Evolution of hierarchical cytoplasmic inheritance in the plasmodial slime mold Physarum polycephalum

A striking linear dominance relationship for uniparental mitochondrial transmission is known between many mating types of plasmodial slime mold Physarum polycephalum. We herein examine how such hierarchical cytoplasmic inheritance evolves in isogamous organisms with many self-incompatible mating types. We assume that a nuclear locus determines the mating type of gametes and that another nuclear locus controls the digestion of mitochondria DNAs (mtDNAs) of the recipient gamete after fusion. We then examine the coupled genetic dynamics for the evolution of self-incompatible mating types and biased mitochondrial transmission between them. In Physarum, a multiallelic nuclear locus matA controls both the mating type of the gametes and the selective elimination of the mtDNA in the zygotes. We theoretically examine two potential mechanisms that might be responsible for the preferential digestion of mitochondria in the zygote. In the first model, the preferential digestion of mitochondria is assumed to be the outcome of differential expression levels of a suppressor gene carried by each gamete (suppression-power model). In the second model (site-specific nuclease model), the digestion of mtDNAs is assumed to be due to their cleavage by a site-specific nuclease that cuts the mtDNA at unmethylated recognition sites. Also assumed is that the mtDNAs are methylated at the same recognition site prior to the fusion, thereby being protected against the nuclease of the same gamete, and that the suppressor alleles convey information for the recognition sequences of nuclease and methylase. In both models, we found that a linear dominance hierarchy evolves as a consequence of the buildup of a strong linkage disequilibrium between the mating-type locus and the suppressor locus, though it fails to evolve if the recombination rate between the two loci is larger than a threshold. This threshold recombination rate depends on the number of mating types and the degree of fitness reduction in the heteroplasmic zygotes. If the recombination rate is above the threshold, suppressor alleles are equally distributed in each mating type at evolutionary equilibrium. Based on the theoretical results of the site-specific nuclease model, we propose that a nested subsequence structure in the recognition sequence should underlie the linear dominance hierarchy of mitochondrial transmission.     

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.     

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.     

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase alpha on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzyme was 30-40% pure and form HS2 enzyme 60% with regard to protein contents of the preparations. Form HS2 enzyme was generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptide in a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as well as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase beta by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

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; )     

Covalent linkage between RNA and nascent DNA in the slime mold, Physarum polycephalum.

When alpha--32 P-labeled deoxyribonucleoside triphosphates are injected into plasmodia of the eukaryotic slime mold, Physarum polycephalum, they are incorporated initially into strands of DNA which are mostly less than 300 nucleotides long. Sixty minutes after injection incorporated deoxyribonucleoside triphosphates are found in much longer strands. If the short strands found two minutes after injection are denatured and centrifuged to equilibrium in a Cs(2)SO(4) density gradient, they migrate to a density slightly greater than that of single-stranded Physarum DNA. When these short strands are treated with alkali to hydrolyze RNA, a small fraction of the incorporated -32P is made acid-soluble and is identified as a mixture of the four ribonucleoside 2',3'-monophosphates. Such transfer of -32P to ribonucleotides occurs when any of the 4 alpha--32P-labeled deoxyribonucleoside triphosphates is used for injection, but the transfer is greatest with [alpha--32P]dGTP. We conclude that very short stretches of RNA are found linked through phosphodiester bonds to nascent DNA chains in Physarum polycephalum and that any of the 16 possible combinations of ribo- and deoxyribonucleotides can occur at the RNA-DNA junction.     

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.     

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.     

DNA replication in Physarum polycephalum: characterization of replication products made in isolated nuclei

Nuclei isolated from synchronous S-phase plasmodia of the myxomycete $\text{Physarum polycephalum}$ were competent in production of low molecular weight DNA replication intermediates. Furthermore, these nuclei showed some competence in joining these fragments into DNA of intermediate molecular weight. The DNA molecules made $\text{in vitro}$ could be correlated with products made $\text{in vivo}$.