NADH Dehydrogenase Genes Research Articles

Mitochondrial NADH-dehydrogenase polymorphisms as sporadic breast cancer risk factor

Breast cancer is the most frequently diagnosed female cancer all over the world. Although the molecular genetics of this disease has been the focus of many projects for over 20 years, the number of prognostic markers used in clinics is still unsatisfactory. Mitochondrial DNA mutations have been reported in many breast cancer studies. To investigate the possible role of mitochondrial inherited polymorphisms in breast cancer development we analyzed the sequence of NADH-dehydrogenase genes in cancer samples and their corresponding normal tissues. We detected increased incidence of mtDNA polymorphisms, in particular very rare polymorphisms such as A4727G, G9947A, A10044G, A10283G, T11233C, and C11503T. Our report supports the notion that mtDNA polymorphisms establish a specific genetic background for breast cancer development and that mtDNA analysis may help in selection of cohorts that should undergo intensive screening and early detection programs.

Adipocyte Differentiation, Mitochondrial Gene Expression and Fat Distribution: Differences between Zidovudine and Tenofovir after 6 Months

Abnormal lipid metabolism and cell oxidative mechanisms are reported in patients on antiretroviral treatment. We compared the expression of several key adipocyte genes in HIV-infected patients randomized to antiretroviral regimens containing zidovudine (AZT) or tenofovir disoproxil fumarate (TDF). Subcutaneous fat was sampled from 32 HIV-positive treatment-naive patients before and 6 months after randomization to AZT/lamivudine/efavirenz (n=15) or TDF/emtricitabine/efavirenz (n=17) plus 15 HIV-negative matched controls. Expression of genes involved in adipocyte differentiation, lipid metabolism, mitochondrial function and glucocorticoid generation were profiled using real-time PCR. Lipoprotein lipase and hepatic lipase activity were assessed. Before treatment, 11beta-hydroxysteroid dehydrogenase expression was down-regulated compared with controls. Following 6 months treatment with AZT, there was a significant increase in visceral adipose tissue (VAT; P=0.02) and the ratio of VAT to subcutaneous adipose tissue (P=0.008), down-regulation of cytochrome B (P=0.003) and cytochrome oxidase (COX)-3 gene expression (P=0.03), up-regulation of NADH dehydrogenase (P=0.008) and nuclear-encoded COX-4 (complex IV) gene expression (P=0.012). Genes involved with adipocyte cortisol generation, fatty acid metabolism and the tricarboxylic acid cycle were up-regulated. In the TDF-treated patients, there was no significant change in regional body fat or mitochondrial genes compared with pretreatment values. Changes in the expression of genes involved with cortisol and fatty acid metabolism were less marked with TDF. Interference with the mitochondrial electron transport chain appears to occur early in an AZT-containing regimen and occurs at a time when there is increased visceral fat and up-regulation of genes involved with adipocyte differentiation and fatty acid flux.

Comparative genomics of Drosophila mtDNA: Novel features of conservation and change across functional domains and lineages.

To gain insight on mitochondrial DNA (mtDNA) evolution, we assembled and analyzed the mitochondrial genomes of Drosophila erecta, D. ananassae, D. persimilis, D. willistoni, D. mojavensis, D. virilis and D. grimshawi together with the sequenced mtDNAs of the melanogaster subgroup. Genomic comparisons across the well-defined Drosophila phylogeny impart power for detecting conserved mtDNA regions that maintain metabolic function and regions that evolve uniquely on lineages. Evolutionary rate varies across intergenic regions of the mtDNA. Rapidly evolving intergenic regions harbor the majority of mitochondrial indel divergence. In contrast, patterns of nearly perfect conservation within intergenic regions reveal a refined set of nucleotides underlying the binding of transcription termination factors. Sequencing of 5' cDNA ends indicates that cytochrome C oxidase I (CoI) has a novel (T/C)CG start codon and that perfectly conserved regions upstream of two NADH dehydrogenase (ND) genes are transcribed and likely extend these protein sequences. Substitutions at synonymous sites in the Drosophila mitochondrial proteomes reflect a mutation process that is biased toward A and T nucleotides and differs between mtDNA strands. Differences in codon usage bias across genes reveal that weak selection at silent sites may offset the mutation bias. The mutation-selection balance at synonymous sites has also diverged between the Drosophila and Sophophora lineages. Rates of evolution are highly heterogeneous across the mitochondrial proteome, with ND accumulating many more amino acid substitutions than CO. These oxidative phosphorylation complex-specific rates of evolution vary across lineages and may reflect physiological and ecological change across the Drosophila phylogeny.

