RESUMO
Myxomycetes (also called Myxogastria or colloquially, slime molds) are worldwide occurring soil amoeboflagellates. Among Amoebozoa, they have the notable characteristic to form, during their life cycle, macroscopic fruiting bodies, that will ultimately release spores. Some 1,000 species have been described, based on the macroscopic and microscopic characteristics of their fruiting bodies. We were interested in Physarum pusillum (Berk. & M.A. Curtis) G. Lister, a very common species described with two variants, each bearing such morphological differences that they could represent two distinct species. In order to test this, we observed key characters in a large selection of specimens attributed to P. pusillum, to its synonyms (in particular Physarum gravidum), and to related species. In addition, the small-subunit ribosomal RNA gene was obtained from seven of these specimens. Based on these data, we provide a comprehensive phylogeny of the order Physarida (Eukaryota: Amoebozoa: Conosa: Macromycetozoa: Fuscisporidia). Morphology and phylogeny together support the reinstatement of P. gravidum Morgan 1896 with a neotype here designated, distinct from P. pusillum, here redefined.
Assuntos
Physarum/classificação , Physarum/fisiologia , Subunidades Ribossômicas Menores de Eucariotos/genética , Análise de Sequência de DNA/métodos , DNA de Protozoário/genética , Microscopia Eletrônica de Varredura , Filogenia , Physarum/ultraestrutura , Esporos de Protozoários/ultraestruturaRESUMO
Identification of the "bean smut" reported in 1998 in abstracts from two conferences was later disseminated by a Plant Disease Note; citations in books, papers, and blogs; and in several official sites, including databases curated by the United States Department of Agriculture and Embrapa-Brazil. After seeing the illustration of the syndrome in 2002, the need became clear for a review of the so-called bean smut. Field collections indicated that it is common in no-till bean and soybean farming in Brazil. Our studies revealed that the "bean smut" attributed to Ustilago sp. or "Ustilago phaseoli" and, later, to "Microbotryum phaseoli" is not a real smut but is Physarum cinereum (Physaraceae, Physarales, Myxomycetes), sporulating superficially on leaves, stems, and pods of dry bean and soybean. To unravel this imbroglio, we produced detailed morphological documentation supported by molecular treatment. This will correct the spread and further incorporation of an error in the literature based upon mistaken taxonomical work related to a plant-associated nonpathogenic organism.
Assuntos
Glycine max/microbiologia , Physarum/isolamento & purificação , Doenças das Plantas/microbiologia , Frutas/microbiologia , Carpóforos , Microscopia Eletrônica de Varredura , Physarum/genética , Physarum/ultraestrutura , Folhas de Planta/microbiologia , Caules de Planta/microbiologia , Esporos FúngicosRESUMO
A new nivicolous species of Physarum was discovered during the study of myxomycetes in the Patagonian Andes of South America. It is described herein under the name Physarum andinum. The species is characterized by stalked sporophores or more rarely sessile sporocarps or short plasmodiocarps. The sporocarps are strikingly large, reaching 2.6 mm tall and 3 mm diam when open, and have a peridium with three layers, the internal layer being clearly visible and opening separately. Physarum andinum was found at five localities in Argentina as well as in herbarium material collected about 100 y ago in Chile. The new species is reminiscent of the non-nivicolous species Physarum brunneolum, but the latter forms smaller sporophores, has darker spores and the three layers of the peridium are adhered and open together. The characters of the new species were examined under stereomicroscope, light microscope and scanning electron microscope and micrographs of relevant details are included.
Assuntos
Physarum/isolamento & purificação , Esporos de Protozoários/isolamento & purificação , Argentina , Chile , Physarum/classificação , Physarum/crescimento & desenvolvimento , Physarum/ultraestrutura , Solo/parasitologia , Esporos de Protozoários/classificação , Esporos de Protozoários/crescimento & desenvolvimento , Esporos de Protozoários/ultraestrutura , Árvores/parasitologiaRESUMO
A new species of Physarum (Myxomycetes), Physarum atacamense is described in this paper, and details are provided on its life cycle as observed in spore-to-spore culture in agar. The new species was collected during studies of the Atacama Desert in Chile. It has been collected directly in the field and isolated in moist chamber cultures prepared with material from an endemic cactus. The combination of characters that make this species unique in the genus are its large fusiform nodes of the capillitium, its long, bicolored stalk and the very dark brown and densely warted angular spores. The morphology of specimens of this myxomycete was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details and life cycle stages are included in this paper. The importance of resistant stages in the life cycle of this myxomycete is stressed, and the close association of this myxomycete with its plant substrates is discussed.
Assuntos
Mixomicetos/classificação , Mixomicetos/ultraestrutura , Physarum/classificação , Physarum/ultraestrutura , Chile , Clima Desértico , Esporos de Protozoários/classificação , Esporos de Protozoários/ultraestruturaRESUMO
A known species, Physarum melleum, was found fruiting on living leaves of Dendrobium candidum, which was collected in China in 2004. Its morphological characters were revealed by light microscopy (LM), environmental scanning electron microscopy (ESEM) and scanning electron microscopy (SEM). Character variations were distinguished by its olive-yellow peridium and its always thinner capillitium containing globulose granular material between the large calcareous nodes. The calcium carbonate granules, deposited on stalks, peridium and hypothallus as well as within stalks, were globose and smooth.
Assuntos
Dendrobium/ultraestrutura , Physarum/ultraestrutura , Animais , China , Microscopia Eletrônica de Varredura , Folhas de Planta/ultraestruturaRESUMO
Exocytosis has been proposed to participate in the formation of pseudopods. Using video-enhanced microscopy, we directly visualized exocytosis of single vesicles in living Physarum plasmodia migrating on a substrate. Vesicles containing slime, the plasmodial extracellular matrix, of approximately 3.5 microm in diameter, shrank at the cell periphery at the average rate of approximately 1 microm/second, and became invisible. Immediately after exocytotic events, the neighboring cell surface extended to form a protrusion. The rate of extension was approximately 1 microm/second. The protrusion showed lamella-like morphology, and contained actin microfilaments. Electron microscopy suggested that the organization of microfilaments in such protrusions may be a random meshwork rather than straight bundles. These morphologies suggest that protruded regions are pseudopods. Importantly, only the slime-containing vesicle preferentially invaded the hyaline layer that consists of dense actin microfilaments while the other vesicular organelles remained in the granuloplasm. Quantitative analysis demonstrated a linear relationship in terms of their surface area, between individual protrusions and single slime-containing vesicles. It is, therefore, likely that most of the plasma membrane of the protrusion was supplied by fusion of the slime-containing vesicle during exocytosis.
Assuntos
Exocitose/fisiologia , Physarum/fisiologia , Pseudópodes/fisiologia , Actinas/análise , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Microscopia de Vídeo , Physarum/química , Physarum/ultraestrutura , Pseudópodes/química , Pseudópodes/ultraestrutura , TempoRESUMO
Lamin-like proteins have been identified as components of the nuclear matrix of the myxomycete Physarum polycephalum. A 67 kDa homologue was detected by immunoblotting of nuclear matrix proteins with affinity-purified anti-lamin antibodies of human autoimmune sera. A 65 kDa lamin B homologue was identified with polyclonal antibodies against turkey erythrocyte lamin B in nuclei and nuclear matrix of Physarum amoebae and plasmodia. Indirect immunofluorescence microscopy revealed that the 65 and 67 kDa proteins are localized in the nuclear lamina of plasmodial nuclei.
Assuntos
Proteínas Fúngicas/análise , Matriz Nuclear/química , Proteínas Nucleares/análise , Physarum/química , Perus/sangue , Animais , Anticorpos Monoclonais , Proteínas Fúngicas/imunologia , Humanos , Soros Imunes , Lamina Tipo B , Laminas , Proteínas Nucleares/imunologia , Physarum/ultraestrutura , RatosRESUMO
Under unfavorable conditions for growth, haploid myxoamoebae of Physarum polycephalum retracted their pseudopodia and changed their cell shape into disk-like form, after which they constructed the cell walls to form microcysts. These morphological changes of haploid cells were associated with changes in intracellular distribution of actin filaments. Staining with phalloidin showed that actin filaments were almost uniformly distributed throughout the cytoplasm of the myxoamoebae. When these cells were transferred to a cyst-inducing medium, the actin structures changed into short rods or dots, after which the rods/dots disappeared in the microcysts. An incubation of the myxoamoebae in the cyst-inducing medium caused the synthesis of several proteins, among which a 66-kD protein was most prominently induced. The morphological changes and the induction of the 66-kD protein was pronounced at elevated temperatures, e.g. 40 degrees C. The 66-kD protein was not induced, however, when plasmodia of the same species were incubated at 40 degrees C. We found that the 66-kD protein was co-precipitated with polymerized actin and bound to ATP-agarose. A double staining of the disk-shaped cells with anti-66-kD protein antibody and phalloidin revealed superimposable localization of the 66-kD protein and actin filaments in the short rods or dots. Although the induction of the 66-kD protein was enhanced at high temperatures, the protein was immunologically unrelated to the common heat shock proteins, HSP70 and HSP90, those are highly conserved during evolution. These results indicate that the 66-kD protein is a novel heat shock protein which is specifically expressed during cyst formation.
Assuntos
Proteínas de Choque Térmico/biossíntese , Physarum/metabolismo , Physarum/fisiologia , Actinas/química , Actinas/metabolismo , Adaptação Fisiológica , Animais , Anticorpos Monoclonais/biossíntese , Precipitação Química , Reações Cruzadas/imunologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Proteínas Fúngicas/biossíntese , Proteínas de Choque Térmico/imunologia , Immunoblotting , Peso Molecular , Fotofluorografia , Physarum/ultraestrutura , Estresse Fisiológico/patologia , Radioisótopos de EnxofreRESUMO
It has been claimed that the plasmodium of the myxomycete Physarum polycephalum constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.
Assuntos
Carbamatos , Microtúbulos/ultraestrutura , Physarum/ultraestrutura , Animais , Benzimidazóis/farmacologia , Citoplasma/ultraestrutura , Imunofluorescência , Griseofulvina/farmacologia , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacosRESUMO
The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.
Assuntos
Divisão Celular , Microtúbulos/ultraestrutura , Mitose , Physarum/citologia , Fuso Acromático/ultraestrutura , Imunofluorescência , Microtúbulos/química , Physarum/análise , Physarum/ultraestrutura , Fuso Acromático/química , Tubulina (Proteína)/análiseRESUMO
An F-actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F-actin bundling protein in Physarum extract was passed through a DEAE-cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S-300 HR followed by chromatography on CM-Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS-PAGE, and the molecular weight was estimated to be 52,000. This F-actin bundling protein is referred to as the 52 kDa protein. Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F-actin and microtubule bundles was Ca(2+)-insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross-reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F-actin bundling protein from HeLa cells) (Yamashiro-Matsumura and Matsumura: J. Biol. Chem. 260:5087-5097, 1985). When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co-bundles composed of both filaments formed. This is the first reported example in which an F-actin bundling protein induced co-bundling of actin filaments and microtubules.
Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/isolamento & purificação , Microtúbulos/metabolismo , Physarum/química , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Células HeLa/imunologia , Humanos , Proteínas dos Microfilamentos/imunologia , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/farmacologia , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Peso Molecular , Physarum/imunologia , Physarum/ultraestruturaRESUMO
The development of uninucleate amoebae into multinucleate plasmodia in myxomycetes is called the amoebal-plasmodial transition (APT). During the APT in Physarum polycephalum the ability to form flagellar axonemes is lost; the astral, open mitosis is replaced by the anastral, closed mitosis; and cytoskeletal microtubules disappear. These changes are accompanied by alterations in the repertoire of expressed tubulins. Using immunofluorescence microscopy we have studied the timing of loss and accumulation of developmentally regulated tubulin isotypes in relation to other cellular events during the APT. We specifically asked whether changes in the composition of microtubules are correlated with changes in their organization. The plasmodium-specific beta 2-tubulin can first be detected in microtubules of uninucleate cells after they become committed to plasmodium formation. However, rare cells are observed that exhibit beta 2-tubulin at earlier or only at later stages of development. Amoeba-specific acetylated alpha 3-tubulin disappears gradually during development. Individual cells differ in the timing of loss of this isotype: alpha 3-tubulin is present in the majority of uninucleate cells, in a fraction of binucleate and quadrinucleate cells, and is absent from larger multinucleate cells. Cytoplasmic microtubules in uninucleate cells are organized by a single microtubule-organizing center (MTOC) juxtaposed to the nucleus. Binucleate cells and quadrinucleate cells exhibit variable numbers of MTOCs. Cytoplasmic microtubules persist during the APT until the stage of plasmodia containing at least 100 nuclei. The lack of a strict correlation between the changes in tubulin composition and changes in organization of microtubular structures indicates that accumulation of beta 2-tubulin and disappearance of alpha 3-tubulin isotypes are not sufficient to bring about reorganization of microtubules during development. Individual cells in a developing population differ not only in the succession of accumulation and loss of developmentally regulated tubulins, but also in the sequences of other cellular changes occurring during the APT.
Assuntos
Microtúbulos/ultraestrutura , Physarum/ultraestrutura , Citoesqueleto/ultraestrutura , Microtúbulos/metabolismo , Physarum/crescimento & desenvolvimento , Physarum/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.
Assuntos
Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Physarum/crescimento & desenvolvimento , Animais , DNA/metabolismo , Microscopia de Fluorescência , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Mitose , Physarum/genética , Physarum/ultraestruturaRESUMO
Directed migration by a cell is a good phenomenon for studying intracellular coordination. Dynamic organization of both ATP and birefringent fibrils throughout the cell was studied in the multinuclear ameboid cell of the Physarum plasmodium during free locomotion and galvanotaxis. In a directionally migrating plasmodium, waves of ATP as well as thickness oscillations propagated from just inside the advancing front to the rear, and ATP concentration was high at the front on the average. In a DC electric field, locomotion was inhibited more strongly, ATP concentration decreased more, and birefringent fibrils were formed more abundantly at the anodal than at the cathodal side. Inside the cell there were a few undulations in the distributions of ATP and birefringent fibrils. In short, birefringent fibrils become abundant where ATP concentration decreases. The possible mechanism of the coordination in the directed migration and the implications of the scaling law are discussed.
