RESUMEN
The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP-HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy.
Asunto(s)
Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , ARN Nucleolar Pequeño/genética , Secuencia de Bases , Línea Celular , Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Células HeLa , Humanos , Conformación de Ácido Nucleico , Unión Proteica , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismoRESUMEN
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Fosfoserina/metabolismo , Secuencia de Aminoácidos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Semivida , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Interfase/efectos de los fármacos , Mitógenos/farmacología , Datos de Secuencia Molecular , Oncogenes , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hypoxia and inflammation are intimately linked. It is known that nuclear factor κB (NF-κB) regulates the hypoxia-inducible factor (HIF) system, but little is known about how HIF regulates NF-κB. Here, we show that HIF-1α represses NF-κB-dependent gene expression. HIF-1α depletion results in increased NF-κB transcriptional activity both in mammalian cells and in the model organism Drosophila melanogaster. HIF-1α depletion enhances the NF-κB response, and this required not only the TAK-IKK complex, but also CDK6. Loss of HIF-1α results in an increased angiogenic response in mammalian cancer cells and increased mortality in Drosophila following infection. These results indicate that HIF-1α is required to restrain the NF-κB response, and thus prevents excessive and damaging pro-inflammatory responses.
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunidad Innata/genética , FN-kappa B/metabolismo , Transducción de Señal/genética , Animales , Línea Celular , Quinasa 6 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/microbiología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/patología , Técnicas de Silenciamiento del Gen , Humanos , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Mamíferos/metabolismo , Neovascularización Fisiológica , Análisis de SupervivenciaRESUMEN
Hypoxia, or low oxygen availability, is an important physiological and pathological stimulus for multicellular organisms. Molecularly, hypoxia activates a transcriptional programme directed at restoration of oxygen homoeostasis and cellular survival. In mammalian cells, hypoxia not only activates the HIF (hypoxia-inducible factor) family, but also additional transcription factors such as NF-κB (nuclear factor κB). Here we show that hypoxia activates the IKK-NF-κB [IκB (inhibitor of nuclear factor κB)-NF-κB] pathway and the immune response in Drosophila melanogaster. We show that NF-κB activation is required for organism survival in hypoxia. Finally, we identify a role for the tumour suppressor Cyld, as a negative regulator of NF-κB in response to hypoxia in Drosophila. The results indicate that hypoxia activation of the IKK-NF-κB pathway and the immune response is an important and evolutionary conserved response.
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , Hipoxia/genética , Proteínas I-kappa B/genética , FN-kappa B/genética , Animales , Línea Celular Tumoral , Enzima Desubiquitinante CYLD , Proteínas de Drosophila/genética , Células HeLa , Humanos , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1ß and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción Paired Box/metabolismo , Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células HEK293 , Células HeLa , Histona Demetilasas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción Paired Box/antagonistas & inhibidores , Factores de Transcripción Paired Box/genética , Fotoblanqueo , Unión Proteica , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción GenéticaRESUMEN
PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Centrosoma/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Oxígeno/farmacología , Secuencia de Aminoácidos , Centriolos/efectos de los fármacos , Centriolos/metabolismo , Proteínas Cromosómicas no Histona/química , Células HeLa , Humanos , Hidroxilación/efectos de los fármacos , Mitosis/efectos de los fármacos , Modelos Biológicos , Datos de Secuencia Molecular , Prolina/metabolismo , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The IKK [inhibitor of NF-κB (nuclear factor κB) kinase] complex has an essential role in the activation of the family of NF-κB transcription factors in response to a variety of stimuli. To identify novel IKK-interacting proteins, we performed an unbiased proteomics screen where we identified TfR1 (transferrin receptor 1). TfR1 is required for transferrin binding and internalization and ultimately for iron homoeostasis. TfR1 depletion does not lead to changes in IKK subunit protein levels; however, it does reduce the formation of the IKK complex, and inhibits TNFα (tumour necrosis factor α)-induced NF-κB-dependent transcription. We find that, in the absence of TfR1, NF-κB does not translocate to the nucleus efficiently, and there is a reduction in the binding to target gene promoters and consequentially less target gene activation. Significantly, depletion of TfR1 results in an increase in apoptosis in response to TNFα treatment, which is rescued by elevating the levels of RelA/NF-κB. Taken together, these results indicate a new function for TfR1 in the control of IKK and NF-κB. Our data indicate that IKK-NF-κB responds to changes in iron within the cell.
