The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

The alpha subunit of the mitochondrial ATP synthetase is mitochondrially made in the unicellular heterotrophPrototheca zopfiii

Summary Prototheca zopfii is a unicellular, colorless alga which grows heterotrophically on several different carbon sources.P. zopfii cells were grown, labeled with [³⁵S] sulfate in the presence of cycloheximide, and the mitochondrial fraction isolated and analyzed by gel electrophoresis and autoradiography. Cycloheximide resistant translation products included a heavily labeled protein with an apparent molecular weight of 60,000. This labeled protein co-fractionated with mitochondria when the mitochondria were purified on Percoll gradients. When F₁-ATPase was isolated from the mitochondria, an active ATPase preparation was obtained which, when analyzed by gel electrophoresis, was found to contain 2 prominent proteins in the 55 kd to 60 kd region of the gel. Autoradiography of this electrophoretically-resolved ATPase preparation revealed that the larger of the two, bands, but not the smaller, was heavily labeled with [³⁵S]. Extraction of ATP synthetase (F₁ -F₀) from mitochondria with the detergent octylglucoside, yielded a preparation containing, as major components, the same two 55 kd to 60 kd proteins seen in the F₁-ATPase preparation. Limited proteolysis of the larger protein, isolated from mitochondria that had been labeled in the presence of cycloheximide, was carried out usingStaphylococcus aureus V8 protease or papain. The pattern of peptide fragments observed by Coomassie Blue staining was essentially identical to that observed by autoradiography. This shows that the labeled protein corresponds to the alpha subunit of the ATPase. Thus the alpha subunit of the mitochondrial ATP synthetase ofP. zopfii is synthesized in the mitochondria.     

Inheritance of mitochondrial DNA in sexual crosses and protoplast cell fusions in Lentinula edodes

By using mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) as genetic markers, the modes of mitochondrial inheritance in sexual crosses and protoplast cell fusions of the higher basidiomycete Lentinula edodes were examined. All newly established dikaryons from reciprocal crosses between compatible monokaryons carrying different mtDNA RFLP phenotypes retained mtDNA genotypes from one of the monokaryons, suggesting that mitochondrial inheritance is principally uniparental. In contrast, it was shown that recombinant mtDNA genomes arose in some dikaryons obtained after protoplast cell fusion. Based on these results, a possible mechanism for mitochondrial inheritance in L. edodes is discussed.     

Relaxation in ferret ventricular myocytes: role of the sarcolemmal Ca ATPase

In ferret ventricular myocytes the rate of intracellular Ca concentration [Ca]_i decline and relaxation is remarkably fast (compared with rabbit and rat) under conditions where both the sarcoplasmic reticulum Ca uptake and Na/Ca exchange are inhibited. Here we explore the possibility that this rapid [Ca]_i decline in ferret cells is attributable to the sarcolemmal Ca ATPase by using carboxyeosin (a potent inhibitor of the sarcolemmal Ca-ATPase). We compare the effects of carboxyeosin with those of elevated extracellular [Ca] ([Ca]_o) (a thermodynamic approach to limit Ca transport by the sarcolemmal Ca ATPase). In rabbit cells, carboxyeosin and high [Ca]_o slowed [Ca]_i decline similarly and both virtually abolished [Ca]_i decline when mitochondrial Ca uptake was also inhibited. In ferret cells, carboxyeosin treatment produced these same effects on [Ca]_i decline, but high [Ca]_o did not mimic them. Moreover, only in carboxyeosintreated ferret cells did additional inhibition of mitochondrial Ca uptake nearly abolish [Ca]_i decline. We conclude that, carboxyeosin loading can inhibit the sarcolemmal Ca-ATPase in intact myocytes; that this pump seems likely to be responsible for the much faster relaxation observed in ferret cells after block of SR Ca accumulation and Na/Ca exchange transport and that the sarcolemmal Ca pump apparently has different characteristics in rabbit and ferret ventricular myocytes. Present address: Centro de Engenharia Biomédica Caixa Postal 6040, Universidade Estadual de Campinas (UNICAMP), 13081 Campinas, SP, Brazil     

Mitochondrial DNA inheritance in sexual crosses of Pleurotus ostreatus

The inheritance of mitochondrial DNA (mtDNA) in sexual crosses was investigated to expand our understanding of the large genetic divergence in mtDNAs among natural populations of the higher basidiomycete Pleurotus ostreatus. Reciprocal crosses were made between compatible monokaryons with distinguishable mtDNA restriction fragment length polymorphisms (PFLPs). Almost all of the dikaryons produced by these crosses had mtDNA genotypes from one of the parental monokaryons. However, for dikaryons isolated from the junction-zone of crossed monokaryons, recombinant mitochondrial genomes commonly appeared. These results showed that P. ostreatus mtDNA can be inherited biparentally, via mtDNA recombination, as well as uniparentally. Further, it was suggested that mtDNA recombination may be an important source of variation in mitochondrial genomes among natural populations of P. ostreatus. Received: 4 June / 14 August 1996     

Extensive mitochondrial DNA variation in somatic tissue cultures initiated from wheat immature embryos

Summary Wheat mitochondria) DNA has been isolated from callus cultures initiated from both immature embryos and the corresponding parental cultivar. A Sall restriction pattern study has shown that the organization of callus culture mitochondria) DNA underwent extensive change, characterized by either the disappearance or the decrease in the relative stoichiometry of several restriction bands. Hybridization of labelled mitochondrial fragments obtained from a recombinant cosmid library to Southern blots of callus and parental line restricted mitochondria) DNAs has shown that a fraction of the mitochondria) genome was lost in callus cultures. Data from a Sall + HindIII restriction map of a defined part of the wheat mitochondria) genome concerned with some of these variations strongly suggest that the observed variations correspond to the disappearance of at least one mitochondria) DNA subgenomic molecule in callus cultures.Abbreviations mtDNA mitochondrial DNA - cpDNA chloroplast DNA - rRNA ribosomal RNA - mRNA messenger RNA - kb kilobase - cv cultivar     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

Translation in a wheat germ cell-free system of RNA from mitochondria of the normal and Texas male-sterile cytoplasms of maize (Zea mays L.)

RNA isolated from etiolated seedling shoot mitochondrial of maize (Zea mays L.) with normal (N) or Texas male-sterile (T) cytoplasm stimulated the incorporation of [³⁵S]-methionine into protein when added to a cell-free protein-synthesizing system from wheat germ. Discrete polypeptides with molecular masses of up to approximately 67 kDa were synthesized, and the pattern of bands was distinct from that obtained with total RNA. Products of translation of T-urf13 RNA were identified by immunoprecipitation, and ofatpA, coxI, andcoxII RNA by hybrid arrest of translation by the cloned gene. several polypeptides were differentially synthesized from N and T mitochondrial RNA; these differences were more extensive than those found when isolated, intact, N and T mitochondria are allowed to synthesize proteins.     

Transmission of mitochondrial DNA in Ustilago violacea

Summary Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrial transmission in the basidiomycete Ustilago violacea. Yeast-like cells of opposite mating types (a₁ and a₂) were mated on 2% water agar and were treated with -tocopherol to induce formation of dikaryotic hyphae. Upon depletion of the -tocopherol, the hyphae budded off haploid cells with parental nuclear genotypes. These cells were examined for mitochondrial RFLP phenotype. In progeny expressing the a₁ mating type, mitochondria from either parent were observed equally frequently. In progeny with the a₂ mating type, mitochondria were almost exclusively (94%) from the a₂ parent.     

An ameiotic mutant of Coprinus cinereus halted prior to pre-meiotic S-phase

Summary Five Coprinus cinereus monokaryons were isolated which bore a dominant mutation designated Mar. Dikaryons formed by mating Mar-bearing monokaryons with normal monokaryons produced aborted fruiting bodies in which the basidia never underwent karyogamy. The Mar mutation probably prevented pre-meiotic DNA replication; extracts of Mar-bearing dikaryons harvested at a stage equivalent to pre-meiotiv S-phase caused little net DNA synthesis in DNA polymerase assays. There was no marked incorporation of 32p into DNA (and low incorporation of 32p into RNA) of cap tissue from Mar-bearing fruiting bodies at a stage equivalent to pre-meiotic S-phase. The aborted fruiting bodies were similar to those resulting on normal cultures following treatment prior to S-phase with cycloheximide, or continuous light at 35 °C. In contrast to normal C. cinereus monokaryons, no Mar-bearing monokaryon formed fruiting bodies when subjected to nutritional stress.     