Assuntos
Trifosfato de Adenosina/análise , Physarum/fisiologia , Trifosfato de Adenosina/metabolismo , Birrefringência , Movimento Celular , Simulação por Computador , Estimulação Elétrica , Microcomputadores , Oscilometria , Physarum/ultraestruturaRESUMO
Microtubule-interacting proteins have been studied in the lower eukaryote Physarum polycephalum. We show for the first time 1) the presence in Physarum amoebal crude extracts of at least six polypeptides that bind specifically to amoebal microtubules, 2) the binding between these proteins and mammalian microtubules, 3) the heat stability of two of these polypeptides (125 and 235 kDa), 4) the functional properties of a fraction containing a heat-soluble 125 kDa polypeptide, and 5) the phosphorylation of the 125 kDa polypeptide during two distinct periods of the cell cycle in Physarum synchronous plasmodia, first at late S/early G2 phase and second at late G2/prophase.
Assuntos
Microtúbulos/metabolismo , Peptídeos/metabolismo , Physarum/metabolismo , Animais , Ciclo Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Peso Molecular , Peptídeos/análise , Fosforilação , Physarum/citologia , Physarum/ultraestrutura , Ligação Proteica/fisiologia , Fase S/fisiologiaRESUMO
The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.
Assuntos
Microtúbulos/ultraestrutura , Physarum/ultraestrutura , Tubulina (Proteína)/análise , Imunofluorescência , Immunoblotting , Microtúbulos/imunologia , Mitose , Physarum/genética , Physarum/crescimento & desenvolvimento , Proteínas , Tubulina (Proteína)/imunologiaRESUMO
We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Citoesqueleto/ultraestrutura , Flagelos/ultraestrutura , Microtúbulos/ultraestrutura , Physarum/ultraestrutura , Cálcio/farmacologia , Citoesqueleto/efeitos dos fármacos , Flagelos/efeitos dos fármacos , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Physarum/efeitos dos fármacos , Ribonucleotídeos/farmacologiaRESUMO
(1) In order to protect the nuclear RNA of Physarum polycephalum plasmodia during cell homogenisation and purification of the nuclei, the following conditions were used: low temperature (-11 degrees C), high pH (8.1-8.9), formaldehyde (2.8%) and spermine (2.3 mM). (2) The efficiency of these RNAase-inhibiting and inactivating conditions is demonstrated by the high molecular weight of the processing products of transcripts from ribosomal genes (11.9, 9.5 and 5.0 kilobases), which were recovered from the isolated nuclei and visualised on agarose gels. (3) Hybridisation experiments with a DNA probe from an actin gene on size-fractionated nuclear RNA (Northern blots) indicate that the transcripts from actin genes are rapidly spliced in P. polycephalum. (4) The nuclear polyadenylated RNA has an average size of about 2.2 kb, which is not significantly larger than the average length of mRNA.
Assuntos
Núcleo Celular/ultraestrutura , Physarum/ultraestrutura , RNA Fúngico/isolamento & purificação , Northern Blotting , Fracionamento Celular/métodos , Sondas de DNA , Proteínas Fúngicas/genética , Genes , Genes Fúngicos , Indicadores e Reagentes , Peso Molecular , Hibridização de Ácido Nucleico , Physarum/genética , Plasmídeos , RNA Fúngico/genéticaRESUMO
We used a phalloidin-gold complex to study the distribution of F-actin in the myxamoebae and macroplasmodia of the slime mold Physarum polycephalum. After incubation of Lowicryl- or Quetol-embedded specimens with this complex, significantly different labeling intensities were found over the various cytoplasmic and nuclear regions of the cells. The nucleoplasm was the most heavily labeled cell compartment, followed in decreasing order of labeling intensity by the cytoplasm, the nucleolus, and the chromocenters. The labeling observed over the latter area did not appear significantly different from that of the background. Sections incubated in the phalloidin-gold complex to which an excess of F-actin was added showed no significant labeling over any of the above-mentioned cell regions. Other control experiments included incubation of the sections with a phalloidin solution followed by the phalloidin-gold complex, PEG-stabilized colloidal gold, and a bovine serum albumin-gold complex. There was no or very little labeling of the preparations.
Assuntos
Actinas/análise , Ouro , Oligopeptídeos , Faloidina , Physarum/análise , Núcleo Celular/análise , Citoplasma/análise , Imuno-Histoquímica , Microscopia Eletrônica , Physarum/ultraestruturaRESUMO
In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.