Repair properties in yeast mitochondrial DNA mutators

Summary After ethy1methanesulfonate mutagenesis of the strain Saccharomyces cerevisiae D273-1013, out of 100,000 survivors, 1,000 were selected for their high production of petite mutants at 36 °C. Among these 1,000 mutators, 5 also showed an increased frequency of spontaneous point mutations measured at 25 °C. Further analysis revealed that in all mutators, except 2, petite accumulation proceeded at 25 °C as well as 36 °C. In these 2 mutants, the production of petite mutants was much higher at 36 °C than at 25 °C. In one of them, however, the mutator and the thermosensitive petite phenotypes were due to mutations in two unlinked nuclear genes. In the other mutants, both traits were the result of a mutation in a single nuclear gene. The mutators fell into three complementation groups (tpm1, tpm2, mup1). No complementation was observed between tpm1 mutants and the gam4 mutant previously described by Foury and Goffeau (1979). From the latter and the present works, only four complementation groups (gam1, gam2, gam4 or tpml, mupl) have been identified and it is likely that the number of genes controlling specifically the spontaneous mutability of the mtDNA is low. The mutators exhibited a variety of responses to damaging agents such as UV light and ethidium bromide; especially in a representative mutant from the complementation group tpm1, the induction of ^– mutants was sensitive to UV light and resistant to ethidium bromide. In addition, we found that in the mutants from this complementation group, the synthesis of mtDNA in isolated mitochondria was low; however their mitochondrial DNA polymerase activity was similar to that of the wild type strain. A relationship might exist between the mutator phenotype and the low mtDNA synthesis in the tpm1 mutants.     

Analysis of the functional domains of the mismatch repair homologue Msh1p and its role in mitochondrial genome maintenance

Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function. Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability. In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function. The mechanisms for which it is essential are unclear, however. Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function. Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair. Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability.     

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.     

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.     

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.     

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.     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

Two dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA

Summary Two dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA. Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate. When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found. Identified as mtDNA-encoded were 2 spots for tRNA^leu, 2 for tRNA^met, and 1 each for tRNA^phe, tRNAtrp and tRNA^tyr. The remaining three were unidentified. Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids. These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytostolic enzymes. Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a nontreated control. Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells. ³H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation. The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions. Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs. These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs.     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.     

Mitochondrial DNA of the filamentous ascomycete Cochliobolus heterostrophus

Summary The mitochondrial chromosome of Cochliobolus heterostrophus is a circle approximately 115 kb in circumference, among the largest known from fungi. A physical map of C. heterostrophus mtDNA was constructed using the restriction enzymes BamHI, EcoRI, and PvulI by DNA-DNA hybridizations with cloned or purified mtDNA BamHI fragments. The following sequences were located on the mtDNA map: (1) the large and small mitochondrial ribosomal RNA genes (identified by heterologous hybridization to cloned Neurospora crassa rRNA genes); (2) the sequence homologous to a mitochondrial plasmid present in one field isolate of C. heterostrophus; and (3) a 1.05 kb EcoRI fragment that functions as an autonomously replicating sequence in Saccharomyces cerevisiae. An examination of mtDNA from 23 isolates of C. heterostrophus collected worldwide revealed polymorphisms in restriction enzyme sites. One such polymorphism, coupled with data on a polymorphism in nuclear rDNA, suggests that there are two genetically distinct but geographically overlapping mating populations of C. heterostrophus in the world.     

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.     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.     

The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning,physical mapping and gene location

Summary The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80 500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.     

The mating type-specific homeodomain genes <Emphasis Type="Italic">SXI1α</Emphasis> and <Emphasis Type="Italic">SXI2a</Emphasis> coordinately control uniparental mitochondrial inheritance in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type α (MATα) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type α (MATα)-specific gene SXI1α, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATα parent, and both the MATa and the MATα parents. In addition, progeny from same-sex mating between MATα strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.     

Inheritance of mitochondrial DNA in sexual crosses and protoplast cell fusions in Lentinula edodes

By using mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) as genetic markers, the modes of mitochondrial inheritance in sexual crosses and protoplast cell fusions of the higher basidiomycete Lentinula edodes were examined. All newly established dikaryons from reciprocal crosses between compatible monokaryons carrying different mtDNA RFLP phenotypes retained mtDNA genotypes from one of the monokaryons, suggesting that mitochondrial inheritance is principally uniparental. In contrast, it was shown that recombinant mtDNA genomes arose in some dikaryons obtained after protoplast cell fusion. Based on these results, a possible mechanism for mitochondrial inheritance in L. edodes is discussed.     

Extrachromosomal genetics of Cephalosporium acremonium

Summary In order to facilitate strain improvement by concerted breeding in the cephalosporin producing imperfect filamentous fungus Cephalo sporium acremonium, it is attempted to develop a eukaryotic vector for molecular cloning based on mitochondrial (mt) DNA.Fragments of mtDNA from C. acremonium were inserted into a yeast/bacterial hybrid plasmid (pDAM1) lacking a eukaryotic replicon. Six hybrid plasmids were obtained each containing a different mt fragment which together comprise about 60% of the total mtDNA. One of these recombinant plasmid vectors (pCP2) showed a high replication efficiency which is comparable to that of vectors containing yeast 2 m DNA. This plasmid therefore fulfills the requirements for practical application.     

Maternal inheritance of mitochondrial DNA in sexual crosses of Pythium sylvaticumm

Summary Fifty single oospore progeny from crosses between opposite mating types of Pythium sylvaticum that contained polymorphisms in mitochondrial DNA (mtDNA) for HindIII restriction sites were analyzed for patterns of mitochondrial inheritance. All progeny retained the morphological form of the oogonial parental type; the antheridial form or recombinant forms of parental mtDNA were not detected. With the techniques used, other forms of mtDNA would have been detected it they had comprised 8% or more of the mitochondrial population.