Trypanosoma cruzi maxicircle heterogeneity in Chagas disease patients from Brazil

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, ∼3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex I) subunit 7 ( ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes ( ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T. cruzi II (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T. cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T. cruzi II cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite.

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes.

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.

A novel mitochondrial heteroplasmic C13806A point mutation associated with Iranian Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by decreased expression of the protein Frataxin. Frataxin deficiency leads to excessive free radical production and dysfunction of chain complexes. Mitochondrial DNA (mtDNA) could be considered a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. It prompted us to focus on the mtDNA and monitor the nucleotide changes of genome which are probably the cause of respiratory chain defects and reduced ATP generation. We searched about 46% of the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE) and DNA fragments showing abnormal banding patterns were sequenced for the identification of exact mutations. In 18 patients, for the first time, we detected 26 mtDNA mutations; of which 5 (19.2%) was novel and 21 (80.8%) have been reported in other diseases. Heteroplasmic C13806A polymorphisms were associated with Iranian FRDA patients (55.5%). Our results showed that NADH dehydrogenase (ND) genes mutations in FRDA samples were higher than normal controls (P < 0.001) and we found statistically significant inverse correlation (r = -0.8) between number of mutation in ND genes and age of onset in FRDA patients. It is possible that mutations in ND genes could constitute a predisposing factor which in combination with environmental risk factors affects age of onset and disease progression.

Fly diversity revealed by PCR‐RFLP of mitochondrial DNA

In this article, we describe an inexpensive, two-session undergraduate laboratory activity that introduces important molecular biology methods in the context of biodiversity. In the first session, students bring tentatively identified flies (order Diptera, true flies) to the laboratory, extract DNA, and amplify a region of the mitochondrial gene NADH dehydrogenase subunit 1. In the second session, the students digest the PCR product with a restriction enzyme, visualize the resulting fragments by agarose gel electrophoresis, and analyze their results with comparison to known sequences. The diversity of flies and their importance as disease vectors, agriculture pests, pollinators, models of speciation, and in the case of Drosophila melanogaster, as a genetic model organism, offer many perspectives with which to appeal to students' interests. The laboratory exercise can be linked as a module to topics in biodiversity, bioinformatics, entomology, evolution, and mutagenesis.

The Mosaic Genome of Anaeromyxobacter dehalogenans Strain 2CP-C Suggests an Aerobic Common Ancestor to the Delta-Proteobacteria

Anaeromyxobacter dehalogenans strain 2CP-C is a versaphilic delta-Proteobacterium distributed throughout many diverse soil and sediment environments. 16S rRNA gene phylogenetic analysis groups A. dehalogenans together with the myxobacteria, which have distinguishing characteristics including strictly aerobic metabolism, sporulation, fruiting body formation, and surface motility. Analysis of the 5.01 Mb strain 2CP-C genome substantiated that this organism is a myxobacterium but shares genotypic traits with the anaerobic majority of the delta-Proteobacteria (i.e., the Desulfuromonadales). Reflective of its respiratory versatility, strain 2CP-C possesses 68 genes coding for putative c-type cytochromes, including one gene with 40 heme binding motifs. Consistent with its relatedness to the myxobacteria, surface motility was observed in strain 2CP-C and multiple types of motility genes are present, including 28 genes for gliding, adventurous (A-) motility and 17 genes for type IV pilus-based motility (i.e., social (S-) motility) that all have homologs in Myxococcus xanthus. Although A. dehalogenans shares many metabolic traits with the anaerobic majority of the delta-Proteobacteria, strain 2CP-C grows under microaerophilic conditions and possesses detoxification systems for reactive oxygen species. Accordingly, two gene clusters coding for NADH dehydrogenase subunits and two cytochrome oxidase gene clusters in strain 2CP-C are similar to those in M. xanthus. Remarkably, strain 2CP-C possesses a third NADH dehydrogenase gene cluster and a cytochrome cbb 3 oxidase gene cluster, apparently acquired through ancient horizontal gene transfer from a strictly anaerobic green sulfur bacterium. The mosaic nature of the A. dehalogenans strain 2CP-C genome suggests that the metabolically versatile, anaerobic members of the delta-Proteobacteria may have descended from aerobic ancestors with complex lifestyles.

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na +/H + antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na +-resistance on an antiporter-deficient E. coli strain, nor did they confer Na +/H + antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.

