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.     

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.     

Rearrangements in the Physarum polycephalum mitochondrial genome associated with a transition from linear mF-mtDNA recombinants to circular molecules

Although mitochondrial DNA (mtDNA) is transmitted to progeny from one parent only in Physarum polycephalum, the mtDNAs of progeny of mF⁺ plasmodia vary in structure. To clarify the mechanisms associated with the mitochondrial plasmid mF that generate mtDNA polymorphisms, 91 progeny of four strains (KM88 × JE8, KM88 × TU111, KM88 × NG111, Je90) were investigated using RFLP analysis, PCR, and pulse-field gel electrophoresis (PFGE). Nine mtDNA rearrangement types were found, with rearrangements occurring exclusively in the mF regions. PFGE revealed that, in the groups containing rearranged mtDNA, the linear mF–mtDNA recombinants had recircularized. Sequencing the rearranged region of one of the progeny suggested that the mF plasmid and the mtDNA recombine primarily at the ID sequences, linearizing the circular mtDNA. Recombination between the terminal region of the mF plasmid and a region about 1 kbp upstream of the mitochondrial/plasmid ID sequence results in a rearranged circular mtDNA, with variations caused by differences in the secondary recombination region.     

Elimination of mitochondrial mutations by sexual reproduction: two Podospora anserina mitochondrial mutants yield only wild-type progeny when mated

In order to understand the transmission of mitochondrial mutations in sexual crosses of Podospora, we attempted to create compatible strains with defined mitochondrial mutations. A previously characterized mutant, Mn19, with a bipartite mitochondrial genome, served as the fertilizing parent in a cross with a mitochondrial deletion mutant, αΔ5. Characterization of the deletion mutant is reported here. All six of the monokaryotic progeny isolated had neither parental defect but instead appeared to have inherited wild-type mitochondrial DNA. One of the progeny had a mitochondrial plasmid derived from intramolecular recombination between an 11-bp repeated mitochondrial sequence. Subsequent analysis using the polymerase chain reaction (PCR) identified rare undeleted wild-type mtDNA sequences in the maternal parent. The uniform inheritance of wild-type mitochondrial DNA suggests either an aggressive repair mechanism or else selective amplification and transmission of rare wild-type mtDNA molecules. Received: 12 December 1995 / 6 May 1996     

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 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.     

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.     

Association between variant plasmid formation and senescence in retroplasmid-containing strains of Neurospora spp.

Serial transfer of Neurospora strains harboring the Mauriceville and Varkud mitochondrial retroplasmids frequently displays erratic growth and senescence. Growth impairment is associated with the formation of variant forms of the retroplasmids that can integrate into the mitochondrial genome, resulting in mtDNA rearrangements and eventual loss of respiratory function. Here, we evaluate the rate at which variant plasmids arise in subcultures of the Mauriceville strain of N. crassa and their association with the senescent phenotype. Although variant plasmid formation preceded senescence, subcultures were found to tolerate variant plasmids for variable lengths of time and no correlation could be made between the specific sequence inserted in the plasmids and the rate or frequency of senescence. In addition, many cultures were found to contain more than one variant plasmid. The lack of concordance between the timing of variant plasmid formation and growth cessation distinguishes these two events, and provides additional insight into the etiology of senescence. We also detected differences in the frequency of senescence between retroplasmid-containing strains of N. crassa and N. intermedia and report the isolation of a strain in which senescence occurs in the absence of variant plasmid formation or detectable alterations in mtDNA. Our findings indicate there are multiple pathways that lead to senescence and suggest there are host-specific mechanisms that suppress the deleterious effects of the variant plasmids. Received: 8 August 2000 / Accepted: 17 November 2000     

Transfer of Neurospora kalilo plasmids among species and strains by introgression

There are four different variants of the kalilo “family” of linear mitochondrial plasmids. This family is found in several heterothallic species and one pseudohomothallic species of Neurospora, as well as in one homothallic species of Gelasinospora. The mode of dispersal of these plasmids is not known. Horizontal transmission has proved difficult to demonstrate. Another possibility is transfer by introgression, and this is modelled in the present paper. We have used introgression and subsequent heterokaryosis to successfully transfer the LA-kalilo plasmid from a Haitian strain of Neurospora crassa to the standard Oak Ridge N. crassa background, the LA-kalilo plasmid from the pseudohomothallic Neurospora tetrasperma to N. crassa, and the kalilo plasmid from N. crassa to N. tetrasperma. Thus, introgression is shown to be a possible avenue of dispersal between species. The recipient strains were all senescent but the mechanism of this senescence is not known. It could be caused by the plasmids, but if so the mechanism is novel since plasmid/mtDNA junction fragments of the type found in the standard mode of mtDNA insertion could not be detected. However, mtDNA changes were observed in the senescent recipients. Received: 15 February / 24 June 1999     

The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene

Summary As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to lie in its single G,C rich cluster, which differs in just one nucleotide from the standard ori ^s sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergencies (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.     

Mitochondrial DNA inheritance in sexual crosses of Pleurotus ostreatus

The inheritance of mitochondrial DNA (mtDNA) in sexual crosses was investigated to expand our understanding of the large genetic divergence in mtDNAs among natural populations of the higher basidiomycete Pleurotus ostreatus. Reciprocal crosses were made between compatible monokaryons with distinguishable mtDNA restriction fragment length polymorphisms (PFLPs). Almost all of the dikaryons produced by these crosses had mtDNA genotypes from one of the parental monokaryons. However, for dikaryons isolated from the junction-zone of crossed monokaryons, recombinant mitochondrial genomes commonly appeared. These results showed that P. ostreatus mtDNA can be inherited biparentally, via mtDNA recombination, as well as uniparentally. Further, it was suggested that mtDNA recombination may be an important source of variation in mitochondrial genomes among natural populations of P. ostreatus. Received: 4 June / 14 August 1996     

Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite,Romanomermis culicivorax

Summary Physical characterization of the mitochondrial genome derived from the obligate mosquito parasite, Romanomermis culicivorax has generated some surprising physical properties regarding the molecular structure of nematode mitochondrial DNA (mtDNA). Restriction enzyme analysis of this mtDNA has revealed a mitochondrial genome size of approximately 26 kb, the largest metazoan mtDNA reported to date. Isofemale lineages are monomorphic for one of three size variants, differing by 500-1,000 base pairs, present in our original field population. Cloned hybridization probes derived from a single region exhibiting a 600 by size polymorphism share strong homology with several spatially separated sites distributed about the mtDNA. This suggests that the homology is a result of repeated DNA sequence elements contained within this mitochondrial genome that contribute to mtDNA size polymorphism.     

Heterokaryotic transmission of senescence plasmid DNA in Neurospora

Summary Heterokaryotic transmission is one of the major techniques for the study of cytoplasmic inheritance and here we have applied it to the senescence-determining plasmids kalilo (Hawaiian) and maranhar (Indian). We have shown that kalilo-based senescence is effectively transmitted by cytoplasmic contact, both in N. crassa and in N. intermedia. In the first place, the heterokaryons themselves are senescent, confirming the suppressivity of the senescence phenotype in mixtures of normal and senescent cytoplasms. Second, senescence is found in new nuclear associations, as shown by analysis of conidial isolates and meiocytes stemming from the heterokaryons. In addition, the free plasmid AR-kalDNA, and its form that is inserted into mtDNA, (mtIS-kalDNA), are both transmitted to new nuclear associations. In a transient fusion between senescent N. intermedia and nonsenescent N. crassa cells, AR-kalDNA was transmitted to N. crassa and mtIS-kalDNA was transmitted to N. crassa mtDNA. A cryptic mitochondrial plasmid, not associated with senescence, was also transmitted very efficiently to N. crassa mitochondria. In mixed kalilo/maranhar fusions, both plasmids coexisted, approximately equally, in the heterokaryons themselves, and in conidial isolates. However, in sexual derivatives, AR-marDNA was in an excess and AR-kalDNA was sometimes absent. The efficient heterokaryotic transmission of these elements suggests that this is one of their natural modes of spread in populations.     

Small interspersed sequences that serve as recombination sites at the cox2 and atp6 loci in the mitochondrial genome of soybean are widely distributed in higher plants

Mitochondrial DNA (mtDNA) fragments that contain cox2 and atp6 were cloned from a wild soybean (Glycine soja, accession `B09002') and from a cultivated soybean (G. max, `Harosoy'). Comparison of these DNAs revealed that two sets of repeated sequences, namely, 299 bp and 23 bp, were present in the 5′ regions of cox2 and atp6. The 299-bp and 23-bp repeats were present close to each other on the 5′ flanking region and the 5′ part of the coding region of cox2 in both `Harosoy' and `B09002', as well as on the 5′ flanking region of atp6 in `Harosoy', while these two repeats were separated by a 706-bp nucleotide sequence that contained a truncated sequence of nad3 at the 5′ flanking region of atp6 in `B09002'. The mtDNA configurations upstream from atp6 and cox2 found in `Harosoy' appeared to have been generated from configurations of cox2 and atp6 found in `B09002' via recombination across the 299-bp or 23-bp repeated sequences, or vice versa, in the mitochondrial genome of the hypothetical progenitor of these plants. The 299-bp sequence was found to be interspersed in the mitochondrial genome. Eight loci were identified that include mtDNA configurations that are inter-convertible with each other via recombination across this sequence in `B09002'. Various loci on the mitochondrial genomes of higher plants that harbor segments of the 299-bp repeats in Glycine were identified. Received: 16 August / 15 December 1997     

Interaction between mitochondrial DNA and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the IrRNA-gene

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pC1K1 and pClT5, to the upstream region of the large ribosomal RNA gene in the mtDNA of three strains (W3, T5 and K) has been investigated in detail to explore the widespread phenomenon of homology between mt plasmids and mtDNA in C. purpurea. Sequence comparison indicates that recombination between free plasmids and mtDNA is the cause of the observed homology. The process is similar to the integration of the structurally related adenoviruses into the mammalian genome. As in other fungi, palindromic sequences seem to be involved in this mitochondrial recombination process.     

Evolutionary changes in the structures of the cox2 and atp6 loci in the mitochondrial genome of soybean involving recombination across small interspersed sequences

Mitochondrial DNA (mtDNA) fragments that contained cox2 or atp6 loci were cloned from three accessions of wild soybean (Glycine soja) in order to understand the evolutionary changes of mitochondrial genomes in the genus Glycine subgenus Soja. Cox2 was cloned as a single configuration, while atp6 was cloned as either one or two configurations from each accession. Structural variations were detected in the 5′ upstream region of cox2 and in both the 5′ upstream and 3′ downstream regions of atp6. These variations appeared to be the results of recombination events. A comparison of the mtDNA fragments previously cloned from a cultivated soybean (G. max) and a wild soybean revealed various sites of recombination, as well as various combinations of the 5′ and 3′ regions, at the cox2 and atp6 loci. Some of the cloned fragments were found to contain a set of repeated sequences, namely 299-bp and 23-bp repeats in the 5′ region of cox2 or atp6, which were interspersed in the mitochondrial genome in the subgenus Soja. Recombination events involving the 299-bp or 23-bp repeated sequences were shown to account for the generation of structural variations in the 5′ regions of these loci. Received: 21 March / 4 August 1998     

The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene

Summary In the ascomycete Podospora anserina strain ageing (senescence) is caused by a mitochondrial plasmid. In juvenile mycelia it is an integral part of the mtDNA and becomes liberated during ageing. The nucleotide sequence of this plasmid and of its flanking regions was determined. It consists of 2,539 by and contains an un identified reading frame (URF) originating in the adjacent mtDNA upstream of excision point 1. Within the URF a putative 48 by autonomously replicating sequence (ars) was identified. At both excision sites of the plasmid there are two short nonidentical interrupted palindromes and a few base pairs apart from these palindromes, both upstream and downstream, two short inverted repeats are localised. The experimental data make it evident that the mt plasmid is an intron of the cytochrome c oxidase gene (subunit I) which may be excised at the DNA level and thus become the mobile infective agent causing senescence. The concept of this mobile intron and current hypotheses concerning the relationship between introns and transposons are stressed.