Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

Complex terminal structure of a linear mitochondrial plasmid from Physarum polycephalum: three terminal inverted repeats and an ORF encoding DNA polymerase

The mitochondria of Physarum polycephalum have a linear plasmid (mF) which promotes mitochondrial fusion. To determine the terminal structure of the mF plasmid, restriction fragments derived from its ends were cloned and sequenced. The sequences showed that the mF plasmid has three kinds of terminal inverted repeats (TIRs). The most characteristic feature is a 144-bp repeating unit which exists between a 205-bp TIR at the extreme ends of the plasmid and another 591-bp TIR. All of the clones showed at least one of these 144-bp repeating units. The GC content of the 205-bp TIR (49%) was higher than those of the other TIRs and of another sequenced region (23%). This TIR can form three thermodynamically-stable hairpin structures based on complex internal palindromic components. Moreover, in the right terminal region of the mF plasmid, there is an open reading frame (ORF) which covers the entire 591-bp TIR and most of one of the 144-bp repeating units. This ORF encodes a 547-amino-acid polypeptide, ORF-547, and shows extensive homology with the polymerization domain of the putative DNA polymerases of linear mitochondrial plasmids from other sources.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Evidence for giant linear plasmids in the ascomycete Podospora anserina

In the extrachromosomal mutant AL2 of the ascomycete Podospora anserina longevity is correlated with the presence of the linear mitochondrial plasmid pAL2-1. In addition to this autonomous genetic element, two types of closely related pAL2-1-homologous molecules were detected in the high-molecular-weight mitochondrial DNA (mtDNA). One of these molecules is of linear and the other of circular structure. Both molecules contain pAL2-1 sequences which appear to be integrated at the same site in the mtDNA. Sequence analysis of a DNA fragment cloned from one of these molecules revealed that it contains an almost full-length copy of pAL2-1. At the site of plasmid integration a 15-nucleotide AT-spacer and long inverted mtDNA sequences were identified. Finally, two giant linear plasmid-like DNAs of about 50 kbp and 70 kbp were detected in pulsed-field gels of mutant AL2. These molecules are composed of mtDNA and pAL2-1-specific sequences and may result from the integration of mtDNA sequences into linear plasmid pAL2-1.     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

Palindrome content of the yeast Saccharomyces cerevisiae genome

Palindromic sequences are important DNA motifs involved in the regulation of different cellular processes, but are also a potential source of genetic instability. In order to initiate a systematic study of palindromes at the whole genome level, we developed a computer program that can identify, locate and count palindromes in a given sequence in a strictly defined way. All palindromes, defined as identical inverted repeats without spacer DNA, can be analyzed and sorted according to their size, frequency, GC content or alphabetically. This program was then used to prepare a catalog of all palindromes present in the chromosomal DNA of the yeast Saccharomyces cerevisiae. For each palindrome size, the observed palindrome counts were significantly different from those in the randomly generated equivalents of the yeast genome. However, while the short palindromes (2–12 bp) were under-represented, the palindromes longer than 12 bp were over-represented, AT-rich and preferentially located in the intergenic regions. The 44-bp palindrome found between the genes CDC53 and LYS21 on chromosome IV was the longest palindrome identified and contained only two C-G base pairs. Avoidance of coding regions was also observed for palindromes of 4–12 bp, but was less pronounced. Dinucleotide analysis indicated a strong bias against palindromic dinucleotides that could explain the observed short palindrome avoidance. We discuss some possible mechanisms that may influence the evolutionary dynamics of palindromic sequences in the yeast genome.     

Mitochondrial DNA variability detected in a single wheat regenerant involves a rare recombination event across a short repeat

The mitochondrial genome of the selfed progeny of a plant regenerated from long-term somatic tissue culture displays specific structural rearrangements characterized by the appearance of novel restriction fragments. A mitochondrial DNA library was constructed from this selfed progeny in the SalI site of cosmid pHC79 and the novel fragments were subsequently studied. They were shown to arise from reciprocal recombination events involving DNA sequences present in the parental plant. The regions of recombination were sequenced and the nucleotide sequences were aligned with those of the presumptive parental fragments. We characterized an imperfect short repeated DNA sequence, 242 bp long, within which a 7-bb DNA repeat could act as a region of recombination. The use of PCR technology allowed us to show that these fragments were present in both parental plants and tissue cultures as low-abundance sequence arrangements.     

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.     

Terminal structure of a Densovirus implies a hairpin transfer replication which is similar to the model for AAV

We cloned the complete sequence of Bombyx DNV (Ina isolate; Bm DNV-1) genome in a bacterial plasmid pUC 119 and determined the nucleotide sequences of both termini, resulting in elucidation of the nucleotide sequence of the complete genomic DNA of DNV. The complete sequence of the DNV DNA (5048 nucleotides) has inverted repeats of 225 nucleotides and the terminal 153 nucleotides are palindromic. The palindromes can fold back on themselves to form a hairpin structure but, unlike AAV, the small internal palindrome which forms a T-shaped conformation was not observed. End-label analysis demonstrated that the palindromic sequences at both termini can exist in either of two orientations (flip or flop) in virion DNA with different frequencies. These data suggest that the hairpin transfer model for AAV replication must be modified to explain the DNV replication. Additionally, a comparison study on the terminal structures of insect, human, and rodent parvoviruses allowed a prediction on the ancestral terminal structure of parvovirus genome.     

Linear mitochondrial genome organization in vivo in the genus Pythium

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.     

R-type plasmids in mitochondria from a single source of Zea luxurians teosinte

Two linear DNA plasmids resembling the R1 and R2 plasmids that are present in the mitochondria of several South American strains of maize were found in mitochondria from a single source of Zea luxurians collected by L. Mazoti. The Mazoti mtDNA is closely related to mtDNAs of other Z. luxurians, but mitochondria derived from the other Z. luxurians sources lack the plasmids. The larger plasmid from Mazoti mitochondria, M1, was cloned and large portions of it were sequenced. Restriction mapping and sequence comparisons showed that approximately 4.9 kb is similar to the S1 plasmid of maize and an additional 2.6 kb is related to R1 sequences integrated into the main mitochondrial genome of N cytoplasm. Therefore, the M1 plasmid appears to be very similar to the R1 plasmid. The inverted repeats at the ends of the M1 plasmid are not identitical. The left end IR is similar to the S-TIRs found at the termini of the S plasmids. The right end IR more closely resembles the integrated R1 sequences, including the variant region of the TIR. Whereas the variant region contains 13 bp in the S-TIRs and 15 bp in an integrated version of R1, it is 16 bp long in M1. The region of M1 that has no homology to the S1 plasmid is expressed at very low levels in Mazoti and RU cytoplasms, but at much higher levels in CMS-S mitochondria, where part of it is present in the main mitochondrial genome.