Systematics and Evolution in the Tityrinae (Passeriformes: Tyrannoidea)

AbstractWe tested the monophyly and determined the phylogenetic relationships of the seven genera (Laniisoma, Laniocera, Iodopleura, Pachyramphus, Schiffornis, Tityra, and Xenopsaris) and 27 of the 31 recognized species of the subfamily Tityrinae using complete gene sequence data from the mitochondrial gene NADH dehydrogenase subunit 2. Monophyly of all seven genera was recovered using both weighted parsimony and Bayesian methods. Intergeneric relationships were nearly identical between the two methods and are largely in concordance with previous studies. Both analyses recovered two basal clades within the Tityrinae: one clade contained Schiffornis, Laniocera, and Laniisoma; the other clade consisted of Iodopleura, Tityra, Xenopsaris, and Pachyramphus. All genera in the Tityrinae that contained multiple species were monophyletic and are concordant with current taxonomy. We present the first phylogeny for Pachyramphus and suggest that Platypsaris is not valid. Character mapping of morphological, nest-construction, and breeding-system data on our phylogeny suggest conservative evolution of most characters. We recommend elevating the Tityrinae to family level.Sistemática y Evolución de los Tityrinae (Passeriformes: Tyrannoidea)

Molecular species identification boosts bat diversity

The lack of obvious morphological differences between species impedes the identification of species in many groups of organisms. Meanwhile, DNA-based approaches are increasingly used to survey biological diversity. In this study we show that sequencing the mitochondrial protein-coding gene NADH dehydrogenase, subunit 1 (nd1) from 534 bats of the Western Palaearctic region corroborates the promise of DNA barcodes in two major respects. First, species described with classical taxonomic tools can be genetically identified with only a few exceptions. Second, substantial sequence divergence suggests an unexpected high number of undiscovered species.

SYSTEMATICS AND EVOLUTION IN THE TITYRINAE (PASSERIFORMES: TYRANNOIDEA)

We tested the monophyly and determined the phylogenetic relationships of the seven genera (Laniisoma, Laniocera, Iodopleura, Pachyramphus, Schiffornis, Tityra, and Xenopsaris) and 27 of the 31 recognized species of the subfamily Tityrinae using complete gene sequence data from the mitochondrial gene NADH dehydrogenase subunit 2. Monophyly of all seven genera was recovered using both weighted parsimony and Bayesian methods. Intergeneric relationships were nearly identical between the two methods and are largely in concordance with previous studies. Both analyses recovered two basal clades within the Tityrinae: one clade contained Schiffornis, Laniocera, and Laniisoma; the other clade consisted of Iodopleura, Tityra, Xenopsaris, and Pachyramphus. All genera in the Tityrinae that contained multiple species were monophyletic and are concordant with current taxonomy. We present the first phylogeny for Pachyramphus and suggest that Platypsaris is not valid. Character mapping of morphological, nest-construction, and breeding-system data on our phylogeny suggest conservative evolution of most characters. We recommend elevating the Tityrinae to family level. Sistemática y Evolución de los Tityrinae (Passeriformes: Tyrannoidea)

Differential transcription profiles in Trypanosoma cruzi associated with clinical forms of Chagas disease: Maxicircle NADH dehydrogenase subunit 7 gene truncation in asymptomatic patient isolates

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2–3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.

AN EXPRESSED SEQUENCE TAG ANALYSIS OF THALLUS AND REGENERATING PROTOPLASTS OFCHONDRUS CRISPUS(GIGARTINALES, RHODOPHYCEAE)1

In order to identify genes involved in cell wall regeneration and stress responses in red algae, expressed sequence tags (ESTs) from protoplasts (2002 ESTs) and thalli (2052 ESTs) from the seaweedChondrus crispus(Stackh.) were studied. Clustering gave 2291 non‐redundant sequences; 50% of the ESTs showed similarity (e&lt;10−4) to known sequences. The fraction of stress‐related ESTs was five‐times higher in the protoplast library than in the thallus library. The ESTs that were statistically over‐represented in protoplasts included: glutathione S‐transferases, heat shock proteins, vanadium bromoperoxidase, and several genes of unknown function; in all 32 transcripts. Over‐represented genes in thallus included: NADH dehydrogenase, a peroxidase, and several genes of unknown function; in all 12 transcripts. In general, the ESTs from the two libraries were very different; for example, only 38% of contigs had members of both catalogues. The approach allowed the identification of numerous stress genes; including 23 different heat shock proteins and molecular chaperones, antioxidative enzymes, and several genes potentially involved in detoxification. Genes potentially involved in the construction of the cell wall or the extracellular matrix included α‐galactosidase, pullulanase, sulfohydrolase, and several sequences with von Willebrand factor type A domains with similarities to cochlin, integrin, and vitrin.

Relationships among 3Kochiaspecies based on PCR-generated molecular sequences and molecular cytogenetics

Forage kochia (Kochia prostrata ssp. virescens 'Immigrant' is native to the arid and semiarid regions of central Eurasia. It was introduced into the United States in 1966 as PI 314929 and released as a perennial forage shrub in 1984. Kochia americana is a perennial native to the United States, whereas Kochia scorparia is an introduced annual species that became a weed. To assess both the breeding potential and the possibility of genetic contamination, relationships among the 3 Kochia species were analyzed using random amplified polymorphic DNA (RAPD) markers, sequence tagged site (STS) marker sequences of the chloroplast NADH dehydrogenase gene (ndhF), genomic in situ hybridization (GISH), and multicolor fluorescence in situ hybridization (MC-FISH). Seventy decamer random primers yielded 458 polymorphic bands from 9 plants of K. americana, 20 plants of K. prostrata, and 7 plants of K. scoparia. Fifty-four and 55 species-specific RAPD markers were identified for K. americana and K. prostrata, whereas 80 RAPD markers were specific to K. scoparia. Based on the presence or absence of informative RAPD markers, the 3 species always grouped into 3 distinct clusters in a NTSYSpc2.01b-generated dendrogram. The same relationships were found among the 3 Kochia species based on ndhF DNA sequence divergence. Using a set of 7 STS markers that can identify each Kochia species, we did not find a single interspecific hybrid from artificial hybridizations among the 3 Kochia species. In GISH studies, chromosomes of 1 species fluoresced in green only when they were probed by genomic DNA of the same species. Cross-hybridization by genomic DNA of another species was not observed. In FISH studies using pTa71 (for 18S-5.8S-26S rDNAs) and pScT7 (for 5S rDNA) as probes, there were 1, 1 and 3 pTa71 sites and 2, 1, and 1 pScT7 sites in each haplome of K. prostrata, K. americana, and K. scoparia, respectively. It is concluded that these 3 Kochia species are so genomically distinct that gene introgression among them would be extremely rare.

Contribution of de novo point mutations to the overall mutational burden in mitochondrial DNA of adult rats

This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tissues from young (6-month) and old (24-month) male F344 rats. Coding sequence mutations in subunit 5 of the NADH dehydrogenase gene were detected after high-fidelity PCR amplification and cloning by denaturing gradient gel electrophoresis (DGGE) assay followed by sequencing of detected mutants. In total, almost a thousand individual clones were analyzed both in brain and muscle samples. On average, mtDNA from brain tissue showed a 66% increase with age in mutation frequencies (2.3±1.9 vs. 3.8±4.5×10 −4 mutations/bp, mean±SD), which failed to reach statistical significance ( p=0.45). Muscle tissues yielded substantially fewer mutants with average mutant frequencies for both young and old rats almost 10 times lower than the corresponding values in the brain tissue (0.3±0.4 and 0.5±0.6×10 −4, respectively). The difference in mutation accumulation between muscle and brain was highly significant in both the younger group (Chi-squared=9.7, p≤0.01) and in older animals (Chi-squared=10.9, p≤0.001). Molecular analysis of the mutated sequences revealed that almost half were identical sequences recurring in different samples and tissues. Our findings indicate that the process of mutation accumulation in mitochondria begins in the germ-line and/or during earlier stages of life, contributing up to half of the total mutational burden, whereas de novo spontaneous formation of point mutations in adulthood is far less than was anticipated.

