Changes in membrane glycoproteins during fruit-body formation inPhysarum polycephalum

Sporangia formation ofPhysarum polycephalum was induced by starvation and illumination, and the morphogenic process during the differentiation was studied by scanning electron microscopy. Plasma membranes were prepared from these differentiating plasmodia and the membrane proteins were analyzed by polyacrylamide gel electrophoresis. Many glycoproteins appeared during the fruit-body formation. Of these a protein of apparent molecular mass of 66 kD was prominent in sporangia forming stage which showed a high affinity to RCA lectin. Inhibition of the glycosylation and processing of these glycoproteins resulted in the prevention of fruit-body formation suggesting that the synthesis of these membrane components is a prerequisite process for the sporangia formation in the slime mold.     

Alkaline phosphatase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> is insoluble

The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg²⁺-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.     

Nuclear matrix proteins of Physarum polycephalum

The proteins of nuclear matrix preparations from Physarum polycephalum were compared with analogous mammalian fractions by gel electrophoresis, DNA-binding studies and immunological tests. Polypeptides of 28 and 36 K dalton, which dominate in Physarum preparations, differed from calf thymus matrix proteins in that they were basic and showed low affinity to DNA. These polypeptides were present at about 1.2 mg per mg of nuclear DNA. Polypeptides of higher molecular weight occurred in the preparation at about 0.5 mg per mg of nuclear DNA. At least some of the latter proteins showed high affinity to DNA and cross-reacted with the antiserum against calf thymus matrix proteins.     

A method of isolation of mitochondrial nucleoid of Physarum polycephalum and evidence for the presence of a basic protein

A large amount of nucleoids could be isolated from mitochondria of the slime mold Physarum polycephalum by treating the mitochondria successively with Triton X-100 and Nonidet P-40 followed by centrifugation. The preparation retained the ultra-structure characteristics of the intact mitochondrial nucleoid. The population of proteins extracted from the nucleoid preparation was analysed by polyacrylamide gel electrophoresis. The result indicated presence of at least one species of basic protein.     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

Structural comparison of deoxyribonucleic acid-dependent ribonucleic acid polymerases I and II from the slime mold Physarum polycephalum

RNA Polymerases I and II have been purified to homogeneity from the slime mold Physarum polycephalum. When subjected to ultracentrifugation in separate sucrose gradients, or together, the two enzymes migrate with S values estimated at 14.5 S. Polyacrylamide gel electrophoresis under denaturing conditions reveals that the two enzymes have distinct but similar subunit structures. The probable subunit structure of polymerase II is as follows: 175,000₁; 140,000₁; 24,000₂; 17,000₁.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

Differential synthesis of an alkaline endonuclease in Physarum polycephalum

During the sclerotization of microplasmodia of Physarum polycephalum in non-nutrient salt medium or in salt medium supplemented by glucose, RNA or nucleotides a 6-fold increase in the specific activity of an alkaline endonuclease was found within 6 h after the induction. The increase was based on de novo synthesis of the enzyme and it was strongly correlated to the sharp drop in the level of cellular RNA in the first hours of the process of scerotization. The induction in exhausted growth medium or in salt medium supplemented by protein or mannitol showed a gradual 2-3-fold increase of the endonuclease in 30 h, parallel to the gradual decrease of the RNA. No changes in the specific activity of the endonuclease were found during logarithmic growth or under conditions of starvation without the induction to sclerotization.The alkaline, polyA-specific endonuclease could possibly regulate the turnover of RNA.     

The life cycle of mitochondria in the true slime mould,Physarum polycephalum

The cytoplasmic aspects of mitochondrial biogenesis have been the focus of much recent attention and a review is presented here of studies on the life cycle of mitochom dria inPhysarum polycephalum. Such studies have focused predominantly on behavior of the mitochondrial genome throughout the mitochondrial life cycle and have been designed to reveal details about (1) the role of the DNA-membrane complex in the segregation of the mitoehondrial genome; (2) the regulation of mitochondrial activity associated with changes in ptoidy of the mitoehondrial nucleus; (3) the hierarchical pattern of transmission of the mitochondrial genome as it relates to the mating-type locus (matA) during the sexual development; and (4) the fusion of mitoehondria that is promoted by a mitochondrial plasmid. The results of such studies contribute significantly to efforts towards a better understanding not only of the mitochondrial life cycle inP. potycephalum but also of the biogenesis of mitoehondria and plastids in many other organisms. Recipient of the Botanical Society Award for Young Scientists, 1988.     

Synthesis of actinomycin-insensitive RNA during the first post-irradiation mitotic cycle,in the synchronously mitotic plasmodia of Physarum polycephalum

A sucrose density gradient analysis of³H-uridine pulse-labelled RNA from the first postirradiation mitotic cycle ofPhysarum polycephalum shows that all the density classes of RNA synthesized during this period are resistant to the peptide-antibiotic, actinomycin D. In fact, the synthesis is found to be greater in the presence of the drug. The heterogenously sedimenting synthetic activity here may represent a single species of RNA and its precursors or more than one kind of RNA. Further characterization of this RNA is meaningful in view of the actinomycin insensitivity of the postirradiation mitotic cycle itself to this antibiotic.     

