A “Stopper” mutant of Neurospora crassa containing two populations of aberrant mitochondrial DNA

Summary [E35], an extranuclear mutant of Neurospora crassa has all the phenotypic characteristics of the stopper mutants (De Vries et al. 1980). In the present work, the mitochondrial DNA as well as the mitochondrial translation products are characterized further. The primary mutational event appears to have been the deletion of about 4 kbp from the wild-type genome. Moreover, after prolonged vegetative growth the mutant accumulates an 8-m circular mtDNA, which was demonstrated both by electronmicroscopy and by restriction enzyme analysis. Hence, the mutant contains two populations of aberrant mitochondrial DNA, the smaller of which is an amplification of the rRNA-tRNA part of the larger. We propose that the primary deletion has generated a signal in the larger DNA which can cause premature termination of replication at the deletion site, and subsequent circularization of the unfinished daughter molecule. Finally, the deleted part may contain a determinant for synthesis of a protein of 11 kDal. The function of this protein, which is not a subunit of the F₀ ATPase, is not yet known.Abbreviations (k)bp (kilo)basepairs - kDal kilodalton - mt mitochondrial     

At least two nuclear-encoded factors are involved together with a mitochondrial factor (MR1) in spontaneous mitochondrial frameshift-suppression of the yeast S. cerevisiae

Summary Earlier genetic analyses have identified a mitochondrial +1 frameshift suppressor (MF1) in the 15S rRNA region of a leaky mitochondrial frameshift mutant and the respective wild-type strain 777-3A (Weiss-Brummer et al. 1987). Further genetic analyses revealed that for the observed spontaneous frameshift suppression in M5631 the mitochondrial factor (MF1) must act together with at least two dominant nuclear-encoded factors.     

Characterization of a variant Neurospora crassa mitochondrial DNA which contains tandem reiterations of a 1.9 kb sequence

Summary We have identified a second variant of Neurospora mtDNA which contains tandem, head-to-tail repeats of sequences at the boundary of Eco RI-4 and 6. This region may contain a. major replication origin of Neurospora mtDNA.     

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome

Summary To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.     

Mapping of mitochondrial DNA from Torulopsis glabrata

Summary Large and small rRNAs have been isolated from mitochondria of the yeast Torulopsis glabrata and have been shown to have lengths of 2,700 bases and 1,400 bases respectively. Construction of a restriction endonuclease site map of mitochondria) DNA has enabled us to position these rRNAs by hybridization of labelled RNA to DNA fragments transferred to nitrocellulose. The large and small mt rRNA genes are separated by a minimum of 1,820 by and a maximum of 2,765 by on the 18,870 by mitochondria1 genome. tRNA genes map within this separating sequence but they are also located distal to both rRNA genes. The implication of these results to the structural relationships of mitochondrial DNAs from yeasts is discussed.     

Chromosome pairing and meiotic recombination in Neurospora crassa spo11 mutants

Some organisms, such as mammals, green plants and fungi, require double-strand breaks in DNA (DSBs) for synapsis of homologous chromosomes at pachynema. Drosophila melanogaster and Caenorhabditis elegans are exceptions, achieving synapsis independently of DSB. SPO11 is responsible for generating DSBs and perhaps for the initiation of recombination in all organisms. Although it was previously suggested that Neurospora may not require DSBs for synapsis, we report here that mutation of Neurospora spo11 disrupts meiosis, abolishing synapsis of homologous chromosomes during pachynema and resulting in ascospores that are frequently aneuploid and rarely viable. Alignment of homologues is partially restored after exposure of spo11 perithecia to ionising radiation. Crossing over in a spo11 mutant is reduced in two regions of the Neurospora genome as expected, but is unaffected in a third.     

An yeast nuclear mutation conferring temperature-sensitivity to the mitochondrial tryptophanyl-tRNA synthetase

Summary The conditional respiratory-deficient Saccharomyces cerevisiae mutant pet-ts2281 was complemented by an yeast genomic DNA library. The gene thus isolated was sequenced and proved to be identical to the known MSW1 sequence encoding mitochondrial tryptophanyl-tRNA synthetase (Myers and Tzagoloff 1985). Compared to the wild-type, the ts2281 mutant allele of MSW1 contained a single TC transition leading to a LeuSer replacement at position 294 of the protein sequence. In addition to this mutational alteration, our sequence data for the wild-type gene differ from the originally published MSW1 sequence at five other DNA positions which affect two locally restricted regions of the polypeptide chain. As expected, at the non-permissive temperature ts2281 cells are specifically defective in mitochondrial trp-tRNA formation and, thus, in overall mitochondrial protein synthesis. In addition, the patterns of cytochrome b mRNA maturation intermediates were distinctly different in ts2281 and wild-type yeast cells. The mutational effect of the observed amino-acid substitution in ts2281 is discussed in terms of weakened hydrogen bonding in the C-terminal half of the MSW1-encoded protein.     

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).     

The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.     

Elimination of mitochondrial mutations by sexual reproduction: two Podospora anserina mitochondrial mutants yield only wild-type progeny when mated

In order to understand the transmission of mitochondrial mutations in sexual crosses of Podospora, we attempted to create compatible strains with defined mitochondrial mutations. A previously characterized mutant, Mn19, with a bipartite mitochondrial genome, served as the fertilizing parent in a cross with a mitochondrial deletion mutant, αΔ5. Characterization of the deletion mutant is reported here. All six of the monokaryotic progeny isolated had neither parental defect but instead appeared to have inherited wild-type mitochondrial DNA. One of the progeny had a mitochondrial plasmid derived from intramolecular recombination between an 11-bp repeated mitochondrial sequence. Subsequent analysis using the polymerase chain reaction (PCR) identified rare undeleted wild-type mtDNA sequences in the maternal parent. The uniform inheritance of wild-type mitochondrial DNA suggests either an aggressive repair mechanism or else selective amplification and transmission of rare wild-type mtDNA molecules. Received: 12 December 1995 / 6 May 1996     

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.     

Isolation and characterization of yeast mitochondrial mutants defective in spore germination

Summary This paper describes a new type of mitochondrial mutation. During germination of ascospores the mutants are blocked at the first budding stage and consequently die. However, vegetative growth on nonfermentable carbon sources and respiration are close to normal. The spores of the mutants which (like the wild type) contain very low amounts of mitochondrial cytochromes, do not synthesize cytochromes b and aa₃ during germination.The mutants show a pleiotropic phenotype during the vegetative phase: they lack carbon catabolite repression of cytochromes on media containing 10% glucose. We discuss here the hypothesis that the mutation is located in a regulatory region on the mitochondrial genome which is needed for the reinitiation of mitochondrial genetic activity during germination of ascospores.     

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia

BACKGROUND: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. METHODS: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men undergoing vasectomy (n = 11) and testicular sperm from men with obstructive azoospermia (n = 25). Nuclear DNA (nDNA) fragmentation was measured by an alkaline gel electrophoresis (comet) assay. RESULTS: Wild-type mtDNA was detected in only 60% of fertile men's testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different, but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (P < 0.02). NDNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. CONCLUSION: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia, the mtDNA deletions in testicular sperm are of a larger scale.     

rRNA genes are located far away from the D-loop region in Peking duck mitochondrial DNA

Summary Peking duck mitochondrial DNA was isolated, digested with restriction endonucleases, electrophoresed and blotted with ³²P-labeled mitochondrial ribosomal RNAs. The genes for these RNAs were thereby shown to lie within a 3.23 kb Bgll fragment approximately 9.5 kb from the D-loop.     

Clinical and molecular characterization of pediatric mitochondrial disorders in south of China

Mitochondrial disorders (MDs) are genetic ailments affecting all age groups. Epidemiological data and frequencies of gene mutations in pediatric patients in China are scarce. This retrospective study assessed 101 patients with suspected MDs treated at the Neurology Department of Children's Hospital, Fudan University, in 2011–2017. Mitochondrial (mtDNA) and nuclear (nDNA) samples were assessed by long-range polymerase chain reaction (PCR)-based whole mtDNA sequencing and whole exome sequencing (WES) for identifying pathogenic mutations. Muscle samples underwent various staining protocols and immunofluorescence for detecting selected proteins. Seventeen mutations in the MT-TL1, MT-COX2, MT-ND4, MT, tRNA TRNE, MT-TN, MT-TK, MT-ATP6, MT-ND6, MT-ND3 and MT-CO3 genes were identified in 39 patients, of which m.3243A > G, m.3303C > T, m.8993T > C/G, m.9176T > C, and m.10191T > C were most common. Mitochondrial myopathy and MELAS were most common for m.3243A > G mutation. Four novel mutations were detected, including m.9478insT, m.5666T > C, m.8265T > C, and m.8380–13600 deletion mutations related to Leigh syndrome, mitochondrial myopathy and KSS, respectively. Thirty-three mutations in the TK2, POLG, IBA57, HADHB, FBXL4, ALDH5A1, FOXRED1, TPK1, NDUFAF5, NDUFAF7, NDUFV1, CARS2, PDHA1, and HIBCH genes were identified in 19 patients, including 23 currently unknown. Higher rates of TK2, POLG, IBA57, and HADHB mutations were found in nDNA-mutated MD compared with the remaining individuals. Besides, IBA57 c.286T > C (p.Y96H), TK2 c.497A > T (p.D166V) founder mutations critically contributed to MDs. Comprehensive genomic analysis plays a critical role in pediatric MD diagnosis. These data summarize the relative frequencies of different gene mutations in a large Chinese population, and identified 23 novel MD-associated nDNA and 4 novel mtDNA mutations.