Mitochondrial Plasmid Research Articles

NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis.

An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast.

Detection of Three DNA‐Binding Proteins from Lupine Mitochondria

To investigate the mechanism of transcription in plant mitochondria, proteins displaying binding activity to mitochondrial plasmid promoters were studied. We have identified a conserved nonanucleotide motif at the 5′ end of a Lupinus albus mitochondrial plasmid transcript. Three proteins with molecular masses of ca. 25, 30, and 48 kD interacted with ds oligonucleotides, representing different parts of the plasmid promoter. Proteins with molecular masses 25 and 30 kD seem to represent two components of a heterodimer. The DNA‐binding activities of these proteins have been confirmed by electrophoretical mobility shift assay (EMSA), SDS‐PAGE, Southwestern, and UV cross‐linking assays. The specificity of DNA‐binding reaction was examined by competition experiments.

Association of mitochondrial plasmids with rejuvenation of the coastal redwood, Sequoia sempervirens (D. Don) Endl.

Repeated grafting of shoot apices from mature Sequoia sempervirens (D. Don) Endl. trees onto young seedling shoots in vitro causes emergence of rejuvenated, or phase-reversed, scion shoots. The phase reversal is indicated by a restored rooting competence and renewed vigor of roots and shoots. We earlier reported of restriction fragment length polymorphism between juvenile or rejuvenated and adult shoot mtDNA (mitochondrial DNA). This investigation of undigested mtDNA disclosed that S. sempervirens shoots contained at least six small mtDNA molecules, four of which were uniquely associated with juvenile and rejuvenated shoots. The small molecules re- mained observable in continuously subcultured shoots. Results of cloning and sequencing indicated they are circular in form; we thus, called them plasmids. Significant sequence homology, 38 to 56%, was found among the molecules, indicating highly conserved regions and possibly common origin. No similar phase-associated relationships were observed for unrestricted cpDNA (chloroplast DNA) or nucDNA (nuclear DNA). The plasmids hybridized with the larger, electrophoretically less mobile mtDNA, but not with cpDNA or nucDNA, suggesting their origin in the master circle mtDNA. The distinctness of these plasmids has remained unchanged after more than 20 years of subculturing.

Mitochondrial plasmids of the pCp family are spread worldwide in Cryphonectria parasitica populations

A worldwide collection of strains of Cryphonectria parasitica was examined to draw a precise picture of the incidence and diversity of mitochondrial plasmids related to the plasmid pUG1. Amplification by specific PCR of 199 strains showed the presence of pUG1-like plasmids in 22% of the populations examined. The entire plasmid molecules were amplified by multiplex PCR and the products showed different RFLP patterns. The variability was mostly in a non-coding region of the molecule that has been sequenced in some representative strains, enabling the molecular evolution of the molecule to be elucidated. The data show that mitochondrial plasmids of C. parasitica comprise an almost homogeneous family (designated pCp) that can be divided into two clusters based on the presence/absence respectively of a 60 nucleotide region in North American and European plasmids.

Transmission rates and phenotypic effects of mitochondrial plasmids and cytotypes in Silene vulgaris.

We investigated the transmission properties and the phenotypic effects of two mitochondrial plasmids in a population of the bladder campion, Silene vulgaris. In reciprocal crosses between plasmid-free and plasmid-carrying plants, no cases of paternal transmission or loss during maternal transmission were recorded. Neither was any transmission via pollen observed when plasmid-carrying plants of S. vulgaris were used to pollinate plasmid-free plants of the closely related species Silene uniflora. The phenotypic effects of the plasmids were investigated by comparing germination rate, early growth properties, and the gender of plants grown from seeds with and without plasmids. A significant association between plasmid status, on the one hand, and germination propensity and offspring gender, on the other, was found. However, because all plants carrying plasmids in the experiment shared the same cytoplasmic background, the exact contribution of the plasmid to the phenotypic variation could not be determined. Taken together, our experiments show that in S. vulgaris the mt-plasmids are not currently involved in any strong genetic conflict, but that they evolve in close association with their mitochondrial host.

Structural features and expression analysis of a linear mitochondrial plasmid in rapeseed (Brassica napus L.).

A linear plasmid molecule about 11 kb in length is present in the mitochondria of some varieties of rapeseed (Brassica napus L.). This plasmid can be inherited from the male parent, through the pollen, as well as by the usual maternal route, although the main mitochondrial genome is maternally inherited in rapeseed. We determined the complete nucleotide sequence of this plasmid DNA and clarified its genetic organization. The length of the linear plasmid is 11,640 bp. At the termini of the plasmid molecule are inverted repeats of 327 bp. The GC content of the plasmid DNA is 30.9%; thus, the plasmid is quite AT-rich compared to the main mitochondrial genome in higher plants. The plasmid has six ORFs, two of which encode a phage-type DNA polymerase and a phage-type RNA polymerase, respectively. RT-PCR analyses revealed that all six ORFs are transcribed, and all four ORFs on the minus strand are probably cotranscribed from a single promoter located in the terminal inverted repeat. We also show here that at least three of the six ORFs are translated into proteins in rapeseed mitochondria, and expressed at relatively high levels in flowers, as shown by Western analysis. These results suggest that this linear DNA molecule is able to replicate as an autonomous replicon and to express the genes it carries in rapeseed mitochondria.

Characterization of a putative fusogen encoded in a mitochondrial plasmid of Physarum polycephalum.

The mitochondrial plasmid mF induces mitochondrial fusion in zygotes and during sporulation in the true slime mold Physarum polycephalum. There are nine open reading frames (ORFs) in the mF plasmid, and it has been suggested that ORF640 encodes the mitochondrial fusogen. We prepared antisera directed against the ORF640 protein (ORF640p) in rabbits, and used it to localize this protein in mitochondria. Western blot analysis showed that ORF640p was produced only in the mitochondria of mF(+) strains, i.e., in cells that carried the mF plasmid. Proteinase K treatment of mitochondria isolated from the mF(+) strain suggested that the C-terminus coiled-coil (CC) region of ORF640p was exported from the matrix to the cytosol. Digitonin treatment confirmed this localization. West-Western blot analysis suggested that the CC region of ORF640p formed multimers and could interfere with an unknown mitochondrial protein in normal mitochondria. These results suggest that ORF640p is related to fusion of the outer mitochondrial membrane.

R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1.

The mitochondrial (mt) plasmid mp1 of Chenopodium album replicates by a rolling-circle (RC) mechanism initiated at two double-stranded replication origins (dso1 and dso2). Two-dimensional gel electrophoresis and electron microscopy of early mp1 replication intermediates revealed novel spots. Ribonucleotide (R)-loops were identified at dso1, which function as a precursor for the RCs in vivo and in vitro. Bacteriophage T4-like networks of highly branched mp1 concatemers with up to 20 monomer units were mapped and shown to be mainly formed by replicating, invading, recombining and resolving molecules. A new model is proposed in which concatemers were separated into single units by a "snap-back" mechanism and homologous recombination. dso1 is a recombination hotspot, with sequence homology to bacterial Xer recombination cores. mp1 is a unique eukaryotic plasmid that expresses features of phages like T4 and could serve as a model system for replication and maintenance of DNA concatemers.

TRANSMISSION RATES AND PHENOTYPIC EFFECTS OF MITOCHONDRIAL PLASMIDS AND CYTOTYPES IN SILENE VULGARIS

We investigated the transmission properties and the phenotypic effects of two mitochondrial plasmids in a population of the bladder campion, Silene vulgaris. In reciprocal crosses between plasmid-free and plasmid-carrying plants, no cases of paternal transmission or loss during maternal transmission were recorded. Neither was any transmission via pollen observed when plasmid-carrying plants of S. vulgaris were used to pollinate plasmid-free plants of the closely related species Silene uniflora. The phenotypic effects of the plasmids were investigated by comparing germination rate, early growth properties, and the gender of plants grown from seeds with and without plasmids. A significant association between plasmid status, on the one hand, and germination propensity and offspring gender, on the other, was found. However, because all plants carrying plasmids in the experiment shared the same cytoplasmic background, the exact contribution of the plasmid to the phenotypic variation could not be determined. Taken together, our experiments show that in S. vulgaris the mt-plasmids are not currently involved in any strong genetic conflict, but that they evolve in close association with their mitochondrial host.

Discrimination of cultivated Flammulina velutipes by mitochondrial DNA and plasmid

Discrimination of cultivated Flammulina velutipes by mitochondrial DNA and plasmid

A Phylogeny of the Genus Agaricus Based on Mitochondrial atp6 Sequences

The genus Agaricus includes the most eco- nomically-important, commercially-cultivated mush- room in the world, A. bisporus. Effective efforts to improve A. bisporus through breeding will require a greater understanding of the evolutionary relation- ships between A. bisporus and other Agaricus species. We present here the complete mitochondrial atp6 gene of A. bitorquis and amplified homologues from 9 other species of Agaricus, including 6 different iso- lates of A. bisporus. Unexpectedly, only 2 variants of atp6 were found among the 6 isolates of A. bisporus, which had been chosen to represent mitochondrial types thought to be highly divergent based on RFLP data. Although interspecific variation was high, par- simony and maximum likelihood analyses resulted in identical phylogenetic trees for the genus: A. bispo- rus, A. subfloccosus and A. subperonatus were tightly clustered as a single subgroup, whereas the remain- ing species were highly divergent. The species rep- resentatives with the closest proximity to the A. bis- porus cluster were A. bitorquis and A. campestris, which is in agreement with published phylogenies de- rived from nuclear sequence data. Incongruency be- tween this mitochondrial gene-based phylogeny and a phylogeny of linear mitochondrial plasmids found in these same species indicates separate evolutionary histories for the plasmids and their host mitochon- dria.

A phylogeny of the genus Agaricus based on mitochondrial atp6 sequences

The genus Agaricus includes the most economically-important, commercially-cultivated mushroom in the world, A. bisporus. Effective efforts to improve A. bisporus through breeding will require a greater understanding of the evolutionary relationships between A. bisporus and other Agaricus species. We present here the complete mitochondrial atp6 gene of A. bitorquis and amplified homologues from 9 other species of Agaricus, including 6 different isolates of A. bisporus. Unexpectedly, only 2 variants of atp6 were found among the 6 isolates of A. bisporus, which had been chosen to represent mitochondrial types thought to be highly divergent based on RFLP data. Although interspecific variation was high, parsimony and maximum likelihood analyses resulted in identical phylogenetic trees for the genus: A. bisporus, A. subfloccosus and A. subperonatus were tightly clustered as a single subgroup, whereas the remaining species were highly divergent. The species representatives with the closest proximity to the A. bisporus cluster were A. bitorquis and A. campestris, which is in agreement with published phylogenies derived from nuclear sequence data. Incongruency between this mitochondrial gene-based phylogeny and a phylogeny of linear mitochondrial plasmids found in these same species indicates separate evolutionary histories for the plasmids and their host mitochondria.

Role of Mitochondrial DNA in the Senescence and Hypovirulence of Fungi and Potential for Plant Disease Control.

The unique coenocytic anatomy of the mycelia of the filamentous fungi and the formation of anastomoses between hyphae from different mycelia enable the intracellular accumulation and infectious transmission of plasmids and mutant mitochondrial DNAs (mtDNAs) that cause senescence. For reasons that are not fully apparent, mitochondria that are rendered dysfunctional by so-called "suppressive" mtDNA mutations proliferate rapidly in growing cells and gradually displace organelles that contain wild-type mtDNA molecules and are functional. The consequence of this process is senescence and death if the suppressive mtDNA contains a lethal mutation. Suppressive mtDNA mutations and mitochondrial plasmids can elicit cytoplasmically transmissible "mitochondrial hypovirulence" syndromes in at least some of the phytopathogenic fungi. In the chestnut-blight fungus Cryphonectria parasitica, the pattern of asexual transmission of mutant mtDNAs and mitochondrial plasmids resembles the pattern of "infectious" transmission displayed by the attenuating virus that is most commonly used for the biological control of this fungus. At least some of the attenuating mitochondrial hypovirulence factors are inherited maternally in crosses, whereas the viruses are not transmitted sexually. The natural control of blight in an isolated stand of chestnut trees has resulted from the invasion of the local population of C. parasitica by a senescence-inducing mutant mtDNA. Moreover, a mitochondrial plasmid, pCRY1, attenuates at least some virulent strains of C. parasitica, suggesting that such factors could be applied to control plant diseases caused by fungi.

Basidiomycetous fungus Flammulina velutipes harbors two linear mitochondrial plasmids encoding DNA and RNA polymerases

Basidiomycetous fungus Flammulina velutipes R15 strain had two linear plasmids in its mitochondria designated pFV1 and pFV2. They were double-stranded DNAs, whose sizes were 8.3 and 8.9 kb, respectively. Sequencing analysis of 7364 bases of the pFV1 and 6861 bases of the pFV2 revealed that the both plasmids had one set of two open reading frames (ORFs) each of that encoded putative DNA and RNA polymerases similar to those of mitochondrial plasmids in other filamentous fungi. In phylogenetic analysis of deduced amino acid sequences of the ORFs and counterparts of other filamentous fungi, the pFV2 was expectedly clustered with plasmids of basidiomycetous fungi, whereas the pFV1 with kalilo plasmid of ascomycetous fungus Neurospora intermedia.

Basidiomycetous fungus Flammulina velutipes harbors two linear mitochondrial plasmids encoding DNA and RNA polymerases.

Basidiomycetous fungus Flammulina velutipes R15 strain had two linear plasmids in its mitochondria designated pFV1 and pFV2. They were double-stranded DNAs, whose sizes were 8.3 and 8.9 kb, respectively. Sequencing analysis of 7364 bases of the pFV1 and 6861 bases of the pFV2 revealed that the both plasmids had one set of two open reading frames (ORFs) each of that encoded putative DNA and RNA polymerases similar to those of mitochondrial plasmids in other filamentous fungi. In phylogenetic analysis of deduced amino acid sequences of the ORFs and counterparts of other filamentous fungi, the pFV2 was expectedly clustered with plasmids of basidiomycetous fungi. whereas the pFV1 with kalilo plasmid of ascomycetous fungus Neurospora intermedia.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64)and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich pallindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mtRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Dynamics of asexual transmission of a mitochondrial plasmid in Cryphonectria parasitica

In the chestnut blight fungus Cryphonectria parasitica, as in most fungi, little is known about the efficiency of the asexual transmission of optional mitochondrial plasmids, vertically through conidia, and horizontally through hyphal anastomoses. In this paper, we show that pCRY1, a circular mitochondrial plasmid, is transmitted vertically with 100%-efficiency through conidia. Moreover, the plasmid is transmitted horizontally through hyphal contact from donor strains to vegetatively compatible and most incompatible strains. An allelic difference between the donor and recipient strain, at only one of the five nuclear incompatibility genes that were tested strongly inhibited, but did not absolutely prevent, the transfer of pCRY1 through hyphal fusions. In contrast, allelic differences in any one or several of the other four heterokaryon-compatibility loci suppressed the transmission of the plasmid only partially or not at all. The plasmid was also transmitted among incompatible strains by protoplast fusion without the concomitant transfer of mitochondrial DNA (mtDNA). A comparison of plasmid-bearing with plasmid-free isogenic strains revealed that pCRY1 significantly diminishes the pathogenic potency of some strains of the fungus, but does not affect the virulence of others. Collectively, the observations indicate that the introduction of deleterious mitochondrial genetic elements into natural populations may be a means for managing fungal pathogens.

A circular mitochondrial plasmid incites hypovirulence in some strains of Cryphonectria parasitica.

In the chestnut-blight fungus Cryphonectria parasitica, a plasmid, pCRY1, occurs in the mitochondria of several strains isolated at various locations in the northeastern United States and Canada. The monomer of this plasmid is a 4.2-kb circular double-stranded DNA that has no detectable sequence homology with the 160-kb mitochondrial DNA of Ep155, a standard virulent laboratory strain of C. parasitica. The circular nature and oligomeric characteristics of the plasmid were deduced from the heterogeneous size of plasmid DNA molecules as detected by one- and two-dimensional gel-electrophoresis, the nature and alignment of restriction fragments, and the lack of detectable termini in the nucleotide sequence. The cytoplasmic location of the plasmid was deduced from its co-purification with mitochondria, uniparental (maternal) transmission in sexual crosses, dissociation from the nuclei of the donor strain during its horizontal transfer between vegetatively compatible strains through hyphal anastomoses, and mitochondrial codon usage (UGA = Try). The pCRY1 plasmid contains a long open reading frame that is transcribed and potentially encodes a unique 1214 amino-acid, B-family DNA polymerase similar to those encoded by the LaBelle and Fiji circular mitochondrial plasmids of Neurospora. In this subgroup of proteins, the DTD motif characteristic of B-family DNA polymerases is replaced by TTD. Amino-acid motifs related to those that are characteristic of the 3'-->5' exonuclease domains of B-family DNA polymerases have been located in the amino-terminal portion of the proteins. A comparison of isogenic plasmid-free and plasmid-containing cultures indicates that pCRY1 is an infectious agent that effects a reduction in the pathogenicity of some, but not all, strains of C. parasitica.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74 A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Possible Benefits of Kalilo Plasmids to Their Neurospora Hosts

Neurospora mitochondrial plasmids are ubiquitous in natural populations, yet many of them are lethal to their host strains or seem to impose a molecular genetic load. Five pairs of strains of Neurospora tetrasperma and N. crassa with and without kalilo-like plasmids were tested under a variety of situations. The purpose was to find possible beneficial effects of plasmids that might offset their disadvantages. We found that, in all cases tested, plasmids conferred an advantage to growth at temperatures close to the top of the range for this fungus. Also, the plasmids improved fertility, as measured by perithecial production. Negative results were obtained for heavy metal resistance and ascospore germination. The results generate the hypothesis that plasmids may have adaptive significance to their hosts.