Telomeric structures in a linear mitochondrial plasmid from Physarum polycephalum

Summary A mitochondrial plasmid was isolated from Physarum polycephalum and characterized by restriction mapping. Cloned fragments of the plasmid were assembled and used to construct a restriction map. This plasmid was a linear molecule with telomeric structures at each end. Southern hybridization with the ends of the plasmid as probes revealed that the plasmid included repeating units at both ends, with each unit being approximately 125 bp in length. The most extensive array of repeats consisted of at least 17 repetitions of the 125-bp unit. The sensitivity of these repeats to Bal31 exonuclease confirmed that they were at, or very near to, the ends of the plasmid. From the extent of the repetitions, the size of the plasmid was estimated to vary from 13.3 kbp to more than 18.3 kbp.     

Rearrangements in the Physarum polycephalum mitochondrial genome associated with a transition from linear mF-mtDNA recombinants to circular molecules

Although mitochondrial DNA (mtDNA) is transmitted to progeny from one parent only in Physarum polycephalum, the mtDNAs of progeny of mF⁺ plasmodia vary in structure. To clarify the mechanisms associated with the mitochondrial plasmid mF that generate mtDNA polymorphisms, 91 progeny of four strains (KM88 × JE8, KM88 × TU111, KM88 × NG111, Je90) were investigated using RFLP analysis, PCR, and pulse-field gel electrophoresis (PFGE). Nine mtDNA rearrangement types were found, with rearrangements occurring exclusively in the mF regions. PFGE revealed that, in the groups containing rearranged mtDNA, the linear mF–mtDNA recombinants had recircularized. Sequencing the rearranged region of one of the progeny suggested that the mF plasmid and the mtDNA recombine primarily at the ID sequences, linearizing the circular mtDNA. Recombination between the terminal region of the mF plasmid and a region about 1 kbp upstream of the mitochondrial/plasmid ID sequence results in a rearranged circular mtDNA, with variations caused by differences in the secondary recombination region.     

Complex terminal structure of a linear mitochondrial plasmid from Physarum polycephalum: three terminal inverted repeats and an ORF encoding DNA polymerase

The mitochondria of Physarum polycephalum have a linear plasmid (mF) which promotes mitochondrial fusion. To determine the terminal structure of the mF plasmid, restriction fragments derived from its ends were cloned and sequenced. The sequences showed that the mF plasmid has three kinds of terminal inverted repeats (TIRs). The most characteristic feature is a 144-bp repeating unit which exists between a 205-bp TIR at the extreme ends of the plasmid and another 591-bp TIR. All of the clones showed at least one of these 144-bp repeating units. The GC content of the 205-bp TIR (49%) was higher than those of the other TIRs and of another sequenced region (23%). This TIR can form three thermodynamically-stable hairpin structures based on complex internal palindromic components. Moreover, in the right terminal region of the mF plasmid, there is an open reading frame (ORF) which covers the entire 591-bp TIR and most of one of the 144-bp repeating units. This ORF encodes a 547-amino-acid polypeptide, ORF-547, and shows extensive homology with the polymerization domain of the putative DNA polymerases of linear mitochondrial plasmids from other sources.     

Constitutive homologous recombination between mitochondrial DNA and a linear mitochondrial plasmid in Physarum polycephalum

Summary In one particular myxamoebal strain (NG7; mF⁺) of Physarum polycephalum, a linear mitochondrial plasmid (mF plasmid) which promotes mitochondrial fusion has been identified. A mating between mF^- strains, that do not carry the mF plasmid, resulted in uniparental inheritance of the mtDNA. In matings between mF⁺ and mF^- strains a recombination occurred between the mtDNA and the mF plasmid, and recombinant mtDNA was generated with the end of the mF plasmid as its ends. The DNA sequences of the recombination site in the mtDNA and the mF plasmid, and of the recombinant mtDNA, revealed that the mF plasmid had a 473-bp sequence that was identical to, but slightly shorter than, a 477-bp sequence of the mtDNA. This so-called identical sequence was found at the junction between unique sequences of the mF plasmid and the mtDNA in the recombinant mtDNA. Thus, the recombination between the mtDNA and the mF plasmid was due to reciprocal crossing-over at the identical sequence.     

Mitochondrial DNA rearrangements associated with mF plasmid integration and plasmodial longevity in Physarum polycephalum

Plasmodial cultures of Physarum polycephalum have defined life spans and undergo a reproducible decline (senescence) before losing the ability to be subcultured. Studies of the mtDNA of a long-lived Physarum strain, which does not undergo senescence, revealed a 7.9-kb insertion in its mtDNA. This insertion is derived from a mitochondrial plasmid which enhances mitochondrial fusion and mtDNA recombination. Four different recombination events are required to convert the parental mtDNA to the form found in the long-lived strain. An additional recombination event, which deletes a 2.4-kb region of the insert from the long-lived strain, has been identified in the mtDNA of a normally senescing strain. These observations imply a mitochondrial involvement in the process of plasmodial senescence and implicate a region of the DNA derived from the mitochondrial plasmid as being necessary for plasmodial longevity. Received: 11 August / 18 November 1997     

Identification,partial sequence and genetic analysis of mlpA,a novel gene encoding a myosin-related protein in Physarum polycephalum

