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.     

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.     

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.     

The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5′ ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3′-CCCCC-5′, similar to those of Escherichia coli phage PRD1. The pMLP1 plasmid harbors two long open reading frames (ORF1 and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase. Received: 9 February 2000 / Accepted: 18 July 2000     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

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.     

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.     

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.     

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.     

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.     

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     

The linear plasmid pMC3-2 from Morchella conica is structurally related to adenoviruses

Summary pMC3-2, one of two linear plasmids localised in the mitochondria of the ascomycete Morchella conica, was completely sequenced. It is 6044 bp in size, contains terminal inverted repeats of 713 and 710 bp length and two open reading frames, ORF1 and ORF2, spanning 2706 bp and 918 bp, respectively. ORF1 probably encodes a viral B-type DNA-polymerase. Concerning ORF2, no homology to any other published protein-or DNA-sequence could be detected. According to the structure of DNA-polymerases, linear plasmids can be grouped into two classes reflecting their localisation either in the cytoplasm or within the mitochondria. In general, the structure of plasmid pMC3-2, as well as of other linear plasmids from filamentous fungi, indicates a close relationship of these genetic elements to adenoviruses.     

Analysis of a linear plasmid isolated from the pathogenic fungus Ceratocystis fimbriata Ell. & Halst.

Summary A linear DNA plasmid, designated pCF637, was isolated from the fungus Ceratocystis fimbriata Ell. & Halst. strain CF637. It has an apparent molecular weight of 5.3 × 10⁶ daltons (8.2 kb). A restriction pattern of pCF637 using the enzymes AvaI, EcoRV and HpaII, was done. That pCF637 was sensitive to exonuclease III, but resistant to -exonuclease, suggest that there might be a protein associated with the 5 termini. The blocking action of the protein on -exonuclease was not eliminated by treatment with either pronase or proteinase K. The plasmid was also checked for homology with pFQ501, a linear plasmid found in strain CF560 of Ceratocystis fimbriata. By Southern hybridization under moderate stringency conditions, no homology was detected. The approximate copy number of the plasmid was estimated to be about 20–30 copies per cell by scanning, with a laser densitometer, a gel electrophoretic Polaroid^TM negative. No function is known for these plasmids.     

The kalilo linear senescence-inducing plasmid of Neurospora is an invertron and encodes DNA and RNA polymerases

Summary The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by intergrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA; (2) small imperfect palindromes that are situated at the termini of the plasmid and are cognate with the active sites for plasmid integration into mtDNA; (3) two large, non-overlapping open-reading frames, ORF-1 and ORF-2, which are located on opposite strands of the plasmid and potentially encode RNA and DNA polymerases, respectively, and (4) a set of imperfect palindromes that coincide with similar structures that have been detected at more or less identical locations in the nucleotide sequences of other linear mitochondrial plasmids. The nucleotide sequence does not reveal a distinct gene that codes for the protein that is attached to the ends of the plasmid. However, a 335-amino acid, cryptic, N-terminal domain of the putative DNA polymersse might function as the terminal protein. Although the plasmid has been co-purifed with nuclei and mitochondria, its nucleotide composition and codon usage indicate that it is a mitochondrial genetic element.     

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.