Amphimeric mitochondrial genomes of petite mutants of yeast. II. A model for the amplification of amphimeric mitochondrial petite DNA

  A model for the recombination-directed replication and amplification of the mtDNA of amphimeric petite mutants of S. cerevisiae is proposed. Replication of an amphimeric master basic unit datA would be initiated in the inverted components a and A. The initiation of replication should be associated with the amphimeric structure of the master basic unit itself, but could be promoted by the presence of ori sequences or of sequences facilitating the initiation of replication in the inverted duplications. The amplification unit of amphimeric genomes is considered to be the double-stranded circular hetero-diamphimer datA-DaTA. Amplification of both diamphimeric strands involves an invasion of the 3′ ends of the newly synthesized strands into symmetrical homologous duplex DNA regions promoting the continuation of replication, and leads to the accumulation of two (``flip' and ``flop') types of multi-amphimers. We consider that this mode of amplification represents a modified rolling-circle mechanism. By analogy, we propose to call our model of amplification the ``rocking-circle model'. This model is likely to apply to other genomes organized as amphimeric structures. Received: 2 November 1995     

Amphimeric mitochondrial genomes of petite mutants of yeast. III. Generation by linking two secondary–structure-dependent illegitimate recombination events

The generation of amphimeric mitochondrial petite genomes of yeast can be explained by a process that links together two illegitimate recombination events, each involving a pair of short inverted repeats. Following “diagonal” double-strand breaks and inter-strand ligations at both possible stem-and-loop structures, a subgenomic single-stranded DNA circle can be excised. This circle comprises four building blocks organized in the so-called datA arrangement where d and t correspond, respectively, to the segments looped out by the upstream and the downstream pair of inverted repeats, a to the sequence separating the two loops, and A to the inverted duplication of segment a. Depending on the different possible “diagonal” recombinations at the inverted repeats, any of four isomeric circles can be excised, representing in its double-stranded form the nascent basic unit of an amphimeric mitochondrial petite genome of yeast. These isomeric basic units differ in the relative orientation of their sequences d and t (called D and T, respectively, when inverted), and are designated datA, DatA, daTA, and DaTA. Any one of these may be replicated to form the previously described regularly arrayed multimeric flip-flop genomes. Our new understanding of the amphimeric mitochondrial petite genomes of yeast emphasizes the role that topological features of DNA can play in mitochondrial genome dynamics. It also permits the re-interpretation of various observations reported in the literature. Some of them, including EtBr-mutagenesis in yeast, are discussed. Received: 13 July / 15 October 1998     

The organization of repeating units in mitochondrial DNA from yeast petite mutants

Summary We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ^– petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ^– petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.     

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences

Summary Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho^–) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho^– mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).Abbreviations mtDNA mitochondrial DNA - bp base pairs - kbp kilobase pairs     

Does mitochondrial DNA length influence the frequency of spontaneous petite mutants in yeasts?

Summary Results from the theory of random walks applied to the random excision hypothesis for production of petite mutants in yeast suggest that frequency of excision should increase as a linear function of mitochondrial DNA length (see appendix). For a series of petite positive yeasts we have determined the spontaneous petite frequency (ranging from about 0.003% to 9%) and length of mtDNA (ranging from about 19 Kbp to c. 108 Kbp) and found that, while the frequency of petite mutants does generally increase with mtDNA length, the relationship is far from linear. Although these results are inconclusive concerning the random excision hypothesis they do indicate that amongst related yeasts other factors have a greater influence than mtDNA length in determining the frequency of petite mutants.     

Supersuppressive “petite” mutants of yeast

Summary A class of suppressive petite mutants of S. cerevisiae, called here supersuppressive, is characterized by a) the fact that their unmodified mitochondrial genomes are the only ones found in the progeny of crosses with wild-type cells; b) very short repeat units (400–900 base pairs) in their mitochondrial genomes. The repeat units of the three supersuppressive petites investigated here share a common 83 nucleotide sequence, which seems to correspond to an initiation site of DNA replication; the multiple copies of this site in the mitochondrial genomes of supersuppressive petites might explain why these genomes can compete out those of wild-type cells.     

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.     

Effect of ethidium bromide on transmission of mitochondrial genomes and DNA synthesis in the petite negative yeast Schizosaccharomyces pomhe

Summary Treatment of haploid strains of the petite negative yeast Schizosaccharomyces pomhe with ethidium bromide prior to mating with untreated cells reduces transmission of mitochondrial markers from the treated strains. This effect is fully reversible after 20 generations of growth in drug free medium before mating. In contrast to the petite positive yeast Saccharomyces cerevisiae, where nuclear DNA synthesis is not affected but mitochondrial DNA is degraded in the presence of 20 g/ml ethidium bromide, the same concentration decreases both nuclear and mitochondrial DNA synthesis in Schizosaccharomyces pomhe. After removal of the drug, nuclear DNA synthesis increases faster than its mitochondrial counterpart in Schizosaccharomyces pomhe.     

Effects of T-2 toxin on induction of petite mutants and mitochondrial function in Saccharomyces cerevisiae

Summary The influence of the trichothecene mycotoxin T-2 on the mitochondria of Saccharomyces cerevisiae was studied. T-2 is a cytotoxic molecule inhibiting growth and macromolecular synthesis in S. cerevisiae. At low concentrations, T-2 toxin arrested yeast growth on glycerol medium and at higher concentrations, it arrested growth on glucose medium. The toxin was not capable itself of inducing petite mutations. Its inhibitory effect on the growth of petite strains, of both chromosomally isogenic and nonisogenic strains was less than that of grande strains. One exception to this was equally low susceptibility of psi ⁺ SUP4-3 strain in both rho ⁺ and rho ^– state. T-2 toxin was also capable of retarding the petite inducing activity of the mutagen, ethidium bromide. T-2 toxin inhibited the polymerization of P-ribosylaminoimidazole in an ade2 strain of S. cerevisiae. These results show that T-2 toxin is capable of interfering with the activity of the mitochondria in addition to its well studied effects on cytoplasmic protein synthesis.     

Starvation for a specific amino acid induces high frequencies of rho− mutants in Saccharomyces cerevisiae

Auxotrophic yeast cells were starved on solid media for their respective essential amino acid in the course of “adaptive mutation” experiments. Thereby, high proportions of mitochondrially respiratory deficient (rho⁻) mutants accumulated among the cells stressed on selective plates. Using a strain with a plus-four frameshift mutation in a chromosomal gene involved in lysine biosynthesis, we observed that many of the revertant colonies which arose late under the selective pressure were composed of mixtures of rho⁺ and rho⁻ cells, indicating that they originated from founder cells containing intact as well as defective mitochondrial genomes. We show that in spite of the slower growth of rho⁻ cells the late-appearing colonies cannot be interpreted as descending from rho⁻ revertants present before selective plating. Received: 7 November 1996 / 2 February 1997     

A controversy concerning the structure of the mitochondrial genome of a yeast petite mutant: resolution by sequence analysis

Summary Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho ^–) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 by segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).     

Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants

 We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2Δ and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Received: 13 October/27 November 1995     

Temperature-sensitive nuclear mutants of Neurospora crassa deficient in mitochondrial ribosomes

Summary We have isolated two non-allelic nuclear mutants of Neurospora crassa that are temperature-sensitive for the production of cytochromes aa ₃ and b. When grown at 23 °C the mutants are virtually indistinguishable from the parent wild-type strains. When grown at 41 °C the mutants have large amounts of KCN-insensitive respiration and lack cytochromes aa ₃ and b. Further examination of the mutants revealed that they were extremely deficient in their capacity for mitochondrial protein synthesis when grown at 41 °C. This protein synthesis deficiency appears to be related to a virtual absence of both small and large mitochondrial ribosomal subunits following growth at 41 °C. Examination of the mitochondrial RNAs of the mutants suggests that mitochondrial rRNAs are synthesized in greatly reduced amounts or that they are misprocessed when these mutants are grown at the non-permissive temperature.Abbreviations mt mitochondrial - SDS sodium dodecylsulfate - EDTA ethylenediaminetetraacetic acid - CAP chloramphenicol - TCA trichloroacetic acid - SHAM salicylhydroxamic acid     

Further characterization of a large inverted repeat in the mitochondrial genomes of Agaricus bisporus (=A. brunnescens) and related species

The mitochondrial (mt) genome of Agaricus bisporus Ag50 (a heterokaryon) is a 136-kilobase (kb) circular molecule which contains a pair of large inverted repeats (IRs). Two large BAMHI fragments (B1 and B2) which contain the IR regions were further mapped. The repeated regions were determined to be approximately 7.7 kb in length. The mt small ribosomal RNA (S rRNA) gene is located adjacent to one of the repeated regions. Orientational isomers, generated by homologous recombination between the repeated regions, were not observed in mtDNA extractions from Ag50 mycelium (liquid culture) or from Ag50 fruit bodies. We also did not observe any orientational isomers in Ag50HA or Ag50HB, two homokaryons somatically isolated from Ag50. DNA homologous to the Ag50 mt repeated regions was observed in ten other isolates of Agaricus including four isolates of A. bisporus, two isolates of A. subperonatus, two isolates of A. subfloccosus, one isolate of A. bitorquis, and one isolate of A. pattersonae. The repeated regions and the small unique regions in two other heterokaryotic strains of A. bisporus, Ag2 and Ag85, were physically mapped. The repeated regions in these two strains are also in the inverted forms. Restriction endonuclease mapping indicated that the two copies of the IR in Ag85 were not identical.     

