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     

Linear,non-mitochondrial plasmids of Alternaria alternata

Summary Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5 ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.     

Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colletotrichum musae (Berk. & Curt.) Arx.]

A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5′ termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1. Received: 27 June 1996 / 2 April 1997     

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.     

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.     

Plasmid RNA polymerase-like mitochondrial sequences inAgaricus bitorquis

A linear mitochondrial plasmid, pEM, found in certain isolates of the basidiomyceteAgaricus bitorquis potentially encodes virus-like DNA and RNA polymerases. Mitochondrial DNA fromAgaricus bisporus that hybridizes to an internal region of pEM contains a fragmented and potentially non-functional version of the carboxy terminal end of the plasmid RNA polymerase. In this study, we present the sequence of the corresponding region of mitochondrial DNA fromA. bitorquis. This sequence contained the same region of the plasmid RNA polymerase gene as was reported for the mitochondrial DNA ofA. bisporus, and the level of similarity between theA. bisporus andA. bitorquis mitochondrial sequences was much higher than the level of similarity between either mitochondrial sequence and the plasmid. We propose that this plasmid RNA polymerase-like sequence was present in theAgaricus mitochondrial genome before the divergence ofA. bisporus andA. bitorquis, and thus is unlikely to be a recent derivative of the plasmid pEM.     

DNA probe for strain typing of Cryptococcus neoformans.

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.     

Characterization and sequence of lupin mitochondrial plasmid-like DNA

Summary Two minicircular DNAs of 1.2 kb (K1) and 1.4 kb (K2) were found in mitochondria of fertile lupin (Lupinus albus). The plasmid-like DNA, K1, was cloned, labelled and hybridized with mitochondrial DNA from three different species of lupin. We have found no evidence for integrated copies of K1 in any of the mitochondrial genomes probed in this study. No sequence homology between plasmid K1 and K2, and no homology of either with chloroplast DNA, has been detected. The K1 DNA is two-fold more abundant than the K2 DNA and about seven-fold more abundant than a unique segment of the mtDNA. The entire nucleotide sequence of the K1 DNA has been determined. This sequence exibits a 340 base pair region with highly organized repeats. The sequence of K1 shows no substantial homology with sequence of other mitochondrial plasmids of higher plants.     

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.     

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.     

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.     

Homologous linear plasmids in mitochondria of three species of wheat bunt fungi,Tilletia caries,T. laevis and T. controversa

Summary All isolates of Tilletia spp. investigated (five isolates of T. caries, including one from Japan, two isolates of T. laevis, and five isolates of T. controversa) contained a linear DNA plasmid ranging in size from 7.2 to 7.6 kb. All plasmids were highly homologous to each other as shown by DNA-DNA hybridization and comparison of restriction enzyme sites. Variability in the size of the plasmid was found to be due to differences within a central region of the plasmid. No homology between the plasmid and mitochondrial or nuclear DNA was found, but the mitochondrial origin of the plasmid was confirmed.     

Outer membrane proteins of nontypable Haemophilus influenzae and reactivity of paired sera from infected patients with their homologous isolates.

Plasmid DNAs obtained from 18 Escherichia coli isolates that hybridized with the heat-stable b (ST-b) enterotoxin gene probe were examined by Southern blot analysis for genes coding for heat-labile, ST-a, and ST-b enterotoxins with specific radiolabeled DNA probes. Four E. coli isolates contained plasmids coding for both heat-labile and ST-b enterotoxins, and one isolate contained a plasmid coding for ST-a and ST-b. Five of 11 isolates of antibiotic-resistant enterotoxigenic E. coli isolates containing ST-b-coding DNA transferred a plasmid coding for both antibiotic resistance and ST-b to E. coli K-12, suggesting that the widespread use of antibiotics could increase the distribution of genes coding for ST-b.     

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.     

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5 termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.     

DNA analysis of Borrelia burgdorferi NCH-1, the first northcentral U.S. human Lyme disease isolate.

The DNA of the first northcentral United States human Lyme disease isolate, Borrellia burgdorferi NCH-1, was characterized and compared with the DNAs of nine other B. burgdorferi isolates. Strain NCH-1 was isolated in August 1989 from a human skin biopsy specimen. DNA was analyzed by pulsed-field gel electrophoresis and restriction endonuclease analysis. Contour-clamped homogeneous electric field pulsed-field gel electrophoresis of in situ-lysed cells was performed to compare the plasmid profiles of the various isolates. The plasmid profile of isolate NCH-1, which included five plasmids of approximately 69, 42, 38, 32, and 23 kb, could be distinguished from those of the other isolates examined. The DNA profile of NCH-1 was most similar to those of strain 297 (human cerebrospinal fluid isolate, Connecticut) and strain PAL (human erythema migrans isolate, New York) and most dissimilar from those of strain P/Gau (human erythema migrans isolate, Germany) and strain IPF (Ixodes persulcatus tick isolate, Japan). These results indicate that genetic diversity exists among B. burgdorferi strains isolated from different geographical areas.     

Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence

Summary Recently, a parvo-like virus was newly isolated from silkworm larvae and the two viral DNAs (VD1 and VD2) with different electro-mobilities were identified. We cloned the viral DNAs in a plasmid pUC119 and demonstrated that these two DNAs were not a bimorphic molecules though they shared a common terminal sequence of 53 nucleotides. In addition, the sequence at the 5 terminus of each strand of the viral DNA was located in inverted form at its 3 terminus. On the other hand, the nucleotide sequences of VD1 and VD2 were different from that of the Bombyx densovirus (Ina isolate) DNA.