RESUMEN
BACKGROUND: Myxomycetes are a group of eukaryotes belonging to Amoebozoa, which are characterized by a distinctive life cycle, including the plasmodium stage and fruit body stage. Plasmodia are all found to be associated with bacteria. However, the information about bacteria diversity and composition in different plasmodia was limited. Therefore, this study aimed to investigate the bacterial diversity of plasmodia from different myxomycetes species and reveal the potential function of plasmodia-associated bacterial communities. RESULTS: The bacterial communities associated with the plasmodia of six myxomycetes (Didymium iridis, Didymium squamulosum, Diderma hemisphaericum, Lepidoderma tigrinum, Fuligo leviderma, and Physarum melleum) were identified by 16S rRNA amplicon sequencing. The six plasmodia harbored 38 to 52 bacterial operational taxonomic units (OTUs) that belonged to 7 phyla, 16 classes, 23 orders, 40 families, and 53 genera. The dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria. Most OTUs were shared among the six myxomycetes, while unique bacteria in each species only accounted for a tiny proportion of the total OTUs. CONCLUSIONS: Although each of the six myxomycetes plasmodia had different bacterial community compositions, a high similarity was observed in the plasmodia-associated bacterial communities' functional composition. The high enrichment for gram-negative (> 90%) and aerobic (> 99%) bacteria in plasmodia suggest that myxomycetes may positively recruit certain kinds of bacteria from the surrounding environment.
Asunto(s)
Mixomicetos , Physarum , Plasmodium , Humanos , Mixomicetos/genética , ARN Ribosómico 16S/genética , Physarum/genética , Bacterias/genéticaRESUMEN
A new plasmodiocarpic and sporocarpic species of myxomycete in the genus Physarum is described and illustrated. This new species appeared on decayed leaves and remains of succulent plants (Agave, Opuntia, Yucca) growing in arid zones. It differs from all other species in the genus in having polyhedral spores linked in chains like a string of beads, a unique feature within all known myxomycetes. Apart from detailed morphological data, partial sequences of both the small-subunit ribosomal RNA and elongation factor 1-alpha genes, generated from four isolates collected in two distant regions, i.e., Mexico and Canary Islands, are also provided in this study. Combined evidence supports the identity of the specimens under study as a new species.
Asunto(s)
Physarum/citología , Physarum/genética , Esporas Protozoarias/citología , Agave/parasitología , Genes Protozoarios/genética , México , Opuntia/parasitología , Physarum/clasificación , España , Especificidad de la Especie , Yucca/parasitologíaRESUMEN
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.
Asunto(s)
Glycine max/microbiología , Physarum/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Frutas/microbiología , Cuerpos Fructíferos de los Hongos , Microscopía Electrónica de Rastreo , Physarum/genética , Physarum/ultraestructura , Hojas de la Planta/microbiología , Tallos de la Planta/microbiología , Esporas FúngicasRESUMEN
Physarum myosin is a Ca(2+)-binding protein and its activity is inhibited by Ca(2+) In the present study, to clarify the light chains (LCs) from the different species (Physarum and scallop) and to determine the specific Ca(2+)-regulated effects, we constructed hybrid myosins with a Physarum myosin heavy chain (Ph·HC) and Physarum and/or scallop myosin LCs, and examined Ca(2+)-mediated regulation of ATPases and motor activities. In these experiments, it was found that Ca(2+) inhibited motilities and ATPase activities of Physarum hybrid myosin with scallop regulatory light chain (ScRLC) and Physarum essential light chain (PhELC) but could not inhibit those of the Physarum hybrid myosin mutant Ph·HC/ScRLC/PhELC-3A which lacks Ca(2+)-binding ability, indicating that PhELC plays a critical role in Ca(2+)-mediated regulation of Physarum myosin. Furthermore, the effects of Ca(2+) on ATPase activities of Physarum myosin constructs are in the following order: Ph·HC/PhRLC/PhELC > Ph·HC/ScRLC/PhELC > Ph·HC/PhRLC/ScELC > Ph·HC/ScRLC/ScELC, suggesting that the presence of PhRLC and PhELC leads to the greatest Ca(2+) sensitivity of Physarum myosin. Although we did not observe the motilities of Physarum hybrid myosin Ph·HC/PhRLC/ScELC and Ph·HC/ScRLC/ScELC, our results suggest that Ca(2+)-binding to the PhELC may alter the flexibility of the regulatory domain and induce a 'closed' state, which may consequently prevent full activity and force generation.