Age-dependent changes in glutamate oxidation by non-synaptic and synaptic mitochondria from rat brain

The oxidation of glutamate by non-synaptic and synaptic mitochondria from brains of 3-, 12- and 24-month-old rats was studied. With glutamate plus malate as substrates, non-synaptic mitochondria showed higher respiration rates than synaptic mitochondria in all the three age groups studied. The rate of oxidation of L-[1-¹⁴C] glutamate and the activities of NAD-glutamate dehydrogenase and aspartate aminotransferase were also higher in non-synaptic mitochondria compared with synaptic mitochondria in three age groups. With glutamate plus malate as substrates, a significant reduction in state 3 respiration was observed in both mitochondrial populations from 12- and 24-month-old rats compared with 3-month-old animals. Although an age-dependent decrease in the oxidation of L-[1-¹⁴C] glutamate was observed in both non-synaptic and synaptic mitochondria from aging rats, the oxidation of [1-¹⁴C]-2-oxoglutarate was unaltered in non-synaptic and synaptic mitochondria from senescent rats. The activity of NAD-glutamate dehydrogenase was decreased with age in both mitochondrial populations, whereas aspartate aminotransferase was not altered with age. The results indicate that the oxidation rate of glutamate in rat brain mitochondria is decreased during aging.     

A mitochondrial reading frame which may code for a second form of ATPase subunit 9 in Aspergillus nidulans

Summary The nucleotide sequence of a 74 codon reading frame from the Aspergillus nidulans mitochondrial genome is presented. The derived amino acid sequence displays typical features of dicyclohexylcarbodiimide (DCCD) binding proteins and is 84% homologous with a mitochondrial reading frame that potentially encodes an ATPase subunit 9 polypeptide in Neurospora crassa. However, in A. nidulans, as in N. crassa, there is strong biochemical and genetic evidence that this subunit is in fact nuclearly-encoded. In both organisms the DCCD-binding protein found in the F₀ complexes of mitochondria from actively-growing cultures is almost certainly the product of this nuclear gene, and definitely not that of the mitochondrial reading frame. The discovery of an intact open reading frame than can code for a DCCD-binding protein in the mitochondrial genome of a second species of filamentous fungus strenghthens the possibility that the presence of a mitochondrial version of this gene has some biological significance.     

Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes

Aim: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca²⁺ mobilizations in cold‐acclimated rats. Methods: A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca²⁺ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied. Results: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca²⁺ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) followed by mitochondrial Ca²⁺ ([Ca²⁺]_mito), the effect being transformed into an increase in [Ca²⁺]_cyto whereas a decrease in [Ca²⁺]_mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca²⁺ release from mitochondria. CCCP induced Ca²⁺ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells. Conclusions: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito, in which H⁺ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca²⁺ signalling in cold‐acclimated brown adipocytes.     

Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoxim-methyl

Several agricultural fungicides related to the antifungal strobilurins act as inhibitors of respiration by binding to mitochondrial cytochrome b. Two types of laboratory mutants resisting higher doses of the strobilurin-related inhibitor kresoxim-methyl were characterized for Venturia inaequalis, the causal agent of apple scab. Selection of mutagenized conidia by kresoxim-methyl yielded mutants altered in the expression of alternative respiration during the stage of conidia germination. Cytochrome b sequences were not affected in the respective mutants. Selection of conidia on media containing the alternative oxidase inhibitor salicylhydroxamic acid in addition to kresoxim-methyl yielded a highly resistant mutant distinguished by a G143A exchange in cytochrome b. The status of mitochondrial cytochrome b genes remained heteroplasmic, and mitochondria containing wild-type cytochrome b returned to high frequencies during cultivation on inhibitor-free medium. However, continuation of the selection process led to a more pronounced replacement of sensitive by mutated mitochondria. The G143A mutation of cytochrome b causing resistance of V. inaequalis to a strobilurin-related inhibitor has been reported previously for mouse mitochondria; and a permanent G143A exchange rendering naturally resistant mitochondria has been reported for the strobilurin-producing basidiomycete Mycena galopoda and for the sea urchin Paracentrotus lividus. At the corresponding position, alanine was also present in chloroplast cytochrome b ₆ exhibiting low binding of strobilurin-related inhibitors. The mutation of cytochrome b reported here for V. inaequalis describes the first example of a mutation in filamentous ascomycetes and is part of an assessment of resistance risks inherent to strobilurin fungicides. Received: 20 March 2000 / Accepted: 26 May 2000     

p-Aminohippurate/2-oxoglutarate exchange in bovine renal brush-border and basolateral membrane vesicles

The transport of the amphiphilic organic anion, P-aminohippurate (PAH), across the luminal (brush-border) and contraluminal (basolateral) membrane of renal proximal tubule cells was studied with membrane vesicles isolated from bovine kidney cortex. On the basis of the enrichment of specific activities of marker enzymes, leucine aminopeptidase and Na⁺/K⁺-ATPase, brush-border and basolateral membrane vesicles can be obtained from bovine kidneys in reasonably pure form. The uptake of [³H]PAH into both brush-border and basolateral membrane vesicles was trans-stimulated by intravesicular PAH and by 2-oxoglutarate. In the absence of Na⁺, [³H]PAH/2-oxoglutarate exchange was cis-inhibited by unlabelled 2-oxoglutarate in the medium. In the presence of an inward Na⁺ gradient, 10 M 2-oxoglutarate, but no other Krebs cycle derivative, cis-stimulated [³H]PAH uptake, indicating that a Na³-coupled dicarboxylate transporter and PAH/2-oxoglutarate exchanger cooperate in both membranes to enhance [³H]PAH uptake. [³H]PAH uptake showed a non-saturable and a saturable component with similar apparent K _m values in brush-border and basolateral membranes. Although one negatively charged PAH molecule exchanges with one doubly negatively charged 2-oxoglutarate molecule the exchange was electroneutral. Probenecid inhibited [³H]PAH/2-oxoglutarate exchange in brush-border and basolateral membrane vesicles with indistinguishable kinetics. We conclude that similar or identical PAH transporters are located in brush-border and basolateral membranes of bovine kidney proximal tubule cells. This arrangement seems species-specific since a Na⁺ gradient plus 2-oxoglutarate caused concentrative [³H]PAH uptake in brush-border membrane vesicles from bovine, but not from rat kidney.     

一氧化氮合酶激活参与烧伤后Ca2+介导的心肌线粒体损伤

目的:探讨线粒体一氧化氮合酶(mtNOS)在严重烧伤早期心肌线粒体损害中的作用。方法:复制30%TBSAⅢ°烧伤大鼠模型,取正常及伤后1、3、6、12、24h大鼠心肌分离线粒体,测其呼吸功能、Ca²⁺浓度(_m)及细胞色素c氧化酶、mtNOS活性。结果:①伤后1h心肌线粒体呼吸控制率(RCR)显著高于正常组,但3、6、12、24h明显低于正常组,Ⅲ态呼吸速率(ST₃)变化与RCR平行,ST₄仅于伤后3h明显升高;②伤后各时点_m均明显高于正常组,尤以3、6h为甚,而mtNOS活性于伤后3、6、12、24h显著高于对照组,细胞色素c氧化酶活性于伤后3、6、12、24h显著低于正常组;③伤后mtNOS活性与_m呈明显正相关,相关系数为0.8945(P＜0.05),RCR与mtNOS活性呈显著负相关,相关系数为-0.9347(P＜0.05)。结论:伤后_m升高激活mtNOS,可能参与严重烧伤早期心肌线粒体损害。     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.     

Respiratory response of malignant and placental cells to changes in oxygen concentration

Malignant cells and foetal tissues are exposed to low oxygen partial pressure (pO₂) in situ due to the limited supply of oxygenated blood. Whether these cells have adapted to low pO₂ or live under constant constraint is not clear. Herein, we compared the respiratory responses of different malignant cell types, maternal and foetal placental leucocytes, and benign cells by incubating them under a gradient of pO₂, from saturation to hypoxia, in a high resolution respirometer. The malignant cells and foetal leucocytes showed higher rates of mitochondrial oxygen uptake compared to the benign cells and maternal leucocytes, respectively. On the other hand, the mitochondrial oxygen uptake rates of the hypoxia adapted cells declined faster than the other cell types during the onset of hypoxia, probably suggesting conformance of aerobic metabolism to the local oxygen concentration. The O₂ consumption rate per million cells (JO₂) of the malignant cells declined only when the O₂ concentration ([O₂]) decreased to values ≤10 μM. On the other hand, the JO₂ of the benign cells declined with the decrease in [O₂] from 200 to 40 μM and ≤10 μM. In the [O₂] ranges outside these values the JO₂ remained constant regardless of the decline of [O₂] in the medium. The JO₂ of foetal leucocytes and malignant cells responded to the change in [O₂] in a similar manner, and may indicate comparable mechanisms of adaptation to hypoxia.