Plasmids of mitochondrial origin in senescent mycelia of Podospora curvicolla

Summary Podospora curvicolla displays symptoms of senescence similar but not quite identical to those reported for Podospora anserina. In Podospora curvicolla single hyphae may escape from death leading to a new growth front and consequently to a mode of growth characterized by alternating phases of growth and non-growth. Restriction analyses and hybridization experiments have revealed that the Podospora curvicolla type of senescence is correlated with plasmids originating from amplification of a single distinct region of the mitochondrial DNA containing the IrRNA gene. In the yeast transformation system sequences of this region may function as autonomously replicating sequences (ARS). Plasmids (pl1, pl2 and pl3) isolated from different, independently aged mycelia are largely homologous to each other but differ in their excision/junction sites and have different sizes: 10.85 kb (pl1), 9.01 kb (p12) and 10.50 kb (pl3). The sequence of the most frequently occurring plasmid in ageing strains of Podospora anserina is absent in Podospora curvicolla either as free plasmid DNA or as an integrated part of the mtDNA. Possibly there is a correlation between the absence of this particular sequence in Podospora curvicolla and the type of senescence displayed in this organism.     

The kalilo linear senescence-inducing plasmid of Neurospora is an invertron and encodes DNA and RNA polymerases

Summary The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by intergrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA; (2) small imperfect palindromes that are situated at the termini of the plasmid and are cognate with the active sites for plasmid integration into mtDNA; (3) two large, non-overlapping open-reading frames, ORF-1 and ORF-2, which are located on opposite strands of the plasmid and potentially encode RNA and DNA polymerases, respectively, and (4) a set of imperfect palindromes that coincide with similar structures that have been detected at more or less identical locations in the nucleotide sequences of other linear mitochondrial plasmids. The nucleotide sequence does not reveal a distinct gene that codes for the protein that is attached to the ends of the plasmid. However, a 335-amino acid, cryptic, N-terminal domain of the putative DNA polymersse might function as the terminal protein. Although the plasmid has been co-purifed with nuclei and mitochondria, its nucleotide composition and codon usage indicate that it is a mitochondrial genetic element.     

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.     

Despite mtDNA polymorphism the mobile intron (plDNA) of the COI gene is present in ten different races of Podospora anserina

Summary Polymorphism of mitochondrial DNA (mtDNA) from ten different geographical races of Podospora anserina was characterized by means of restriction and hybridization analysis. Our data reveals that the mobile intron (plDNA), first characterized by DNA sequencing in strain s (Osiewacz and Esser 1984), is present in all of the races investigated. Therefore, we conclude that the mobile intron is not an optional intron of the COI gene in wild type strains. In addition, the detailed physical and genetical mapping of mtDNA from three different races with different lifespans leads us to propose that length polymorphism can result due to the lack or presence of intron sequences of structural mitochondrial genes. In view of our results we discuss the function of the mobile intron during senescence in P. anserina.     

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.     

Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron

Summary Recently, the nucleotide sequences for three mitochondrial plasmids associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian URF1 gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.     

Anatomy of amplified mitochondrial DNA in “Ragged” mutants of Aspergillus amstelodami: Excision points within protein genes and a common 215 bp segment containing a possible origin of replication

Summary The extranuclearly-inherited ragged growth phenotype (Rgd) of Aspergillus amstelodami is always accompanied by excision and head-to-tail amplification of mtDNA sequences. In one mutant strain (Rgd1) the amplified mtDNA segment (rgd1 DNA, monomeric length 0.9 kb) maps downstream of the large subunit ribosomal RNA gene (Region 1), whereas in all other strains analyzed the amplified sequences (rdg3-7DNA) are located in Region 2 between genes coding for cytochrome b and ATPase subunit 6. The various region 2 sequences differ in lengths (1.5 to 2.7 kb) but have in common a 215 bp sequence mapping between an. unidentified protein gene (corresponding to URF4 of human mtDNA) and an arginine tRNA gene. This common sequence may contain an origin of replication, because a looped-out hairpin structure similar to that of yeast and human mitochondrial origin sequences can be formed. Furthermore, Region 2 DNA suppresses replication of Region 1 DNA, indicating that the former group of molecules contains the more efficient origin. The nucleotide sequence of the rgd6 repeat unit starts and ends within protein genes of mtDNA, and no homologies were found between heads and tails or their flanking sequences.Abbreviations mtDNA DNA isolated from DNase — treated mitochondria - Rgd ragged mutant strain - rgdDNA highly-reiterated DNA sequences isolated simultaneously with the wild-type genome from mitochondria of ragged mutants - bp base pairs - kb kilobase pairs - URF unassigned reading frame     

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.     

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.     

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.     

A potential impact of DNA repair on ageing and lifespan in the ageing model organism Podospora anserina: Decrease in mitochondrial DNA repair activity during ageing

The free radical theory of ageing states that ROS play a key role in age-related decrease in mitochondrial function via the damage of mitochondrial DNA (mtDNA), proteins and lipids. In the sexually reproducing ascomycete Podospora anserina ageing is, as in other eukaryotes, associated with mtDNA instability and mitochondrial dysfunction. Part of the mtDNA instabilities may arise due to accumulation of ROS induced mtDNA lesions, which, as previously suggested for mammals, may be caused by an age-related decrease in base excision repair (BER).Alignments of known BER protein sequences with the P. anserina genome revealed high homology. We report for the first time the presence of BER activities in P. anserina mitochondrial extracts. DNA glycosylase activities decrease with age, suggesting that the increased mtDNA instability with age may be caused by decreased ability to repair mtDNA damage and hence contribute to ageing and lifespan control in this ageing model.Additionally, we find low DNA glycosylase activities in the long-lived mutants grisea and ΔPaCox17::ble, which are characterized by low mitochondrial ROS generation.Overall, our data identify a potential role of mtDNA repair in controlling ageing and life span in P. anserina, a mechanism possibly regulated in response to ROS levels.     

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.     

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.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

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.     

A Short Insert in the Leader Sequence of RNA 3L,A Long Variant of <Emphasis Type="Italic">Alfalfa mosaic virus</Emphasis> RNA3, Introduces Two Unidentified Reading Frames

N20-RNA 3L, a large form of RNA 3 associated with Alfalfa mosaic virus (AMV) strain N20 comprises 2281nt and has approximately 97% overall sequence similarity to the longest previously described RNA 3 of AMV strain YSMV (YSMV-RNA 3; 2188nt). Compared with YSMV-RNA 3, N20-RNA 3L contains an additional 97nt in the 5' leader upstream of the open reading frames for movement protein (MP) and coat protein (CP). Two overlapping unidentified reading frames (URF1 and URF2) result from this modification, each of which code for putative translation products of 21 amino acids. The URF1 putative peptide has a hydrophilic N-terminus and a hydrophobic C-terminus, indicating a possible association with both host cell membrane and cytosol whereas the putative URF2 product is predominantly hydrophobic. A further structural modification found in N20-RNA 3L is a new tandem repeat of 243nts which overlaps with the MP open reading frame.     

DNA sequence and organization of the mitochondrial ND1 gene from Podospora anserina: analysis of alternate splice sites

Summary Earlier, we reported that the ND1 mitochondrial gene of Podospora anserina is mosaic, containing at least three class I introns. We have now completed the sequence of the ND1 gene and have determined that it contains four class I introns of 1,820, 2,631, 2,256 and 2,597 by with the entire gene complex containing 10,505 bp, only 1,101 of which are exon sequences. Introns 1 and 3 appear to be related in that their open reading frames (ORFS) exhibit extensive amino acid sequence similarity and like the URFN sequence from Neurospora crassa have multiple sequence repetitions. Introns 2 and 4 are similar in that both appear to be mosaic introns. Where intron 2 has many short ORFS, intron 4 has two, 391 and 262 as respectively. The first ORF has some patch work sequence similarity with one of the intron 2 ORFs but the second ORF is strikingly similar to the single intron ORF in the ND1 gene of N. crassa. Just upstream of the sequences necessary to form the central core of the P. anserina intron 4 secondary structure, there is a 17 bp sequence which is an exact replica of the exon sequence abutting the 5 flank of the 1,118 by N. crassa ND1 intron. Secondary structure analysis suggests that the 2,597 by intron 4 can fold as an entity but a similar structure can be constructed just from an 1,130 bp portion by utilizing the 17 bp element as an alternate splice site. Detailed structural analysis suggests that intron 4 (as well as the single ND1 intron from N. crassa) can utilize helical configurations which bring the downstream open reading frame into juxtaposition with the exon sequences.     

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.