Asunto(s)
Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Pectinidae/metabolismo , Physarum/metabolismo , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Calcio/metabolismo , Modelos Moleculares , Movimiento , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Pectinidae/genética , Physarum/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A new widespread myxomycete species, Physarum pseudonotabile, inhabiting the arid regions of the Eurasia, South and North America is described and illustrated. Tentatively assigned to Ph. notabile T. Macbr., a phylogeny based on the small ribosomal subunit (SSU) and elongation factor 1 alpha (EF1a) genes placed the new species in a clade far from Ph. notabile. Ph. pseudonotabile was found to be frequent in surveys based on the moist chamber culture technique with samples of litter, bark and herbivore dung collected in dry steppe and deserts of the Caspian lowland (Russia), Kazakhstan, Mongolia, China, Spain, Argentina and USA. The main morphological difference between Ph. pseudonotabile and Ph. notabile lies in spore ornamentation. Spores of the former species display irregularly distributed verrucae, whereas the latter species possesses spores with dense and regularly arranged spinulae. In addition, the ecological preferences of the two species differ. Ph. pseudonotabile inhabits the bark of living plants and ground litter in arid regions, whereas Ph. notabile is found on coarse woody debris in boreal and temperate forests. Although the new species appears to be closest to Ph. notabile morphologically, the phylogenetic analysis reveals Ph. pusillum and Ph. nivale as the closest relatives. In addition, the molecular investigations revealed a considerable amount of hidden diversity within species of Physarum with gray lime flakes. Currently we have only sufficient material to assess the morphological variation of Ph. pseudonotabile but expect that more taxa within this clade may emerge within studies combining morphological and molecular analyses.
Asunto(s)
Filogenia , Physarum/clasificación , Physarum/aislamiento & purificación , Clima Desértico , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica/genética , Physarum/genética , Physarum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Federación de Rusia , Esporas Protozoarias/clasificación , Esporas Protozoarias/genética , Esporas Protozoarias/crecimiento & desarrollo , Esporas Protozoarias/aislamiento & purificaciónRESUMEN
BACKGROUND: Physarales represents the largest taxonomic order among the plasmodial slime molds (myxomycetes). Physarales is of particular interest since the two best-studied myxomycete species, Physarum polycephalum and Didymium iridis, belong to this order and are currently subjected to whole genome and transcriptome analyses. Here we report molecular phylogeny based on ribosomal DNA (rDNA) sequences that includes 57 Physarales isolates. RESULTS: The Physarales nuclear rDNA sequences were found to be loaded with 222 autocatalytic group I introns, which may complicate correct alignments and subsequent phylogenetic tree constructions. Phylogenetic analysis of rDNA sequences depleted of introns confirmed monophyly of the Physarales families Didymiaceae and Physaraceae. Whereas good correlation was noted between phylogeny and taxonomy among the Didymiaceae isolates, significant deviations were seen in Physaraceae. The largest genus, Physarum, was found to be polyphyletic consisting of at least three well supported clades. A synapomorphy, located at the highly conserved G-binding site of L2449 group I intron ribozymes further supported the Physarum clades. CONCLUSIONS: Our results provide molecular relationship of Physarales genera, species, and isolates. This information is important in further interpretations of comparative genomics nd transcriptomics. In addition, the result supports a polyphyletic origin of the genus Physarum and calls for a reevaluation of current taxonomy.
