RESUMEN
In flowering plants, RNA editing is a post-transcriptional process that selectively deaminates cytidines (C) to uridines (U) in organellar transcripts. Pentatricopeptide repeat (PPR) proteins have been identified as site-specific recognition factors for RNA editing. Here, we report the map-based cloning and molecular characterization of the defective kernel mutant dek504 in maize. Loss of Dek504 function leads to delayed embryogenesis and endosperm development, which produce small and collapsed kernels. Dek504 encodes an E+-type PPR protein targeted to the mitochondria, which is required for RNA editing of mitochondrial NADH dehydrogenase 3 at the nad3-317 and nad3-44 sites. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the mitochondrial NADH dehydrogenase complex I activity, indicating that the alteration of the amino acid sequence at nad3-44 and nad3-317 through RNA editing is essential for NAD3 function. Moreover, the amino acids are highly conserved in monocots and eudicots, whereas the events of C-to-U editing are not conserved in flowering plants. Thus, our results indicate that Dek504 is essential for RNA editing of nad3, which is critical for NAD3 function, mitochondrial complex I stability, and seed development in maize.
Asunto(s)
Edición de ARN , Zea mays , Complejo I de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica de las Plantas , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Zea mays/metabolismoRESUMEN
Nad complex plays a very important role during cellular respiration. nad3 (nad dehydrogenase subunit 3) is one of the biggest subunits in this complex. Four cDNAs of nad3 gene were characterized in Hordeum vulgare subsp. spontaneum at exposed to four periods of 500 mM salinity, 0 h or control (accession no. MN066165), after 2 h (accession no. MN066166), after 12 h (accession no. MN066167) and after 24 h (accession no. MN066168) using RNA-seq raw data. Seventeen RNA editing sites were found in positions (or nucleotide nos. C5, C39, C44, C61, C62, C79, C80, C147, C185, C190, C191, C208, C209, C275, C317, C344, C349) within the nad3 coding region. These alterations represent differential editing at four exposure times. The maximum editing rate was revealed 2 and 12 h after salinity exposure. However, these edits were disrupted after 24 h probably due to the initiation of program cell death machinery. We found that RNA editing not only improved protein function but also may improve codon bias by altering the nucleotide without any change in amino acid. Characterization of pentatricopeptide repeat-containing protein At4g13650 (PPRSp1) in wild barley helped us to understand the behavior of editing sites C190 and C191 under salinity. Position - 6 in cis-element upstream editing sites of C155, C190 and C191 may be vital to the editing process in these sites by PPRSp1 protein. The differential editing of this gene under salinity led to a relationship between RNA editing and cellular respiration regulation.
Asunto(s)
Hordeum/genética , NADH Deshidrogenasa/genética , Proteínas de Plantas/genética , Edición de ARN/genética , Secuencia de Aminoácidos , Secuencia de Bases/genética , Respiración de la Célula/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Hordeum/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , NAD/genética , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , Sistemas de Lectura Abierta , ARN/genética , Salinidad , Transcripción GenéticaRESUMEN
The pentatricopeptide repeat (PPR) protein family is a large family characterized by tandem arrays of a degenerate 35-amino-acid motif whose members function as important regulators of organelle gene expression at the post-transcriptional level. Despite the roles of PPRs in RNA editing in organelles, their editing activities and the underlying mechanism remain obscure. Here, we show that a novel DYW motif-containing PPR protein, PPS1, is associated with five conserved RNA-editing sites of nad3 located in close proximity to each other in mitochondria, all of which involve conversion from proline to leucine in rice. Both pps1 RNAi and heterozygous plants are characterized by delayed development and partial pollen sterility at vegetative stages and reproductive stage. RNA electrophoresis mobility shift assays (REMSAs) and reciprocal competition assays using different versions of nad3 probes confirm that PPS1 can bind to cis-elements near the five affected sites, which is distinct from the existing mode of PPR-RNA binding because of the continuity of the editing sites. Loss of editing at nad3 in pps1 reduces the activity of several complexes in the mitochondrial electron transport chain and affects mitochondrial morphology. Taken together, our results indicate that PPS1 is required for specific editing sites in nad3 in rice.
