogel 1.5 m.

The method is described for separation and purification by chromatography on Biogel 1.5 m of subnucleosomal nucleoprotein particles obtained by extensive digestion of calf thymus nuclei with micrococcal nuclease.

The presence of serine protease in pea embryo chromatin.

1. It has been shown that chromatin from pea seedlings contains a proteolytic enzyme, similar to that of mammalian chromatin. 2. The protease was isolated from chromatin by acid extraction and partly characterized. It is a serine-type enzyme, sensitive to DFP, of low mol. wt. (about 18 000 - 20 000), with optimum pH at about 8 with [3H]N-acetylated histone as a substrate. 3. In chromatin complex, histones fl and f3 are preferentially degraded.

Similarity in active site arrangement of neutral protease from calf thymus chromatin and trypsin.

1. Susceptibility to inhibitors of neutral protease from calf thymus chromatin has been compared with that of trypsin. The chromatin protease reacts stoichiometrically with the inhibitors specific for trypsin (diisopropylfluorophosphate, tosyl-lysyl chloromethane, soybean trypsin inhibitor and Kunitz basic inhibitor from pancreas), but not with the inhibitor specific for chymotrypsin (tosyl-phenylalanyl chloromethane). 2. Chromatin protease, similarly as trypsin, cleaves Lys-X and Arg-X peptide bonds. 3. It is concluded that the structure of active site region of both enzymes is very similar.

Transcriptionally active chromatin can be selectively released by DNase I from Physarum polycephalum genome.

In a simple eukaryote Physarum polycephalum about 13% of the genome is transcribed into abundant cytoplasmic RNA as shown by S1 nuclease digestion of DNA-RNA hybrids. Mild digestion of isolated Physarum nuclei with DNase I liberates a fraction of chromatin 3.5-fold enriched in sequences hybridizing by Physarum poly(A)+ RNA. This fraction is similarly enriched in histone H4 and actin genes known to be actively transcribed in Physarum. High content (about 45%) of actively transcribed sequences in DNase-I-released fraction of Physarum chromatin makes it particularly well suited for studying the structural basis of transcriptional activation in eukaryotes.

PPRAS1PPRAS2 AND PPRAP1 GENES, MEMBERS OF A RAS GENE FAMILY FROM THE TRUE SLIME MOLD PHYSARUM POLYCEPHALUM ARE DEVELOPMENTALLY REGULATED

The expression patterns of two true ras genes, Ppras1 and Ppras2and one rap gene, Pprap1were examined in four Physarum polycephalum developmental stages: uninucleate amoebae, plasmodia (multinucleate syncytia), spherules (a vegetative, dormant stage) and fruiting bodies. Ppras1 and Pprap1 are expressed in all stages examined with the maximum levels of their transcripts in amoebae and fruiting bodies, respectively, and the minimum levels in plasmodia, whereas the Ppras2 transcript is only detectable in amoebae and fruiting bodies. The results obtained indicate that P. polycephalum is an organism possessing a developmentally regulated ras gene family and presents a convenient system to study the role of ras/rap genes in control of growth and differentiation of lower eukaryotic organisms.Copyright 1997 Academic Press Limited Copyright 1997Academic Press Limited