Gene Encoding NADH Dehydrogenase Research Articles

System-wide analysis of groundnut's salinity resilience: Integrating plant-cell interactions with environmental stress dynamics through cutting-edge transcriptomics

Salinity stress is a major concern in regions where irrigation relies on saline water. This study aimed to investigate the relative water content (RWC), electrolytic leakage (EL), total chlorophyll content, free amino acid content, and total soluble sugar content were analyzed in different groundnut species subjected to various salinity treatments. The results showed that salinity stress significantly reduced the RWC in groundnut leaves, with A. duranensis (wild type) exhibiting higher RWC values compared to the Arachis hypogaea species. RNA sequencing was performed to identify differentially expressed genes (DEGs) during salt stress. A total of 9079 DEGs were identified, with 1372 genes upregulated and 2509 genes downregulated. Genes belonging to transcription factor families, such as WRKY, MYB, bHLH, E2F, and Auxin efflux carrier proteins, were induced under salt stress in the tolerant genotype. Conversely, genes encoding NADH dehydrogenase, glutathione S-transferase, protein kinases, UDP-glycosyltransferase, and peroxidase were downregulated. Gene ontology and pathway analyses revealed several enriched categories and metabolic pathways associated with salt stress response, including catalytic activity, response to salt stress, ATP-dependent activity, and oxidative phosphorylation. The findings of this study provide insights into the physiological and molecular responses of groundnut to salinity stress. A. duranensis exhibited better salinity tolerance than Arachis hypogaea, as indicated by higher RWC values, lower electrolytic leakage, and differential gene expression patterns. These results contribute to our understanding of the mechanisms underlying salt stress tolerance in groundnut and may guide future efforts to develop salinity-tolerant groundnut species, ultimately improving crop yield in saline-affected regions.

Plastome evolution and phylogenomics of Impatiens (Balsaminaceae)

This study reported seven new plastomes from Impatiens and observed three highly variable regions for phylogeny and DNA barcoding, which resolved the relationships among sections of subgenus Impatiens. Impatiens L. (Balsaminaceae, Ericales) is one of the largest and most diverse genera of angiosperms, widely known for its taxonomic difficulty. In this study, we reevaluated the infrageneric relationships within the genus Impatiens, using complete plastome sequence data. Seven complete plastomes of Impatiens (representing 6 species) were newly sequenced and characterized along with 20 previously published plastomes of other Impatiens species, plus 2 plastomes of outgroups (Hydrocera triflora, Balsaminaceae; Marcgravia coriacea, Marcgraviaceae). The total size of these 29 plastomes ranged from 151,538bp to 152,917bp, except 2 samples of Impatiens morsei, which exhibited a shorter length and lost some genes encoding NADH dehydrogenase subunits. Moreover, the number of simple sequence repeats (SSRs) ranged from 51 to 113, and the number of long repeats from 17 to 26. In addition, three highly variable regions were identified (trnG-GCC (The previous one), ndhF-rpl32-trnL-UGA-ccsA, and ycf1). Our phylogenomic analysis based on 80 plastome-derived protein-coding genes strongly supported the monophyly of Impatiens and its two subgenera (Clavicarpa and Impatiens), and fully resolved relationships among the six (out of seven) sampled sections of subgenus Impatiens. Overall, the plastome DNA markers and phylogenetic results reported in this study will facilitate future identification, taxonomic and DNA barcoding studies in Impatiens as well as evolutionary studies in Balsaminaceae.

The Mitochondrial Genome of the Sea Anemone Stichodactyla haddoni Reveals Catalytic Introns, Insertion-Like Element, and Unexpected Phylogeny

A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.

Carriage of Shiga toxin phage profoundly affects Escherichia coli gene expression and carbon source utilization

BackgroundEnterohemorrhagic Escherichia coli (E. coli) are intestinal pathogenic bacteria that cause life-threatening disease in humans. Their cardinal virulence factor is Shiga toxin (Stx), which is encoded on lambdoid phages integrated in the chromosome. Stx phages can infect and lysogenize susceptible bacteria, thus either increasing the virulence of already pathogenic bacterial hosts or transforming commensal strains into potential pathogens. There is increasing evidence that Stx phage-encoded factors adaptively regulate bacterial host gene expression. Here, we investigated the effects of Stx phage carriage in E. coli K-12 strain MG1655. We compared the transcriptome and phenotype of naive MG1655 and two lysogens carrying closely related Stx2a phages: ϕO104 from the exceptionally pathogenic 2011 E. coli O104:H4 outbreak strain and ϕPA8 from an E. coli O157:H7 isolate.ResultsAnalysis of quantitative RNA sequencing results showed that, in comparison to naive MG1655, genes involved in mixed acid fermentation were upregulated, while genes encoding NADH dehydrogenase I, TCA cycle enzymes and proteins involved in the transport and assimilation of carbon sources were downregulated in MG1655::ϕO104 and MG1655::ϕPA8. The majority of the changes in gene expression were found associated with the corresponding phenotypes. Notably, the Stx2a phage lysogens displayed moderate to severe growth defects in minimal medium supplemented with single carbon sources, e.g. galactose, ribose, L-lactate. In addition, in phenotype microarray assays, the Stx2a phage lysogens were characterized by a significant decrease in the cell respiration with gluconeogenic substrates such as amino acids, nucleosides, carboxylic and dicarboxylic acids. In contrast, MG1655::ϕO104 and MG1655::ϕPA8 displayed enhanced respiration with several sugar components of the intestinal mucus, e.g. arabinose, fucose, N-acetyl-D-glucosamine. We also found that prophage-encoded factors distinct from CI and Cro were responsible for the carbon utilization phenotypes of the Stx2a phage lysogens.ConclusionsOur study reveals a profound impact of the Stx phage carriage on E. coli carbon source utilization. The Stx2a prophage appears to reprogram the carbon metabolism of its bacterial host by turning down aerobic metabolism in favour of mixed acid fermentation.

Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212g/L L-lactic acid with a 97.9% yield and 264g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.

Sex and Mitonuclear Adaptation in Experimental Caenorhabditis elegans Populations

To reveal phenotypic and functional genomic patterns of mitonuclear adaptation, a laboratory adaptation study with Caenorhabditis elegans nematodes was conducted in which independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1 Following 60 generations of evolution in large population sizes with competition for food resources, two distinct classes of lines representing different degrees of adaptive response emerged: a low-fitness class that exhibited minimal or no improvement compared to the gas-1 mutant ancestor, and a high-fitness class containing lines that exhibited partial recovery of wild-type fitness. Many lines that achieved higher reproductive and competitive fitness levels were also noted to evolve high frequencies of males during the experiment, consistent with adaptation in these lines having been facilitated by outcrossing. Whole-genome sequencing and analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome and could be classified into a small number of functional genomic categories. A highly nonrandom pattern of mitochondrial DNA mutation was observed within high-fitness gas-1 lines, with parallel fixations of nonsynonymous base substitutions within genes encoding NADH dehydrogenase subunits I and VI. These mitochondrial gene products reside within ETC complex I alongside the nuclear-encoded GAS-1 protein, suggesting that rapid adaptation of select gas-1 recovery lines was driven by fixation of compensatory mitochondrial mutations.

Development of a direct PCR assay to detect Taenia multiceps eggs isolated from dog feces

Taenia multiceps is a tapeworm that leads to the death of livestock, resulting in major economic losses worldwide. The adult stage of this parasite invades the small intestine of dogs and other canids. In the present study, we developed a direct PCR assay to detect T. multiceps eggs isolated from dog feces to help curb further outbreaks. The genomic DNA was rapidly released using a lysis buffer and the PCR reaction was developed to amplify a 433-bp fragment of the T. multiceps mitochondrial gene encoding NADH dehydrogenase subunit 5 (nad5) from eggs isolated from dog feces. The procedure could be completed within 3 h, including flotation. The sensitivity of the assay was determined by detecting DNA from defined numbers of eggs, and the specificity was determined by detecting DNA from other intestinal tapeworm and roundworm species that commonly infect dogs. In addition, 14 taeniid-positive fecal samples determined by the flotation technique were collected and further evaluated by the regular PCR and our direct PCR. The results showed that the direct PCR developed herein was sensitive enough to detect the DNA from as few as 10 T. multiceps eggs and that no cross-reactions with other tapeworm and roundworm were observed, suggesting its high sensitivity and specificity for T. multiceps detection. Moreover, 14 taeniid-positive samples were screened by the regular PCR and direct PCR, with detection rates of 78.6% and 85.7%, respectively. In conclusion, the direct PCR assay developed in the present study has high sensitivity and specificity to identify T. multiceps eggs isolated from dog feces and therefore could represent an invaluable tool to identify T. multiceps outbreaks and would contribute to future clinical applications.

