RESUMEN
Insulin resistance is the primary pathophysiology underlying metabolic syndrome and type 2 diabetes1,2. Previous metagenomic studies have described the characteristics of gut microbiota and their roles in metabolizing major nutrients in insulin resistance3-9. In particular, carbohydrate metabolism of commensals has been proposed to contribute up to 10% of the host's overall energy extraction10, thereby playing a role in the pathogenesis of obesity and prediabetes3,4,6. Nevertheless, the underlying mechanism remains unclear. Here we investigate this relationship using a comprehensive multi-omics strategy in humans. We combine unbiased faecal metabolomics with metagenomics, host metabolomics and transcriptomics data to profile the involvement of the microbiome in insulin resistance. These data reveal that faecal carbohydrates, particularly host-accessible monosaccharides, are increased in individuals with insulin resistance and are associated with microbial carbohydrate metabolisms and host inflammatory cytokines. We identify gut bacteria associated with insulin resistance and insulin sensitivity that show a distinct pattern of carbohydrate metabolism, and demonstrate that insulin-sensitivity-associated bacteria ameliorate host phenotypes of insulin resistance in a mouse model. Our study, which provides a comprehensive view of the host-microorganism relationships in insulin resistance, reveals the impact of carbohydrate metabolism by microbiota, suggesting a potential therapeutic target for ameliorating insulin resistance.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Microbioma Gastrointestinal , Resistencia a la Insulina , Animales , Humanos , Ratones , Diabetes Mellitus Tipo 2/metabolismo , Microbioma Gastrointestinal/fisiología , Resistencia a la Insulina/fisiología , Monosacáridos/metabolismo , Insulina/metabolismo , Síndrome Metabólico/metabolismo , Heces/química , Heces/microbiología , MetabolómicaRESUMEN
Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.
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Tejido Adiposo Pardo , Obesidad , Ratones , Animales , Obesidad/genética , Obesidad/metabolismo , Tejido Adiposo Pardo/metabolismo , Aumento de Peso/genética , Tejido Adiposo Blanco/metabolismo , Metabolismo Energético/genética , Fenotipo , Ratones Endogámicos C57BL , Dieta Alta en Grasa , Ratones NoqueadosRESUMEN
Because of limited free diffusion in the cytoplasm, viruses must use active transport mechanisms to move intracellularly. Nevertheless, how the plant single-stranded DNA begomoviruses hijack the host intracytoplasmic transport machinery to move from the nucleus to the plasmodesmata remains enigmatic. Here, we identified nuclear shuttle protein (NSP)-interacting proteins from Arabidopsis (Arabidopsis thaliana) by probing a protein microarray and demonstrated that the cabbage leaf curl virus NSP, a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA, interacts in planta with an endosomal vesicle-localized, plant-specific syntaxin-6 protein, designated NSP-interacting syntaxin domain-containing protein (NISP). NISP displays a proviral function, unlike the syntaxin-6 paralog AT2G18860 that failed to interact with NSP. Consistent with these findings, nisp-1 mutant plants were less susceptible to begomovirus infection, a phenotype reversed by NISP complementation. NISP-overexpressing lines accumulated higher levels of vDNA than wild-type. Furthermore, NISP interacted with an NSP-interacting GTPase (NIG) involved in NSP-vDNA nucleocytoplasmic translocation. The NISP-NIG interaction was enhanced by NSP. We also showed that endosomal NISP associates with vDNA. NISP may function as a docking site for recruiting NIG and NSP into endosomes, providing a mechanism for the intracytoplasmic translocation of the NSP-vDNA complex toward and from the cell periphery.
Asunto(s)
Arabidopsis , Begomovirus , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virología , Begomovirus/fisiología , Núcleo Celular/metabolismoRESUMEN
Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.