Effect of the underwater torque on the energy cost,drag and efficiency of front crawl swimming

Underwater torque (T′) is defined as the product of the force with which the swimmer's feet tend to sink times the distance between the feet and the centre of volume of the lungs. It has previously been shown that experimental changes ofT′, obtained by securing around the swimmer's waist a plastic tube filled, on different occasions, with air, water or 2-kg lead, were accompanied by changes in the energy cost of swimming per unit of distance (C_S) at any given speed. The aim of this study was to investigate whether the observed increases of C_S withT′ during front crawl swimming were due to an increase of active body drag (D_b), a decrease of drag efficiency (η_d) or both. The effect of experimental changes ofT′ on C_S, D_b and η_d were therefore studied on a group of eight male elite swimmers at two submaximal speeds (1.00 and 1.23 m · s⁻¹). To compare different subjects and different speeds, the individual data for C_S, D_b,η_d andT′ were normalized dividing them by the corresponding individual averages. These were calculated from all individual data (of C_S, D_b, η_d andT′) obtained from that subject at that speed. It was found that, between the two extremes of this study (tube filled with air and with 2-kg lead),T′ increased by 73% and that C_S, D_b and η_d increased linearly withT′. The increase of C_S between the two extremes was intermediate ( ≈ 20%) between that of D_b (≈ 35%) and of η_d ( ≈ 16%). Thus, the actual strategy implemented by the swimmers to counteractT′, was to tolerate a large increase of D_b. This led also to a substantial (albeit smaller) increase of did, the effect of which was to reduce the increase of C_S that would otherwise have occurred.     

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.     

Sequences and gene organization of the mitochondrial genomes of the liver flukes Opisthorchis viverrini and Clonorchis sinensis (Trematoda)

Opisthorchis viverrini and Clonorchis sinensis are important trematodes infecting humans and animals, belonging to the family Opisthorchiidae. In the present study, we sequenced the nearly complete mitochondrial (mt) DNA (mtDNA) sequences of O. viverrini from Laos, obtained the complete mtDNA sequences of C. sinensis from China and Korea, and revealed their gene annotations and genome organizations. The mtDNA sequences of O. viverrini, C. sinensis (China isolate), C. sinensis (Korea isolate) were 13,510, 13,879, and 13,877 bp in size, respectively. Each of the three mt genomes comprises 36 genes, consisting of 12 genes coding for proteins, two genes for rRNA, and 20 genes (O. viverrini) or 22 genes (C. sinensis) for tRNA. The gene content and arrangement are identical to that of Fasciola hepatica, and Paragonimus westermani, but distinct from Schistosoma spp. All genes are transcribed in the same direction and have a nucleotide composition high in T. The contents of A + T of the mt genomes were 59.39% for O. viverrini, 60.03% for C. sinensis (China isolate), and 59.99% for C. sinensis (Korea isolate). Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms [maximum parsimony, maximum likelihood, and Bayesian analysis], all revealed distinct groups with high statistical support, indicating that O. viverrini and C. sinensis represent sister taxa. These data provide additional novel mtDNA markers for studying the molecular epidemiology and population genetics of the two liver flukes and should have implications for the molecular diagnosis, prevention, and control of opisthorchiasis and clonorchiasis in humans and animals.     

The in vivo effect of acriflavine on mitochondrial functions in the petite negative yeast Hansenula saturnus

Summary In the petite negative yeast Hansenula saturnus, acriflavine determined a decrease of cell yield and of the total Q_O2,the disappearance of the cytochromes aa ₃ and b and the inhibition of in vivo mitochondrial protein synthesis without affecting the cell survival. The restriction enzymes analysis of mitDNA shows that no specific fragmentation occurred after acriflavine treatment.     

The mitochondrial DNA of the yeast Hansenula petersonii: genome organization and mosaic genes

Summary The mitochondrial genomes of yeasts are circular DNA molecules that vary greatly in size in different species. The mitochondrial DNA of the yeast H. petersonii is about 42 kbp in length, about one half the size of the corresponding genome in S. cerevisiae. Sequences homologous to protein-encoding genes from S. cerevisiae have been identified and localized on this genome by hybridization with DNA from petite mutants. The comparison between the mitochondrial genomes of H. petersonii and S. cerevisiae showed differences in the overall genome organization, but both include genes with mosaic organization. In fact, sequences homologous to the first intron of the S. cerevisiae cob short gene are found in (or adjacent to) the cob and cox1 genes present in the genome of H. petersonii. Moreover, an intron homologous to that present in the 21S rRNA gene of S. cerevisiae seems to have been conserved in the large ribosomal RNA gene of H. petersonii, in a similar position.     

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.