Genetic and biological diversity among populations of Leishmania major from Central Asia, the Middle East and Africa

Evidence is provided for genetic and biological variation among Leishmania major strains that correlates with their geographical origin. The host–parasite relationship also appears to be specific. Great gerbils, Rhombomys opimus, and fat sand rats, Psammomys obesus, are the main reservoir hosts in Central Asia and the Middle East, respectively. However, the Central Asian parasite failed to infect the Middle Eastern rodent host in the laboratory, and vice versa. A permissively primed intergenic polymorphic (PPIP)-PCR and a single-stranded conformation polymorphism (SSCP)-PCR exposed genetic polymorphism among 30 strains of L. major from different geographical regions. This was verified by subsequent sequencing of DNA from the same strains using four genomic targets: (a) the NADH-dehydrogenase (NADH-DH) gene, (b) the 6-phosphogluconate dehydrogenase (6PGD) gene, (c) the ribosomal internal transcribed spacers, and (d) an anonymous DNA sequence originally amplified with random primers. All the genetic markers indicated that the nine Central Asian strains were a separate homogenous genetic group. The Middle Eastern strains formed another geographical group that displayed heterogeneity corresponding with their different Middle Eastern locations. Molecular markers and host–parasite relationships confirmed that Central Asian and Middle Eastern strains are genetically and biologically distinct sub-populations of L. major. Three African strains of L. major were genetically closer to the Middle Eastern strains, and a representative one did infect fat sand rats, but they had distinct permissively primed inter-genic polymorphic PCR patterns and internal transcribed spacer 2 types.

A single nucleotide polymorphism map of the mitochondrial genome of the parasitic nematode Cooperia oncophora.

The 13,636 bp mitochondrial (mt) genome sequence of the trichostrongylid nematode Cooperia oncophora was determined. Like the mt genomes of other nematodes it is AT rich (76.75%) and cytidine is the least common nucleoside in the coding strand. There are 2 ribosomal RNA (rrn) genes, 22 transfer RNA (trn) genes and 12 protein coding genes. The relatively short AT-rich region (304 bp) and the lack of a non-coding region between two of the NADH dehydrogenase genes, nad3 and nad5, makes the mt genome of C. oncophora one of the smallest known to date, having only 525 bp of non-coding regions in total. The majority of the C. oncophora protein encoded genes are predicted to end in an abbreviated stop codon like T or TA. In total, 426 single nucleotide polymorphisms (SNP) were mapped on the mt genome of C. oncophora, which is an average of 1 polymorphism per 32 bp. The most common SNPs in the mt genome of C. oncophora were G/A (59.2%) and C/T (28.4%) transitions. Synonymous substitutions (86.4%) were favoured over non-synonymous substitutions. However, the degree of sequence conservation between individual protein genes of different parasitic nematode species did not always correspond to the relative number of non-synonymous SNPs. The mt genome sequence of C. oncophora presents the first mt genome of a member of the Trichostrongyloidea and will be of importance in refining phylogenetic relationships between nematodes. The, still limited, SNP map presented here provides a basis for obtaining insight into the genetic diversity present in the different protein coding genes, trn, rrn and non-coding regions. A more detailed study of the more variable regions will be of use in determining the population genetic structure of C. oncophora. Ultimately this knowledge will add to the understanding of the host-parasite relationship.

The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria

A DNA sequence homologous to non-proton-pumping NADH dehydrogenase genes was found in the genome of Neurospora crassa encoding a polypeptide of 577 amino acid residues, molecular mass of 64,656 Da, with a putative transmembrane domain. Analysis of fungal mitochondria fractionated with digitonin indicates that the protein is located at the outer face of the inner membrane of the organelle (external enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. Mitochondria from the resulting null-mutant nde2 are highly deficient in the oxidation of cytosolic NADH and NADPH. A triple mutant nde1/nde2/ndi1, lacking mitochondrial alternative NAD(P)H dehydrogenases, was obtained, indicating that these proteins are not essential in N. crassa. However, crosses between the nde2 mutant strain and complex I-deficient mutants yielded no viable double mutants. Transcription of the nde-2 gene, as well as of ndi-1 (internal enzyme), is repressed in the late exponential phase of fungal growth.

PCR-RFLP identification of endemic Chilean species of Rhagoletis (Diptera: Tephritidae) attacking Solanaceae.

Four species of the genus Rhagoletis are native to Chile: R. nova (Schiner), R. conversa, (Brèthes), R. penela Foote and R. tomatis Foote. Currently, identification of these species is based on morphological criteria, but their strong similarity makes precise recognition difficult. To clarify species separation for quarantine purposes, a reliable method based on a PCR-RFLP procedure is reported. A DNA region containing mitochondrial NADH dehydrogenase genes was selected as a target sequence for the analysis. The amplification products (c. 1 kb) were digested with either SspI or DdeI, yielding specific patterns that differentiated each of the endemic species. Complete nucleotide sequences were determined, confirming empirical restriction maps. This report updates information on the geographical distribution of Rhagoletis species in Chile.