Asunto(s)
Antígenos CD/metabolismo , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Receptores de Transferrina/metabolismo , Transducción de Señal/fisiología , Línea Celular Tumoral , Células HEK293 , Humanos , Quinasa I-kappa B/fisiología , Hierro/química , Hierro/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/fisiología , Unión Proteica/fisiología , Receptores de Transferrina/deficienciaRESUMEN
The hypoxia-inducible factor (HIF) is a master regulator of the cellular response to hypoxia. Its levels and activity are controlled by dioxygenases called prolyl-hydroxylases and factor inhibiting HIF (FIH). To activate genes, HIF has to access sequences in DNA that are integrated in chromatin. It is known that the chromatin-remodeling complex switch/sucrose nonfermentable (SWI/SNF) is essential for HIF activity. However, no additional information exists about the role of other chromatin-remodeling enzymes in hypoxia. Here we describe the role of imitation switch (ISWI) in the cellular response to hypoxia. We find that unlike SWI/SNF, ISWI depletion enhances HIF activity without altering its levels. Furthermore, ISWI knockdown only alters a subset of HIF target genes. Mechanistically, we find that ISWI is required for full expression of FIH mRNA and protein levels by changing RNA polymerase II loading to the FIH promoter. Of interest, exogenous FIH can rescue the ISWI-mediated upregulation of CA9 but not BNIP3, suggesting that FIH-independent mechanisms are also involved. Of importance, ISWI depletion alters the cellular response to hypoxia by reducing autophagy and increasing apoptosis. These results demonstrate a novel role for ISWI as a survival factor during the cellular response to hypoxia.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , Oxigenasas de Función Mixta/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Antígenos de Neoplasias/metabolismo , Apoptosis , Autofagia , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/metabolismo , Hipoxia de la Célula , Proliferación Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Transportador de Glucosa de Tipo 3/metabolismo , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Luciferasas/biosíntesis , Luciferasas/genética , Proteínas de la Membrana/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Polimerasa II/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Hypoxia inducible factor (HIF) is the major transcription factor involved in the regulation of the cellular response to hypoxia, or low oxygen tensions. Even though HIF-1 function is mostly studied following hypoxic stress, well oxygenated areas of several diseased tissues have detectable levels of this transcription factor. Therefore, it is surprising how little is known about the function of HIF in normoxia. This study seeks to fill this gap. Using transient HIF-1α knockdown, as well as, stable cell lines generated using short hairpin RNAs (shRNA), we have further characterized the role of HIF-1α in normoxia. Our data reveals that knockdown of HIF-1α results in a significant increase in cells in the G1 phase of the cell cycle. We find that HIF-1α depletion increases the protein and mRNA of both p21 and p27. p21 is induced via, at least in part, p53-independent but SP1-dependent mechanisms. Interestingly, HIF-1α knockdown also alters the cellular response to chemotherapeutic agents. These data have important implications in not only for the further understanding of HIF-1α, a major transcription factor, but also for the use of HIF-targeted and combination therapies in cancer treatment.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia , Receptores Inmunológicos/metabolismo , Factores de Transcripción/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Biomarcadores de Tumor/genética , Ciclo Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/genética , Femenino , Silenciador del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Terapia Molecular Dirigida , ARN Mensajero/análisis , ARN Interferente Pequeño/metabolismo , Receptores Inmunológicos/genética , Factores de Transcripción/genética , TransfecciónRESUMEN
The cellular response to hypoxia relies on the activation of a specific transcriptional program. Although, most of the attention is focused on the transcription factor HIF, other transcription factors are also activated in hypoxia. We have recently described the mechanism for hypoxia induced NFκB. We have demonstrated the crucial dependency on the IKK complex as well as in the upstream IKK kinase TAK1. TAK1 and IKK activation is dependent upon the calcium calmodulin kinase, CaMK2 and requires Ubc13 as the E2 ubiquitin conjugation enzyme. We report a role for XIAP as the possible E3-ubiquitin ligase for this system. Interestingly, hypoxia induced IKK mediated phosphorylation of IκBα, does not lead to degradation. Hypoxia prevents IκBα de-sumoylation of Sumo-2/3 chains on critical lysine residues, normally required for K-48 linked polyubiquitination. Our results define a novel pathway regulating NFκB activation.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Humanos , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/genética , Fosforilación , Transducción de Señal , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1ß mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1ß result in modulation of HIF-2α protein. HIF-1ß overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1ß (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Regulación de la Expresión Génica , FN-kappa B/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fibroblastos/efectos de los fármacos , Redes Reguladoras de Genes/genética , Células HEK293 , Células HeLa , Humanos , Hipoxia/genética , Ratones , FN-kappa B/genética , ARN Interferente Pequeño/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease.