Asunto(s)
Mitocondrias/metabolismo , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Edición de ARN/genética , Secuencias de Aminoácidos , Secuencia de Bases , Núcleo Celular/metabolismo , Secuencia Conservada , Transporte de Electrón , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Mitocondrias/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Oryza/ultraestructura , Fenotipo , Polen/metabolismo , Polen/ultraestructura , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Metacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data. METHODS: Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Stúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the 'barcode' region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene. RESULTS: Only lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and 'D. mergi Lineage 2', and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of 'D. mergi Lineage 2'. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and 'D. mergi Lineage 2' (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene. CONCLUSIONS: This first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.
Asunto(s)
Código de Barras del ADN Taxonómico , ADN Mitocondrial/genética , Enfermedades de los Peces/parasitología , Variación Genética , Trematodos/genética , Animales , Oftalmopatías/parasitología , Oftalmopatías/veterinaria , Peces/parasitología , Hungría/epidemiología , Cristalino/parasitología , Filogenia , Ríos , Eslovaquia/epidemiología , Trematodos/aislamiento & purificaciónRESUMEN
The genus Spirometra belongs to the family Diphyllobothriidae and order Pseudophyllidea, and includes intestinal parasites of cats and dogs. In this study, a plerocercoid labeled as Spirometra mansonoides from the USA was examined for species identification and phylogenetic analysis using 2 complete mitochondrial genes, cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit 3 (nad3). The cox1 sequences (1,566 bp) of the plerocercoid specimen (USA) showed 99.2% similarity to the reference sequences of the plerocercoid of Korean Spirometra decipiens (GenBank no. KJ599679), and 99.1% similarity in regard to nad3 (346 bp). Phylogenetic tree topologies generated using 4 analytical methods were identical and showed high confidence levels with bootstrap values of 1.00, 100%, 100%, and 100% for Bayesian inference (BI), maximum-likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methods, respectively. Representatives of Diphyllobothrium and Spirometra species formed a monophyletic group, and the sister-genera status between these species was well supported. Trapezoic proglottids in the posterior 1/5 region of an adult worm obtained from an experimentally infected cat were morphologically examined. The outer uterine loop of the uterus coiling characteristically consisted of 2 complete turns. The results clearly indicated that the examined Spirometra specimen from the USA matched to S. decipiens very well, and indicated possible presence of the life cycle of this species in this region.
Asunto(s)
Complejo IV de Transporte de Electrones/genética , NADH Deshidrogenasa/genética , Filogenia , Spirometra/clasificación , Spirometra/aislamiento & purificación , Estructuras Animales/anatomía & histología , Animales , Análisis por Conglomerados , Microscopía , Análisis de Secuencia de ADN , Homología de Secuencia , Spirometra/anatomía & histología , Spirometra/enzimología , Estados UnidosRESUMEN
Several tapeworm species in the genus Diphyllobothrium Cobbold, 1858 have uncertain taxonomic positions, leading to taxonomic confusion as well as misdiagnosis of infections. Taxonomic revision based on DNA sequence analysis is considered necessary to resolve the taxonomy of several cases, including that between Diphyllobothrium stemmacephalum, the type species of the genus, and Diphyllobothrium yonagoense. Diphyllobothrium yonagoense was synonymized with D. stemmacephalum based on morphological observations by Andersen (1987), however no molecular studies have been undertaken to verify the validity of this synonymization. In the present study, the first human case confirmed molecularly as D. stemmacephalum infection is reported, and the validity of the synonymization of D. yonagoense with D. stemmacephalum was assessed based on molecular phylogenetics. Diphyllobothrium stemmacephalum and D. yonagoense grouped into the same clades with high bootstrap confidence values for both cox1 and nad3. Genetic distances between the two taxa were very small (0.000-0.012 and 0.000-0.017 for cox1 and nad3, respectively) and were considered to fall within the range of intraspecific variation. Using these molecular analyses, this study verified molecularly that D. yonagoense is a junior synonym of D. stemmacephalum. Further, the closer phylogenetic relationship between D. stemmacephalum and Diplogonoporus species rather than other diphyllobothriids, including Diphyllobothrium nihonkaiense and Diphyllobothrium latum, was corroborated. The genus name for D. nihonkaiense and D. latum is also discussed.
