Genetic analysis of the products of a cross involving a suppressive ‘petite’ mutant of S. cerevisiae

Summary A genetically defined highly suppressive petite yeast strain ( ^–cob⁺A^sE^oC^oO^oP^o) was crossed with a grande strain carrying a multiply marked mitochondrial genome ( ⁺A^rE^rC^rO rp^r). Petite diploid progeny, isolated from individual zygotic clones consisting either of wholly petite or mixtures of grande and petite cells, were characterised genetically by crossing to grande haploids. The diploid petites were found to closely resemble the petite parent and in general not to carry mitochondrial markers from the grande parent. In the petites from the mixed clones recombination was detected, but only within the region of homology between the genomes. These observations are inconsistent with models of suppressiveness based on destructive recombination and suggest that the petite genome eliminates the grande genome from zygotic progeny through being preferentially replicated. The most plausible model to explain the observed pattern of zygotic clones postulates a limited number of mDNA replication sites in zygotes, competition for sites between input mDNA molecules and an advantage in this competition for suppressive ^– mDNA.     

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     

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates ^– and ⁰ cells. Here we demonstrate that, in ^– yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric ^– mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1–5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1 yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.     

Pathogenic role of mtDNA duplications in mitochondrial diseases associated with mtDNA deletions

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination.     

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).     

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5 termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.     

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.     

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.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

Brief Communication: Role of Nuclear Background and in vivo Environment in Variable Segregation Behavior of the Aging-Dependent T414G Mutation at Critical Control Site for Human Fibroblast mtDNA Replication

Previous work had shown a large accumulation (up to 50% of mtDNA) of a noninherited T414G transversion at a critical control site for mtDNA replication in skin fibroblasts from the majority of human subjects above 65 years old, and its absence in younger individuals. In the present studies, long-term in vitro culture of several fibroblasts populations carrying the heteroplasmic T414G mutation revealed an outgrowth of the mutant cells by wild-type cells. This observation supported the previous conclusion that the mutation accumulation is an in vivo phenomenon, while, at the same time, indicating intrinsic physiological differences between mutant and wild-type cells. Furthermore, subcloning experiments revealed a striking mosaic distribution of the mutation in the original fibroblasts populations, as shown by its presence, in heteroplasmic or homoplasmic form, in a fraction (18–32%) of the fibroblasts, and its absence in the others. In other investigations, transfer of mitochondria from mutation-carrying fibroblasts into mtDNA-less 143B.TK^–0 206 cells revealed the persistence of the mosaic distribution of the mutation, however, with a near-complete shift to homoplasmy. The generality of the latter phenomenon would exclude a founder effect by one or few mitochondria in the transformation experiments, and would rather point to the important role of the nuclear background in the in vitro behavior of the T414G mutation. The stability of the homoplasmic mutation in ⁰ cell transformants provides a powerful tool for analyzing its biochemical effects.     

The origin of inverted tandem duplications,and phenotypic effects of tandem duplication of the X chromosome long arm

Tandem repeats of chromosome material can arise as inverted or as direct duplications. Such duplications of the X chromosome are instructive regarding X-linked genetic determinants of phenotype. We describe a 40-year-old woman with a direct duplication Xq13.3 to Xq27.2, short stature, gonadal dysgenesis, and secondary amenorrhea. Comparison of her phenotype with that of two other women with a direct duplication of part of Xq confirms the existence of statural determinants within the region X13 to Xq21, determinants of ovarian function within X22 to X27, and the X inactivation center within or proximal to band Xq13.3. In humans, direct duplications are more frequent than inverted, but both forms are rare. The mean age of parents is normal in subjects with direct duplications, but is advanced in subjects with inverted duplications. An inverted duplication can arise from a three-break rearrangement that includes a U-type exchange; a similar origin (two breaks and a U-type exchange) and a parental age association can be postulated for dicentric inverted duplications including dicentric isochromosome X.     

Temporospatial Heterogeneity of Myocardial Perfusion and Blood Volume in the Porcine Heart Wall

Spatial heterogeneity of myocardial perfusion has been recognized for many years. Whether this is primarily the result of heterogeneity of parameters such as myocardial metabolism, of intramyocardial mechanical forces, or of vasomotor function within the myocardial microcirculation, is not clear. A practical problem is that it has been almost impossible to measure any two of these parameters simultaneously in the same piece of myocardium so that an unambiguous correlation, much less a cause-and-effect relationship, has been difficult to establish. In this study of six anesthetized pigs, we propose that whole-body computed tomography is a method for providing the simultaneous measurement of heterogeneity of myocardial perfusion (F) and myocardial blood volume (). The first finding was that the empirical relationship =AF+BF^0.5 between myocardial blood flow (F) and intramyocardial blood volume () is maintained over a range of sizes of regions of interest (approximately 1 to 0.125 cm³) within the myocardium of each individual animal despite the spatial heterogeneity of the F and the values. The value of A ranges from 0.014 to 0.021 min and of B ranges from 0.061 to 0.076 ml^0.5 g^–0.5 min^0.5. A second finding was that the pattern of spatial heterogeneity of F and of remained reasonably stable over at least a 1 h period. © 1998 Biomedical Engineering Society. PAC98: 8745Ft, 8759Fm     

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the ⁺/^– state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.     

Fiber-fluorescence in situ hybridization analyses as a diagnostic application for orientation of microduplications

Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by standard fluorescence in situ hybridization (FISH), our data indicates that fiber-FISH analysis has the potential to reveal the orientation of duplicated and triplicated segments of chromosomes. Recurrent microduplications reciprocal to microdeletions show tandem orientations of the duplicated segments, which is consistent with a non-allelic homologous recombination mechanism. Several random duplications showed tandem configurations and inverted duplications are rare. Further analysis is required to fully elucidate the basic mechanisms underlying such duplications/triplications.     

Structural polypeptides of California encephalitis virus: BFS-283

Summary The polypeptides of California encephalitis virus (BFS-283) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four polypeptides were detected in virions grown in both BHK-21 and LLC-MK₂ cell cultures with molecular weights of 17,500, 30,000, 38,000, and 82,000 (VP-1, VP-2, VP-3, and VP-4, respectively). Viral proteins 2, 3, and 4 were glycoproteins and appeared to be associated with the envelope of the virus. Treatment of virions (=1.18 g/cm³) with then non-ionic detergent, NP-40, allowed detection of a RNA-rich fraction (=1.26/cm³) with contained the smallest polypeptide (VP-1).With 8 Figures     

Postzygotic telomere capture causes segmental UPD,duplication and deletion of chromosome 8p in a patient with intellectual disability and obesity

Using SNP array and FISH analysis, a patient with moderate intellectual disability and obesity was found to harbour an atypical 1.6 Mb inverted duplication on 8p23.1, directly flanked by a distally located interstitial deletion of 2.3 Mb and a terminal segmental uniparental disomy. The duplicated and deleted regions lie exactly between the two segmental duplication regions.These segmental duplications on chromosome 8p23.1 are known to be involved in chromosomal rearrangements because of mutual homology and homology to other genomic regions. Genomic instability mediated by these segmental duplications is generally caused by non-allelic homologous recombination, resulting in deletions, reciprocal duplications, inversions and translocations.Additional analysis of the parental origin of the fragments of this atypical inverted duplication/interstitial deletion shows paternal contribution in the maternal derivate chromosome 8. Combined with the finding that the normal chromosome 8 carries an inversion in 8p23.1 we hypothesize that a double strand break in 8p23.1 of the maternal chromosome was postzygotically repaired with the paternal inverted copy resulting in a duplication, deletion and segmental uniparental disomy, with no particular mediation of the 8p23.1 segmental duplication regions in recombination.     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

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     

Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast

Large-scale changes (gross chromosomal rearrangements [GCRs]) are common in genomes, and are often associated with pathological disorders. We report here that a specific pair of nearby inverted repeats in budding yeast fuse to form a dicentric chromosome intermediate, which then rearranges to form a translocation and other GCRs. We next show that fusion of nearby inverted repeats is general; we found that many nearby inverted repeats that are present in the yeast genome also fuse, as does a pair of synthetically constructed inverted repeats. Fusion occurs between inverted repeats that are separated by several kilobases of DNA and share >20 base pairs of homology. Finally, we show that fusion of inverted repeats, surprisingly, does not require genes involved in double-strand break (DSB) repair or genes involved in other repeat recombination events. We therefore propose that fusion may occur by a DSB-independent, DNA replication-based mechanism (which we term “faulty template switching”). Fusion of nearby inverted repeats to form dicentrics may be a major cause of instability in yeast and in other organisms.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicolresistant (CAP^R)human × mouse hybrids and one CAP^R cybrid derived from CAP^R HeLa cells and CAP^S mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with ³² P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [³²P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited reverse chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human mouse hybrid is to retain human mitochondrial DNA.