Asunto(s)
Hipoxia de la Célula/fisiología , FN-kappa B/biosíntesis , Animales , Secuencia de Bases , Hipoxia de la Célula/genética , Línea Celular , ADN/genética , ADN/metabolismo , Técnicas de Inactivación de Genes , Humanos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Interleucina-8/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Fosfohidrolasa PTEN/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Proteína SUMO-1/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Activación TranscripcionalRESUMEN
In response to replication stress, Claspin mediates the phosphorylation and activation of Chk1 by ATR. Claspin is not only necessary for the propagation of the DNA-damage signal, but its destruction by the ubiquitin-proteosome pathway is required to allow the cell to continue the cell cycle allowing checkpoint recovery. Here, we demonstrate that both the NF-kappaB family of transcription factors and their upstream kinase IKK can regulate Claspin levels by controlling its mRNA expression. Furthermore, we show that c-Rel directly controls Claspin gene transcription. Disruption of IKK and specific NF-kappaB members impairs ATR-mediated checkpoint function following DNA damage. Importantly, hyperactivation of IKK results in a failure to inactivate Chk1 and impairs the recovery from the DNA checkpoint. These results uncover a novel function for IKK and NF-kappaB modulating the DNA-damage checkpoint response, allowing the cell to integrate different signalling pathways with the DNA-damage response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Técnicas de Inactivación de Genes , Humanos , Quinasa I-kappa B/genética , Mutagénesis Insercional , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transducción de Señal , Transducción GenéticaRESUMEN
Hypoxia induces a variety of cellular responses such as cell cycle arrest, apoptosis, and autophagy. Most of these responses are mediated by the hypoxia-inducible factor-1alpha. To induce target genes, hypoxia-inducible factor-1alpha requires a chromatin environment conducive to allow binding to specific sequences. Here, we have studied the role of the chromatin-remodeling complex SWI/SNF in the cellular response to hypoxia. We find that SWI/SNF is required for several of the cellular responses induced by hypoxia. Surprisingly, hypoxia-inducible factor-1alpha is a direct target of the SWI/SNF chromatin-remodeling complex. SWI/SNF components are found associated with the hypoxia-inducible factor-1alpha promoter and modulation of SWI/SNF levels results in pronounced changes in hypoxia-inducible factor-1alpha expression and its ability to transactivate target genes. Furthermore, impairment of SWI/SNF function renders cells resistant to hypoxia-induced cell cycle arrest. These results reveal a previously uncharacterized dependence of hypoxia signaling on the SWI/SNF complex and demonstrate a new level of control over the hypoxia-inducible factor-1alpha system.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Ciclo Celular/fisiología , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , HumanosRESUMEN
Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and beta-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Hordeum/genética , Hordeum/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alelos , Empalme Alternativo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Clonación Molecular , Canales Catiónicos Regulados por Nucleótidos Cíclicos , ADN de Plantas/genética , Genes de Plantas , Mutación , Fenotipo , Enfermedades de las Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Especificidad de la EspecieRESUMEN
More than 2,000 genome-wide barley single nucleotide polymorphisms (SNPs) were developed by resequencing unigene fragments from eight diverse accessions. The average genome-wide SNP frequency observed in 877 unigenes was 1 SNP per 200 bp. However, SNP frequency was highly variable with the least number of SNP and SNP haplotypes observed within European cultivated germplasm reflecting effects of breeding history on genetic diversity. More than 300 SNP loci were mapped genetically in three experimental mapping populations which allowed the construction of an integrated SNP map incorporating a large number of RFLP, AFLP and SSR markers (1,237 loci in total). The genes used for SNP discovery were selected based on their transcriptional response to a variety of abiotic stresses. A set of known barley abiotic stress QTL was positioned on the linkage map, while the available sequence and gene expression information facilitated the identification of genes potentially associated with these traits. Comparison of the sequenced SNP loci to the rice genome sequence identified several regions of highly conserved gene order providing a framework for marker saturation in barley genomic regions of interest. The integration of genome-wide SNP and expression data with available genetic and phenotypic information will facilitate the identification of gene function in barley and other non-model organisms.
Asunto(s)
Genes de Plantas , Ligamiento Genético , Hordeum/genética , Polimorfismo de Nucleótido Simple , Etiquetas de Secuencia ExpresadaRESUMEN
A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes.