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PubMed2006

The complete mitochondrial genome sequence and characterization of single-nucleotide polymorphisms in the control region of the Asian seabass (Lates calcarifer).

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.

Animals↗
PubMed2026

Alterations of endothelial cell bioenergetics in congenital diaphragmatic hernia.

BACKGROUND: Pulmonary vascular development in congenital diaphragmatic hernia (CDH) is characterized by impaired angiogenesis and pathologic remodeling that contribute to pulmonary hypertension/hypoplasia. Mitochondria regulate endothelial energy, redox balance, and angiogenic signaling, suggesting a role in CDH vascular disease. METHODS: Endothelial cells (ECs) were isolated from umbilical veins of healthy and CDH newborns. Mitochondrial bioenergetics and glycolytic acidification were assessed by extracellular flux. Oxidative stress, mitochondrial membrane potential, and mitochondrial mass were measured by flow cytometry, while mitochondrial DNA copy number (mtDNA-CN) and morphology were evaluated by qPCR and microscopy. RESULTS: CDH ECs exhibited increased maximal respiratory capacity with elevated proton leak and reduced ATP coupling efficiency. Basal glycolytic activity was elevated. These changes were accompanied by increased mitochondrial superoxide and cellular reactive oxygen species and by severity-associated loss of membrane potential. Despite reduced MitoTracker Green, mtDNA-CN was unchanged, and confocal imaging revealed a highly branched, peripherally distributed network. CONCLUSIONS: These data define a distinct endothelial mitochondrial phenotype marked by metabolic activation, bioenergetic inefficiency, and oxidative stress, with concurrent upregulation of glycolysis and oxidative phosphorylation rather than a glycolytic shift. Structural remodeling with preserved mitochondrial content further indicates qualitative changes. Collectively, these findings link mitochondrial dysfunction to vascular pathology in CDH. IMPACT: Defines a distinct mitochondrial state in CDH endothelium, characterized by metabolic activation with inefficient oxidative phosphorylation, redox imbalance, and structural reorganization in primary human cells. Demonstrates that mitochondrial alterations in CDH occur without changes in mitochondrial content, supporting a model of qualitative remodeling. Provides rare human, cell-based data in CDH, addressing a major gap in a field largely reliant on animal models and indirect measures. Links mitochondrial alterations to clinical severity, supporting relevance to disease burden and heterogeneity. Establishes a framework for mitochondrial involvement in CDH vascular disease, with potential implications for future biomarker development and therapeutic targeting.

Journal Article↗
PubMed2000

Quantitative analysis, by ultrastructural in situ hybridization, of mitochondrial genomes and their expression in mid-gut and ovarian cells of a mutant strain of Drosophila subobscura.

In the mitochondrial deletion mutant strain studied here, two types of DNA coexist (heteroplasmy): intact mtDNA (15.9 kb) and mutant mtDNA (10.9 kb), which represents about 80% of the mitochondrial genomes in somatic tissues. The heteroplasmy level is lower in ovary (63%). Mutation is transmitted unchanged through generations. Quantitative analysis of in situ DNA hybridization demonstrated that for the 12SrDNA probe, of a gene outside the deletion, the mitochondrial DNA cellular content in the studied cells of the mutant strain is 1.5 times higher than in the wild-type strain. For the probe encoding Cyto b, a mitochondrial gene affected by the mutation, the ratios (mutant versus wild-type content) differ according to cell type: close to 0.4 in MGE cells and 0.7 in ovary cells. These values indicate heteroplasmic levels of about 72% in MGE cells and 50% in stage 10 oocytes, which is lower than that previously reported for stage 14 oocytes (60%) and embryos (69%). Analysis of in situ RNA hybridization showed that for the 12SrDNA probe, the transcript concentrations do not differ significantly between MGE cells and cells of germinal origin from the two strains. For the Cyto b probe, the mutant RNA/wild-type RNA ratios are lower in somatic cells than in stage 10 nurse cells and oocytes, but in each case less than expected. These studies indicate that the progressive heteroplasmy increase may be related to intense phases of mitochondria biogenesis and that different compensatory phenomena may exist.

Animals↗
PubMed2001

Yeast population dynamics in spontaneous fermentations: comparison between two different wine-producing areas over a period of three years.

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.

DNA, Mitochondrial↗
PubMed1995

Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea.

The complete sequence (14,130 bp) of the mitochondrial DNA (mtDNA) of the land snail Albinaria coerulea was determined. It contains 13 protein, two rRNA and 22 tRNA genes. Twenty-four of these genes are encoded by one and 13 genes by the other strand. The gene arrangement shares almost no similarities with that of two other molluscs for which the complete gene content and arrangement are known, the bivalve Mytilus edulis and the chiton Katharina tunicata; the protein and rRNA gene order is similar to that of another terrestrial gastropod, Cepaea nemoralis. Unusual features include the following: (1) the absence of lengthy noncoding regions (there are only 141 intergenic nucleotides interspersed at different gene borders, the longest intergenic sequence being 42 nucleotides) (2) the presence of several overlapping genes (mostly tRNAs), (3) the presence of tRNA-like structures and other stem and loop structures within genes. An RNA editing system acting on tRNAs must necessarily be invoked for posttranscriptional extension of the overlapping tRNAs. Due to these features, and also because of the small size of its genes (e.g., it contains the smallest rRNA genes among the known coelomates), it is one of the most compact mitochondrial genomes known to date.

Animals↗
PubMed2005

HmtDB, a human mitochondrial genomic resource based on variability studies supporting population genetics and biomedical research.

BACKGROUND: Population genetics studies based on the analysis of mtDNA and mitochondrial disease studies have produced a huge quantity of sequence data and related information. These data are at present worldwide distributed in differently organised databases and web sites not well integrated among them. Moreover it is not generally possible for the user to submit and contemporarily analyse its own data comparing them with the content of a given database, both for population genetics and mitochondrial disease data. RESULTS: HmtDB is a well-integrated web-based human mitochondrial bioinformatic resource aimed at supporting population genetics and mitochondrial disease studies, thanks to a new approach based on site-specific nucleotide and aminoacid variability estimation. HmtDB consists of a database of Human Mitochondrial Genomes, annotated with population data, and a set of bioinformatic tools, able to produce site-specific variability data and to automatically characterize newly sequenced human mitochondrial genomes. A query system for the retrieval of genomes and a web submission tool for the annotation of new genomes have been designed and will soon be implemented. The first release contains 1255 fully annotated human mitochondrial genomes. Nucleotide site-specific variability data and multialigned genomes can be downloaded. Intra-human and inter-species aminoacid variability data estimated on the 13 coding for proteins genes of the 1255 human genomes and 60 mammalian species are also available. HmtDB is freely available, upon registration, at http://www.hmdb.uniba.it. CONCLUSION: The HmtDB project will contribute towards completing and/or refining haplogroup classification and revealing the real pathogenic potential of mitochondrial mutations, on the basis of variability estimation.

Computational Biology↗
PubMed2006

The complete mitochondrial genome of the helmet catfish Cranoglanis bouderius (Siluriformes: Cranoglanididae) and the phylogeny of otophysan fishes.

The complete sequence of the 16,539 nucleotide mitochondrial genome from the single species of the catfish family Cranoglanididae, the helmet catfish Cranoglanis bouderius, was determined using the long and accurate polymerase chain reaction (LA PCR) method. The nucleotide sequences of C. bouderius mitochondrial DNA have been compared with those of three other catfish species in the same order. The contents of the C. bouderius mitochondrial genome are 13 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, and a non-coding control region, the gene order of which is identical to that observed in most other vertebrates. Phylogenetic analyses for 13 otophysan fishes were performed using Bayesian method based on the concatenated mtDNA protein-coding gene sequence and the individual protein-coding gene sequence data set. The competing otophysan topologies were then tested by using the approximately unbiased test, the Kishino-Hasegawa test, and the Shimodaira-Hasegawa test. The results show that the grouping ((((Characiformes, Gymnotiformes), Siluriformes), Cypriniformes), outgroup) is the most likely but there is no significant difference between this one and the other alternative hypotheses. In addition, the phylogenetic placement of the family Cranoglanididae among siluriform families was also discussed.

Animals↗
PubMed2004

Mitochondrial DNA and STR analyses of maggot crop contents: effect of specimen preservation technique.

DNA analysis of maggot crop contents can be used to identify a missing body or aid entomologists with interpreting evidence used for PMI estimations. Entomological evidence is often collected and preserved to keep identifiable external features intact. The preservation methods currently in use may not be suitable for preserving DNA in the maggot crop for later analysis. In this study, carrion maggots raised on human tissue were preserved under the following 8 preservation conditions: no fluid at -70 degrees C, no fluid at 4 degrees C, no fluid at 24 degrees C, 70% ethanol at 4 degrees C, 70% ethanol at 24 degrees C, 95% ethanol at 24 degrees C, Kahle's solution at 24 degrees C and formaldehyde at 24 degrees C. Maggots were dissected following 2 weeks, 8 weeks and 6 months of preservation. The maggot crops were extracted, human DNA was quantitated, and an attempt was made at amplifying mitochondrial DNA (mtDNA) and short tandem repeat (STR) loci. Both mtDNA and STRs were successfully amplified from maggots stored in ethanol or without any preservation fluid. Formalin-containing preservation solutions reduced the recovery of DNA. The best results were observed from maggots stored without any preservation fluid at -70 degrees C.

Animals↗
PubMed2006

A new activating role for CO in cardiac mitochondrial biogenesis.

To investigate a possible new physiological role of carbon monoxide (CO), an endogenous gas involved in cell signaling and cytotoxicity, we tested the hypothesis that the mitochondrial generation of reactive oxygen species by CO activates mitochondrial biogenesis in the heart. In mice, transient elevations of cellular CO by five- to 20-fold increased the copy number of cardiac mitochondrial DNA, the content of respiratory complex I-V and interfibrillar mitochondrial density within 24 hours. Mitochondrial biogenesis is activated by gene and protein expression of the nuclear respiratory factor 1 (NRF1) and NRF2, of peroxisome proliferator-activated receptor gamma co-activator-1alpha, and of mitochondrial transcription factor A (TFAM), which augmented the copy number of mitochondrial DNA (mtDNA). This is independent of nitric oxide synthase (NOS), as demonstrated by the identical responses in wild-type and endothelial NOS (eNOS)-deficient mice, and by the inhibition of inducible NOS (iNOS). In the heart and in isolated cardiomyocytes, CO activation involved both guanylate cyclase and the pro-survival kinase Akt/PKB. Akt activation was facilitated by mitochondrial binding of CO and by production of hydrogen peroxide (H(2)O(2)). Interference with Akt activity by blocking PI 3-kinase and by mitochondrial targeting of catalase to scavenge H(2)O(2) prevented binding of NRF1 to the Tfam promoter, thereby connecting mitochondrial H(2)O(2) to the pathway leading to mtDNA replication. The findings disclose mitochondrial CO and H(2)O(2) as new activating factors in cardiac mitochondrial biogenesis.

Animals↗
PubMedDate not supplied

Unusual clinical presentation of a patient carrying a novel single 1.8 kb deletion of mitochondrial DNA.

Kearns-Sayre syndrome (KSS) is a mitochondrial encephalomyopathy characterized by progressive external ophthalmoplegia (PEO), pigmentary retinopathy and onset before the age of 20 years. Cerebellar ataxia, as well as short stature and increased protein content in the cerebrospinal fluid, are frequent additional symptoms. A single large mitochondrial (mt) DNA deletion of 4,977 bp is the most common molecular defect in KSS. Recently, different mutations have also been associated with incomplete, KSS-like phenotypes. We describe the unusual clinical presentation of a patient carrying a novel 1,814-bp deletion of mtDNA. In contrast with typical KSS, the clinical picture of this patient did not include either palpebral ptosis or PEO and was dominated by an ataxic syndrome.

Adult↗
PubMed2005

Possible ways nucleoside analogues can affect mitochondrial DNA content and gene expression during HIV therapy.

In recent years, research into nucleoside reverse transcriptase inhibitor (NRTI)-related mitochondrial (mt) toxicity in HIV therapy has led to conflicting results and many unanswered questions regarding the molecular mechanisms that lead to such toxicity. From the early hypothesis that inhibition of the human mt polymerase gamma by NRTls was responsible for the drugs' mt toxicity, an increasingly complex picture is emerging that probably involves multiple mt pathways. Results have been presented suggesting that NRTIs affect not only mtDNA but also mtRNA, nucleotide phosphorylation and the mt respiratory chain. Based on the current level of knowledge, this overview addresses some of the potential mechanisms through which NRTIs could affect mitochondria and ultimately cause the toxicity symptoms observed in HIV patients receiving NRTI-containing antiretroviral therapy.

Anti-HIV Agents↗
PubMed1998

The role of mitochondrial glutathione in DNA base oxidation.

The objective of this study was to elucidate the role of mitochondrial GSH in the reactions leading to mitochondrial DNA oxidative damage in terms of 8-hydroxy-desoxyguanosine (8-HOdG) accumulation. With this purpose, tightly coupled mitochondria depleted of matrix GSH were used and the effects of H2O2 (generated during the oxidation of substrates) on 8-HOdG levels were investigated. Mitochondrial integrity, assessed by O2 uptake, respiratory control and P/O ratios, was conserved upon depletion of GSH up to 95%. The rates of H2O2 production linked to the oxidation of endogenous substrates by control and GSH-depleted mitochondria were similar. Succinate (in the absence or presence of antimycin A) enhanced the rate H2O2 production to a similar extent in both control and GSH-depleted mitochondria. These rates of H2O2 production accounted for 1.5-2.5% of the rate of O2 uptake. The levels of 8-HOdG in GSH-depleted mitochondria were 35-50% lower than those in control mitochondria, when measured at different H2O2 production rates. Conversely, in experiments carried out with calf thymus DNA with different Cu/Fe content, GSH increased 1.4-2.4-fold the accumulation of 8-HOdG. These values were further enhanced (44-50%) by superoxide dismutase and decreased by catalase. The lower levels of 8-HOdG in GSH-depleted mitochondria and the higher levels in GSH-supplemented calf thymus DNA suggest a role for the non-protein thiol in the reactions leading to mtDNA oxidative damage. These findings are interpreted in terms of the redox transitions involving O2, GSH, and metal catalysts bound to DNA. A mechanism is proposed by which GSH plays a critical role in the reduction of DNA-Cu complexes and decays by free radical pathways kinetically regulated by superoxide dismutase.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Interpretation of variability of mitochondrial genomes in the species Aspergillus carbonarius.

For interpretation of intraspecific polymorphism and the considerable differences in the size of mtDNAs among three groups of A. carbonarius, restriction maps were constructed from several enzymes. Functional maps were also developed to compare genome organisations and gene content. The appearance of various mtDNAs of A. carbonarius strains are different in size, but their gene content is almost identical. The 1.1 kb size difference between two closely related subgroups (1a, 1b) can be attributed to the presence or absence of an intron in cox2 gene. This phenomenon demonstrates that the migration of introns is possibly responsible for the development of variable mitochondrial genomes in nature. The striking differences in size and restriction patterns between two main mtDNA groups might derive from both the intronal variations and the altered intergenic organisation.

Aspergillus↗
PubMed2000

Regulation of the expression of the sea urchin mitochondrial D-loop binding protein during early development.

The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA, may control the content of this protein in the following stages of embryogenesis.

Animals↗
PubMed1998

The 'evolutionary signal' of homoplasy in protein-coding gene sequences and its consequences for a priori weighting in phylogeny.

To analyse independently homoplasy for the six possible types of substitution (i.e., A-G, C-T, A-C, A-T, C-G and G-T) at each of the three codon-positions of the cytochrome b gene, two approaches were used: the first is based on the consistency index which measures the amount of homoplasy, and the second is based on the saturation analysis which describes graphically the distribution of homoplasy within the taxonomic sampling. The results obtained from a data set of 32 sequences of Artiodactyla indicate that evolution of the cytochrome b is governed by differential constraints: 1) between the six substitutions-types, 2) between the three codon-positions, and 3) between the two mtDNA strands. Moreover, we find that non-synonymous sites can be more homoplastic than synonymous sites when the possibilities of substitutions are severely restricted because of the functional requirements of hydrophobicity. Most weighting schemes applied to protein-coding genes are elaborated from unjustified assumptions. We propose to weight each substitution-type at each codon-position according to its homoplasy content evaluated either with the consistency index or with an index representing the level of mutational saturation.

Amino Acid Sequence↗
PubMed2006

A complete mitochondrial genome sequence of Asian black bear Sichuan subspecies (Ursus thibetanus mupinensis).

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16,868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%).

Animals↗
PubMed2003

Heteroplasmic ratio of the A3243G mitochondrial DNA mutation in single pancreatic beta cells.

AIMS/HYPOTHESIS: To examine whether there is a high content of mutated mitochondrial DNA in individual pancreatic beta cells from a patient with the A3243G mitochondrial DNA mutation. METHODS: Tissues were available from a patient with diabetes and the A3243G mutation including pancreatic tissue. We quantified the amount of mutated mitochondrial DNA in tissue homogenates and single pancreatic beta cells using hot last cycle PCR. RESULTS: The percentage ratio of mutated to wild-type mtDNA was high in tissues such as muscle and brain (>60%), but surprisingly low in both pancreatic islets and in individual beta cells from these islets. The islets were smaller in the patient than in control subjects in keeping with a decreased beta-cell mass. CONCLUSIONS/INTERPRETATION: These observations suggest that either the beta cells show increased sensitivity to the effects mtDNA mutations on respiratory chain function, and/or cells with a high mutant load are preferentially removed leading to a progressive decrease in the islet beta-cell mass.

Brain↗
PubMed2006

Metabolic capacity regulates iron homeostasis in endothelial cells.

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.

Blotting, Western↗