Subunit Of NADH-ubiquinone Oxidoreductase Research Articles

Genomic Differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, Identified by Suppression Subtractive Hybridization

Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B12 for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.

Investigations of the potential effects of phosphorylation of the MWFE and ESSS subunits on complex I activity and assembly

There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant.

Decreased level of mitochondrial RNA by glutamate in cultured cortical neurons

Mitochondrial injury is induced by a decline in mitochondrial function as well as by damaged mitochondrial DNA. In this study, we evaluate the effects of glutamate exposure on the level of mitochondrial mRNA in cultured cortical neurons of mice. Glutamate exposure for 15 min significantly reduced cell viability 24 h later. Under these experimental conditions, glutamate was effective in reducing the level of mitochondrial mRNAs, especially the mRNAs of NADH-ubiquinone oxidoreductase subunits (nd1 and nd6), 6 h after the exposure. Southern blot analysis, however, revealed no significant change in that of the mitochondrial DNA at any time after glutamate exposure. These results suggest that the activation of glutamate signals negatively regulated the expression of mitochondrial mRNA, without affecting the level of mitochondrial DNA.

Lessons from an estivating frog: sparing muscle protein despite starvation and disuse

Long (6- to 9-mo) bouts of estivation in green-striped burrowing frogs lead to 28% atrophy of cruralis oxidative fibers (P < 0.05) and some impairment of in vitro gastrocnemius endurance (P < 0.05) but no significant deficit in maximal twitch force production. These data suggest the preferential atrophy of oxidative fibers at a rate slower than, but comparable to, laboratory disuse models. We tested the hypothesis that the frog limits atrophy by modulating oxidative stress. We assayed various proteins at the transcript level and verified these results for antioxidant enzymes at the biochemical level. Transcript data for NADH ubiquinone oxidoreductase subunit 1 (71% downregulated, P < 0.05) and ATP synthase (67% downregulated, P < 0.05) are consistent with mitochondrial quiescence and reduced oxidant production. Meanwhile, uncoupling protein type 2 transcription (P = 0.31), which is thought to reduce mitochondrial leakage of reactive oxygen species, was maintained. Total antioxidant defense of water-soluble (22.3 +/- 1.7 and 23.8 +/- 1.5 microM/microg total protein in control and estivator, respectively, P = 0.53) and membrane-bound proteins (31.5 +/- 1.9 and 42.1 +/- 7.3 microM/microg total protein in control and estivator, respectively, P = 0.18) was maintained, equivalent to a bolstering of defense relative to oxygen insult. This probably decelerates muscle atrophy by preventing accumulation of oxidative damage in static protein reserves. Transcripts of the mitochondrially encoded antioxidant superoxide dismutase type 2 (67% downregulated, P < 0.05) paralleled mitochondrial activity, whereas nuclear-encoded catalase and glutathione peroxidase were maintained at control values (P = 0.42 and P = 0.231), suggesting a dissonance between mitochondrial and nuclear antioxidant expression. Pyruvate dehydrogenase kinase 4 transcription was fourfold lower in estivators (P = 0.11), implying that, in contrast to mammalian hibernators, this enzyme does not drive the combustion of lipids that helps spare hypometabolic muscle.

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.

Intragenic Inversion of mtDNA: A New Type of Pathogenic Mutation in a Patient with Mitochondrial Myopathy

We report an unusual molecular defect in the mitochondrially encoded ND1 subunit of NADH ubiquinone oxidoreductase (complex I) in a patient with mitochondrial myopathy and isolated complex I deficiency. The mutation is an inversion of seven nucleotides within the ND1 gene, which maintains the reading frame. The inversion, which alters three highly conserved amino acids in the polypeptide, was heteroplasmic in the patient's muscle but was not detectable in blood. This is the first report of a pathogenic inversion mutation in human mtDNA.

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.

Phylogenetic relationships of the suckermouth minnows, genus Phenacobius, inferred from parsimony analyses of nucleotide sequence, allozymic and morphological data (Cyprinidae: Cypriniformes)

Abstract We investigated the phylogenetic relationships among the five species of the genus Phenacobius (Cyprinidae: Cypriniformes) using a combined analysis of molecular and morphological characters. The nucleotide characters were comprised of 208 potentially informative sites among a total 1773 aligned base pairs from several segments of mitochondrial genes that encode the large and small RNA subunits, the valine transfer RNA, and the NADH ubiquinone oxidoreductase subunit 2. Allelic data from 23 polymorphic allozyme loci and nine morphological characters taken from the literature were also included in the total evidence analysis. Third position codon characters were demonstrated to be saturated for transitional changes and unsuitable for phylogeny estimation among the species studied here. The total evidence phylogeny is isomorphic with Mayden’s (1989) morphological hypothesis. Phenacobius mirabilis was found to be basal, followed in ascending order by P. teretulus, P. catostomus and a clade comprised of P. crassilabrum and P. uranops .

Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone.

Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.

Living in the cold: freeze-induced gene responses in freeze-tolerant vertebrates.

1. Winter survival for numerous cold-blooded animals includes freeze tolerance: the ability to endure the conversion of as much as 65% of total body water into extracellular ice. Selected molecular adaptations underlying freeze tolerance (e.g. cryoprotectants, ice nucleating proteins) have been widely studied, but the full range of metabolic adjustments needed for freeze endurance remains unknown. 2. Recent studies using gene screening techniques are providing a different approach to the search for biochemical responses that support freezing survival by identifying genes and proteins that are up-regulated by freezing or thawing in freeze-tolerant amphibians and reptiles. 3. Screening of a cDNA library from wood frog liver revealed the freeze-induced up-regulation of genes coding for the alpha- and gamma-subunits of fibrinogen (a plasma clotting protein), the mitochondrial ADP/ATP translocase and a novel 10 kDa protein containing a nuclear exporting sequence. 4. Northern blotting revealed that these genes were differentially responsive to two of the component stresses of freezing (dehydration and anoxia), indicating that different genes are induced by signals radiating either from cell volume change or oxygen deprivation during freezing. 5. Freeze up-regulation of fibrinogen synthesis in liver and other organs appears to be a damage repair response that anticipates a need for enhanced plasma clotting capacity to deal with ice crystal damage to capillary beds. 6. Up-regulation of ADP/ATP translocase in frog liver is linked with ischaemia resistance and studies with freeze-tolerant turtles have shown that other genes encoding proteins involved in mitochondrial energetics (NADH-ubiquinone oxido-reductase subunit 5, cytochrome C oxidase subunit 1) are also up-regulated by both anoxia and freezing exposures. 7. These studies are making major advances in our understanding of freeze tolerance as a natural phenomenon and also highlight new key areas that can be targeted by applied interventions for the optimization of medical cryopreservation techniques for cells, tissues and organs.

Heterologous biosynthesis and characterization of the [2Fe-2S]-containing N-terminal domain of Clostridium pasteurianum hydrogenase.

The primary structure of Clostridium pasteurianum hydrogenase I appears to be composed of modules suggesting that the various iron-sulfur clusters present in this enzyme might be segregated in structurally distinct domains. On the basis of this observation, a gene fragment encoding the 76 N-terminal residues of this enzyme has been expressed in Escherichia coli. The polypeptide thus produced contains a [2Fe-2S]n+ cluster of which the oxidized level (n = 2) has been monitored by UV-visible absorption, circular dichroism, and resonance Raman spectroscopy. This cluster can be reduced by dithionite or electrochemically to the n = 1 level which has been investigated by EPR and by low-temperature magnetic circular dichroism. The redox potential of the +2 to +1 transition is -400 mV (vs the normal hydrogen electrode). The spectroscopic and redox results indicate a [2Fe-2S]2+/+ chromophore coordinated by four cysteine ligands in a protein fold similar to that found in plant- and mammalian-type ferredoxins. Among the five cysteines present in the N-terminal hydrogenase fragment, four (in positions 34, 46, 49, and 62) are conserved in other sequences and are therefore the most likely ligands of the [2Fe-2S] site. The fifth cysteine, in position 39, can be dismissed on the grounds that the Cys39Ala mutation does not alter any of the properties of the iron-sulfur cluster. The spectroscopic signatures of this chromophore are practically identical with some of those reported for full-size hydrogenase. This confirms that C. pasteurianum hydrogenase I contains a [2Fe-2S] cluster and indicates that the polypeptide fold around the metal site of the N-terminal fragment is very similar, if not identical, to that occurring in the full-size protein. The N-terminal sequence of this hydrogenase is homologous to sequences of a number of proteins or protein domains, including a subunit of NADH-ubiquinone oxidoreductase of respiratory chains. From that, it can be anticipated that the structural domain isolated and described here is a building block of electron transfer complexes involved in various bioenergetic processes.

The mitochondrial ND8 gene from Crithidia oncopelti is not pan-edited

RNA editing in trypanosomatid mitochondria is a process involving the insertion and deletion of uridine residues within the coding region of maxicircle messenger RNA transcripts. Twelve of the 17 known genes need editing to produce functional molecules. We have analyzed the predicted editing sites for the Crithidia oncopelti mitochondrial NADH-ubiquinone oxidoreductase subunit 8 (ND8) gene based on known mRNAs from other trypanosomatid species. All studied ND8 mRNAs undergo editing throughout the coding (and 3′ non-coding) sequences (pan-editing). The 5′ part of the C. oncopelti ND8 gene undergoes editing (like in Leishmania tarentolae and Trypanosoma brucei) while the 3′ part of the pre-edited gene corresponds to the 3′ part of edited ND8 mRNAs from other organisms. The organization of the ND8 gene in C. oncopelti mitochondrial DNA differs from all organisms investigated so far – this gene is not pan-edited. We have also localized the guide RNA for cytochrome b between 9S rRNA and the ND8 gene. This RNA shows high homology to the gCYb-II gene of L. tarentolae and the gCyb gene of Crithidia fasciculata. A hypothetical editing pattern for the cytochrome b gene in C. oncopelti maxicircles is proposed.

The 49-kDa subunit of NADH-ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone-related inhibitors

Piericidin is a potent inhibitor of the mitochondrial and bacterial type I NADH-ubiquinone oxidoreductases (Complex I) and is considered to bind at or close to the ubiquinone binding site(s) of the enzyme. Piericidin-resistant mutants of the bacterium Rhodobacter capsulatus have been isolated and the present work demonstrates that a single missense mutation at the level of the gene encoding the peripheral 49-kDa/NUOD subunit of Complex I is definitely associated with this resistance. Based on this original observation, we propose a model locating the binding site for piericidin (and quinone) at the interface between the hydrophilic and hydrophobic domains of Complex I.

Identification and Primary Structure of Five Human NADH-Ubiquinone Oxidoreductase Subunits

The multi-subunit NADH-ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria. A small number of NADH-ubiquinone oxidoreductase subunits are the products of mitochondrial genes (subunits 1-7), while the remainder are nuclear encoded and imported from the cytoplasm. We have isolated and sequenced five subunits of the human complex I from a human heart λZAP Express cDNA library. Comparison of the deduced amino acid sequences of the human subunits with the corresponding bovine sequences revealed greater than 80% amino acid identity. The high degree of similarity between human and bovine sequences suggests functional conservation of these subunits in the complex I.In silicoNorthern analysis revealed that two of the subunits were expressed ubiquitously while the remainder may have more restricted patterns of expression.

Anoxia-induced gene expression in turtle heart. Upregulation of mitochondrial genes for NADH-ubiquinone oxidoreductase subunit 5 and cytochrome c oxidase subunit 1.

A cDNA library constructed from heart of anoxia-exposed adult turtles (Trachemys scripta elegans) was differentially screened with 32P-labeled single-stranded cDNA probes from heart of control versus anoxic animals to clone genes induced by anoxia stress. Four cDNA clones, pBTaR20, pBTaR34, pBTaR63 and pBTaR914 were obtained and confirmed to be upregulated in response to anoxic submergence (20 h in N2-bubbled water at 7 degrees C). Two clones, pBTaR20 and pBTaR63, were characterized by sequence analysis and in vivo expression. The clone pBTaR20 had a 1597-bp cDNA sequence and pBTaR63 contained a 1837-bp sequence. The pBTaR20 sequence contained a single open reading frame that was very close to full length and could potentially encode a polypeptide with 508 amino acids. The deduced polypeptide sequence showed approximately 83% of the residues identical with the sequence of cytochrome c oxidase subunit 1 (CO1) that is encoded by a mtDNA gene Cox1. The clone pBTaR63 contained a single potentially full-length open reading frame that could encode a polypeptide of 591 residues. This was similar to another mitochondrial protein, NADH-ubiquinone oxidoreductase subunit 5 (ND5), which is encoded by mtDNA gene Nad5. Analysis of the time course of expression of Cox1 and Nad5 by northern hybridization analysis showed that mRNA transcripts for both accumulated rapidly (within 1 h) in response to anoxia exposure. Both showed similar increases in their transcript content after 1 h of anoxia but with longer anoxia exposures (5 or 20 h) Nad5 mRNA levels remained high whereas Cox1 mRNA content declined somewhat. Northern-blot hybridization also revealed differential expression of these two genes in five other organs of T. s. elegans during anoxia exposure (brain, kidney, liver, red and white skeletal muscle), with a particularly large increase in mRNA transcript levels of both genes in anoxic red muscle. Organ-specific analysis of these genes in a freeze-tolerant turtle species (Chrysemys picta marginata) also showed that differential expression of these genes occurred in response to the ischemia induced by plasma freezing.

The Human B22 Subunit of the NADH-Ubiquinone Oxidoreductase Maps to the Region of Chromosome 8 Involved in Branchio-Oto-Renal Syndrome

To identify candidate genes for Branchio-oto-renal (BOR) syndrome, we have made use of a set of cosmids that map to 8q13.3, which has previously been shown to be involved in this syndrome. These cosmids were used as genomic clones in the attempts to isolate corresponding cDNAs using a modified hybrid selection technique. cDNAs from the region were identified and used to search for sequence similarity in human or other species. One cDNA clone was found to have 89% sequence similarity to the bovine B22 subunit of NADH-ubiquinone oxidoreductase, a mitochondrial protein in the respiratory electron transport chain. Given the history of other mitochondrial mutations being involved in hearing loss syndromes, this gene should be considered a strong candidate for involvement in BOR.

Structural Organization and Chromosomal Localization of the Human Nuclear Gene (NDUFV2) for the 24-kDa Iron-Sulfur Subunit of Complex I in Mitochondrial Respiratory Chain

The structural organization of the entire human nuclear encoded gene for the 24-kDa iron-sulfur subunit of mitochondrial NADH-ubiquinone oxidoreductase (Complex I) and its chromosomal localization were determined. The gene contains 8 exons spanning 31.5 kb. The 5′ flanking region sequenced lacks typical CAAT and TATA boxes but contains three putative GC boxes and there is one GC box at the beginning of the first intron. The sequences matching completely with the NRF-1 binding site and Mt elements were not identified in the flanking region. This gene was assigned to human chromosome 18 at region p11.3, by fluorescent in situ hybridization.

Organization and transcription of the mitochondrial ATP synthase genes in the yeast Yarrowia lipolytica.

Mitochondrial gene organization was studied in a dimorphic yeast, Yarrowia lipoltica. The gene order in a sequenced 6.6-kilobase region closely resembles that of the human mitochondrial genome in that ATP synthase subunit 8 and 6 genes are followed by genes for cytochrome c oxidase subunit 3 (which contains an intron), NADH-ubiquinone oxidoreductase subunit 4, and ATP synthase subunit 9. This region also contains tRNA genes decoding AUA, UGA, CUN and CCN codons, suggesting a unique mitochondrial translation. All the above genes are transcribed from the same DNA strand into multigenic RNAs, starting from a nonanucleotide sequence, 5'-ATATAAATA-3', similar to other yeast mitochondrial promoters.

A chicken genomic DNA fragment that hybridizes to the murine Hox-3.1 homeobox is likely to encode the NADH ubiquinone oxidoreductase subunit B15

A chicken DNA fragment, which was isolated from a cosmid genomic library using a murine homeobox probe, is shown here to be a likely candidate for encoding a protein homologous to the bovine B 15 subunit of NADH ubiquinone oxidoreductase. The genomic sequence suggests the presence of an intron separating two coding regions. An alignment between the bovine NADH B 15 protein and its putative avian homolog implies the presence of conserved domains, including a transmembrane helix, tyrosine sulfatation site, and a phosphorylation consensus site.

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H+-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request.