Assembly of the mitochondrial membrane system. Organization of yeast mitochondrial DNA in the Oli1 region

Summary The mitochondrial DNA (mtDNA) of a cytoplasmic petite mutant (DS401) of Saccharomyces cerevisiae genetically marked for the ATPase proteolipid, serine tRNA and varl genes has been characterized by restriction endonuclease analysis and DNA sequencing. The DS401 mtDNA segment is 5.3 kb long spanning the region between 79.1 and 86.8 units of the wild type genome. Most of the DS401 mtDNA consists of A+T rich sequences. In addition, however, there are ten short sequences with a high content of G+C and two sequences that have been identified as the ATPase proteolipid and the serine tRNA genes. The two genes map at 81 and 83 units and are transcribed from the same DNA strand. Even though there are other possible coding sequences in the DNA segment, none are sufficiently long to code for a gene product of the size of the varl protein. Based on the relative organization of the G+C rich clusters and genes, a model has been proposed for the processing of mitochondria) RNA. This model postulates the existence of mitochondrial double strand specific RNases that cleave the RNA at the G+C clusters.     

Recombination of mitochondrial DNA without selection pressure among compatible strains of the Aspergillus niger species aggregate

Previous mitochondrial transmission experiments between oligomycin-resistant and oligomycin-sensitive incompatible strains of the A. niger aggregate bearing various mtDNA RFLP profiles resulted in a great variety of mitochondrial recombinants under selection pressure. Apart from the recombinant mtDNAs, resistant clones harbouring unchanged RFLP profiles of resistant donor mtDNAs with the recipient nuclear backgrounds were rarely isolated. These strains were anastomosed with nuclearly isogenic oligomycin-sensitive recipient partners and the mitochondria of the resulting progeny were examined under non-selective conditions. These experiments provide insights into events which are possibly similar to those occurring in nature. The heterokaryons obtained formed both oligomycin-resistant and -sensitive sectors, most of which were found to be homoplasmons. Progenies harbouring oligomycin-resistant and -sensitive mtDNAs may originate either from individual recombination events or be due to parental segregation. MtDNA recombination might take place in the heterokaryons without selection by oligomycin. The most frequent recombinant types of mtDNA RFLP profiles were indistinguishable from those recombinant mtDNAs which were frequently obtained under selection pressure from directed transfer experiments between incompatible strains. We present evidence that mixed mitochondrial populations may influence the compatibility reactions in the presence of an isogenic nuclear background, that recombination may take place without selection pressure, and that the process does not require specific nuclear sequences of both parental strains. Received: 20 October 1997 / 13 January 1998     

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.     

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     

Intramolecular recombination of a mitochondrial minicircular plasmid-like DNA of date-palm mediated by a set of short direct-repeat sequences

A molecular clone containing the complete sequence of a mitochondrial circular plasmid-like DNA (the R plasmid) isolated from the date-palm variety V3DP was used as a probe in Southern analyses of mitochondrial DNA prepared from other varieties. Another circular structure (the S plasmid) was detected in some of these varieties, and sequenced from variety V2DP. It appears that the R plasmid could have arisen from the S plasmid by an intermolecular recombination event at a set of 26-bp imperfect short direct repeats.     

Recombination initiated by double-strand breaks

The HO endonuclease was used to introduce a site-specific double-strand break (DSB) in an interval designed to monitor mitotic recombination. The interval included the trp1 and his3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Mitotic recombination was monitored in a diploid carrying heteroalleles of trp1 and his3. The normal recognition sites for the HO endonuclease were mutated at the MAT alleles and a synthetic recognition site for HO endonuclease was placed between trp1 and his3 on one of the chromosomes. HO-induced cleavage resulted in efficient recombination in this interval. Most of the data can be explained by double-strand gap repair in which the cut chromosome acts as the recipient. However, analysis of some of the recombinants indicates that regions of heteroduplex were generated flanking the site of the cut, and that some recombinants were the result of the cut chromosome acting as the genetic donor.By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government and its agents and contractors to retain a nonexclusive, royalty-free license in and to any copyright covering the article     

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     

Recombination between parental mitochondrial DNA following protoplast fusion can occur in a region which normally does not undergo intragenomic recombination in parental plants

Summary A novel restriction fragment, which was not present in either parent's mitochondrial DNA, was cloned from the mitochondrial genome of a somatic hybrid Petunia line. This fragment resulted from interspecific recombination between homologous mitochondrial DNA regions of the parental plants, Petunia line 3704 and line 3688. Hybridization with a cloned Petunia atp9 gene revealed that the regions involved in recombination carry atp9 coding sequences. Intragenomic recombination within the parental mitochondrial genomes was not detected between the atp9 regions which recombined following protoplast fusion.     

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.     

Maize mitochondrial DNA rearrangements between the normal type,the Texas male sterile cytoplasm,and a fertile revertant cms-T regenerated plant

Summary The maize mitochondrial DNA (mtDNA) from the cytoplasmic male sterile type T (cms-T) contains a 6.6 kb XhoI fragment not found in the normal type (N) or the fertile revertant (V3). Analysis of the fragment in cms-T mtDNA and homologous regions in N and V3 mtDNAs reveals that a number of events are necessary to explain the formation of the 6.6 kb XhoI fragment in T mtDNA and its subsequent loss in V3 mtDNA. This includes the formation and the loss of a 4 kb repeat and various recombinational events. It is interesting to note that a recombinational event that has occurred in the fertile revertant genome reconstitutes a sequence arrangement identical to one found in the normal genome.     

The absence of introns in yeast mitochondria does not abolish mitochondrial recombination

Summary The respiratory competency of a yeast strain devoid of mitchondrial introns is quite normal. However, it may be asked whether intron-encoded proteins participate in metabolisms other than those of mitochondrial introns. Using strains without mitochondrial introns we have answered two questions. The first was: does the absence of intron-encoded proteins abolsh mitochondrial recombination? The second was: do mitochondrial introns and intron-encoded proteins play a part in mitochondrial DNA rearrangements induced by ethidium bromide (rho^- production)? We have shown that the introns and intron-encoded proteins are not essential essential components of either phenomenon.     

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.     

DNA synthesis catalyzed in vitro by yeast extracts using A 2 μm DNA containing plasmid as template for enzymatic DNA synthesis

Summary Partially purified cell-free extracts prepared from growing cells of the yeast Saccharomyces cerevisiae catalyze DNA synthesis directed by the chimeric plasmid BTYP-2 containing the whole 2 m DNA sequence. The biochemical properties of the reaction have been examined and inactivation studies indicate that DNA synthesis is only due to protein factors. Analysis of the products of the DNA synthesis reaction demonstrates that part of the in vitro synthesized DNA is not covalently linked to the template, thus suggesting initiation of new DNA chains. The analysis of the distribution of the labelled products at early times of reaction showed a preferential synthesis on the 2 m portion of the BTYP-2 DNA template.Other abbreviations used are BSA bovine serum albumin - DTT dithiotreitol - EDTA ethylendiaminetetraacetate - EGTA ethylenglycol-bis(-aminoethyl-ether)N,N-tetraacetic acid PhcMeSO₂F, phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate Abbreviations: The abbreviations used are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN).     

Generation of lymphokine-activated killer cells does not require DNA synthesis.

We studied the role of DNA synthesis in the induction of lymphokine-activated killer (LAK) cells by recombinant interleukin-2 (IL-2) and the dependence of this phenomenon on DNA synthesis. Doses of gamma-irradiation (1000-5000 rads) that profoundly reduced DNA synthesis in human peripheral blood mononuclear leucocytes (PBL) also effectively suppressed the development of cytotoxic activity in the absence of IL-2. However, the same doses of irradiation affected the induction of LAK activity by IL-2 to a much lesser extent. Blocking the formation of deoxyribonucleotides by hydroxyurea, which resulted in a complete inhibition of DNA synthesis in PBL or purified T lymphocytes, had virtually no effect on the generation of LAK cells. These results indicate that the expression of LAK activity is not dependent on DNA synthesis.     

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.     

The effect of hydroxyurea on the mechanism of DNA synthesis in the yeast Saccharomyces cerevisiae

Summary Newly synthesised DNA molecules the same size as replicons (7 million-60 million daltons) accumulate in yeast cells treated with hydroxyurea. During prolonged incubation in low concentrations of the drug, there is a large accumulation of these molecules without any corresponding increase in their molecular weight. On release from the inhibtion the molecules are converted to large molecular weight DNA. These observations are consistent with an inhibition by hydroxyurea of the joining of completed replicons. In addition, newly synthesised DNA molecules the size of yeast Okazaki fragments also accumulate in cells treated with hydroxyurea.     

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.