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.     

Prevalence of Toxoplasma gondii infection in HIV-infected patients and food animals and direct genotyping of T. gondii isolates,Southern Ghana

Toxoplasma gondii is of public health and veterinary importance causing severe diseases in immunocompromised individuals including HIV/AIDS patients and in congenital cases and animals. There is limited information on the epidemiology of T. gondii infection in humans, particularly HIV patients and food animals and the parasite genotypes in Ghana. A total of 394 HIV-infected patients from three hospitals were screened for T. gondii anti-IgG and IgM using ELISA. DNAs from blood samples of seropositve participants and 95 brain tissues of food animals were PCR assayed to detect Toxoplasma gra6. DNA positive samples were genotyped using multilocus nested polymerase chain reaction restriction fragment length polymorphism at 10 loci: sag1, alt.sag2, sag3, btub, gra6, l358, c22-8, c29-2, pk1, and apico. The overall seroprevalence was 74.37% (293/394). Toxoplasma DNAs were detected in 3.07% of the seropositive participants and 9.47% of the animals. Six of the human DNA positive samples were partly typed at sag3: 33.33, 50, and 16.67% isolates had type I, II, and III alleles, respectively. All nine isolates from food animals typed at nine loci except apico were atypical: six isolates were identical to ToxoDB #41 and #145, and one was identical to TgCkBrRj2 all identified in Brazil. The genotype of two isolates has not been reported previously and was named as TgCtGh1. T. gondii seroprevalence is high among the HIV-infected individuals with T. gondii circulating in Ghana being genetically diverse.

     

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     

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.     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

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.     

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.     

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.     

Sequence analysis of a Papaver somniferum L. mitochondrial DNA fragment promoting autonomous plasmid replication in Saccharomyces cerevisiae and Kluyveromyces lactis

The minimal fragment of mitochondrial DNA from Papaver somniferum L. (poppy) able to promote autonomous plasmid replication in the yeast Saccharomyces cerevisiae was sequenced. Sequence analysis of the 917-bp MK4/8 DNA fragment revealed a high AT content, and the presence of two 12-bp sequences differing from the ARS core consensus of S. cerevisiae only by a T and C insertion, respectively. The mitochondrial insert contains a further six 11-bp sequences with one mismatch to the S. cerevisiae core consensus, more then 20 related sequences with two base pair exchanges, numerous direct and inverted repeats, and many copies of a sequence motif called the ARS box. The original 4.2-kb mitochondrial DNA fragment, as well as the minimal 917-bp subfragment in vector pFL1-E (a variant of YIP5, lacking an origin of replication in yeast), were then tested for their ability to replicate autonomously in another fungus, Kluyveromyces lactis.     

Characterization and prevalence of a circular mitochondrial plasmid in senescence-prone isolates of Neurospora intermedia

Genetic and molecular analyses of the phenomenon of senescence—i.e., irreversible loss of growth and reproductive potential upon subculturing—in Neurospora intermedia strain M1991-60A, collected from Maddur in southern India, showed the presence of plasmid pMaddur1, which is homologous to the senescence-inducing circular mitochondrial plasmid, pVarkud. Maternal inheritance of senescence in M1991-60A correlated to the formation of variant pMaddur1, its subsequent insertion into mitochondrial (mt)DNA and the accumulation of defective mtDNA with the pMaddur1insert. PCR-based analyses for similar plasmids in 147 natural isolates of Neurospora from Maddur showed that nearly 40% of the strains had pMaddur1 or pMaddur2 that shared 97–98% sequence homology with pVarkud and pMauriceville. Nearly 50% of the strains that harbored either pMaddur1 or pMaddur2, also contained a circular Varkud satellite plasmid (pVS). Size polymorphism maps to the cluster of PstI sites in the non-coding region. Whereas senescence of nearly 40% of N. intermedia strains may be due to pMaddur, the presence in seven strains of pVS but not pMaddur and the absence of either of these two plasmids in other senescence-prone isolates suggests yet undiscovered mechanisms of senescence in the Maddur strains.     

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.     

Circular plasmid DNAs from the red alga Gracilaria chilensis

Summary Total cellular DNA extracted from eight red algal species (from the genera Gracilaria, Gracilariopsis, Porphyra and Gymnogongrus) was centrifuged on Hoechst dye/CsCl gradients. In five species, plasmid-like DNAs banded with the A+T rich organellar DNAs in the CsCl gradients. Based on their electrophoretic migration in different agarose gels, the plasmid-like DNAs are circular. This is the first report of putative plasmid DNAs in the red algae outside the genus Gracilaria. Two similar Gracilaria chilensis plasmid-like DNAs of 3.8 and 3.4 kb (GC2 and GC3) were cloned in pUC19. The cloned GC2 DNA did not hybridize to either the organellar or nuclear genomes of G. chilensis, suggesting that GC2 is a true plasmid. GC2 did hybridize to the plasmid of one other red algal species, Gracilaria sordida.     

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     

The mitochondrial DNAs of Agaricus: heterogeneity in A. bitorquis and homogeneity in A. brunnescens

Summary Mitochondrial DNAs were isolated from four cultivated strains of the commercial two-spored mushroom Agaricus brunnescens (bisporus) and from ten isolates of the four spored mushroom Agaricus bitorquis. Digestion of the fungal mitochondrial DNA with restriction endonucleases yielded numerous fragments. Summation of the fragment sizes gave a mitochondrial DNA size of 98.3 ± 2.4 kilobases (kb) (64.9 x 10⁶ daltons) for A. brunnescens. The size of the mitochondrial DNA ranged from 148.5 ± 10.8 kb (98.0 x 10⁶ daltons) to 176.3 ± 12.0 kb (116.4 x 10⁶ daltons) for A. bitorquis. The restriction patterns, produced by a variety of endonucleases, were identical for all four isolates of A. brunnescens. The ten isolates of A. bitorquis demonstrated extensive restriction pattern heterogeneity and have been tentatively assigned into four groups. Approximately 60% of the A. bitorquis mitochondrial DNA restriction fragments show sequence homology with A. brunnescens mitochondrial DNA based on DNA — DNA hybridizations.     

Characterization of bovine septicemic, bovine diarrheal, and human enteroinvasive Escherichia coli that hybridize with K88 and F41 accessory gene probes but do not express these adhesins

Certain DNA probes derived from accessory genes of cloned K88 and F41 determinants hybridize with Escherichia coli strains that express K88 or F41 and with certain other E. coli strains that do not express these antigens. We found that these probes hybridized with human enteroinvasive E. coli, and with bovine E. coli isolates which produced a fatal septicemia in experimentally infected piglets. These strains did not hybridize with probes derived from the structural subunit genes encoding the K88 and F41 antigens. E. coli strains isolated from turkeys with septicemia, Shigella and Salmonella strains did not hybridize to the K88 and F41 accessory gene probes. The K88 and F41 accessory gene probes hybridized with a 200 kb plasmid which is required for invasion by human enteroinvasive E. coli. The K88 and F41 accessory gene homology in the bovine isolates was located on a 150 kb transmissible plasmid but was unrelated to plasmids encoding aerobactin, Vir, or colicin V, which are suspected virulence factors in septicemic E. coli. A common plasmid-encoded antigen was associated with bovine isolates that hybridized with the K88 and F41 accessory gene probes. This included strains which express CS31A, a surface antigen associated with bovine septicemic E. coli, which also hybridized with the K88 and F4 accessory gene probes. The results suggest that the K88 and F41 accessory gene probes hybridized with sequences that may be associated with a common mechanism of pilus expression in distinct groups of E. coli pathogens.     

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.     

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.     

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.     

Mitochondrial DNA in the aquatic fungus Allomyces

Summary The mitochondrial DNA from seven species of the aquatic phycomycete Allomyces has been isolated and characterized by restriction enzyme analysis. Comparison of the mitochondrial DNA restriction enzyme fragmentation patterns showed pronounced differences not only among species but also among four isolates of A. arbuscula. The mitochondrial DNAs range in size from 39 kbp in A. neo-moniliformis to 56 kbp in A. macrogynus.A physical map of the mitochondrial DNA of Allomyces arbuscula strain Costa Rica 21 has been constructed. The genome is circular and has a size of 49.2 kbp. The genes coding for the small and large ribosomal RNAs, cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, and ATPase subunits 6 and 9 were localized in the mitochondria) DNA by heterologous hybridization with specific mitochondria) gene probes from Saccaromyces cerevisiae and Neurospora crassa. Comparison of the gene map of the closely related species Blastocladiella emersonii with that of A. arbuscula indicates a similar gene order in the two organisms.