A method for separation of pigments from plasmodia of the true slime molds, Physarum polycephalum and Physarum nudum

Plasmodia of the true slime molds Physarum polycephalum and Physarum nudum were grown on agar semidefined medium (J. W. Daniel and H. Baldwin (1964) in Methods in Cell Physiology (Precsott, D. M., Ed.), Vol. 1, pp. 9-44, Academic Press, New York.) and pigments were extracted from 8-day-old plasmodia with the same solvent mixture that was used later as a mobile phase in the HPLC analysis. The separation was carried out on a strong anion exchanger column in a methanol/borate buffer solution. Under these conditions eight pigments from P. polycephalum and eight from P. nudum were revealed. This method can be used for detailed studies of plasmodial pigments in these species.     

Identification of the telomeric sequence of the acellular slime molds Didymium iridis and Physarum polycephalum.

We have determined the telomeric DNA sequence of the acellular slime molds Didymium iridis and Physarum polycephalum. In both organisms the telomeres consist of tandem repeats of the hexamer 5'(TTAGGG)3'. This sequence was determined by cloning and sequencing the telomeric fragment of the linear extrachromosomal ribosomal DNA from Didymium, as well as direct end labeling and sequencing the rDNA from both organisms. Interestingly, this sequence is identical to the telomeric DNA sequence of the flagellated protozoan Trypanosoma brucei, and suggests that despite the diversity of telomeric sequences previously determined in lower eukaryotes, the necessity to create functional telomeres has led to constraints on these sequences.     

Serological investigations of cell adhesion in the slime molds,Dictyostelium discoideum,Dictyostelium purpureum,and Polysphondylium violaceum

1. Antibodies to slime molds were produced by injecting D. discoideum and D. purpureum amebas from 48 hour cultures into rabbits. 2. Anti-D. discoideum and anti-D. purpureum sera caused agglutination of homologous amebas from 24 to 26 hour cultures, agglutination of certain heterologous amebas from 30 to 36 hour cultures, and agglutination of all heterologous amebas from 43 to 48 hour cultures. 3. The data show that new surface antigens are formed in cultures after 26 hours and it is suggested that the new antigens are concerned with cell adhesion. 4. The probable role of surface antigens in the interaction of cells of different species of slime molds was discussed.     

Nuclear plasmids in the Dictyostelium slime molds

Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2-microns circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3-27 kb and in copy number from 50-300 molecules per cell. Plasmids have been identified in approximately one-fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10(-4) per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro.

Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.     

Threshold phenomena in chemoreception and taxis in slime mold Physarum polycephalum

The plasmodium of Physarum polycephalum reacts to various kinds of chemicals substances and moves towards or away from them. Threshold concentration of recognition of chemicals was examined in terms of membrane potential and of the averaged motive force of tactic movement by using a double-chamber method, i.e., a single plasmodium was placed between two compartments through a narrow ditch, and differences in membrane potential and in pressure between two compartments were measured. Results are summarized as follows: (a) By increasing the concentration of various substances in one compartment, the membrane potential started to change at a certain threshold concentration, C-th, for each chemical. Chemotactic movement of the plasmodium took place at the same threshold concentration. These results held both for attractants (glucose, galactose, phosphates, pyrophosphates, ATP, c-AMP, etc) and for repellents (various inorganic salts, sucrose, fructose, etc.). (b) The threshold concentration, Cth, for inorganic salts decreased remarkably with increase of the valences of cations, zeta, and was proportional to Z-6, I.E., THE Shultze-Hardy rule known in the field of colloid chemistry was found to be applicable. (c) The plasmodium distinguished the species of monovalent cations in the following order: H(Li(K(Na(Rb(Cs(NH-4 Plots of log Cth against the lyotropic number of anion fell on different straight lines for each monovalent cation species. (d) Plots of log Cth, against the reciprocal of the absolute tempe lines were almost the same and gave a value of 12 kcal/mol for the enthalpy change. These results suggest that the recognition of chemical substances appears as the result of a structural change of the membrane at the threshold point, and that the change in membrane structure is transmitted simultaneously to the motile system of the plasmodium.     

Comparison of base specificity and other enzymatic properties of two protozoan ribonucleases from Physarum polycephalum and Dictyostelium discoideum

Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5'-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3'-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A > or = G > C > U, and A > or = G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.     

Identification of mRNA in the slime mold Physarum polycephalum.

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.     

Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

Responses of the slime mold Physarum polycephalum to changing accelerations

Every cell is probably able to respond to gravity (g) via an unknown gravireceptor mechanism (supposed general gravisensitivity of cells). To investigate this mechanism a free-living ameboid cell, which uses gravity for its spatial orientation (geotaxis), was selected as a model system: the acellular slime mold Physarum polycephalum (Myxomycetes). In this paper results of 0 g-simulation experiments will be compared to results obtained in the Spacelab IML-1 Physarum experiment to stress the reliability of experiments performed on the fast-rotating clinostat.     

Coordination of macromolecular synthesis in the slime mould Physarum polycephalum.

Microplasmodia of P. polycephalum were grown either in batch culture, in both complex and defined media to give a 3-4 fold variation in growth rate, or in a chemostate. The protein/DNA ratio of batch cultures was almost invariant, whilst the RNA/DNA ratio increased as a non-linear function of growth rate. The amount of ribosomal RNA, expressed as a fraction of total RNA, showed little variation and this was also true for the proportion of ribosomes found in polyribosomes. Calculation of the rate of protein synthesis per ribosome shows that this parameter increases by approximately 50% over the range of growth rates studied, although it should be emphasized that the effect of protein turnover has not yet been taken into account. Enrichment of batch cultures growing in a defined medium produced an increase in the rate of RNA synthesis. Data obtained with chemostat cultures differed in several respects from those described above for batch cultures, especially at low growth rates, and are discussed in relation to the early stages of differentiation of microplasmodia to spherules.     

Cytoplasmic control of mitosis in true slime mold, Physarum polycephalum

To resolve the problem of whether mitosis is controlled by anuclear or cytoplasmic stimulus, plasmodia from various periodsof the mitotic cycle were fused with one another. Mitosis inthe fused plasmodiura occurred about midway between mitosesof the donors. Treatment with cyclohcximide during the G₂-period.delayed the next mitosis for a period equal to the time of treatment. (Received March 2, 1971; )