Diploid Plasmodia Research Articles

CDNA Cloning and Life-Cycle Stage-Specific Expression of Coronin fromPhysarum polycephalum

Coronin cDNA was cloned from the plasmodia of Physarum polycephalum. The amino acid sequence deduced from the cDNA was comprised of 449 residues and showed 60% identity to that of Dictyostelium discoideum coronin. Southern blot analysis suggested that the coronin gene present in the P. polycephalum genome might be a single copy. Coronin was expressed in diploid plasmodia, while it was not detected in haploid amoebae or spores.

Heat Stress Induces a Glycosylation of Membrane Sterol in Myxoamoebae of a True Slime Mold, Physarum polycephalum

To know the very early events occurring after heat shock, the changes of membrane lipids were examined. Heat stress induced the production of a certain glycolipid in the myxoamoebae of Physarum polycephalum in a few minutes. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies that was previously reported to be expressed during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723). The activity of UDP-glucose:poriferasterol glucosyltransferase (Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415) was also expressed rapidly after heat shock. Thus, the activation of sterol glucosyltransferase and the production of sterol-glucoside were considered to be important events that were involved in the signal transduction system to induce some succeeding heat-shock responses, such as the synthesis of heat-shock proteins.

Rapid disappearance of one parental mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum

Five haploid amoebal strains of the myxomycete Physarum polycephalum, each with a distinct mitochondrial genotype, were crossed in all pairwise combinations. The mitochondrial genotype in the diploid plasmodia resulting from these isogamous matings were found to be transmitted uniparentally. This uniparental inheritance could be arranged in a dominant hierarchical order. Time-course analysis of the presence of mitochondrial genotypes in the zygotes and young developing plasmodia show that elimination of one parental mitochondrial genotype is virtually completed during the first two nuclear cycles in the zygote/differentiating plasmodium. To our knowledge this is the first report indicating an active mechanism involving the degradation of mitochondrial genomes in sexual crosses.

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum. In the amoeboid stage, the main components of phospholipid fraction were phosphatidylethanolamine (PE, 43.3%), phosphatidylcholine (PC, 28.8%) and phosphatidylinositol (PI, 8.0%), but in the plasmodial stage, PC was dominant (40.7%) and other main components were PE (31.5%) and phosphatidic acid (PA, 11.0%). The specific activity of phospholipase D in the plasmodia was 5.7-times higher than that in the myxoamoebae when measured in the presence of Ca 2+ at the alkaline pH. In the amoeboid stage, phospholipase A activity (A 1 or A 2) was detected at the alkaline pH with Ca 2+. Phospholipase D activity in the plasmodia was characterized: pH optimum was 6.0; Ca 2+ was required for the reaction and Ba 2+ could substitute partly for Ca 2+; PE was the best substrate for the hydrolytic activity and PC and PI were not appreciably hydrolyzed; and all detergents tested inhibited the enzyme activity.

Expression of UDP-glucose: poriferasterol glucosyltransferase in the process of differentiation of a true slime mold, Physarum polycephalum

An expression of UDP-glucose: poriferasterol glucosyltransferase activity associated with differentiation of a true slime mold, Physarum polycephalum, from haploid myxoamoebae to diploid plasmodia was demonstrated. In the haploid cells, this enzyme activity was not detected, but after conjugation of the myxoamoebae, the enzyme activity was expressed and increased definitely. In the plasmodial stage, high enzyme activity was maintained constantly. The enzyme was partially purified (35-fold purification, and 28% yield), and molecular weight of 72 000 pH optimum of 7.0, and some characteristics were demonstrated.

Expression of poriferasterol monoglucoside associated with differentiation of Physarum polycephalum.

A glycolipid which was expressed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum, has been examined. In the amoeboid stage, cells did not contain this glycolipid, but after conjugation of the haploid cells, this substance appeared and increased in its amount. From structural studies of the purified glycolipid, it has been identified as poriferasterol monoglucoside.

Sterol contents of haploid myxoamoebae of Physarum polycephalum. Comparison with those of diploid plasmodia.

The sterol contents of the myxoamoebae of Physarum poly-cephalum were examined and compared to those of the diploid plasmodia of Physarum. Most of the sterol components were unsaturated, the major species being poriferasterol. The myxoamoebae also contained Δ5-ergostenol and 22-dihydroporiferasterol. The molar ratio of these three species of unsaturated sterols was 80 : 15 : 5.

The Effect of Ploidy on the Stability of Plasmodial Heterokaryons in Physarum polycephalum

The stability of actively growing heterokaryons made by fusing haploid and diploid plasmodia of Physarum polycephalum has been investigated with the aid of genetic markers affecting plasmodial colour and amino acid requirements. All heterokaryons initially expressed the dominant alleles present in both component plasmodia, but after a few subcultures every heterokaryon synthesized from a haploid plus a diploid plasmodium changed to express the recessive alleles carried by the haploid component. In contrast, heterokaryons synthesized from combinations of haploid plasmodia remained stable throughout many subcultures. No evidence was found for the segregation of incompatibility alleles responsible for heterokaryon instability, although the possibility could not be excluded that a gene closely linked to matA was involved. A direct test of the effect of ploidy on heterokaryon stability was carried out, in which the use of isogenic haploid and diploid plasmodia allowed the effect of incompatibility genes to be eliminated. Again the phenotype of every haploid plus diploid heterokaryon changed to that of the haploid component, whereas haploid plus haploid heterokaryons remained stable. Variations in the relative sizes of the haploid and diploid plasmodia altered the speed, but not the direction, of the changes observed. Analysis of progeny indicated that the changes in phenotype were due to loss of diploid nuclei from the heterokaryons.

Three-CloneDidymium IridisCrosses and Plasmodial Incompatibility Phenotype

Heterokaryon formation between diploid plasmodia ofthe myxomycete Didymium iridis is controlled by a number of incompatibility loci. Normally, only plasmodia phenotypically identical for these loci will fuse to form a heterokaryon. Also, multiple myxamoebal (gametic) crosses which produce a number of plasmodia of different incompatibility phenotypes usually result in the final production of only a single phenotype (that displaying the strongest incom? patibility reaction). However, if late log phase myxamoebae are crossed in specific proportions novel phenotypes can be obtained for a number of gametic combinations. These phenotypes are apparently the combined expression of two normally incompatible phenotypes.

A novel acid protease from haploid amoebae of Physarum polycephalum, and its changes during mating and subsequent differentiation into diploid plasmodia.

Haploid myxamoebae of the true slime mold, Physarum poly-cephalum contained an acid protease that had a highly acidic pH optimum of 1.4. This enzyme was not considered to have-SH groups, DAN-Cu2+ reactive-carboxyl groups, or an aspartic acid moiety in its active center. Its activity appeared to decrease with time during mating and the subsequent differ-entiation into the diploid, multinuclear plasmodia.

Non-selfing mutants from selfing (Het−) strains ofPhysarum polycephalum

SummaryPlasmodial formation in the MyxomycetePhysarum polycephalumis under heterothallic control by a mating type (mt) locus. In natural isolates only amoebae with differentmtalleles are able to cross to form diploid plasmodia. A class of mutants isolated from heterothallic amoebae, together with the variant strainCL, is able to form plasmodia in pure clones, designated as selfing. Non-selfing mutants have been isolated fromCLand from other selfing amoebae.This paper reports the isolation and analysis of 64 non-selfing derivatives (designated Npf−) from seven selfing (Het−) strains. The Npf−mutants could be grouped into eight classes on the basis of their crossing and complementation patterns. The possible significance of these mutants is discussed.

Cell fusion competence and its induction in Physarum polycephalum and Didymium iridis

In heterothallic Myxomycetes, diploid plasmodia arise when haploid amoebae of two different mating types are cultured together. In this mating process, the amoebae fuse in pairs, and the resulting zygotes develop directly into plasmodia. It has been shown previously that plasmodia start to form in this fashion only when the growing amoebae in a mixed culture reach a critical density. We have investigated the cellular basis of this phenomenon by growing amoebae of different mating type separately from one another and then mixing them to test their mating ability. Amoebae from cultures above and below the critical density were, respectively, competent and incompetent to mate. Furthermore, both partners had to be competent in order for mating to occur. No binucleate cells were formed in mixtures of incompetent amoebae, indicating that they failed to fuse with one another. Incompetent amoebae growing at low density on filters with 0.2-μm pores became competent when the filters were placed on dense cultures of amoebae. We suggest that amoebae release a filter-transmissible material that accumulates during growth and induces the cells to become fusion competent.

Aging of plasmodial heterokaryons in Didymium iridis

Isogenic diploid plasmodia of different ages were produced by crossing clonal gamete populations of the myxomycete Didymium iridis. These plasmodia were then used to produce “age” heterokaryons to study the timing of death in comparison to the senescence of the two original homokaryons. A majority of the age heterokaryons died concurrently with the oldest nuclear component which indicates that the control of aging may be a “dominant” or active process in the true slime molds. Age heteroplasmons using transfilter contact between two plasmodia across 1 μ pore size Nucleopore membrane filters were also produced which indicates that senescence may not be a cytoplasmic infectious particle.

COMPLEMENTATION OF AMOEBAL-PLASMODIAL TRANSITION MUTANTS IN PHYSARUM POLYCEPHALUM

Amoebae of the Myxomycete Physarum polycephalum differentiate to yield plasmodia in two ways: in crossing, haploid amoebae of appropriate genotypes fuse to form diploid plasmodia; in selfing, plasmodia form without amoebal fusion or increase in ploidy. Amoebae carrying the mating-type allele matAh (formerly mt(h)) self efficiently, but occasionally give rise to mutants that self at very low frequencies. Such "amoebal-plasmodial transition" mutants were mixed in pairs to test their ability to complement one another in the formation of plasmodia by crossing. The pattern of crossing permitted 33 mutants to be assigned to four complementation groups (aptA(-), npfA(-), npfB(-) and npfC(-)). Similar tests had previously proved only partially successful, as crossing had occurred only rarely in mixtures of compatible strains. The efficiency of complementation was greatly increased in the current work by mixing strains that carried different alleles of a newly-discovered mating-compatibility locus, matB; this locus had no effect on the specificity of complementation. A possible interpretation of the complementation behavior of the mutants is suggested.

Viability of Physarum polycephalum spores and ploidy of plasmodial nuclei.

Amoebae of Physarum polycephalum carrying the mth mating-type allele may differentiate into plasmodia in the absence of mating. Such plasmodia are haploid and, upon sporulation, produce mainly inviable spores. We have asked whether the viable spores arise from meiotic or mitotic divisions. Using a microfluorometric measurement of the deoxyribonucleic acid content of individual nuclei, we found the fraction of viable spores to be correlated with the proportion of rare, diploid nuclei containing in the generally haploid plasmodium. When homozygous diploid plasmodia were created by heat shocking, spore viability increased dramatically. We suggest that viable spores are produced via meiosis in mth plasmodia, that the mth allele has no effect on sporulation per se, and that the normal source of viable haploid spores is a small fraction of diploid nuclei ubiquitous in haploid plasmodia.

Isolation and Analysis of Amoebal–Plasmodial Transition Mutants in the MyxomycetePhysarum polycephalum

SUMMARYPlasmodium formation in the MyxomycetePhysarum polycephalumnormally involves fusion of haploid amoebae, carrying different alleles at the mating type (mt) locus, to give diploid plasmodia. Strains carrying themthallele are capable of undergoing the amoebal–plasmodial transition with high efficiency within amoebal clones, resulting in the formation of haploid plasmodia. NMG mutagenesis ofmthamoebae, followed by an enrichment procedure, was used to isolate mutants in which such clonal plasmodium formation was either delayed or absent. Thirteen mutants of the second type were analysed. Three of these were temperature-sensitive for plasmodium formation. All thirteen mutants were able to form diploid crossed plasmodia when mixed with amt1strain. Three new genes were identified and designatednpfA,npfB andnpfC. A mutant allele ofnpfA renderedclonalplasmodium formation temperature-sensitive, but did not prevent crossing at the non-permissive temperature with derived strains carrying the same mutant allele. No recombination was detected betweennpfB ornpfC andmt, butnpfA was unlinked tomtand a locus (apt-1) shown in a previous study to be involved in plasmodium formation. The genesnpfB andnpfC were distinguished by complementation analysis. Strains of the genotypenpfB−;npfC+behaved in the same way as strains carrying themt2allele. The nature of the mutants and the role of the mating-type locus in the initiation of plasmodium formation are discussed.

Developmental mutants in a homothallic strain ofPhysarum polycephalum

SUMMARYA derivative line of the homothallic Colonia strain ofPhysarum polycephalumhas been isolated which produces plasmodia with high efficiency within clones of amoebae. Using the synergistic effect of ultraviolet light and caffeine, mutants of this line have been isolated which fail to undergo the developmental transition between haploid amoebae and diploid plasmodia (aptmutants). They are isolated by selecting for amoebae which fail to produce plasmodia within clones. Complementation tests of four mutants have shown that they are mutants of four different loci and they are recessive to wild-type. A further analysis of one mutant reveals that theapt-1locus is unlinked to three other known markers. Crosses of this mutant with heterothallic strains yield progeny which are homothallic indicating that the lesion is not a revertant from a homothallic to a heterothallic mating-type. The use of this system in isolating developmental mutants is discussed.

GENETICS OF SOMATIC FUSION IN PHYSARUM POLYCEPHALUM: THE PpII STRAIN

Plasmodial (somatic) fusion in a strain of Physarum polycephalum, a true slime mold, is controlled by four loci, each of which displays simple dominance. Two diploid plasmodia fuse with each other only if they are phenotypically or genotypically identical for all four fusion loci.

THE ULTRASTRUCTURE OF MITOSIS IN MYXAMOEBAE AND PLASMODIA OF PHYSARUM FLAVICOMUM

Electron microscopy of glutaraldehyde‐osmium‐fixed samples of haploid myxamoebae and diploid plasmodia of the myxomycete Physarum flavicomum Berk. reveal dissimilar spindle apparatus during mitosis in the two cell types. Myxamoebae exhibit an astral type of mitosis with centrioles at the poles and nuclear envelope breakdown during prophase. Plasmodial nuclei lack centrioles at mitosis and have an intranuclear spindle, with nuclear envelope persisting during the entire division. Coated vesicles are noted during prophase and telophase in myxamoebae and their role in spindle formation and dispersion is suggested.

The Ultrastructure of Mitosis in Myxamoebae and Plasmodia of Physarum flavicomum

Electron microscopy of glutaraldehyde-osmium-fixed samples of haploid myxamoebae and diploid plasmodia of the myxomycete Physarum flavicomum Berk. reveal dissimilar spindle apparatus during mitosis in the two cell types. Myxamoebae exhibit an astral type of mitosis with centrioles at the poles and nuclear envelope breakdown during prophase. Plasmodial nuclei lack centrioles at mitosis and have an intranuclear spindle, with nuclear envelope persisting during the entire division. Coated vesicles are noted during prophase and telophase in myxamoebae and their role in spindle formation and dispersion is suggested.