Asunto(s)
Mixomicetos/genética , Filogenia , Physarum/genética , ADN Protozoario/genética , ADN Ribosómico/genética , Intrones , Mixomicetos/clasificación , Physarum/clasificaciónRESUMEN
We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.
Asunto(s)
Calcio/metabolismo , Miosinas/metabolismo , Physarum/metabolismo , Animales , Calcio/farmacología , Células Cultivadas , Mutación , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Physarum/efectos de los fármacos , Physarum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Mitochondrial RNAs in the myxomycete Physarum polycephalum differ from the templates from which they are transcribed in defined ways. Most transcripts contain nucleotides that are not present in their respective genes. These "extra" nucleotides are added during RNA synthesis by an unknown mechanism. Other differences observed between Physarum mitochondrial RNAs and the mitochondrial genome include nucleotide deletions, C to U changes, and the replacement of one nucleotide for another at the 5' end of tRNAs. All of these alterations are remarkably precise and highly efficient in vivo. Many of these editing events can be replicated in vitro, and here we describe both the in vitro systems used to study editing in Physarum mitochondria and the assays that have been developed to assess the extent of editing of RNAs generated in these systems at individual sites.
Asunto(s)
Bioquímica/métodos , Physarum/genética , Physarum/metabolismo , Edición de ARN , Animales , Enzimas de Restricción del ADN/metabolismo , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Nucleótidos/química , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Transcripción GenéticaRESUMEN
RNAs in Physarum: mitochondria contain extra nucleotides that are not encoded by the mitochondrial genome, at least in the traditional sense. While it is known that insertion of non-encoded nucleotides is linked to RNA synthesis, the exact nature of this relationship remains unclear. Here we demonstrate that the efficiency of editing is sensitive not only to the concentration of the nucleotide that is inserted, but also to the concentration of the nucleotide templated just downstream of an editing site. These data strongly support a co-transcriptional mechanism of Physarum: RNA editing in which non-encoded nucleotides are added to the 3' end of nascent RNAs. These results also suggest that transcription elongation and nucleotide insertion are competing processes and that recognition of editing sites most likely involves transient pausing by the Physarum: mitochondrial RNA polymerase. In addition, the pattern of nucleotide concentration effects, the context of editing sites and the accuracy of the mitochondrial RNA polymerase argue that the mechanism of Physarum: editing is distinct from that of other co-transcriptional editing systems.
Asunto(s)
Mitocondrias/genética , Physarum/genética , Edición de ARN , ARN/metabolismo , Ribonucleótidos/metabolismo , Adenosina Trifosfatasas/genética , Animales , Secuencia de Bases , Citidina Trifosfato/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Complejo IV de Transporte de Electrones/genética , Datos de Secuencia Molecular , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , ARN Mitocondrial , Transcripción GenéticaRESUMEN
The dissection of RNA editing mechanisms in PHYSARUM: mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.
Asunto(s)
Mitocondrias/genética , Physarum/genética , Edición de ARN , ARN Protozoario/biosíntesis , ARN Protozoario/genética , Transcripción Genética , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Extractos Celulares , Sistema Libre de Células , Mitocondrias/química , Mitocondrias/enzimología , Datos de Secuencia Molecular , Nucleótidos/genética , Nucleótidos/metabolismo , Physarum/química , Physarum/citología , Physarum/enzimología , Plásmidos/genética , Edición de ARN/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Ribonucleasas/metabolismo , Solubilidad , Transcripción Genética/genéticaRESUMEN
Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5'-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3'-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A > or = G > C > U, and A > or = G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.
Asunto(s)
Dictyostelium/enzimología , Physarum/enzimología , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Dicroismo Circular , Dictyostelium/genética , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Physarum/genética , ARN Protozoario/química , Ribonucleasas/química , Ribonucleasas/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G. C base pair. Uridine insertions have been identified in the T loop of tRNALys from Didymium and tRNAGlu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTPsiC (Psi = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.