Asunto(s)
Anticuerpos Antiidiotipos/aislamiento & purificación , Desmogleína 3/aislamiento & purificación , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/aislamiento & purificación , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Química Clic , Cicloparafinas/química , Desmogleína 3/genética , Desmogleína 3/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Luciferasas/química , Oligonucleótidos , Proteínas/genética , Proteínas/inmunologíaRESUMEN
Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Análisis por Matrices de Proteínas , Mapeo de Interacción de ProteínasRESUMEN
The gaseous plant hormone ethylene can trigger myriad physiological and morphological responses in plants. While many ethylene signaling pathway components have been identified and characterized, little is known about the function of the integral membrane protein ETHYLENE-INSENSITIVE2 (EIN2), a central regulator of all ethylene responses. Here, we demonstrate that Arabidopsis thaliana EIN2 is a protein with a short half-life that undergoes rapid proteasome-mediated protein turnover. Moreover, EIN2 protein accumulation is positively regulated by ethylene. We identified two F-box proteins, EIN2 TARGETING PROTEIN1 (ETP1) and EIN2 TARGETING PROTEIN2 (ETP2), that interact with the EIN2 C-terminal domain (EIN2-CEND), which is highly conserved and sufficient to activate most ethylene responses. Overexpression of ETP1 or ETP2 disrupts EIN2 protein accumulation, and these plants manifest a strong ethylene-insensitive phenotype. Furthermore, knocking down the levels of both ETP1 and ETP2 mRNAs using an artificial microRNA (amiRNA) leads to accumulation of EIN2 protein, resulting in plants that display constitutive ethylene response phenotypes. Finally, ethylene down-regulates ETP1 and ETP2 proteins, impairing their ability to interact with EIN2. Thus, these studies reveal that a complex interplay between ethylene, the regulation of ETP1/ETP2 F-box proteins, and subsequent targeting and degradation of EIN2 is essential for triggering ethylene responses in plants.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Etilenos/metabolismo , Proteínas F-Box/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas F-Box/química , Expresión Génica , Técnicas de Silenciamiento del Gen , Semivida , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Mapping protein interaction complexes in their natural state in vivo is arguably the Holy Grail of protein network analysis. Detection of protein interaction stoichiometry has been an important technical challenge, as few studies have focused on this. This may, however, be solved by artificial intelligence (AI) and proteomics. Here, we describe the development of HaloTag-based affinity purification mass spectrometry (HaloMS), a high-throughput HaloMS assay for protein interaction discovery. The approach enables the rapid capture of newly expressed proteins, eliminating tedious conventional one-by-one assays. As a proof-of-principle, we used HaloMS to evaluate the protein complex interactions of 17 regulatory proteins in human adipocytes. The adipocyte interactome network was validated using an in vitro pull-down assay and AI-based prediction tools. Applying HaloMS to probe adipocyte differentiation facilitated the identification of previously unknown transcription factor (TF)-protein complexes, revealing proteome-wide human adipocyte TF networks and shedding light on how different pathways are integrated.
RESUMEN
Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.
Asunto(s)
Endospermo/crecimiento & desarrollo , Oryza/genética , Proteínas de Almacenamiento de Semillas/metabolismo , Almidón/análisis , Amilopectina/análisis , Amilosa/análisis , Mapeo Cromosómico , Clonación Molecular , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Glucanos/análisis , Calor , Datos de Secuencia Molecular , Mutación , Oryza/metabolismo , Filogenia , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas de Almacenamiento de Semillas/genéticaRESUMEN
Atopic dermatitis (AD) is a skin disease that is heterogeneous both in terms of clinical manifestations and molecular profiles. It is increasingly recognized that AD is a systemic rather than a local disease and should be assessed in the context of whole-body pathophysiology. Here we show, via integrated RNA-sequencing of skin tissue and peripheral blood mononuclear cell (PBMC) samples along with clinical data from 115 AD patients and 14 matched healthy controls, that specific clinical presentations associate with matching differential molecular signatures. We establish a regression model based on transcriptome modules identified in weighted gene co-expression network analysis to extract molecular features associated with detailed clinical phenotypes of AD. The two main, qualitatively differential skin manifestations of AD, erythema and papulation are distinguished by differential immunological signatures. We further apply the regression model to a longitudinal dataset of 30 AD patients for personalized monitoring, highlighting patient heterogeneity in disease trajectories. The longitudinal features of blood tests and PBMC transcriptome modules identify three patient clusters which are aligned with clinical severity and reflect treatment history. Our approach thus serves as a framework for effective clinical investigation to gain a holistic view on the pathophysiology of complex human diseases.
Asunto(s)
Dermatitis Atópica , Humanos , Dermatitis Atópica/genética , Transcriptoma , Leucocitos Mononucleares , Piel , FenotipoRESUMEN
High-quality datasets are needed to understand how global and local properties of protein-protein interaction, or 'interactome', networks relate to biological mechanisms, and to guide research on individual proteins. In an evaluation of existing curation of protein interaction experiments reported in the literature, we found that curation can be error-prone and possibly of lower quality than commonly assumed.
