Extrachromosomal genetics of Claviceps purpurea

Summary Claviceps purpurea strain K1 contains several linear mitochondrial plasmids comparable to those of higher plants (Tudzynski et al. 1983). Screening of ten Claviceps wild strains from different geographic origin revealed the presence of similar plasmids in at least five strains, indicating a widespread occurrence of these genetic traits. Whereas in strain K1 plasmids have no homology to high-molecular-weight mtDNA, in two of the other wild strains (W3 and W7) sequences homologous to plasmids of strain K1 are integrated in the mt genomic DNA. Physical and genetic maps of strains W3, W7 and K1 were established. Despite completely different physical maps, the relative orientation of 6 mt genes is identical. A DNA sequence homologous to plasmids of strain K1 (termed ) was found to be integrated in both W3 and W7 at the same site: at the 5-part of the 1rRNA gene. A second region () was localized near the srRNA gene of W7 only. These findings are discussed with respect to possible transposon-like properties of the Claviceps purpurea plasmids.     

The Claviceps purpurea glyceraldehyde-3-phosphate dehydrogenase gene: cloning,characterization, and use for the improvement of a dominant selection system

The GPD 1 gene of Claviceps purpurea coding for glyceraldehyde-3-phosphate dehydrogenase was cloned and sequenced, including 1,800 bp of its 5 upstream region. This gene shows an identical structure to the gpd gene of Podospora anserina and Cryphonectria parasitica (one intron at an identical position) with high homology at both the DNA and amino-acid levels. Two fragments of the promoter spanning from the ATG to-500 bp and to-1,400 bp were fused to the phleomycin-resistance gene. Both constructs transformed C. purpurea at a high rate. The enhanced expression of the long vector construct indicates the presence of additional elements between-500 bp and-1,400 bp upstream of the initiation codon.     

Nucleotide-sequence analysis indicates that a DNA plasmid in a diseased isolate of Ophiostoma novo-ulmi is derived by recombination between two long repeat sequences in the mitochondrial large subunit ribosomal RNA gene

The nucleotide sequence of a mitochondrial plasmid (2234 bp) in a diseased isolate of Ophiostoma novo-ulmi, and sequences of the mitochondrial DNA that overlap and flank the plasmid end-points, have been determined. The plasmid was shown to be derived from the O. novo-ulmi mitochondrial large subunit ribosomal RNA gene and contained most of intron 1, the whole of exon 2, and probably the first part of intron 2. Within intron 1 there is an open reading frame with the potential to encode a 323 amino-acid polypeptide which contained dodecapeptide sequences typical of RNA maturases and DNA endonucleases. The endpoints of the plasmid in the mtDNA were located within two 90-bp direct imperfect repeat sequences, one of which comprised the last 7 bp of exon 1 and the first 83 bp of intron 1 whilst the other comprised the last 7 bp of exon 2 and the first 83 bp of intron 2. It is proposed that the Ld plasmid was generated by intramolecular recombination between these two repeats with the crossover point probably within the last 15 bp.     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

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.     

Electron microscopic investigation of mitochondrial DNA fromChenopodium album (L.)

DNA molecules from mitochondria of whole plants and a suspension culture ofChenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0.3–183 kb represented 23–26% of all mtDNA molecules in the preparations from the suspension culture and 13–15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rollingcircle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.     

Intramolecular recombination of a mitochondrial minicircular plasmid-like DNA of date-palm mediated by a set of short direct-repeat sequences

A molecular clone containing the complete sequence of a mitochondrial circular plasmid-like DNA (the R plasmid) isolated from the date-palm variety V3DP was used as a probe in Southern analyses of mitochondrial DNA prepared from other varieties. Another circular structure (the S plasmid) was detected in some of these varieties, and sequenced from variety V2DP. It appears that the R plasmid could have arisen from the S plasmid by an intermolecular recombination event at a set of 26-bp imperfect short direct repeats.     

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.     

De-novo generation of mitochondrial DNA plasmids following cytoplasmic transmission of a degenerative disease in Ophiostoma novo-ulmi

A mitochondrial DNA plasmid was detected in an isolate of Ophiostoma novo-ulmi with a degenerative disease. The DNA plasmid was shown to be derived from the mitochondrial DNA and to map to a region corresponding to the large ribosomal RNA coding region. The DNA plasmid was not transmitted into sexual (ascospore) progeny, irrespective of whether the diseased isolate acted as the female or male parent. Transmission of the disease to healthy, plasmid-free, recipient isolates by hyphal anastomosis was not accompanied by transfer of mitochondrial DNA or DNA plasmid from the diseased donor isolate, but resulted in de-novo generation of different plasmids, derived from the recipient's mitochondrial DNA.     

Distribution of seven homology groups of mitochondrial plasmids in Neurospora: evidence for widespread mobility between species in nature

A survey of mitochondrial DNAs from over 225 Neurospora and related fungal isolates from around the world uncovered three new homology groups of mitochondrial plasmids, two divergent subgroups of the Fiji plasmid family, and extended previous data about plasmid distribution patterns. Newly-discovered circular plasmids, Java and MB1, and the linear Moorea plasmids, were found in relatively-few isolates. A large proportion of isolates (51%) were found to have these or previously-discovered plasmids in the Varkud, kalilo, LaBelle, or Fiji families. Plasmids in most families were found in isolates world-wide and distributed nearly randomly with respect to species. As many as three types of plasmids were found in single isolates, and plasmids typically were found alone or in pairs in a random, independent pattern. The regional clustering of some plasmids was independent of species. providing a strong argument that horizontal transfer of plasmids occurs frequently in nature. Some plasmid families were much more diverse than others. The Fiji plasmids are a superfamily composed of distinct subgroups defined by degrees of cross-hybridization. Between some subgroups there were large regions of non-homology.     

Maize mitochondrial DNA rearrangements between the normal type,the Texas male sterile cytoplasm,and a fertile revertant cms-T regenerated plant

Summary The maize mitochondrial DNA (mtDNA) from the cytoplasmic male sterile type T (cms-T) contains a 6.6 kb XhoI fragment not found in the normal type (N) or the fertile revertant (V3). Analysis of the fragment in cms-T mtDNA and homologous regions in N and V3 mtDNAs reveals that a number of events are necessary to explain the formation of the 6.6 kb XhoI fragment in T mtDNA and its subsequent loss in V3 mtDNA. This includes the formation and the loss of a 4 kb repeat and various recombinational events. It is interesting to note that a recombinational event that has occurred in the fertile revertant genome reconstitutes a sequence arrangement identical to one found in the normal genome.     

Mitochondrial DNA of Chenopodium album (L): a comparison of leaves and suspension cultures

Summary The mitochondrial genome (mt genome) of Chenopodium album was analysed. It was found to have a size of about 270 kb and a GC content of 46%. The genomes of plant cells and suspension cultures were compared. Restriction fragment pattern analyses and hybridization experiments revealed quantitative and qualitative alterations. However, a comparison of the restriction fragment patterns of two independently established in vitro cultures did not reveal differences as far as the mt chromosomal DNA is concerned. Therefore, alterations in mtDNA induced by in vitro culture seem not to be caused by an entirely undirected process in Chenopodium. A plasmid-like DNA molecule was amplified in only one of the cultures investigated and not in the other or in the plant cells. This molecule has a length of 1,083 by and is referred to as Ca1 mp1.     

Molecular analysis of the mitochondrial genome of Phytophthoraa

Summary The organization of the mitochondrial genomes from two morphologically similar Phytophthora isolates, P. megasperma f. sp. glycinea (Pmg) and P. megasperma f. sp. medicaginis (Pmm), and the morphologically different species, P. parasitica var. nicotianae (Ppn), has been studied. The mtDNAs are circular, and their estimated sizes are 45.3 kb, 41 kb, and 39.5 kb for Pmg, Pmm, and Ppn, respectively. Physical maps were constructed for restriction endonuclease sites. Four genes (l-rRNA, s-rRNA, oxi-2, and cob) were found to have the same order in the three mtDNAs.     

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