Acceleration of mitosis induced by mitotic stimulators of Physarum polycephalum

Extracts of the myxomycete Physarum polycephalum exhibit an accelerating effect on nuclear division which fluctuates during the synchronous nuclear division cycle. Extracts from late G2 phase plasmodia can advance mitosis in recipient test plasmodia by up to 30% of the length of the control cycle. The advancing capacity of extracts is heat- and ammonium sulphate-precipitable, non-dialysable and destroyed by pronase, suggesting that the active substance is a protein. The advance of mitosis is in strong correlation with the applied dose of stimulatory material.     

Actin co-purifies with RNA polymerase II.

RNA polymerase II, [EC2.7.7.6], from the slime mold Physarum polycephalum, purified over 4000-fold can contain a protein with an apparent molecular weight of 46,000. This protein is separated from the putative subunits of RNA polymerase II by polyacrylamide gel electrophoresis under non-denaturing conditions, and by chromatography on phosphocellulose. In this report we identify the protein as actin, and we point out that polypeptides of this apparent molecular weight which have been found associated with RNA polymerase II purified from other sources may also be actin from these organisms.     

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.     

Centriole maturation in the amoebae ofPhysarum polycephalum

Summary The precise geometry of pro-centriole formation has been studied inPhysarum polycephalum amoebae. The spatial references used were the posterior and the anterior kinetosomes which are unequivocally defined by the presence of the posterior para-kinetosomal structure, the microtubular array 4 and the microtubular arrays 1, 2, and 3. The observations made suggest that pro-centrioles follow a maturation process. A pro-centriole formed during the n^th cell cycle becomes the posterior kinetosome during the (n + 1)^th cell cycle and the anterior one during all the following cell cycles. Pro-centriole formation occurs late in the cell cycle. This observation disagrees with a role of pro-centriole formation in the regulation of S phase in contrast to what has been suggested in other eucaryotic cells.     

Subunit structure of chromosomes in mitotic nuclei of Physarum polycephalum

We have investigated the subunit structure of mitotic chromosomes of the acellular slime mould Physarum polycephalum, using the nuclease susceptibility of isolated mitotic nuclei as a probe. A characteristic pattern of DNA digestion products is obtained, containing approximately integral multiples of a basic 140 base pair DNA segment that resembles very closely the pattern in G₂ phase nuclei of Physarum and of calf lymphocyte nuclei. These results demonstrate that during the process of chromosome condensation there is no alteration at the primary level of chromatin structure that is responsible for the characteristic DNA digestion pattern.     

Multiple effects of 5 mM sodium butyrate on Physarum polycephalum macroplasmodia

Summary In a Physarum polycephalum macroplasmodium, nuclei naturally divide synchronously. Thus, it offers an opportunity to study growth and mitosis within a true organism. The effects of 5 mM sodium-butyrate on these processes have been examined. When this material is added to the culture medium during mitosis, the butyrate acts like a fixative on condensed chromosomes. During interphase, this short fatty acid stops growth and immediately inhibits DNA synthesis. Furthermore, it prevents differentiation in macroplasmodia induced to spherulate. All these modifications are readily reversible after transfer to a medium lacking butyrate.     

Identification of a birefringent structure which appears and disappears in accordance with the shuttle streaming inPhysarum plasmodia

Summary R-HMM (rhodamine-heavy meromyosin) stained the birefringent fibrous structure which appears and disappears cyclically in parallel with the periodic shuttle streaming in the plasmodium ofPhysarum polycephalum. In addition, 0.6 M KI readily made the birefringent fibrils fade away. These results clearly show that the birefringent fibrils are composed of actin filaments and prove the possibility of actin filaments to alter in the aggregation state during the cyclic production of the motive force responsible for the cytoplasmic streaming.     

DNA-membrane association during the mitotic cycle of Physarum polycephalum

Association of DNA with the nuclear membrane was studied in the naturally synchronous slime mold Physarum polycephalum. Sucrose gradient analyses showed that DNA is bound to the membrane during all stages of interphase. The association is labile, but can be stabilized by spermine, which presumably acts on the membrane. Experiments with pulse-labeled DNA suggest that replication does not occur at a membranal site.     

Newly-initiated DNA isolated from Physarum in early S phase consists of nascent-nascent duplexes

We have studied the nature of newly initiated DNA released during DNA isolation at the beginning of S phase of Physarum polycephalum. The released DNA was separated from the bulk DNA by sedimentation through sucrose gradients. Gentle shearing strongly enhanced the release of newly initiated DNA. The additionally released material had a larger average molecular weight. Buoyant density analysis after labelling with bromodeoxyuridine (BrdU) revealed that the released DNA consisted of nascent-nascent duplexes for more than 90%. This indicates that the release of newly initiated DNA occurs by branch migration. We conclude that shearing enhances branch migration by destabilization of the double helix.