Studies of motility in Physarum polycephalum have concentrated on the well-defined actomyosin system in plasmodia. It is clear from recent genetic studies in lower eukaryotes that myosin is involved in a number of physiological processes in addition to the contractile functions previously asciibed to the classical type II myosins. Moreover, the myosin protein family has proved to be more complex than anticipated, with an increasing number of reported specialized isoforms. Although a myosin type II activity has been identified in both amoebae and plasmodia of P. polycephalum, and it has been inferred that these proteins undergo a phasespecific isoform switch during development, this phenomenon has not been analysed genetically. In an effort to understand the putative developmental expression of actomyosin-associated proteins, we isolated a 180-kDa protein from amoebae which is highly enriched, along with actin and myosin, in actomyosin preparations in the presence of mM concentrations of Mg⁺⁺ ions and 10 mM of ATP. Using polyclonal antisera raised against pl80 we have cloned and sequenced a partial cDNA encoding a protein whose predicted amino-acid sequence indicates some similarity with the Dictyostelium discoideum myosin heavy-chain tail domain. Southern-blot and RFLP analyses indicate that the gene involved, designated mlpA (myosin-like protein), occurs in a single copy in the genome, is a novel Physarum gene and is expressed during amoebal and plasmodial growth and in the dormant forms of both these cell types.     

Identification of an autonomously replicating sequence near a histone gene of Physarum polycephalum

Summary Fragments of DNA with function as autonomous replication sequences in yeast were cloned from Physarum polycephalum. The ars activity is located in a 1.2 kbp fragment extanding 1.5 kbp to 2.7 kbp upstream of the 5 end of a histone H4 gene. Our recent finding that a replication origin is located at a distance less than 3 kbp of this histone gene suggests that the ars element identified coincides with a specialized replication origin and can be used to direct chromosome replication in Physarum polycephalum.     

Different developmental programs for amoebal and plasmodial encystment in Physarum polycephalum

Summary Amoebal and plasmodial encystment (spherulation) of Physarum polycephalum were compared. Encystment of amoebae was induced by the addition of glucose to a dense culture. A change in cellular proteins during the amoeba to rnicrocyst transition was demonstrated by SDS-polyacrylamide gel electrophoresis. We also observed that the encystment of amoebae did not involve the accumulation of the proteins which accompany plasmodial encystment. A cDNA library was constructed from poly(A)⁺ RNA of encysted amoebae and was screened by differential hybridization. Two different mRNAs, specific to mature microcysts were identified. These mRNAs were not found in encysted plasmodia. Likewise, four encysted plasmodial mRNAs were absent from microcysts. These results show that different developmental programs are used for amoebal and plasmodial encystment.     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

Transient expression of a chloramphenicol acetyltransferase gene following transfection of Physarum polycephalum myxamoebae

Summary A plasmid was constructed containing a replication origin sequence from the Physarum ribosomal DNA molecule, and a bacterial chloramphenicol acetyltransferase (CAT) gene linked to a putative promoter of the long terminal repeat (LTR) of the Physarum HpaII-repeat element. The plasmid was transfected into Physarum myxamoebae either by electroporation or CaCl₂ treatment. In both cases significant transient levels of CAT gene expression were detected. Results were compared with those obtained with plasmids in which CAT gene expression was driven by eukaryotic virus promoters.     

Detection and analysis of “CpG-rich” islands in the genome of Physarum polycephalum

Summary A small fraction (Physarum polycephalum contains a high-density of cleavage sites for the restriction endonuclease HpaII. This component can be distinguished from the bulk of the DNA by ³²P-end-labelling followed by size-fractionation using agarose-gel or polyacrylamide-gel electrophoresis. In contrast to the situation in mammalian-cell DNA, where the majority of such small HpaII DNA fragments are derived from CpG-rich islands within diverse single-copy sequences located in the proximity of housekeeping genes, most of the Physarum small HpaII DNA fragments form an array of distinct bands when analysed on polyacrylamide gels, indicating that they are repetitive DNA sequences. Direct sequence analysis shows that the majority of these sequences are derived from the Physarum rDNA minichromosome.     

Nucleotide sequence of the Physarum polycephalum small subunit ribosomal RNA as inferred from the gene sequence: secondary structure and evolutionary implications

Summary The nucleotide sequence of the Physarum polycephalum small subunit ribosomal RNA (SSU rRNA) gene has been determined. Sequence data indicate that the mature 19S SSU rRNA is 1,964 nucleotides long. A complete secondary structure model for P. polycephalum SSU rRNA has been constructed on the basis of the Escherichia coli 16S rRNA model and data from comparative analyses of 28 different eukaryotic sequences. A four-helix model is presented for the central domain variable region. This model can be applied both to vertebrate and most lower eukaryotic SSU rRNAs. The increased size of P. polycephalum SSU rRNA relative to the smaller SSU rRNAs from such other lower eukaryotes, as Dictyostelium, Tetrahymena or Saccharomyces is due mainly to three G+C-rich insertions found in two regions known to be of variable length in eukaryotes. In a phylogenetic tree constructed from pairwise comparisons of eukaryotic SSU rRNA sequences, the acellular myxomycete P. polycephalum is seen to diverge before the appearance of the cellular mycomycete Dictyostelium discoideum.     

A luciferase expression system for Physarum that facilitates analysis of regulatory elements

We have developed a transient expression system for the protist Physarum polycephalum based on firefly luciferase. We demonstrate the utility of this system for comparing the activities of different promoters in Physarum amoebae, and also for detecting genetic elements that affect the level of gene expression. This system is likely to facilitate improvements in the stable transformation of this organism.     

Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

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

Summary 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 plasmodial genotypes 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.     

Genetic regulation of plasmid transfer

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 7, pp. 6–12, July, 1995