Role of genetics in resistance to type 1 diabetes

A single nucleotide polymorphism in the mitochondrial gene encoding NADH dehydrogenase subunit 2 (mt-ND2) has been associated with reduced incidence of human type 1 diabetes (T1D). We identified the orthologue of this mitochondrial single nucleotide polymorphism in mouse and using NOD mouse models linked this genetic polymorphism to T1D resistance. The mechanism how this single nucleotide polymorphism affects the development of diabetes is studied using mouse models and beta cell lines. The impact of this single nucleotide polymorphism on mitochondrial function and resistance to reactive oxygen species was assessed. For these studies we measured oxygen consumption by isolated mitochondria under different doses of nitric oxide. In addition, alloxan sensitivity of beta cell lines was tested using the MTT method to measure cell survival. mt-Nd2a is associated with protection against mouse T1D and alloxan-induced diabetes. Mice with mt-Nd2a exhibited resistance to transfer of diabetes by single clone of diabetogenic CD4+ or CD8+ T cells. Beta cell line with mt-Nd2a resist in vitro attack of diabetogenic CD8+ cytotoxic T cells, as well as free radicals generated by alloxan; isolated mitochondria with mt-Nd2a showed lower reactive oxygen species production and were more resistant to nitric oxide. mt-Nd2a protects against T1D in mouse models. The protection is at beta cell level and is associated with resistance against reactive oxygen species-mediated damage and death.

Phylogeography of the Brownback Salamander reveals patterns of local endemism in Southern Appalachian springs

The Appalachian Mountains of eastern North America are characterized by high faunal diversity and many endemic species, especially in the unglaciated southern latitudes where lineages have been accumulating for tens of millions of years. The Brownback Salamander, Eurycea aquatica, is an enigmatic species that dwells in isolated springs in southeastern North America. Eurycea aquatica have often been dismissed as simply robust spring-adapted ecomorphs of the widespread and more gracile species Eurycea cirrigera. We sequenced the mitochondrial gene encoding NADH dehydrogenase subunit-2 ( ND2; 753 bp) and the nuclear recombination activating gene-1 ( Rag1; 1201 bp) for E. aquatica ( ND2 n = 72; Rag1 n = 17) from across their presumed distribution and compared them to E. cirrigera ( ND2 n = 23; Rag1 n = 10) from nearby populations. Using phylogenetic and morphological analyses we explicitly test if E. aquatica in the Southern Appalachians is simply a local spring-adapted ecomorph of E. cirrigera or a single lineage that resulted from fragmentation of (or dispersal to) spring habitats. We found that E. aquatica from isolated springs form a well-supported monophyletic group that is nested among E. cirrigera, E. wilderae, and E. junaluska. Furthermore, we uncovered three very divergent lineages of E. aquatica that we estimate have been isolated from each another since the early Pliocene to late Miocene (2.5–6.1 Myr) and may each represent distinct species. The distribution of these lineages is coincident with the distribution of other endemic spring-dwelling vertebrates, and suggests that this region may be a relictual habitat for an unexpected diversity of unrecognized endemics.

Molecular cloning and expression of a bush related CmV1 gene in tropical pumpkin

A bush-type plant was selected from tropical pumpkin 'cga' (Cucurbita moschata Duchesne) in order to study the vine development in C. moschata. In this study, a novel gene encoding NADH dehydrogenase was isolated from the vine line (cgaV) of C. moschata, that was not expressed in the near isogenic bush line (cgaBu). This gene, designated as CmV1 (C. moschata vine 1), was 545 bp in length and was composed of a 477 bp open reading frame, which had 99% nucleotide similarity to the chloroplast ndhJ gene for NADH dehydrogenase subunit J from Brassica oleracea. The deduced amino acid sequence of CmV1 had 99% similarity to NADH dehydrogenase subunit J from Arabidopsis and had 98% similarity to NADH dehydrogenase subunit from Barbarea verna. Analysis of the basic characteristics of the CmV1 protein revealed that it has one Respiratory chain NADH dehydrogenase 30 kD subunit signature, three N-myristoylation sites, one Casein kinase II phosphorylation site, and one Protein kinase C phosphorylation site. Reverse transcriptase-PCR analysis showed that CmV1 was expressed at a high level in the internodes and hypocotyls and was expressed stronger in elongating internodes than in fully expanded internodes. In conclusion, results obtained in the present study suggest that CmV1 gene might play important roles in vine elongation of tropical pumpkin.

Novel Nicotine Oxidoreductase-Encoding Gene Involved in Nicotine Degradation by Pseudomonas putida Strain S16

There are quite a few ongoing biochemical investigations of nicotine degradation in different organisms. In this work, we identified and sequenced a gene (designated nicA) involved in nicotine degradation by Pseudomonas putida strain S16. The gene product, NicA, was heterologously expressed and characterized as a nicotine oxidoreductase catalyzing the initial steps of nicotine metabolism. Biochemical analyses using resting cells and the purified enzyme suggested that nicA encodes an oxidoreductase, which converts nicotine to 3-succinoylpyridine through pseudooxynicotine. Based on enzymatic reactions and direct evidence obtained using H(2)(18)O labeling, the process may consist of enzyme-catalyzed dehydrogenation, followed by spontaneous hydrolysis and then repetition of the dehydrogenation and hydrolysis steps. Sequence comparisons revealed that the gene showed 40% similarity to genes encoding NADH dehydrogenase subunit I and cytochrome c oxidase subunit I in eukaryotes. Our findings demonstrate that the molecular mechanism for nicotine degradation in strain S16 involves the pyrrolidine pathway and is similar to the mechanism in mammals, in which pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen, is produced.

NADH Dehydrogenase Activity and Expression of mRNA of Complex I (ND1, 51kDa, and 75kDa) in Heart Mitochondria of klotho Mouse

Mitochondrial enzyme activities and ultrastructure of mitochondria prepared from klotho mutant mice were compared with those in wild-type mice. We also measured the levels of expression of ND1, 51kDa, and 75kDa mRNA associated with the genes encoding NADH dehydrogenase and complex I and that of alpha cardiac myosin heavy chain mRNA in both groups. Mitochondrial NADH oxidoreductase activity was higher in klotho mutant mice during aging than that in wild-type mice. The area of mitochondria per unit area (300 microm2) of cell was almost constant from 4 to 7 weeks of age in both groups. A few large mitochondria were scattered between numerous small mitochondria with compact cristae and myofibrils in klotho mice from 5 weeks of age. The levels of ND1 and 75kDa mRNA were slightly high from 7 weeks of age in klotho mutant mice, whereas they were almost constant in wild-type mice, in spite of reduced expression of alpha cardiac myosin heavy chain mRNA. Our results indicate that klotho protein indirectly plays a role in diminished functional adaptability of enzymes in aged heart muscle, and is required for hypertrophy of cardiac mitochondria.

The Identification of Primary Sites of Superoxide and Hydrogen Peroxide Formation in the Aerobic Respiratory Chain and Sulfite Reductase Complex of Escherichia coli

The fitness of organisms depends upon the rate at which they generate superoxide (O-2) and hydrogen peroxide (H2O2) as toxic by-products of aerobic metabolism. In Escherichia coli these oxidants arise primarily from the autoxidation of components of its respiratory chain. Inverted vesicles that were incubated with NADH generated O-2 and H2O2 at accelerated rates either when treated with cyanide or when devoid of quinones, implicating an NADH dehydrogenase as their source. Null mutations in the gene encoding NADH dehydrogenase II averted autoxidation of vesicles, and its overproduction accelerated it. Thus NADH dehydrogenase II but not NADH dehydrogenase I, respiratory quinones, or cytochrome oxidases formed substantial O-2 and H2O2. NADH dehydrogenase II that was purified from both wild-type and quinone-deficient cells generated approximately 130 H2O2 and 15 O-2 min-1 by autoxidation of its reduced FAD cofactor. Sulfite reductase is a second autoxidizable electron transport chain of E. coli, containing FAD, FMN, [4Fe-4S], and siroheme moieties. Purified flavoprotein that contained only the FAD and FMN cofactors had about the same oxidation turnover number as did the holoenzyme, 7 min-1 FAD-1. Oxidase activity was largely lost upon FMN removal. Thus the autoxidation of sulfite reductase, like that of the respiratory chain, occurs primarily by autoxidation of an exposed flavin cofactor. Great variability in the oxidation turnover numbers of these and other flavoproteins suggests that endogenous oxidants will be predominantly formed by only a few oxidizable enzymes. Thus the degree of oxidative stress in a cell may depend upon the titer of such enzymes and accordingly may vary with growth conditions and among different cell types. Furthermore, the chemical nature of these reactions was manifested by their acceleration at high temperatures and oxygen concentrations. Thus these environmental parameters may also directly affect the O-2 and H2O2 loads that organisms must bear.

Phylogeny of Japanese papilionid butterflies inferred from nucleotide sequences of the mitochondrial ND5 gene.

Phylogenetic relationships among the Japanese papilionid butterflies were analyzed by comparing 783 nucleotide sequences of the mitochondrial gene encoding NADH dehydrogenase subunit 5 (ND5). Phylogenetic trees of the representative species from each family in the superfamily Papilionoidea revealed that the species of the family Papilionidae and those of all other families formed distinct clusters, with a few species of the family Hesperiidae (Hesperioidea) as an outgroup. In the phylogenetic trees of most Japanese species of the family Papilionidae with Nymphalis xanthomelas (Nymphalidae) as an outgroup, the tribe Parnassiini (Parnassiinae) formed a cluster, and the rest formed the other cluster in which the tribe Zerynthiini (Parnassiinae) and the subfamily Papilioninae formed different subclusters. In the Papilioninae cluster, the tribes Troidini and Graphiini formed a subcluster, and the tribe Papilionini formed the other subcluster. These results generally agree with the traditional classification of the papilionid butterflies based on their morphological characteristics and support the proposed evolutionary genealogy of the butterflies based on their morphology, behavior, and larval host plants, except that the tribes Parnasiini and Zerynthiini (both Parnassiinae) are not in the same cluster.

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes.

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.

The rapeseed mitochondrial gene for subunit 2 of the NADH dehydrogenase complex: a trans-spliced structure is conserved in one of the smallest plant mitochondrial genomes.

The single-copy gene encoding NADH dehydrogenase subunit 2 (nad2) has been identified in the mitochondrial genome of rapeseed (Brassica napus L.). The rapeseed nad2 gene has the same gene organization in Oenothera and wheat: it consists of five exons located in two loci encoding the two first exons and the last three exons respectively. All exons are separated by group-II introns. A trans-splicing event is required to join exons B and C. Putative splicing intermediates were identified by transcriptional analysis of the nad2 gene. The complexity of organization of the nad2 gene is completely conserved even in one of the smallest mitochondrial genomes of higher plants.

RNA editing status of nad7 intron domains in wheat mitochondria.

The most highly conserved structures of group II introns are the helical domains V and VI near the 3'splice site. Within this region of each of the four introns in the wheat mitochondrial nad7 gene encoding NADH dehydrogenase subunit 7, there are A-C mispairs. To determine whether C-to-U type RNA editing restores conventional A-U pairing, we sequenced RT-PCR products from partially-spliced nad7 template RNA and gel-fractionated, excised intron RNA. We examined transcripts from germinating wheat embryos and seedlings because these two stages of development show pronounced differences in steady state levels of nad7 intronic RNAs. We observed editing at only two of the six predicted sites, and they were located at homologous positions within domain V of the third and fourth introns. A third site was found to be edited within the unmodelled domain VI loop of the fourth intron. Similar patterns of RNA editing were seen in wheat embryos and seedlings. These observations, and the presence of other non-conventional base pairs particularly within domain V of plant mitochondrial introns, indicate weaker helical core structure than in ribozymic group II introns. Moreover, the incompleteness or absence of editing in wheat nad7 excised intron RNA suggests that, although editing may contribute to splicing efficiency, it is not essential for splicing.

Differential Display and Cloning of Shear Stress-Responsive Messenger RNAs in Human Endothelial Cells

To investigate the effect of shear stress on endothelial gene expression, we performed differential display of mRNAs from cultured human umbilical vein endothelial cells either incubated under static conditions or exposed to shear stress (15 dynes/cm2) for 6 h in a flow-chamber. Around 4% of the total number of mRNAs detected were either up- or down-regulated by shear stress. DNA sequencing of some of these shear stress-responsive mRNAs revealed homology of several clones to known gene sequences and many other clones for unknown genes. Known genes, including those for human laminin B1 chain, H+-ATP synthase coupling factor 6, lysyl oxidase, myosin light chain kinase, and interleukin-8 receptor, were up-regulated by shear stress, while the gene encoding NADH dehydrogenase was down-regulated. The present results suggest that shear stress can change the expression of numerous genes in endothelial cells, far more than reported to date, and that mRNA differential display is quite useful for cloning known and unknown shear stress-responsive genes.

The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria

The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

Multiple trans-splicing events are required to produce a mature nad1 transcript in a plant mitochondrion.

The mitochondrial gene encoding NADH dehydrogenase subunit 1 (nad1) in Petunia hybrida is split into five exons, a, b, c, d, and e. With the use of a complete restriction map of the 443-kb Petunia mitochondrial genome, we have cloned these exons and mapped their location. Exon a is located 130 kb away from and in the opposite orientation from exons b and c. Exon d maps 95 kb away and in the opposite orientation from exons b and c. Exons d and e are separated by 190 kb. By performing the polymerase chain reaction on Petunia cDNAs, we have shown that transcripts from these five exons are joined via a series of cis- and trans-splicing events to create a mature nad1 transcript. In addition, we have found 23 C----U RNA edit sites in Petunia nad1. RNA editing changes 19 of the amino acids predicted by the genomic sequence.