Asunto(s)
Mixomicetos/genética , Physarum/genética , Edición de ARN/genética , ARN Protozoario/genética , ARN de Transferencia/genética , ARN/genética , Animales , Secuencia de Bases , Citidina/genética , ADN Complementario/genética , ADN Mitocondrial/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mitocondrial , Alineación de Secuencia , Uridina/genéticaRESUMEN
The cDNAs encoding haemagglutinin I from plasmodia of Physarum polycephalum have been cloned using PCR protocols. The composite haemagglutinin I cDNA sequence, derived from several overlapping clones from PCR fragments, spans 408 nt and the 315 bp ORF encodes a polypeptide of 104 aa without a typical signal sequence. The putative molecular mass deduced from the amino acid sequence (10,760.76 Da) corresponds exactly to that determined by electrospray ionization MS (10,759.86 +/- 0.15 Da), suggesting that haemagglutinin I is not subject to post-translational modification. Haemagglutinin I lacks sulphur amino acids and has a beta-sheet as the major secondary structure. Expression of the coding sequence in Escherichia coli yielded a product that exhibits the same sugar-binding specificity as natural haemagglutinin I. The deduced amino acid sequence shows little similarity to that of any known lectins and thus apparently represents a novel type of lectin.
Asunto(s)
Proteínas Fúngicas/genética , Hemaglutininas/genética , Physarum/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Cromatografía Líquida de Alta Presión , ADN Complementario/biosíntesis , ADN de Hongos/biosíntesis , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Hemaglutininas/química , Lectinas , Datos de Secuencia Molecular , Physarum/genéticaRESUMEN
The Mycetozoa include the cellular (dictyostelid), acellular (myxogastrid), and protostelid slime molds. However, available molecular data are in disagreement on both the monophyly and phylogenetic position of the group. Ribosomal RNA trees show the myxogastrid and dictyostelid slime molds as unrelated early branching lineages, but actin and beta-tubulin trees place them together as a single coherent (monophyletic) group, closely related to the animal-fungal clade. We have sequenced the elongation factor-1alpha genes from one member of each division of the Mycetozoa, including Dictyostelium discoideum, for which cDNA sequences were previously available. Phylogenetic analyses of these sequences strongly support a monophyletic Mycetozoa, with the myxogastrid and dictyostelid slime molds most closely related to each other. All phylogenetic methods used also place this coherent Mycetozoan assemblage as emerging among the multicellular eukaryotes, tentatively supported as more closely related to animals + fungi than are green plants. With our data there are now three proteins that consistently support a monophyletic Mycetozoa and at least four that place these taxa within the "crown" of the eukaryote tree. We suggest that ribosomal RNA data should be more closely examined with regard to these questions, and we emphasize the importance of developing multiple sequence data sets.
Asunto(s)
Evolución Biológica , Proteínas Fúngicas/genética , Genes Fúngicos , Mixomicetos/clasificación , Factores de Elongación de Péptidos/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Dictyostelium/genética , Evolución Molecular , Intrones , Datos de Secuencia Molecular , Mixomicetos/genética , Factor 1 de Elongación Peptídica , Filogenia , Physarum/genética , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Maturation of Physarum mitochondrial RNA involves the highly specific insertion of nonencoded nucleotides at multiple locations. To investigate the mechanism(s) by which this occurs, we previously developed an isolated mitochondrial system in which run-on transcripts are accurately and efficiently edited by nucleotide insertion. Here we show that under limiting concentrations of exogenous nucleotides the mitochondrial RNA polymerases stall, generating a population of nascent RNAs that can be extended upon addition of limiting nucleotide. Several of these RNA species have been characterized and were found to be fully edited, indicating that nascent RNA is a substrate for nucleotide insertion in isolated Physarum mitochondria. Remarkably, these RNAs are edited at positions located within 14-22 nucleotides of the polymerase active site, suggesting that insertional editing may be physically or functionally associated with transcription. The absence of unedited RNA in these experiments indicates that large tracts of RNA downstream of editing sites are not required for nucleotide addition, and argues that insertional editing in Physarum occurs with a 5' to 3' polarity. These data also provide strong evidence that insertional editing in Physarum is mechanistically distinct from editing in kinetoplastid systems.
Asunto(s)
Mitocondrias/genética , Physarum/genética , Edición de ARN , Precursores del ARN/metabolismo , ARN/metabolismo , Animales , Secuencia de Bases , Fosfatos de Dinucleósidos/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , ARN Mitocondrial , Especificidad de la Especie , Transcripción GenéticaRESUMEN
Two types of RNA editing have been reported in the mitochondria of Physarum; extensive insertions of nucleotides and single-base substitutions. In the Ng strain of P. polycephalum and its derivatives, mitochondria have a specific plasmid (mF) that promotes fusion of mitochondria. We examined the editing of transcripts derived from the mF plasmid. For analysis, we selected three regions of the plasmid, including a DNA fragment that corresponded to missing conserved domains of the RNA polymerase. In contrast to the mitochondrial DNA (mtDNA), no RNA editing of the transcripts of the mF plasmid was detected. Our results suggest that the mechanism of transcription of the mitochondrial plasmid is independent of that of mtDNA, indicating that the plasmid has a different evolutionary origin from the mtDNA.
Asunto(s)
Mitocondrias/genética , Physarum/genética , Plásmidos/genética , Edición de ARN , Transcripción Genética , Animales , Evolución Biológica , ADN Mitocondrial/genética , Datos de Secuencia Molecular , Physarum polycephalum/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNAsunto(s)
Clonación Molecular/métodos , Endonucleasas , Saccharomyces cerevisiae/enzimología , Animales , Chlamydomonas/enzimología , Chlamydomonas/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Physarum/enzimología , Physarum/genética , Plásmidos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Physarum polycephalum rRNA genes are found on extrachromosomal 60 kb linear palindromic DNA molecules. Previous work using electron microscope visualization suggested that these molecules are duplicated from one of four potential replication origins located in the 24 kb central non-transcribed spacer [Vogt and Braun (1977) Eur. J. Biochem., 80, 557-566]. Considering the controversy on the nature of the replication origins in eukaryotic cells, where both site-specific or delocalized initiations have been described, we study here Physarum rDNA replication by two dimensional agarose gel electrophoresis and compare the results to those obtained by electron microscopy. Without the need of cell treatment or enrichment in replication intermediates, we detect hybridization signals corresponding to replicating rDNA fragments throughout the cell cycle, confirming that the synthesis of rDNA molecules is not under the control of S-phase. The patterns of replication intermediates along rDNA minichromosomes are consistent with the existence of four site-specific replication origins, whose localization in the central non-transcribed spacer is in agreement with the electron microscope mapping. It is also shown that, on a few molecules, at least two origins are active simultaneously.
Asunto(s)
Replicación del ADN , ADN Ribosómico/genética , Physarum/genética , Animales , Ciclo Celular , Origen de Réplica/genéticaRESUMEN
The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD delta 1, of the ardD actin gene; ardD delta 1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD+ with ardD delta 1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD delta 1 in addition to ardD+, but in two (5%), ardD+ was replaced by a single copy of ardD delta 1. This is the first example of homologous gene replacement in Physarum. ardD delta 1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD delta 1 on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene.
Asunto(s)
Actinas/genética , Genes Fúngicos/genética , Genes Protozoarios/genética , Physarum/genética , Transformación Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Vectores Genéticos , Meiosis , Datos de Secuencia Molecular , Mutagénesis , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Selección Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes.