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.     

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.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.     

Interactions between the yeast mitochondrial and nuclear genomes: isogenic suppressive and hypersuppressive petites differ in their resistance to the alkaloid lycorine

Summary In a previous paper we have shown that the alkaloid lycorine inhibits growth of rho ⁺, mit ^- and rho ^-, strains of Saccharomyces cerevisiae, whereas strains devoid of mitochondrial DNA (rho ^o) are resistant to more than 200 g/ml of the alkaloid. In this report we show that hypersuppressive petites are almost as resistant as rho ^o mutants, whereas isogenic rho ^- petites, which have retained tained longer segments of the genome, are sensitive to the drug.     

Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.     

Effects of the kar gene on cytoplasmic mixing and mitochondrial genome suppressiveness,and consequences for cytoduction of petite DNA in Saccharomyces cerevisiae

Summary During a series of cytoduction experiments to transfer Saccharomyces cerevisiae mitochondrial genomes from one nuclear background to another, using the karl-1 nuclear fusion mutation, one of the five petite genomes used proved difficult to transfer. This genome, ^- F13, was highly suppressive (90%) in its original nuclear background. Molecular and genetic studies on the putative karl-1 ^–F13 cytoductant were done to discover the nature of this difficulty. They showed that while the ^–F13 was maintained in a karl-l background, zygotes from a mating with a ⁰ strain showed poor cytoplasmic mixing and therefore inefficient ^–F 13 DNA transfer into first zygotic buds. This also caused a reduction of ^–F13 suppressiveness to 20–30% in crosses with different ⁺ strains. The effect was genome specific since another highly suppressive petite in the karl-l background did not show suppressiveness reduction when crossed to ⁺. The nature of suppressiveness modulation is discussed. Since the ^–F13 genome was eventually transferred using a modification of the original scheme, the problems were not caused by the inability of the acceptor nuclear background to maintain the ^–F13 genome.     

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     

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     

Mechanical properties of mammalian single smooth muscle cells I. A low cost large range microforce transducer

Summary A transducer has been developed for measuring the minute forces generated during isometric contractions (1.0–10.0N) of single smooth muscle cells from the pig urinary bladder and the human uterus. In addition to its high sensitivity, resolution and stability (100 mVN^–1, <0.1N and <2.0N h^–1), the transducer features a very wide range (100–140N) with good linearity, enabling measurement of contractions as well as passive force-length characteristics within one uninterrupted measurement session. Since the transducer features an independent and interchangeable force to displacement conversion system, different force ranges can be realized by inserting force conversion systems with different compliances.     

Physical organization of the 18S and 5S ribosomal RNA genes in the mitochondrial genome of rye (Secale cereale L.)

Summary The mitochondrial 18S and 5S ribosomal RNA (rRNA) genes of rye, plus a total of about 90 kilobase pairs of flanking DNA, have been cloned and maps of restriction enzyme cleavage sites have been constructed. Like their homologs from hexaploid wheat, the rye genes are closely linked and are part of a three-copy family of recombining repeats (the 18S/5S repeat). The rye repeat probably also contains a mitochondrial tRNA^fMet gene, which the wheat repeat is known to carry. However, despite the overall organizational similarity between the wheat and rye 18S/5S repeats in the immediate vicinity of their coding regions, extensive rearrangement of flanking sequences has taken place during evolutionary divergence of the two species. Our data provide additional support for an emerging picture of plant mitochondrial genomes as evolving much more rapidly in structure than in sequence.     

Channel-induced apoptosis of infected host cells—the case of malaria

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca²⁺-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca²⁺ and Na⁺, both of which are required for intracellular growth of the pathogen. The entry of Ca²⁺ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte apoptosis that may represent a host defence mechanism serving to eliminate infected erythrocytes.     

Absence of long-term modulation of ventilation by dead-space loading during moderate exercise in humans

The stability of arterial PCO₂ (P_aCO₂) during moderate exercise in humans suggests a CO₂-linked control that matches ventilation (_E) to pulmonary CO₂ clearance (CO₂). An alternative view is that _E is subject to long-term modulation (LTM) induced by hyperpnoeic history. LTM has been reported with associative conditioning via dead-space (V_D) loading in exercising goats (Martin and Mitchell 1993). Whether this prevails in humans is less clear, which may reflect differences in study design (e.g. subject familiarisation; V_D load; whether or not _E is expressed relative to CO₂; choice of P_aCO₂ estimator). After familiarisation, nine healthy males performed moderate constant-load cycle-ergometry (20 W-80 W-20 W; <lactate threshold, _L): day 1, pre-conditioning, n=3; day 2, conditioning (V_D=1.59 l, doubling _E at 20 W and 80 W), n=8 with 10 min rest between tests; and, after 1 h rest, post-conditioning, n=3. Gas exchange was determined breath-by-breath. Post-conditioning, neither the transient [phase 1, phase 2 (1, 2)] nor steady-state _E exercise responses, nor their proportionality to CO₂, differed from pre-conditioning. For post-conditioning trial 1, steady-state _E was 28.1 (4.7) l min^–1 versus 29.1 (3.8) l min^–1 pre-conditioning, and mean-alveolar PCO₂ (a validated P_aCO₂ estimator) was 5.53 (0.48) kPa [41.5 (3.6) mmHg] versus 5.59 (0.49) kPa [41.9 (3.7) mmHg]; the 1 _E increment was 4.2 (2.9) l min^–1 versus 5.2 (1.9) l min^–1; the 2 _E time-constant () was 64.4 (24.1) s versus 64.1 (25.3) s; _E/CO₂ was 1.12 (0.04) versus 1.10 (0.04); and the _E-CO₂ slope was 21.7 (3.4) versus 21.2 (3.2). In conclusion, we could find no evidence to support ventilatory control during moderate exercise being influenced by hyperpnoeic history associated with dead-space loading in humans.     

Polymorphism for ribosomal RNA gene arrangement in the mitochondrial genome of fall rye (Secale cereale L.)

A restriction-fragment-length polymorphism (RFLP) in mitochondrial DNA (mtDNA) was detected between varieties of fall rye (Secale cereale L.) by Southern hybridization with rrn18, the gene encoding the mitochondrial 18S ribosomal RNA. Restriction mapping showed that the RFLP is based on differing numbers of genomic contexts (one vs three) for a recombining-repeat element (the 18S/5S repeat). From examination of other Secale species, we conclude that the one-context state arose relatively recently, putatively by deletion of two of an ancestral set of three distinct genomic loci containing the mitochondrial 18S/5S repeat. This is consistent with our earlier conclusion that the 18S/5S repeat has probably existed in at least two genomic copies throughout much of the history of the grass family (at least 40 million years). Interestingly, the intervarietal difference in the number of distinct rrn18 loci is not accompanied by a major difference in the number of rrn18 copies per unit mass of mtDNA. This suggests the existence of a mechanism that can compensate rather precisely for differences in mitochondrial gene dosage, perhaps by over-replication or stabilization of specific subgenomic molecules.     

Molecular structure of the SWA2 gene encoding an AMY1-related α-amylase from Schwanniomyces occidentalis

A 2.1-kb DNA fragment containing the SWA2 gene determining an -amylase from Schwanniomyces occidentalis has been sequenced. It contains an open reading frame of 1521 bp which has the potential to encode a 507 amino-acid protein of Mr 55966. Its deduced aminoacid sequence shows significant similarities to the sequence of other studied -amylases. These similarities identify a consensus sequence, F(LIV)(ED)NHD, which is shared in addition by most maltases, invertases and glucoamylases.     

De-novo generation of mitochondrial DNA plasmids following cytoplasmic transmission of a degenerative disease in Ophiostoma novo-ulmi

A mitochondrial DNA plasmid was detected in an isolate of Ophiostoma novo-ulmi with a degenerative disease. The DNA plasmid was shown to be derived from the mitochondrial DNA and to map to a region corresponding to the large ribosomal RNA coding region. The DNA plasmid was not transmitted into sexual (ascospore) progeny, irrespective of whether the diseased isolate acted as the female or male parent. Transmission of the disease to healthy, plasmid-free, recipient isolates by hyphal anastomosis was not accompanied by transfer of mitochondrial DNA or DNA plasmid from the diseased donor isolate, but resulted in de-novo generation of different plasmids, derived from the recipient's mitochondrial DNA.     

Structure of the cloned Locusta migratoria mitochondrial genome: restriction mapping and sequence of its ND-1 (URF-1) gene

Summary We have cloned the entire mitochondrial genome of Locusta migratoria in four fragments and characterised by restriction mapping. In addition, we have sequenced a 1,095 kb region containing the ND-1 (URF-1) gene. The inferred primary structure of the protein is highly homologous to its Drosophila counterpart (68%). The gene is flanked at the 5 end by the tRNA _CUN ^leu gene, interrupted by the sequence TTG. The 3 end is flanked by the tRNA _ser ^UCN gene, followed by a sequence homologous to the 3 end of D. yakuba cytochrome b. The relative position of the genes is conserved between Locusta and Drosophila, thus indicating conservation of mitochondrial gene order in insects.     

Differential inhibition of neutrophil superoxide generation by nonsteroidal antiinflammatory drugs

In order to further characterize the effects of nonsteroidal antiinflammatory drugs on neutrophil superoxide (O₂ ^–) generation, human neutrophils were incubated in the presence of sulfinpyrazone, phenylbutazone, and indomethacin prior to exposure to a variety of oxidative stimuli. Stimuli used included the chemotactic peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP, 5.0 × 10^–7 M), NaF (20 mM), phorbol myristate acetate (PMA, 3.2 × 10^–7 M), and opsonized zymosan (250g/ml). superoxide release induced by FMLP was inhibited by all three drugs with half-maximal inhibition (K_i50) at 2.5, 30, and 120M for sulfinpyrazone, phenylbutazone, and indomethacin, respectively. This inhibition was not due to drug interference with the assay system since comparable inhibition was not observed in a cell-free O₂ ^–-generating system. The neutrophil's response to NaF was blunted by sulfinpyrazone (K _i50=400M) and phenylbutazone (K _i50=65M), but was unaffected by indomethacin. A similar inhibitory pattern was observed when zymosan was used as the oxidative stimulus. Sulfinpyrazone and phenylbutazone inhibited the response to zymosan (K _i50s of 425 and 32M, respectively), whereas indomethacin augmented it. PMA stimulation evoked O₂ ^– production which was inhibited by phenylbutazone (K _i50=350M) but not by sulfinpyrazone or indomethacin in concentrations up to 1 mM. The results support the hypothesis that the enzyme system responsible for neutrophil O₂ ^– generation can be activated by more than one mechanism. The results also emphasize the need to evaluate pharmacologie modulation of neutrophil responses in light of the stimulus used to evoke the response.This work was supported by National Institutes of Health grant T32-AMO7186 and National Institute of Allergy and Infectious Diseases grant AI-17950.     

Two Extramitochondrial Circular DNA Species in the Petite Negative Yeast Schizosaccharomyces pombe: Relative abundance and size determination by electron microscopy

Summary In this paper we present the electron microscopic analysis of two distinct extramitochondrial circular DNA species in the fission yeast Schizosaccharomyces pombe (S. pombe). Both DNA species can be isolated from mitochondrial fractions, but disappear after DNase treatment of mitochondria, demonstrating their extramitochondrial location. The size of these molecular species is 3.08 ± 0.18 m and 2.00 ± 0.09 m (standard deviation). They are present in a ratio of approximately 9:1 in the DNA preparations analyzed.     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid