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     

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.     

Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes

  The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast. An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE. Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome. Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50–100 kb, but weakly to the larger DNA from mitochondria of these two strains. For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50–100 kb, but a smear without bands was found for the circular-mapping strain. Received: 28 June 1995 / 15 January 1996     

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.     

Cutaneous leishmaniasis: a model for analysis of the immunoregulation by accessory cells

  In the mammalian host, Leishmania are obligate intracellular parasites and invade macrophages and Langerhans cells. The accessory functions of both types of host cells are important for regulation of the specific cellular immune response and involve the following activities: infiltration into the site of infection, initiation of a T cell response, maintenance of immunity and the effector mechanisms that control intracellular parasite replication. Received: 31 August 1995     

A rapid and reliable method for PCR-based amplification of chromosomal and mitochondrial DNA from intact yeast cells

In this paper we describe a method to amplify mitochondrial and chromosomal DNA from yeasts by PCR using intact cells.     

Mitochondrial DNA amplification in senescent cultures of Podospora anserina: Variability between the retained,amplified sequences

Summary The non-nuclear DNA of a number of independent senescent cultures of Podospora anserina was extracted and studied. In all cases, a specific repetitive DNA (SEN-DNA) arranged in multimeric sets of circular molecules, was identified. Depending on the senescent culture, the SEN-DNA was found either in a band of about same density as the mitochondrial DNA from young mycelia (1.694 g/cm³) or in a band of higher density (1.699 g/cm³). Electron microscopy, restriction enzyme analysis and Southern hybridization experiments allowed us to establish that: (1) SEN-DNAs obtained from independent senescent cultures, both from the same strain and from different strains, can differ in the size of their monomer unit (from 2.5 to 6.3 kb). (2) All SEN-DNAs hybridize with mitochondrial DNA of a young culture and not with nuclear DNA. (3) These SEN-DNAs belong to two classes which hybridize with two non-overlapping regions of the mitochondrial chromosome.     

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.     

The peptidolytic capacity of the spirochete system

  Relatively scant chemical information has been available on the proteinases and peptidases of spirochetes in spite of the association of spirochetes with several serious infections known to plague humans and other animal species. This situation has partly resulted from difficulties in growing some spirochetes under laboratory conditions. The cells of Treponema denticola, a spirochete suggested to be associated with periodontal infections, have turned out to be a good source of new chemical information on those enzymes. Latest studies suggest that the outer cell en-velope or the periplasmic space of T. denticola contains several novel proteinases and peptidases (hence called ``ectoenzymes') which may contribute to the chronicity of periodontal infections. Some of the oligopeptidases discovered are specific for proline-containing host tissue peptides such as substance P, bradykinin, neurotensin, etc., and possibly small collagen fragments. The only spirochetal peptidases purified to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been obtained from T. denticola. One particular peptidase, suggested to be similar to the oligopeptidase B (EC 3.4.21.83) of Escherichia coli seems to be present in the cell envelope or in the periplasmic space at quite large concentrations. The presence of this and several other peptidases in the outer cell structures of the treponemes suggests that such enzymes are important for the nutrition of these highly motile and invasive organisms. The biological role of these enzymes can thus be envisaged in the peptidolytic processing of host tissue proteins and peptides to gradually smaller molecules to fulfill the nutritional requirements of these organisms. Although the genetic similarity between T. denticola and some other treponemes and spirochetes can be hotly debated, it is nevertheless now possible to use T. denticula enzymes as suitable objects for comparison when the chemistry of other spirochetes is studied. Received: 20 Decemer 1995     

Partial sequence of the mitochondrial genome of the crustacean Daphnia pulex

 A 4062-nucleotide (nt) fragment of the mitochondrial genome of the crustacean Daphnia pulex was sequenced and found to contain the complete genes for eight tRNAs and five proteins (ATP6, ATP8, COII, COIII, ND3) and the partial sequence of COI. In combination with data described previously, approximately 50% of the D. pulex mitochondrial genome has been sequenced. The gene order in this half of the genome is identical to that of Drosophila yakuba which differs from that of the other completely sequenced crustacean mitochondrial genome, Artemia franciscana. Comparison of seven mitochondrial proteins among D.␣pulex, A. franciscana, D. yakuba, Anopheles gambiae, Locusta migratoria and Apis mellifera showed that, with one exception, the D. pulex proteins are most similar in length and sequence to the proteins of the dipteran insects. Conversely, patterns of nucleotide bias at third codon positions in fourfold degenerate codon families are very similar in the two crustaceans but differed substantially from the insects. Received: 15 March / 23 September 1996     

Preferential amplification of mitochondrial DNA fragments in somatic hybrids of the Gramineae

Summary Somatic hybrid cell lines of Pennisetum americanum + Panicum maximum, and of Pennisetum americanum + Saccharum officinarum display unique mitochondrial DNA (mtDNA) restriction patterns suggestive of mitochondrial fusion and recombination. Apparent recombinant fragments of the hybrids were recovered, cloned, and hybridized to parental and somatic hybrid mtDNAs. In each somatic hybrid, novel fragments were found to be present at low copy number in one or both of the parental mtDNAs, and amplified 15–30 times in the hybrids. In pearl millet-sugarcane somatic hybrid cells, the amplification does not appear to involve enhanced recombination. The presumably amplified restriction fragment of the pearl millet-Guinea grass somatic hybrids is a junction fragment of a repeat, present in low copy number in both parents, and in high copy number in the hybrids. Thus protoplast and probable mitochondrial fusion results in a marked shift in the direction of mtDNA recombination events. We conclude that amplification of parental mtDNA fragments is a common event in somatic hybrid cells of these Gramineae.     

A rapid and simple method for extracting yeast mitochondrial DNA

Summary A rapid method for the extraction of yeast mitochondrial DNA (mtDNA) is described. In comparison with previous methods, it simplifies several steps, does not require either the isolation of mitochondria or phenol treatment and is less time consuming. This protocol gives a high yield of pure mtDNA (50–120 g from a 100-ml culture), which can be directly used in various molecular applications: restriction enzyme digestion, electrophoresis, blotting, labeling, cloning and sequencing.     

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155

 In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155. Received: 19 September 1995/4 January 1996     

Over-expression of the NUD1-coded endo-exonuclease in Saccharomyces cerevisiae enhances DNA recombination and repair

  The NUD1(=NUC2) gene of Saccharomyces cerevisiae has been subcloned and over-expressed in multi-copy plasmids. Enhanced expression of this nuclear endo-exonuclease gene was confirmed by Northern hybridization (>10-fold increase), and increased enzymatic activity (2.4-fold increase) was demonstrated by direct immunological assay using antibody raised against the purified Neurospora crassa endo-exonuclease. We found that increased expression of NUD1 was associated with an increase in cell surivival after irradiation treatment with gamma rays, and an increase in radiation-induced mitotic recombination frequencies between duplicated gene sequences. The results presented here are consistent with previous biochemical data and confirm a role for the NUD1 gene product in recombination/repair processes. Received: 10 November 1995 / 16 January 1996     

Nonsyndromic sensorineural deafness associated with the A1555G mutation in the mitochondrial small subunit ribosomal RNA in a Balinese family

 Sensorineural deafness associated with increased sensitivity to aminoglycoside antibiotics as the consequence of an A1555G mutation in the mitochondrial DNA (mtDNA) in a highly conserved region of the small (12S) rRNA gene has been reported in Caucasian, Chinese, and Japanese individuals. We report here a large family of Balinese Indonesian origin with progressive/congenital sensorineural deafness who carry the A1555G mutation. The pedigree shows a generally maternal inheritance pattern with some exceptions, which is the result of an unusual multiple entry of the mutation into the pedigree. A complete mtDNA genome sequence from three Balinese individuals revealed a relatively large number of single- nucleotide polymorphisms (20) not previously reported, and confirmed the genetic distance of Southeast Asian populations from those of Caucasians and Japanese. The biochemical expression of the A1555G mutation under the influence of this mtDNA background was investigated. Examination of respiratory enzyme activities showed a significant decrease in respiratory complex I activity, particularly in symptomatic family members. Received: July 12, 2002 / Accepted: November 22, 2002 Acknowledgments We thank all the family members that have participated in the present study. We would like to thank Dr. Helena Suryadi for her expert assistance in the fieldwork, and Ms. Neny Sitorus for tissue culture work. This work was supported by grants from PT Krakatau Steel and PT Inti through the Agency for Strategic Industries (Indonesia) and by a generous Development Fund from the National Development Planning Agency (BAPPENAS) of the Republic of Indonesia. Correspondence to:S. Marzuki     

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.     

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.     

ABL amplification in a patient with lymphoid blast crisis of chronic myelogenous leukaemia

 Although chronic phase myelogenous leukaemia (CML) is characterised by the Philadelphia (Ph) chromosome leading to a fusion of the BCR and ABL genes, additional genetic alterations involved in blast crisis are poorly understood. We report an at least 15-fold amplification of the ABL oncogene in a 29-year-old male patient with a variant Ph-positive t(19;22)(p13;q11.2) CML who presented in lymphoid blast crisis. Our finding suggests that an amplification of the ABL oncogene might play a part in the appearance of an aggressive phenotype in some cases of CML. Received: 27 July 1998 / Accepted: 26 October 1998     

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.