Second-site antibiotic resistance mutations in the ribosomal region of yeast mitochondrial DNA

Summary A large proportion of the spontaneous erythromycin resistant mutants isolated from a strain carrying a previously-induced chloramphenicol resistance mutation at cap3 do not map at ery1, the locus most often associated with mitochondrial erythromycin resistance. Most of the new mutations are also nonallelic at spil, spi2, and other known antibiotic resistance loci within the 21S rRNA gene; they are allelic with each other and define the new locus, ery2. Induced second-site erythromycin resistant mutants from the cap ^r3 strain, as well as spontaneous or induced mutants from strains carrying a cap ^r 1 mutation, all tend to map at eryl. The cap ^r3 mutation is apparently necessary for the expression of erythromycin resistance resulting from a second mutation at ery2.     

Recombination of yeast mitochondrial DNA does not require mitochondrial protein synthesis

Summary Mitochondrial genes recombine extensively in yeast zygotes. In heteropolar crosses (⁺ × ^–) in which the ^– allele consists of an insertion, there is preferential recovery of ⁺ and markers closely linked to it. This polarity has been postulated to be a consequence of one-way gene conversion beginning at the locus (- to ⁺). We have shown that most or all mitochondrial recombination in homopolar and heteropolar crosses, and the phenomenon of polarity itself, does not require products of protein synthesis on mitochondrial ribosomes. (i) Yeast strains were grown and mated, and the zygotes plated and grown, on glucose medium with erythromycin to inhibit and dilute out the products of mitochondrial protein synthesis. Recombination frequencies and polarity at the cap1 and oli1 loci were normal compared to controls in some homopolar (⁺ × ^–) and heteropolar crosses. Apparent changes in recombination frequencies and polarity were seen in other crosses but are attributable to locus-specific petite induction by erythromycin. (ii) Homopolar (⁺ × ⁺) and heteropolar crosses between pairs of petite mutants retaining the cap1, ery1, and oli1 loci also showed nearly normal recombination at the cap1 and oli1 loci, as determined by test-crossing the petite progeny. The petite mutants and zygotes cannot do mitochondria) protein synthesis. These results support the recombinational model of polarity.     

A mitochondrial frameshift suppressor maps in the tRNASer-var1 region of the mitochondrial genome of the yeast S. cerevisiae

Summary A polypeptide chain-terminating mutation (M5631) previously has been shown to be a +1T insertion in the yeast mitochondrial gene oxi1, coding for subunit II of the cytochrome c oxidase. A spontaneously arisen frameshift suppressor (mfs-1) that is mitochondrially inherited suppresses this mutation to a considerable extent. The suppressor mutation was mapped by genetic and molecular analyses in the mitochondrial tRNA^Ser-var1 region of the mitochondrial genome of the yeast S. cerevisiae. Genetic analyses show that the suppressor mfs-1 does not suppress other known mitochondrial frameshift mutations, or missense and nonsense mutations.     

Isolation of a nuclear yeast gene involved in the mitochondrial import of cytoplasmically synthesized precursor proteins

Summary Several nuclear mutants of the yeast, Saccharomyces cerevisiae, have been characterized which synthesize only the higher-molecular weight precursor but not the mature subunit VI of the mitochondrial ubiquinol cytochrome c oxidoreductase. The mutants belong to different complementation groups and vary in the extent of their being simultaneously deficient in other components of the mitochondrial inner membrane. From a yeast genomic DNA library the plasmid pTS2326 was isolated which complements the defect in one of these mutants, ts2326. The cloned DNA fragment, 2.3 kilobases in length, was sequenced. It contains two open reading frames, ORF1 and ORF2, consisting of 723 and 417 base pairs, respectively. By selective deletion of either reading frame it was shown that only ORF1 containes the information necessary to complement the ts2326 mutation. The ORF1 coding sequence is not the structural gene of subunit VI. The postulated gene product of ORF1 has a molecular weight of 27.114 daltons. It exhibits several sequence characteristics typical of proteins which are internalized by the mitochondrial membrane systems. It is proposed that ORF1 is involved in the import and processing of cytoplasmically synthesized mitochondrial precursors.Abbreviations ts temperature-sensitive - YEP yeast extract/peptone/sucrose media - ORF open reading frame - bc ₁-complex ubiquinol cytochrome c oxidoreductase Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Mechanisms of mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae

The transfer of organelle nucleic acid to the nucleus has been observed in both plants and animals. Using a unique assay to monitor mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae, we previously showed that mutations in several nuclear genes, collectively called yme mutants, cause a high rate of mitochondrial DNA escape to the nucleus. Here we demonstrate that mtDNA escape occurs via an intracellular mechanism that is dependent on the composition of the growth medium and the genetic state of the mitochondrial genome, and is independent of an RNA intermediate. Isolation of several unique second-site suppressors of the high rate of mitochondrial DNA-escape phenotype of yme mutants suggests that there are multiple independent pathways by which this nucleic acid transfer occurs. We also demonstrate that the presence of centromeric plasmids in the nucleus can reduce the perceived rate of DNA escape from the mitochondria. We propose that mitochondrial DNA-escape events are manifested as unstable nuclear plasmids that can interact with centromeric plasmids resulting in a decrease in the number of observed events. Received: 21 April / 7 June 1999     

Partial pedigree analysis of the segregation of yeast mitochondrial genes during vegetative reproduction

Summary A three-factor cross of Saccharomyces cerevisiae involving the cap1, ery1, and oli1 loci was done, with partial pedigree analyses of 117 zygotes. First, second, and third buds were removed and the genotypes of their diploid progeny determined, along with those of the residual zygote mother cell. Results were analyzed in terms of frequencies of individual alleles and of recombinant genotypes in the dividing cells. There is a gradual increase in the frequency of homoplasmic cells and in gene frequency variance during these three generations, as would result from stochastic partitioning of mtDNA molecules between mother and bud, probably coupled with random drift of gene frequencies in interphase cells. These phenomena are more pronounced for buds than for mothers, suggesting that buds receive a smaller sample of molecules. End buds are more likely to be homoplasmic and have a lower frequency of recombinant genotypes than do central buds; an end bud is particularly enriched in alleles contributed by the parent that formed that end of the zygote. Zygotes with first central buds produce clones with a higher recombination frequency than do those with first end buds. These results confirm previous studies and suggest that mixing of parental genotypes occurs first in the center of the zygote. If segregation were strictly random, the number of segregating units would have to be much smaller than the number of mtDNA molecules in the zygote. On the other hand, there is no evidence for a region of the molecule (attachment point) which segregates deterministically.     

Partial mitochondrial DNA sequence of the crustaceanDaphnia pulex

A 3667-base pair (bp) fragment of the mitochondrial genome of the crustaceanDaphnia pulex has been sequenced and found to contain the complete genes for the small subunit ribosomal RNA, ND2, seven tRNAs and the control region. This organization is identical to that found inDrosophila yakuba mtDNA yetD. pulex mtDNA exhibits several unique features when compared to other mitochondrial sequences. The sequenced fragment is only 62.6% A+T which is much lower than that of any other arthropod mtDNA sequenced to date.D. pulex mtDNA also exhibits length conservation having shorter coding and non-coding regions. The putative control region is 689 bp in length and includes a sequence that has the potential to fold into a hairpin structure with a perfect 20-bp pair stem and a 22-base loop.     

Maintenance of chloroplast-derived sequences in the mitochondrial DNA of Gramineae

Evidence for the transfer of DNA from the chloroplast to the mitochondrion has been reported in many higher plants and, in most cases, the transferred chloroplast genes do not have the ability to encode functional products as a consequence of base substitutions and/or multiple rearrangements. We reported previously that the sequence of one end of a chloroplast-derived (ct-derived) fragment of DNA that contained the rps19 and trnH genes has been maintained in most gramineous plants and that its presence seems to be correlated with gene expression in this region. In the present study, we have investigated whether or not the ct-derived sequences in mitochondrial DNA (mtDNA) from some gramineous plants and species of Oryza are conserved, and whether or not such conservation is related to gene expression in these regions. We identified two junctions between ct-derived and mitochondrial sequences that were conserved among some gramineous plants. Around these regions, we found a ct-derived gene for tRNA and the promoter of a mitochondrial gene on the ct-derived sequences, respectively, and these regions were transcribed through the junctions. This result indicates that the junctions and/or regions that are transcribed and functional in mitochondria have been strongly conserved and maintained during their evolution. In Oryza, some junctions between ct-derived and mitochondrial sequences were conserved and other junctions were not. These variations seem to have been caused by deletions and/or rearrangements, and appear to be specific to the type of genome. In the case of Oryza, the timing of deletions and/or rearrangements of ct-derived sequences is likely to have coincided with the divergence of the various genome types. Received: 24 July / 13 August 1997     

Preparation of yeast mitochondrial DNA for direct sequence analysis

We describe two simple protocols for preparation of templates for direct sequencing of yeast mitochondrial DNA (mtDNA) by automatic DNA analyzers. The protocols work with a range of yeast species and yield a sufficient quantity and quality of the template DNA. In combination with primer-walking strategy, they can be used either as an alternative or a complementary approach to shot-gun sequencing of random fragment DNA libraries. We demonstrate that the templates are suitable for re-sequencing of the mtDNA for comparative analyses of intraspecific variability of yeast strains as well as for primary determination of the complete mitochondrial genome sequence.     