Asunto(s)
Difilobotriosis/transmisión , Diphyllobothrium/clasificación , Diphyllobothrium/genética , Adolescente , Adulto , Anciano , Animales , Secuencia de Bases , Ciclooxigenasa 1/genética , ADN de Helmintos/genética , ADN Mitocondrial/genética , Difilobotriosis/parasitología , Femenino , Humanos , Japón , Masculino , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Filogenia , Análisis de Secuencia de ADNRESUMEN
The genus Spirometra belongs to the family Diphyllobothriidae and order Pseudophyllidea, and includes intestinal parasites of cats and dogs. In this study, a plerocercoid labeled as Spirometra mansonoides from the USA was examined for species identification and phylogenetic analysis using 2 complete mitochondrial genes, cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit 3 (nad3). The cox1 sequences (1,566 bp) of the plerocercoid specimen (USA) showed 99.2% similarity to the reference sequences of the plerocercoid of Korean Spirometra decipiens (GenBank no. KJ599679), and 99.1% similarity in regard to nad3 (346 bp). Phylogenetic tree topologies generated using 4 analytical methods were identical and showed high confidence levels with bootstrap values of 1.00, 100%, 100%, and 100% for Bayesian inference (BI), maximum-likelihood (ML), neighbor-joining (NJ), and maximum parsimony (MP) methods, respectively. Representatives of Diphyllobothrium and Spirometra species formed a monophyletic group, and the sister-genera status between these species was well supported. Trapezoic proglottids in the posterior 1/5 region of an adult worm obtained from an experimentally infected cat were morphologically examined. The outer uterine loop of the uterus coiling characteristically consisted of 2 complete turns. The results clearly indicated that the examined Spirometra specimen from the USA matched to S. decipiens very well, and indicated possible presence of the life cycle of this species in this region.
Asunto(s)
Adulto , Animales , Gatos , Perros , Humanos , Diphyllobothrium , Complejo IV de Transporte de Electrones , Genes Mitocondriales , Estadios del Ciclo de Vida , NADH Deshidrogenasa , Parásitos , Plerocercoide , Spirometra , Árboles , Estados Unidos , ÚteroRESUMEN
The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A+T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif 'ATACTA' between trnS2(UCN) and nad1, a 7-bp motif "AGC(T)CTTA" between trnW and trnC and a 6-bp motif "ATGATA" of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A+T-rich region contained an 'ATAGT(A)'-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites '(TA)12' and '(TA)9' were observed in the A+T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.
Asunto(s)
Genoma Mitocondrial , Mariposas Nocturnas/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico/genética , ARN de Transferencia/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of +1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the "out-of-frame pairing" model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA - possibly a result of their low growth rates and deep-water lifestyle - has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.
Asunto(s)
ADN Mitocondrial/genética , Sistema de Lectura Ribosómico , Poríferos/genética , Secuencia de Aminoácidos , Animales , ADN Mitocondrial/metabolismo , Evolución Molecular , Mutación del Sistema de Lectura , Orden Génico , Genes Mitocondriales , Genoma Mitocondrial , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Poríferos/clasificación , Poríferos/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Selección Genética , Alineación de SecuenciaRESUMEN
Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16,141 bp, 16,236 bp and 16,183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A+T-rich region. The genomes were highly compact with only 60-68 bp of non-coding intergenic spacer. However, considerable differences in the A+T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.