Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The effect of food supply on the onset of asexual and sexual plasmodium formation in Physarum polycephalum was studied. Asexual differentiation occurs readily in amoebae carrying the matAh mating type allele. The density at which these amoebae begin to differentiate is influenced by the ind locus, which controls the production of a diffusible inducer. The alleles ind-1 and ind-2 are known. Strains carring the ind-1 allele begin plasmodium formation at a low amoebal density (rapid differentiation), while strains carring the ind-2 allele differentiate at a higher amoebal density (slow differentiation). The onset of differentiation is characteristic of the strain and did not change with a 20-fold variation in the number of food bacteria available. Sexual differentiation occurs between compatible amoebal strains. For a given pair of amoebal strains the onset of plasmodium formation occurs at a characteristic cell density that is determined by the genetic backgrounds of the strains. The ind locus is one of the genes that influences this cell density. Plasmodia are formed at a lower cell density in crosses involving compatible amoebae carrying the ind-1 allele than they are in crosses with strains carrying the ind-2 allele. As was found for asexual differentiation, an approximate 20-fold variation in the food supply did not affect the initiation of sexual plasmodium formation. These results suggest that in most cases starvation does not trigger the differentiation of amoebae into plasmodia. The time of onset of plasmodium formation is determined largely by genetic factors.     

Change in ATP-pyrophosphohydrolase activity during spherule formation of Physarum polycephalum.

The activity of Ca2+-dependent ATP pyrophosphohydrolase was found to fluctuate during spherule formation of the acellular slime mold Physarum polycephalum under starving incubation. The enzyme activity increased up to 16-fold at the 3rd day of the starvation, then decreased drastically to less than its original level. Column chromatography of the enzyme preparation suggested that the increase in the activity was due to de novo synthesis of a new isozyme. Cycloheximide inhibited the synthesis. The two isozymes were different in their Ca2+ sensitivity, the new one being less sensitive.     

Cycloheximide resistance of Physarum polycephalum.

In the presence of cycloheximide, wild-type plasmodia of Physarum polycephalum exhibit an immediate decrease in deoxyribonucleic acid synthesis, a reduction in the incorporation of [3H]thymidine into thymidine triphosphate, and an increase in the level of thymidine triphosphate, as well as a decrease in protein synthesis. In this study, we have utilized a cycloheximide-resistant (Cycr) amoebic strain selected from a population of cells mutagenized with nitrosoguanidine. Segregation data indicate that the resistance is due to a single mutation. We have used this Cycr mutant to construct Cycr plasmodial strains. Ribosomes isolated from such Cycr plasmodia showed resistance to cycloheximide in vitro, in contrast to ribosomes isolated from wild-type plasmodia. The Cycr plasmodia showed none of the cycloheximide-induced biochemical effects. Plasmodia heterozygous for the resistance marker were sensitive to cycloheximide with regard to growth but showed an intermediate response in the biochemical parameters. Heterokaryons formed by fusion of various proportions of the sensitive and resistant plasmodia showed a resistance with regard to both growth and biochemical parameters which was directly related to the fraction of Cycr plasmodia present in the heterokaryons. The data are consistent with the hypothesis that the effects of cycloheximide on deoxyribonucleic acid synthesis and nucleoside metabolism are secondary to the effect of the drug on protein synthesis in this organism.     

Induction of spherule formation in Physarum polycephalum by polyols

A method has been developed for inducing spherule formation (spherulation) in the myxomycete Physarum polycephalum by transferring the culture to synthetic medium containing 0.5 m mannitol or other polyols. This morphogenetic process occurred within 12 to 35 hr after the inducer was added. The mature spherules existed as distinct morphogenetic units, in contrast to the clusters of spherules formed during starvation. Ninety per cent of the spherules germinated by 24 hr in synthetic medium. The changes in the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein during plasmodial growth, spherulation, and germination of spherules are described. When spherule formation was completed, RNA, protein, and DNA decreased, compared with the values at the beginning of the conversion. The incorporation of (3)H-uridine into trichloroacetic acid-insoluble material was different in each of these periods, and this incorporation was sensitive to actinomycin D. The amount of glycogen increased during growth, whereas it decreased during spherulation. (14)C-glucose could be taken up by the cells in the presence of the inducer, and mannitol could not replace glucose as a source of energy. The mode of action of mannitol and its mechanism of induction are discussed.     

Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.     

The effect of alfalfa saponins on growth and lysis of Physarum polycephalum

The prelytic events associated with the interaction of saponins with Physarum polycephalum membrane components were studied. It was found that alfalfa saponins form interaction products with membranal sterols, proteins and phospholipids. The interaction of saponins with proteins affect also certain membranal enzymic activities such as NADH oxidase and Malate dehydrogenase. It is suggested that although the interaction of the saponin with sterols is much more specific than with other membranal components, the lysis of plasmodia of P. polycephalum should be attributed to a concerted attack on the various membrane constituents. In continuation of these interactions, the changes of permeability of plasmodia membrane were expressed by increment of inorganic sodium ions and water influx, traced by lysis, while no efflux of ions was observed.Killed in action in the October War, October 22, 1973.     

The subunit structure of chromatin from Physarum polycephalum.

Nucleosome DNA repeat lengths in Physarum chromatin, determined by nuclease digestion experiments, are shorter than those observed in most mammalian chromatin and longer than those reported for chromatin of certain other lower eukaryotes. After digestion with staphylococcal nuclease for short periods of time an average repeat length of 190 base pairs is measured. After more extensive digestion an average repeat length of 172 base pairs is measured. Upon prolonged digestion DNA is degraded to an average monomer subunit length of 160 base pairs, with only a small amount of DNA found in lengths of 130 base pairs or smaller. Mathematical analysis of the data suggests that the Physarum nucleosome DNA repeat comprises a protected DNA segment of about 159 base pairs with a nuclease-accessible interconnecting segment which ranges from 13 to 31 base pairs. The spacing data are compatible with measurements from electron micrographs of Physarum chromatin.     

The development of sporulation competence in Physarum polycephalum.

Plasmodial cells of the slime mold Physarum polycephalum become “competent” for sporulation following a prolonged period of dark starvation in the presence of nicotinamide. Sporulation can then be induced by illumination. Plasmodia are found to release into the medium during starvation one or more cellular products that promote sporulation. These products exert their effect specifically during the dark starvation period, rather than during the final phase of fruiting body construction. The sporulation control factor(s) (SCF) is nondialyzable and can stimulate the development of sporulation competence in the absence of nicotinamide.     

Defined and semi-defined media for the growth of amoebae of Physarum polycephalum.

Amoebae of the true slime mould Physarum polycephalum were cultured in two fully-defined liquid media containing amino acids, glucose, three vitamins and a buffered salts solution. Absolute requirements were demonstrated for methionine, haematin, thiamine and biotin, all of which were known to be specific requirements of the plasmodial stage. Methods are described for large-scale culture in three semi-defined media.     

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.     

Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum.

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH₂[³²P]O₄ and [³H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.     

Behavior of phosphatase isoforms during sclerotium formation in Physarum polycephalum

The behavior of phosphatase isoforms under dark-starvation from plasmodium of Physarum polycephalum were investigated to determine their possible roles in sclerotium formation. Two and a half days after dark-starvation, approximately 95% of plasmodia plates formed sclerotia. Specific phosphatase activity increased markedly up to ca. two-fold within the first day of starvation, after which the enzymatic activity decreased rapidly to a level less than the initial level within 2 days of the starvation period. Among the two isoforms of enzyme detected just before sclerotization under dark-starvation conditions, the enzymatic activity of the major isoform (Rm value of 0.6) decreased gradually within 1.5 days of starvation, then linearly to less than 20% of that at the beginning of the observation. Those of other major isoform (Rm value of 0.7) increased up to ca. two-fold within the first day of starvation, then decreased linearly to levels less than that of the first 2 days of the starvation period. Behavior of this isoform strongly suggests that it initiates the formation of sclerotium under dark-starvation conditions.     

ADP-ribosyltransferase in isolated nuclei during the cell cycle of Physarum polycephalum.

ADP-ribosyltransferase was measured in isolated nuclei of Physarum polycephalum. Activity was determined with and without exogenous DNA and histones. During the synchronous cell cycle the activity measured with exogenous substrates exhibited a typical peak enzyme pattern with a maximum of activity in S-phase, whereas activity measured without exogenous substrates displayed a step enzyme pattern. Both activities doubled in each cell cycle.