Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, ψnad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)⁺ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this ψnad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.     

The complete mitochondrial genome sequence and gene organization of the mud crab (Scylla paramamosain) with phylogenetic consideration

The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824 bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNA^His differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73 bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy.     

The complete mitochondrial genome of Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) and comparison with other Brachyuran crabs

Mitochondrial DNA (mtDNA) is an extrachromosomal genome which can provide important information for evolution and phylogenetic analysis. In this study, we assembled a complete mitogenome of a crab Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) from next generation sequencing reads at the first time. P. pictum is a mudflat crab, belonging to the Sesarmidae family (subfamily Sesarminae), which is perched on East Asia. The 15,716 bp mitogenome covers 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and one control region (CR). The control region spanns 420 bp. The genome composition was highly A+T biased 75.60% and showed negative AT-skew (−0.03) and negative GC-skew (−0.47). Compared with the ancestor of Brachyura, the gene order of Sesarmidae has several differences and the gene order of P. pictum is typical for mitogenomes of Sesarmidae. Phylogenetic tree based on nucleotide sequences of mitochondrial 13 PCGs using BI and ML determined that P. pictum has a sister group relationship with Parasesarma tripectinis and belongs to Sesarmidae.     

An NADH nitrate reductase gene copy appears to have been deleted in barley

Cultivated barley,Hordeum vulgare L., has a single NADH nitrate reductase (NR) gene while diploid wheat,Triticum monococcum, and cultivated hexaploid wheat,Triticum aestivum L., have two NADH NR genes. To determine whether the NADH NR gene was duplicated since the divergence ofTriticum fromHordeum or was deleted from barley, theT. Monococcum NADH NR gene heme-hinge regions were sequenced and compared with the barley NADH NR gene sequence. Sequence identity and phylogenetic analyses showed that one of theT. Monococcum NADH NR genes is more-closely related to the barley NADH NR gene than to the otherT. Monococcum NADH NR gene. The heme-hinge region of all three NR genes appeared to have evolved at a constant rate. These results suggest that the NADH NR gene duplicated before the divergence ofTriticum andHordeum and that a deletion resulted in the loss of one NADH NR gene from cultivated barley.     

Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes

In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD⁺ ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD⁺ ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.     

Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes.     

SPATIAL RELATIONSHIP AND GENE FLOW PATHS BETWEEN POPULATIONS OF THE ALPINE SNAIL ARIANTA ARBUSTORUM (PULMONATA: HELICIDAE)

The alpine snail Arianta arbustorum (Pulmonata, Helicidae) was sampled along an 8-km contour line in the Swiss Alps orthogonal to well-known altitudinal clines in life history and shell traits. The allele frequencies and the morphs of 16 populations were compared with habitats. Two hypotheses about the recolonization of the mountain slopes after the last glaciation were tested against the present population structure. The allele frequencies are best explained by the spatial structure whereas the morphs are better explained by the habitats. The genotypic distances point to a historical explanation by recolonization processes, the phenotypical dissimilarities to a selectionistic interpretation of the present distribution of the populations. The following conclusions are from the study of the spatial relationship and the gene flow paths for A. arbustorum at Mount Martegnas: (1) Streams are the main gene flow paths; gene flow is low, allowing differentiation of local populations. (2) Morphs and habitats are correlated, but the interpretation has to be made carefully. The spatial dependence of habitats has to be included into the analysis of selection. (3) The random assumption for gene flow of A. arbustorum is not a spatial isolation-by-distance model, but a functional isolation-by-distance model, assuming gene flow over the drainage system.     

Effect of dietary lysine on performance and expression of electron transport chain genes in the pectoralis major muscle of broilers

The aim of this study was to evaluate the effect of dietary lysine on performance, protein deposition and respiratory chain gene expression in male broilers. A total of 252 Cobb 500 broilers were distributed, in a completely randomized design, into four treatments with seven replicates of nine birds per experimental unit. Experimental treatments consisted of diets based on corn and soybean meal, with four levels of digestible lysine: 1.016%, 1.099%, 1.182% and 1.265%. The increase in the level of digestible lysine in the diet provided higher weight gains, feed efficiency and body protein deposition. Birds fed the lowest level of dietary lysine (1.016%) showed a lower expression of genes such as NADH dehydrogenase subunit I (ND1), cytochrome b (CYTB) and cytochrome c oxidase subunits I (COX I), II (COX II) and III (COX III), displaying the worst performance and body protein deposition. This demonstrates the relationship existing between the expression of the evaluated genes and the performance responses. In conclusion, results indicate that broilers fed diets with higher levels of digestible lysine have increased messenger RNA expression of some genes coded in the mitochondrial electron transport chain (ND1, CYTB, COX I, COX II and COX III). It may be stated that diets with proper levels of digestible lysine, within the ‘ideal protein’ concept, promote the expression of genes, which increases the mitochondrial energy, thereby fostering body protein deposition and the performance of broilers in the starter phase.     

RNA interference in the Colorado potato beetle,Leptinotarsa decemlineata: Identification of key contributors

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using ³²P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.     

Complete mitochondrial genome sequences of the three pelagic chaetognaths Sagitta nagae,Sagitta decipiens and Sagitta enflata

The complete nucleotide sequences of the mitochondrial genomes were determined for the three pelagic chaetognaths, Sagitta nagae, Sagitta decipiens, and Sagitta enflata. The mitochondrial genomes of these species which were 11,459, 11,121, and 12,631 bp in length, respectively, contained 14 genes (11 protein-coding genes, one transfer RNA gene, and two ribosomal RNA genes), and were found to have lost 23 genes that are present in the typical metazoan mitochondrial genome. The same mitochondrial genome contents have been reported from the benthic chaetognaths belonging to the family Spadellidae, Paraspadella gotoi and Spadella cephaloptera. Within the phylum Chaetognatha, Sagitta and Spadellidae are distantly related, suggesting that the gene loss occurred in the ancestral species of the phylum. The gene orders of the three Sagitta species are markedly different from those of the other non-Chaetognatha metazoans. In contrast to the region with frequent gene rearrangements, no gene rearrangements were observed in the gene cluster encoding COII–III, ND1–3, srRNA, and tRNA^met. Within this conserved gene cluster, gene rearrangements were not observed in the three Sagitta species or between the Sagitta and Spadellidae species. The gene order of this cluster was also assumed to be the ancestral state of the phylum.     

Analysis of bacteriophage T7 early RNAs and proteins on slab gels

The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn &; Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.