Asunto(s)
Bases de Datos de Proteínas , Proteínas/metabolismo , Animales , Bases de Datos Factuales , Humanos , Unión Proteica , Proteínas/análisis , Proteínas/química , Reproducibilidad de los Resultados , Proyectos de InvestigaciónRESUMEN
Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification methods. This novel and efficient conjugation method can be used to prepare HaloTag-barcoded proteins using a click chemistry-based labeling technique. Here, we describe the preparation of protein-DNA complexes and detection of protein-protein interactions which can be used in a HaloTag protein barcode assay to detect an antibody. The protocol includes procedures for preparing the ligand-oligonucleotide complex, plasmid DNA preparation for protein expression, and preparation of the protein-oligonucleotide complex. The described click reaction-based protocols simplify the conventional amine-ester reaction methods which require additional steps for chromatography purification.
RESUMEN
The recent explosion in available genome sequence data has ushered in an era in which analysis of a whole genome can be performed in a single experiment. While DNA microarrays have long been the established technology for measuring gene expression levels, standard expression arrays use relatively few probes for each gene and are typically biased toward known and predicted gene structures. Recently, with the availability of complete genome sequences for many organisms, very-high-density oligonucleotide-based microarrays that span the entire genome have emerged as the preferred platform for genomic analysis. Whole-genome tiling microarrays can be employed for a myriad of purposes, including empirical annotation of the transcriptome, chromatin immunoprecipitation-chip studies, analysis of alternative RNA splicing, characterization of the methylation state of cytosine bases throughout a genome (methylome), and DNA polymorphism discovery. Here, we review several applications of whole-genome technology to obtain a variety of genomic-scale information in plants.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Genoma de Planta/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Empalme Alternativo , Inmunoprecipitación de Cromatina , Modelos Genéticos , Transcripción GenéticaRESUMEN
With the availability of complete genome sequences for a growing number of organisms, high-throughput methods for gene annotation and analysis of genome dynamics are needed. The application of whole-genome tiling microarrays for studies of global gene expression is providing a more unbiased view of the transcriptional activity within genomes. For example, this approach has led to the identification and isolation of many novel non-protein-coding RNAs (ncRNAs), which have been suggested to comprise a major component of the transcriptome that have novel functions involved in epigenetic regulation of the genome. Additionally, tiling arrays have been recently applied to the study of histone modifications and methylation of cytosine bases (DNA methylation). Surprisingly, recent studies combining the analysis of gene expression (transcriptome) and DNA methylation (methylome) using whole-genome tiling arrays revealed that DNA methylation regulates the expression levels of many ncRNAs. Further capture and integration of additional types of genome-wide data sets will help to illuminate additional hidden features of the dynamic genomic landscape that are regulated by both genetic and epigenetic pathways in plants.
Asunto(s)
Genoma de Planta , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Plantas/genética , Metilación de ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las PlantasRESUMEN
Physical interactions between proteins and other molecules can be evaluated at a proteome scale using protein arrays, a type of high-throughput pulldown assay. We developed a modified in situ protein array known as the nucleic acid programmable protein assay (NAPPA) that allows the screening of thousands of open reading frames (ORFs) at a lower cost, with less labor, and in less time than conventional protein arrays. The HaloTag-NAPPA protein array can efficiently capture proteins expressed in situ on a glass slide using the Halo high-affinity capture tag. Here, we describe the fabrication of the array using publicly available resources and detection of protein-protein interactions (PPIs) that can be used to generate a protein interactome map. The Basic Protocol includes procedures for preparing the plasmid DNA spotted on glass slides, in situ protein expression, and PPI detection. The supporting protocols outline the construction of vectors and preparation of ORF clones. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Análisis por Matrices de Proteínas/métodos , Proteoma , Vectores Genéticos , Sistemas de Lectura AbiertaRESUMEN
The Rice Genome Research Project in Japan performs genome sequencing and comprehensive expression profiling, constructs genetic and physical maps, collects full-length cDNAs and generates mutant lines, all aimed at improving the breeding of the rice plant as a food source. The National Institute of Agrobiological Sciences in Tsukuba, Japan, has accumulated numerous rice biological resources and has already successfully produced a high-quality genome sequence, a high-density genetic map with 3000 markers, 30,000 full-length cDNAs, over 700 expression profiles with a 9000 cDNA microarray and 15,000 flanking sequences with Tos17 insertions in about 3765 mutant lines from about 50,000 transposon insertion lines. These resources are available in the public domain. A new unification tool for functional genomics, called Rice PIPELINE, has also been developed for the dynamic collection and compilation of genomics data (genome sequences, full-length cDNAs, gene expression profiles, mutant lines, cis elements) from various databases. The mission of Rice PIPELINE is to provide a unique scientific resource that pools publicly available rice genomic data for search by clone sequence, clone name, GenBank accession number, or keyword. The web-based form of Rice PIPELINE is available at http://cdna01.dna.affrc.go.jp/PIPE/.
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Bases de Datos Genéticas , Genoma de Planta , Genómica , Oryza/genética , Biología Computacional , ADN Complementario/genética , Perfilación de la Expresión Génica , Almacenamiento y Recuperación de la Información , Internet , Japón , MutaciónRESUMEN
Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.
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Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Ácidos Linoleicos/metabolismo , Transducción de Señal , Arabidopsis/genética , Péptidos y Proteínas de Señalización Intracelular , Unión Proteica , Mapeo de Interacción de Proteínas , Nicotiana/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
We collected and completely sequenced 32,127 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. "Nipponbare." Mapping of these clones to genomic DNA revealed approximately 20,500 transcriptional units (TUs) in the rice genome. For each TU, we selected 60-mers using an algorithm that took into account some DNA conditions such as base composition and sequence complexity. Using in situ synthesis technology, we constructed oligonucleotide arrays with these TUs on glass slides. We targeted RNAs prepared from normally grown rice callus and from callus treated with abscisic acid (ABA) or gibberellin (GA). We identified 200 ABA-responsive and 301 GA-responsive genes, many of which had never before been annotated as ABA or GA responsive in other expression analysis. Comparison of these genes revealed antagonistic regulation of almost all by both hormones; these had previously been annotated as being responsible for protein storage and defense against pathogens. Comparison of the cis-elements of genes responsive to one or antagonistic to both hormones revealed that the antagonistic genes had cis-elements related to ABA and GA responses. The genes responsive to only one hormone were rich in cis-elements that supported ABA and GA responses. In a search for the phenotypes of mutants in which a retrotransposon was inserted in these hormone-responsive genes, we identified phenotypes related to seed formation or plant height, including sterility, vivipary, and dwarfism. In comparison of cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response as Arabidopsis specific and for protein storage as rice specific.
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Ácido Abscísico/farmacología , Regulación de la Expresión Génica de las Plantas , Giberelinas/farmacología , Oryza/genética , Reguladores del Crecimiento de las Plantas/farmacología , ARN de Planta/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Células Cultivadas , Elementos Transponibles de ADN , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Oryza/efectos de los fármacos , Oryza/metabolismo , Fenotipo , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacosRESUMEN
We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.
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Ácido Abscísico/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Giberelinas/farmacología , Oryza/genética , Reguladores del Crecimiento de las Plantas/farmacología , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oryza/efectos de los fármacos , Elementos de Respuesta/genética , Factores de Transcripción/genéticaRESUMEN
Apomixis is an intriguing asexual mode of reproduction, because it produces maternal clones that permit vegetative reproduction through seeds. Guineagrass (Panicum maximum) has both facultative aposporous apomixis and obligate sexual modes of reproduction. Despite the importance of apomixis in guineagrass, expressed sequence tags (ESTs) for this condition have not been studied in this species. We constructed a guineagrass cDNA library from two aposporous strains, Ku5954 and GM64-3A, and utilized them as microarray probes. To find genes uniquely expressed in the immature pistils of apomicts, we performed a microarray analysis using target RNA from another apomict, OKI64. Of the 4608 probes in the microarray, only 394 showed clear gene expression in the immature pistils. Of the 394 expressed probes, 196 were successfully sequenced. Of these, 181 had significant homology with other species, including 10 ESTs with matches in a pistil cDNA library from another aposporous species, Cenchrus ciliaris. Of the remaining ESTs, three showed significant homology only with animal database sequences and the other 12 ESTs showed no homology with any previously registered sequence. In reverse-transcriptase PCR and real-time quantitative PCR, nine ESTs reliably detected ovary-specific gene expression. Of these, three revealed aposporous ovary-specific genes expressed in the early developmental stage, suggesting that these could be apomixis-related genes.
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Etiquetas de Secuencia Expresada , Panicum/genética , Panicum/fisiología , Flores/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Hojas de la Planta/genética , Reproducción/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes.