Presence of a linear plasmid-like DNA molecule in the fungal pathogen Ceratocystis fimbriata Ell. & Halst.

Summary A plasmid-like molecule was detected in a strain of the ascomycete Ceratocystis fimbriata Ell. & Halst., a pathogenic fungus of Populus spp. The DNA replicon, designated pFQ501, was found to have a linear structure with a length of 6.0 kb (3.9 × 10⁶ daltons) and a density of 1.685 g/cc. This molecule was found to be associated with the mitochondria and was isolated from the gel; its restriction map was deduced from single and double digestions.     

Proteins are attached to the ends of a linear plasmid in the filamentous fungus Ascobolus immersus

Summary In seven out of eleven wild strains of the Ascomycete Ascobolus immersus plasmid DNA was found. There was great variability with respect to size and number of the plasmids in the strains concerned. For a further analysis two plasmids originating from one wild strain were submitted to restriction analysis and electron microscopy. Both turned out to be linear having different molecular weights (pAIl = 7.9 kb, pAI2 = 5.6 kb). Denaturation of pAI2 and subsequent renaturation revealed the presence of inverted repeats (0.7 kb) at both ends. After treatment with proteinase K and 5 and 3 specific exonucleases it became evident that the 5 ends of pAI2 are linked with proteins. In this respect it is similar in structure to other linear genetic elements such as the linear plasmids found in Zea mays and the genomes of adenoviruses.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

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     

Novel linear megaplasmid from Brevibacterium sp. isolated from extreme environment

Brevibacterium sp. Ap13, isolated from flamingo's feces in Laguna Aparejos, a high‐altitude lake located at approximately 4,200 m in the northwest of Argentina was previously found to be resistant to multiple antibiotics, and was therefore screened for plasmids that may be implicated in antibiotic resistance. Brevibacterium sp. Ap13 was found to contain two plasmids of approximately 87 and 436 kb, designated pAP13 and pAP13c, respectively. Only pAP13 was stably maintained and was extensively characterized by pulsed‐field gel electrophoresis to reveal that this plasmid is linear and likely has covalently linked terminal proteins associated with its 5′ ends. This is the first report of a linear plasmid in the genus Brevibacterium and may provide a new tool for genetic manipulation of this commercially important genus. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

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.     

The complete nucleotide sequence and characterization of the linear DNA plasmid pRS64-2 from the plant pathogenic fungus Rhizoctonia solani

The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures. Received: 3 February / 1 August 1997     

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.     

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.     

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.     

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomou sreplication of hybrid plasmids in Saccharomyces cerevisiae

Summary By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.     

Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani

Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1–5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300–1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and-5, indicating that the plasmids do not play any role in pathogenicity.     

Induction of longevity by cytoplasmic transfer of a linear plasmid inPodospora anserina

InPodospora anserina the longevity inducing linear plasmid pAL2-1 was transferred from the extrachromosomal long-lived mutant AL2 to the shor-tlived wild-type strain A. The resulting strain, AL2-IV, exhibited the long-lived phenotype. In the short-lived progeny of crosses between this strain and wild-type strain A, the plasmid was absent. In contrast, all long-lived progeny contained both the autonomous plasmid as well as copies of it integrated in the mitochondrial DNA (mtDNA). Molecular analysis revealed that the integrated plasmid copies most likely resulted from ade novo integration of the autonomous element and the generation of AT-linker sequences at the integration site. We conclude that once the plasmid is present in mitochondria of a particular genetic background, it is able to integrate into the mtDNA and to induce longevity.     

Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo

Mitochondrial RNA was isolated from the morel strain Morchella conica 3 harbouring the linear plasmid pMC3-2 and subjected to gel electrophoresis followed by a Northern analysis using cloned fragments of the plasmid pMC3-2 as probes. Hybridization was obtained only with central parts of pMC3-2 and specific bands of mtRNA. The hybridization bands (2.8 kb and 1.0 kb) correspond in size to the length of the two ORFs of pMC3-2 which were deduced from nucleotide-sequence data. Thus, both ORFs, one encoding a DNA polymerase and the other a yet unknown protein, are transcribed in the mitochondria of the plasmid-bearing Morchella conica strain.     

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.     

Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species ofGelasinospora. The plasmid genome is 8231 by long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of theNeurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.     

Structure of a linear plasmid of the yeast Kluyveromyces lactis; Compact organization of the killer genome

Summary Some strains of the yeast Kluyveromyces lactis contain a pair of linear DNA plasmids, k1 and k2, 8.8 and 13.8 kilobase pairs long, respectively. Simultaneous presence of the two plasmids confer a killer phenotype on the cell by producing a toxin which blocks the growth of sensitive yeast species. Previous genetic studies have suggested that the toxin protein is coded by the k1 plasmid. We have now determined the total nucleotide sequence of k1 DNA. The genome is 8,874 base pairs in length. It contains four protein-coding reading frames, three transcribed from one strand and the fourth transcribed from the complementary strand and has terminal inverted repeats of 202 base pairs. Nuclease S1 mapping confirmed this arrangement and showed that these genes are transcribed. The terminal repeats and the four genes form an extremely compact genome, with some overlapping of genes. All four genes use highly biased codons, 86% of them having A or T at the wobble position, reminiscent of yeast mitochondrial genes. Three genes share a very similar 5 leader sequence. The nature of gene products is discussed in the light of what is known of the excreted toxin protein.     

Evidence for giant linear plasmids in the ascomycete Podospora anserina

In the extrachromosomal mutant AL2 of the ascomycete Podospora anserina longevity is correlated with the presence of the linear mitochondrial plasmid pAL2-1. In addition to this autonomous genetic element, two types of closely related pAL2-1-homologous molecules were detected in the high-molecular-weight mitochondrial DNA (mtDNA). One of these molecules is of linear and the other of circular structure. Both molecules contain pAL2-1 sequences which appear to be integrated at the same site in the mtDNA. Sequence analysis of a DNA fragment cloned from one of these molecules revealed that it contains an almost full-length copy of pAL2-1. At the site of plasmid integration a 15-nucleotide AT-spacer and long inverted mtDNA sequences were identified. Finally, two giant linear plasmid-like DNAs of about 50 kbp and 70 kbp were detected in pulsed-field gels of mutant AL2. These molecules are composed of mtDNA and pAL2-1-specific sequences and may result from the integration of mtDNA sequences into linear plasmid pAL2-1.     

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.