Nuclear suppressors of mitochondrial chloramphenicol resistance in Baker's yeast: their use for the isolation of novel mutants

Summary Strains that are genotypically sensitive to chloramphenicol and also contain one of the nuclear suppressors of mitochondrial chloramphenicol resistance (Waxman et al. 1979) were constructed. A manganese mutagenesis on such a strain produced chloramphenicol resistant mutants, most of which resulted from mutations in nuclear genes. These mutants may be either dominant or recessive, and they probably do not code for membrane proteins. The few mitochondrial mutants fall into several classes, but all result from mutations in the 21S rRNA gene. The suppressor allele effectively prevents the appearance of the most common group of mitochondrial mutants (those that map at cap1), and thereby enhances the selection of novel mutants in the region.     

A mitochondrial frameshift-suppressor (+) of the yeast S. cerevisiae maps in the mitochondrial 15S rRNA locus

Summary The first case of a +1 extrageneic frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported. The suppressor was identified by genetic analyses in a leaky mitochondrial oxi1 frameshift mutant and the respective wild-type strain 777-3A of the yeast S. cerevisiae. This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree. MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A. These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al. 1984).     

Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region

We studied the influence of homology size and polymorphism on the integration of circular plasmids into the yeast CYC1 region. The plasmids used also contained the URA3 gene, and the proportion of Ura⁺ transformants resulting from plasmid integration into the CYC1 region was determined by Southern-blot analysis. A size-dependent decrease in integration into the CYC1 region was observed from 858 bp to 363 bp of homology. However, with a homology size of 321, 259 or 107 bp, about 2% of the transformants still contained plasmid molecules integrated in the CYC1 region. A single point mutation in the 858-bp fragment decreased the proportion of integrations to the CYC1 gene, but the presence of additional mutations did not have a cumulative effect. For plasmids isolated in a single-stranded (ss) form, the presence of two or six point mutations did not influence integration. These results were compared with those obtained in other assays designed to study substrate requirements for homologous recombination. Received: 18 October / 15 December 1999     

Amphimeric mitochondrial genomes of petite mutants of yeast. I. Flip-flop amphimers make up the mitochondrial genomes of ``palindromic' petite mutants of yeast

 The mitochondrial (mt) genomes of three spontaneous cytoplasmic ``palindromic' petite mutants of yeast were studied by restriction-enzyme analysis. These mt genomes were shown to be made up of an amplified ``master basic unit' consisting of two inverted segments (a and A) and of two different unique segments (d and t) separating them. The basic unit was called ``amphimeric', this term having been first proposed for certain lambda-phage mutants. We propose that in the mt genomes of the petite mutants studied, the four possible variants of the amphimeric basic unit form two – ``flip' and ``flop' – tetra-amphimeric repeat units datA-datA-DaTA-DaTA and DatA-DatA-daTA-daTA, respectively. These repeat units make two types of ``amphimeric' mt genomes which exist in equal proportions in the cell. In each mt genome, the duplicated segment regularly alternates in its direct and inverted orientation (a…A…a…A…), whereas the unique segments are arranged twice in tandem fashion and twice in inverted fashion (d…d…D…D…d…d… and t…t…T…T…t…t….). The only difference between flip and flop amphimeric mt petite genomes is the different relative orientation of the unique segments in the mono-amphimers. In the mono-amphimers of flip mt genomes, both unique segments are arranged in the same direction (d…t and D…T), whereas in the mono-amphimers of flop mt genomes, both unique segments are arranged in opposite directions (D…t and d…T). Control experiments on one spontaneous petite mutant (which was an ancestor of the mutants studied here) and on three independent, previously investigated, EtBr-induced mutants showed that all of them were, in fact, organized in the same way. Analysing our experimental data and the results published by others, we conclude that amphimeric organization is a general feature of mt petite genomes of yeast previously called ``palindromic' or ``rearranged'. Received: 2 November 1995     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number

Summary A gene bank of a yeast wild type DNA in the high copy number vector YEp 13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a –1 by deletion in the first intron of the mitochondrial COB gene (bIl). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bll, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.     

A direct study of the relative synthesis of petite and grande mitochondrial DNA in zygotes from crosses involving suppressive petite mutants of Saccharomyces cerevisiae

Summary Work in recent years has produced indirect evidence to support the view that the phenomenon of suppressiveness in yeast is the result of the ability of the petite mtDNA to out-replicate the wild-type genome. We have developed a method, based on fluorography of gels containing restriction fragments of radioactively labelled zygotic mtDNA, by which it has been possible to follow directly the incorporation of label into the two mtDNA species and hence their relative synthesis. Four petite isolates of 70%, 43%, 23% and 12% suppressiveness were tested by this method in crosses with a grande strain. Only the mtDNA from the 70% suppressive petite showed a replicative advantage over the grande mtDNA. The mtDNA from the 43% and 23% suppressive actually appeared to undergo, if anything, less replication in the zygote than the grande mtDNA. It is concluded that while some petites may exhibit suppressiveness as a result of enhanced replicative efficiency of their mtDNA, this cannot be the explanation for all suppressive petite strains.