New primers for amplifying and sequencing the mitochondrial ND4/ND5 gene region of the Cypriniformes (Actinopterygii: Ostariophysi)

We provide 15 new primers for amplifying and sequencing the mitochondrial ND4/ND5 gene region of the Cypriniformes in an attempt to resolve relationships of this diverse group of freshwater fishes with extensive taxonomic sampling. Sequences from this region have the following desirable characteristics for phylogenetic analyses, some of which are lacking from the more commonly used cyt b and 12S/16S rRNA genes: they are (1) easy to align, (2) relatively long (ca. 3.4 kb), and (3) contain more phylogenetically informative variation at 1st and 2nd codon positions. Moreover, the ND4/ND5 gene region is easy to amplify and sequence when employing the protocol suggested herein.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

Structure and expression of the overlapping ND4L and ND5 genes of Neurospora crassa mitochondria

Summary Genes homologous to the mammalian mitochondrial NADH dehydrogenase subunit genes ND4L and ND5 were identified in the mitochondrial genome of the filamentous fungus Neurospora crassa, and the structure and expression of these genes was examined. The ND4L gene (interrupted by one intervening sequence) potentially encodes an 89 residue long hydrophobic protein that shares about 26% homology (or 41% homology if conservative amino acid substitutions are allowed) with the analogous human mitochondrial protein. The ND5 gene (which contains two introns) encodes a 715 residue polypeptide that shares 23% homology with the human analogue; a 300 amino acid long region is highly conserved (50% homology) in the two ND5 proteins. The stop codon of the ND4L gene overlaps the initiation codon of the downstream ND5 gene, and the two genes are contranscribed and probably cotranslated. A presumed mature dicistronic (ND4L plus ND5) RNA was detected. The postulated mRNA (about 3.2 kb) contains 5 and 3 non-coding regions of about 86 and 730 nucleotides, respectively; this species is generated from very large precursor RNAs by a complex processing pathway. The ND4L and ND5 introns are all stable after their excision from the precursor species.Abbreviations bp base pairs - rRNA ribosomal RNA - ND NADH dehydrogenase - URF unidentified reading frame - kDal kilodaltons; a.a., amino acid     

Three class I introns in the ND4L/ND5 transcriptional unit of Neurospora crassa mitochondria

The overlapping ND4L and ND5 genes of Neurospora crassa mitochondria are interrupted by one and two intervening sequences, respectively, of about 1,490, 1,408 and 1,135 bp in length. All three intervening sequences are class I introns and as such have the potential to fold into the conserved secondary structure that has been proposed for the majority of fungal mitochondrial introns. They contain long open reading frames (ORFs; from 306 to 425 codons long) that are continuous and in frame with the upstream exon sequences. These ORFs contain the conserved decapeptide-encoding sequences that are characteristic of the ORFs present in most class I introns. Extensive homology exists among the ORFs encoded by the ND4L intron, ND5 intron 1, and the second intron of the N. crassa oli2 gene. Also, internal repeats of about 130 amino acid residues are present twice in each of these three ORFs, suggesting that a duplication event may have occurred in the formation of these ORFs. The ND4L intron shares extensive homology (at the levels of both primary and proposed secondary structures) with the self-splicing intervening sequence present in the Tetrahymena nuclear rRNA gene. This homology includes but is not limited to the core secondary structure, as peripheral structural elements are also conserved in the two introns.     

基于ND4和ND5基因序列分析的鳅超科鱼类系统发育关系

ND4和ND5是线粒体基因组中编码NADH脱氢酶亚基4和亚基5的两个蛋白质编码基因.该研究以鳅超科鱼类为研究对象,新测定了10个物种的ND4和ND5基因全序列以及中间的3个tRNA基因共212 bp的序列,结合从GenBank 下载的15个物种的15条序列进行序列比较和系统发育关系分析.结果显示:鳅超科鱼类ND4基因全长1380～1387 bp,以ATG为起始密码子,终止密码子为不完全终止信号;ND5基因全长1821～1839bp,同样起始密码子为ATG,终止密码子为TAA或TAG;ND4和ND5基因之间插入了3个tRNA基因,分别编码携带组氨酸、丝氨酸、亮氨酸的tRNA.ND4和ND5基因(包含3个tRNA基因)中A、T、G、C的平均含量分别为30.4%、27.3%、14.2%、28.1%,A+T(57.7%)的含量高于G+C(42.3%)的含量.转换与颠换比(Ti/Tv)平均值为1.586.选取斑马鱼和鲤鱼作为外类群,采用最大简约法(MP)、最大似然法(ML)和贝叶斯推断法(BI)进行系统发育树的重建.三种方法的系统发育分析结果都显示:花鳅亚科、条鳅亚科、沙鳅亚科、平鳍鳅科及Vaillantellidae分别构成单系;它们的系统发育关系为:(Vaillantellidae+(沙鳅亚科+(花鳅亚科+(条鳅亚科+平鳍鳅科).这与线粒体全基因组和某些核基因(如RAG1基因)的研究结果类似,且支持率较高,表明ND4和ND5基因用于鳅超科鱼类的系统发育分析是可行的;但是该研究的结果有别于其他线粒体基因的分析结果,如基于cytb和D-loop基因进行的系统发育分析表明,条鳅亚科和花鳅亚科聚为姐妹群,再和平鳍鳅科聚在一起.这种差异可能是由于使用的基因长度差异造成的,长度越长,信息量越大,所反映的系统发育结果可能更加接近真实情况.     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

Review: properties and assembly mechanisms of ND10, PML bodies, or PODs

Nuclear domain 10 (ND10), also referred to as PML bodies or PODs, are discrete interchromosomal accumulations of several proteins including PML and Sp100. We describe here developments in the visualization of ND10 and the mechanism of ND10 assembly made possible by the identification of proteins that are essential for this process using cell lines that lack individual ND10-associated proteins. PML is critical for the proper localization of all other ND10-associated proteins under physiological conditions. Introducing PML into a PML -/- cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10, attesting to its essential nature in ND10 formation. This recruitment includes Daxx, a protein that can bind PML and is highly enriched in condensed chromatin in the absence of PML. The segregation of Daxx from condensed chromatin to ND10 by increased accumulation of Sentrin/SUMO-1 modified PML suggests the presence of a variable equilibrium between these two nuclear sites. These findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML. Additional adapter proteins are suggested to exist by the behavior of Sp100, and Sp100 will provide the basis for their identification. Further information about the dynamic balance of proteins between ND10 and the actual site of functional activity of these proteins will establish whether ND10 function as homeostatic regulators or only in storage of excess proteins destined for turnover.     

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

Isoforms of the promyelocytic leukemia protein differ in their effects on ND10 organization

The PML protein is a defining constituent of subnuclear structures known as ND10. PML is expressed as a series of primary sequence isoforms through alternative RNA processing. Expression of each of six distinct PML isoforms that differed in their C-terminal domains caused reproducible differences in the number, size, and shape of ND10 in both transformed cell lines and diploid fibroblasts. In each case, PML from the endogenous genes was reorganized to participate with the exogenously expressed PML in the new configuration of ND10. Variation in ND10 number is known to occur during the cell cycle; however, the cell cycle distribution of the transfected cells that displayed these altered ND10 was similar for all six PML isoforms. Given our findings, the precise level of expression of the different PML isoforms under particular physiological conditions will be an important determinant of ND10 organization and function and is a potential point of regulation of PML/ND10 function.     

Phylogenetic relationships of catostomid fishes (Actinopterygii: Cypriniformes) based on mitochondrial ND4/ND5 gene sequences

Family Catostomidae is a diverse group of benthic freshwater fishes that are distributed across North America and in parts of East Asia. In this study, the phylogenetic relationships of Catostomidae is examined using 3436 nucleotides of mitochondrial ND4 and ND5 protein coding genes and intervening tRNAs. All 13 genera and 60 species of catostomids were sampled to represent diversity of the family. Catostomidae and its four subfamilies were found to be monophyletic; however, relationships of the subfamilies are not strongly supported with bootstrapping. The analysis provides strong support for recognizing four tribes in subfamily Catostominae.     

Phylogenetic relationships within the genus Pimephales as inferred from ND4 and ND4L nucleotide sequences

In phylogenetic analyses, conducted on the ND4L gene and part of the ND4 gene from species of the genus Pimephales , maximum parsimony yielded four trees, with the strict consensus providing no resolution of relationships among species. Maximum likelihood and minimum evolution methods yielded identical tree topologies, which differed from previous hypotheses of relationships for these species. If this topology is correct, it implies independent evolution of morphological characters, possibly associated with convergent trophic specialization.     

Phylogeny and evolution of the Drosophila nasuta subgroup based on mitochondrial ND4 and ND4L gene sequences

The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup. The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genes is strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D. sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D. nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous.     

Complete genome sequence of Streptococcus thermophilus strain ND03

Streptococcus thermophilus strain ND03 is a Chinese commercial dairy starter used for the manufacture of yogurt. It was isolated from naturally fermented yak milk in Qinghai, China. We present here the complete genome sequence of ND03 and compare it to three other published genomes of Streptococcus thermophilus strains.     

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.     

Simian virus 40-specific polypeptides in AD2+ ND1- and Ad2+ ND4-infected cells.

A comparison of the proteins synthesized in human cells at late times after infection with adenovirus (Ad2) and with the adeno-simian virus 40 (SV40) hybrid viruses revealed polypeptides of 30,000 and 92,000 molecular weight specific for the hybrid viruses Ad2+ND1 and Ad2+ND4, respectively. Cell-free translation of SV40-specific mRNA, prepared from these cells by hybridization of total cytoplasmic RNA to SV40 DNA, showed that the mRNA's specifying these two polypeptides were at least partially encoded by the SV40 portion of the hybrid viruses. Cell-free translation of SV40-specific mRNA prepared from monkey cells infected with SV40 produced polypeptides of 40,000, 43,000, 48,500, and 92,000 molecular weight. The SV40 and Ad2+ND4 92,000-molecular-weight polypeptides made in vitro were very similar in electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to the polypeptide precipitated by Tegtmeyer (1974) with SV40 anti-T serum.     

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.     

The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes

Leber's Hereditary Optic Neuropathy (LHON) was the first maternally inherited mitochondrial disease identified and is now considered the most prevalent mitochondrial disorder. LHON patients harbor mutations in mitochondrial DNA (mtDNA). In about 90% of cases, the genes involved encode proteins of the respiratory chain complex I. Even though the molecular bases are known since 20 years almost all remains to be done regarding physiopathology and therapy. In this study, we report a severe decrease of complex I activity in cultured skin fibroblasts isolated from two LHON patients harboring mutations in ND4 or ND1 genes. Most importantly, we were able to restore sustainably (a) the ability to grow on galactose, (b) the ATP synthesis rate and (c) the complex I activity, initially impaired in these cells. Our strategy consisted of forcing mRNAs from nuclearly-encoded ND1 and ND4 genes to localize to the mitochondrial surface. The rescue of the respiratory chain defect observed was possible by discreet amounts of hybrid mRNAs and fusion proteins demonstrating the efficiency of their mitochondrial import. Hence, we confirmed here for two mitochondrial genes located in the organelle that the optimized allotopic expression approach represents a powerful tool that could ultimately be applied in human therapy for LHON.     

Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees.

Biochemical and molecular genetic evidence is presented that in six independent pedigrees the development of Leber hereditary optic neuropathy (LHON) is due to the same primary mutation in the mitochondrial ND1 gene. A LHON family from the Newcastle area of Great Britain was analyzed in depth to determine the mitochondrial genetic etiology of their disease. Biochemical assays of mitochondrial electron transport in organelles isolated from the platelet/white-blood-cell fraction have established that the members of this family have a substantial and specific lowering of flux through complex I (NADH-ubiquinone oxidoreductase). To determine the site of the primary mitochondrial gene mutation in this pedigree, all seven mitochondrial complex I genes were sequenced, in their entirety, from two family members. The primary mutation was identified as a homoplasmic transition at nucleotide 3460, which results in the substitution of threonine for alanine at position 52 of the ND1 protein. This residue occurs within a very highly conserved hydrophilic loop, is invariantly alanine or glycine in all ND1 proteins, and is adjacent to an invariant aspartic acid residue. This is only the second instance in which both a biochemical abnormality and a mitochondrial gene mutation have been identified in an LHON pedigree. The sequence analysis of the ND81 gene was extended to a further 11, unrelated LHON pedigrees that had been screened previously and found not to carry the mitochondrial ND4/R340H mutation. The ND1/A52T mutation at nucleotide 3460 was found in five of these 11 pedigrees. In contrast, this sequence change was not found in any of the 47 non-LHON controls. The possible role of secondary complex I mutations in the etiology of LHON is also addressed in these studies.