RESUMEN
Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.
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Histonas , Pez Cebra , Animales , Cromatina , ADN , Histonas/metabolismo , Humanos , Síndrome , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
ATP1A3 encodes the α3 subunit of the sodium-potassium ATPase, one of two isoforms responsible for powering electrochemical gradients in neurons. Heterozygous pathogenic ATP1A3 variants produce several distinct neurological syndromes, yet the molecular basis for phenotypic variability is unclear. We report a novel recurrent variant, ATP1A3(NM_152296.5):c.2324C>T; p.(Pro775Leu), in nine individuals associated with the primary clinical features of progressive or non-progressive spasticity and developmental delay/intellectual disability. No patients fulfil diagnostic criteria for ATP1A3-associated syndromes, including alternating hemiplegia of childhood, rapid-onset dystonia-parkinsonism or cerebellar ataxia-areflexia-pes cavus-optic atrophy-sensorineural hearing loss (CAPOS), and none were suspected of having an ATP1A3-related disorder. Uniquely among known ATP1A3 variants, P775L causes leakage of sodium ions and protons into the cell, associated with impaired sodium binding/occlusion kinetics favouring states with fewer bound ions. These phenotypic and electrophysiologic studies demonstrate that ATP1A3:c.2324C>T; p.(Pro775Leu) results in mild ATP1A3-related phenotypes resembling complex hereditary spastic paraplegia or idiopathic spastic cerebral palsy. Cation leak provides a molecular explanation for this genotype-phenotype correlation, adding another mechanism to further explain phenotypic variability and highlighting the importance of biophysical properties beyond ion transport rate in ion transport diseases.
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Ataxia Cerebelosa , Discapacidad Intelectual , Humanos , Mutación/genética , Síndrome , Discapacidad Intelectual/genética , Ataxia Cerebelosa/genética , Fenotipo , Espasticidad Muscular/genética , Cationes , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.
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Predisposición Genética a la Enfermedad , Pruebas Genéticas , Humanos , Pruebas Genéticas/métodos , Genómica , Exoma/genética , América del NorteRESUMEN
De novo variants in FOXP4 were recently associated with a neurodevelopmental disorder characterized by speech and language delay, growth abnormalities, hypotonia, and variable congenital abnormalities, including congenital diaphragmatic hernia, cervical spine abnormalities, strabismus, cryptorchidism, and ptosis. The variant spectrum in this small cohort was limited to de novo missense except for one frameshift, the inheritance of which was unknown. Variants tested in vitro exhibited reduced repressor transcriptional activity, indicating loss of function is the likely mechanism of disease, but only one frameshift variant was reported. Here, we report four affected individuals from two unrelated families heterozygous for a nonsense variant, c.1893C > G, p.Tyr631*, in FOXP4. The phenotype of the affected children includes developmental delay, feeding difficulties in infancy, and similar facial features. In both cases, the variant was inherited from a parent with mild or even subclinical features. Interestingly, one patient presented with congenital diaphragmatic hernia, as reported in two other FOXP4 patients. This report implicates FOXP4 truncating variants in human disease and highlights the wide phenotypic spectrum and variable expressivity.
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Factores de Transcripción Forkhead , Hernias Diafragmáticas Congénitas , Trastornos del Neurodesarrollo , Niño , Humanos , Masculino , Factores de Transcripción Forkhead/genética , Mutación del Sistema de Lectura , Hernias Diafragmáticas Congénitas/genética , Discapacidad Intelectual/genética , Hipotonía Muscular/genética , FenotipoRESUMEN
The DEAD/DEAH box RNA helicases are a superfamily of proteins involved in the processing and transportation of RNA within the cell. A growing literature supports this family of proteins as contributing to various types of human disorders from neurodevelopmental disorders to syndromes with multiple congenital anomalies. This article presents a cohort of nine unrelated individuals with de novo missense alterations in DDX23 (Dead-Box Helicase 23). The gene is ubiquitously expressed and functions in RNA splicing, maintenance of genome stability, and the sensing of double-stranded RNA. Our cohort of patients, gathered through GeneMatcher, exhibited features including tone abnormalities, global developmental delay, facial dysmorphism, autism spectrum disorder, and seizures. Additionally, there were a variety of other findings in the skeletal, renal, ocular, and cardiac systems. The missense alterations all occurred within a highly conserved RecA-like domain of the protein, and are located within or proximal to the DEAD box sequence. The individuals presented in this article provide evidence of a syndrome related to alterations in DDX23 characterized predominantly by atypical neurodevelopment.
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Trastorno del Espectro Autista/genética , ARN Helicasas DEAD-box/genética , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , Trastorno del Espectro Autista/complicaciones , Trastorno del Espectro Autista/epidemiología , Trastorno del Espectro Autista/fisiopatología , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Inestabilidad Genómica/genética , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/epidemiología , Discapacidad Intelectual/fisiopatología , Masculino , Mutación Missense/genética , Trastornos del Neurodesarrollo/complicaciones , Trastornos del Neurodesarrollo/epidemiología , Trastornos del Neurodesarrollo/fisiopatología , Empalme del ARN/genética , ARN Bicatenario/genética , Convulsiones/complicaciones , Convulsiones/genética , Convulsiones/fisiopatologíaRESUMEN
Congenital heart disease (CHD) is a major cause of morbidity in the pediatric population yet its genetic and molecular causes remain poorly defined. Previously, we identified AGMO as a candidate heterotaxy disease gene, a disorder of left-right (LR) patterning that can have a profound effect on cardiac function. AGMO is the only known alkylglycerol monooxygenase, an orphan tetrahydrobiopterin dependent enzyme that cleaves the ether linkage in alkylglycerols. However, whether AGMO plays a role in LR patterning was unexplored. Here we reveal that Agmo is required for correct development of the embryonic LR axis in Xenopus embryos recapitulating the patient's heterotaxy phenotype. Mechanistically, we demonstrate that Agmo is a regulator of canonical Wnt signaling, required during gastrulation for normal formation of the left - right organizer. Mutational analysis demonstrates that this function is dependent on Agmo's alkylglycerol monooxygenase activity. Together, our findings identify Agmo as a regulator of canonical Wnt signaling, demonstrate a role for Agmo in embryonic axis formation, and provide insight into the poorly understood developmental requirements for ether lipid cleavage.
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Tipificación del Cuerpo/genética , Oxigenasas de Función Mixta/metabolismo , Vía de Señalización Wnt/genética , Animales , Tipificación del Cuerpo/fisiología , Análisis Mutacional de ADN/métodos , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Metabolismo de los Lípidos , Lípidos/fisiología , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/fisiología , Éteres Fosfolípidos/metabolismo , Vía de Señalización Wnt/fisiología , Xenopus/embriología , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance. We investigated how codon identity and translation affect mRNA stability during development and homeostasis. We show that the codon triplet contains translation-dependent regulatory information that influences transcript decay. Codon composition shapes maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila, and gene expression during homeostasis across human tissues. Some synonymous codons show consistent stabilizing or destabilizing effects, suggesting that amino acid composition influences mRNA stability. Codon composition affects both polyadenylation status and translation efficiency. Thus, the ribosome interprets two codes within the mRNA: the genetic code which specifies the amino acid sequence and a conserved "codon optimality code" that shapes mRNA stability and translation efficiency across vertebrates.
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Codón , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/genética , Cigoto/crecimiento & desarrollo , Animales , Drosophila , Humanos , Ratones , Ribosomas/metabolismo , Xenopus , Pez CebraRESUMEN
The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.
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ADN Ribosómico/genética , Proteínas Nucleares/metabolismo , Cráneo/embriología , Transcripción Genética , Xenopus/embriología , Animales , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Humanos , Disostosis Mandibulofacial/metabolismo , Disostosis Mandibulofacial/patología , Ratones , Cresta Neural/embriología , Proteínas Nucleares/genética , Especificidad de Órganos , ARN Mensajero/genética , Ribosomas/metabolismo , Cráneo/metabolismo , Xenopus/genética , Xenopus/metabolismoRESUMEN
BACKGROUND: Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step. RESULTS: Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping. CONCLUSION: Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.
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Exoma , Exones , Mutación , Polimorfismo de Nucleótido Simple , Proteínas de Xenopus/genética , Xenopus/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Células Clonales , Ligamiento Genético , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Meiosis/genética , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de ADNRESUMEN
With a surprisingly complex genome and an ever-expanding genetic toolkit, the sea anemone Nematostella vectensis has become a powerful model system for the study of both development and whole-body regeneration. Here we provide the most current protocols for short-hairpin RNA (shRNA )-mediated gene knockdown and CRISPR/Cas9-targeted mutagenesis in this system. We further show that a simple Klenow reaction followed by in vitro transcription allows for the production of gene-specific shRNAs and single guide RNAs (sgRNAs) in a fast, affordable, and readily scalable manner. Together, shRNA knockdown and CRISPR/Cas9-targeted mutagenesis allow for rapid screens of gene function as well as the production of stable mutant lines that enable functional genetic analysis throughout the Nematostella life cycle.
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Anémonas de Mar , Animales , Técnicas de Silenciamiento del Gen , Genoma , Mutagénesis , ARN Interferente Pequeño/genética , Anémonas de Mar/genéticaRESUMEN
Fructans are fructose polymers synthesized in a wide range of species such as bacteria, fungi and plants. Fructans are synthesized by fructosyltransferases (FTs) and depolymerized by fructan exohydrolases (FEHs). Bromus pictus is a graminean decaploid species from the Patagonian region of Argentina, which accumulates large amounts of fructans even at temperate temperatures. The first gene isolated from B. pictus fructan metabolism was a putative sucrose:fructan 6-fructosyltransferase (6-SFT). Here, a complete cDNA of the first fructan exohydrolase (FEH) from B. pictus (Bp1-FEHa) was isolated using RT-PCR strategies. The Bp1-FEHa encoding gene is present as a single copy in B. pictus genome. Functional characterization in Pichia pastoris confirmed Bp1-FEHa is a fructan exohydrolase with predominant activity towards beta-(2-1) linkages. Its expression was analyzed in different leaf sections, showing the highest expression levels in the second section of the sheath and the tip of the blade. Bp1-FEHa expression was studied along with FEH and FT activities and fructan accumulation profile in response to chilling conditions during a 7-day time course experiment. Bp1-FEHa expression and FEH activity followed a similar pattern in response to low temperatures, especially in basal sections of the sheaths. In these sections the FEH and FT activities were particularly high and they were significantly correlated to fructan accumulation profile, along with cold treatment.
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Adaptación Fisiológica , Bromus/enzimología , Bromus/genética , Frío , Glicósido Hidrolasas/genética , Secuencia de Aminoácidos , Southern Blotting , Clonación Molecular , Fructanos/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Glicósido Hidrolasas/química , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Datos de Secuencia Molecular , Filogenia , Pichia , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Hox genes encode conserved developmental transcription factors that govern anterior-posterior (A-P) pattering in diverse bilaterian animals, which display bilateral symmetry. Although Hox genes are also present within Cnidaria, these simple animals lack a definitive A-P axis, leaving it unclear how and when a functionally integrated Hox code arose during evolution. We used short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9 mutagenesis to demonstrate that a Hox-Gbx network controls radial segmentation of the larval endoderm during development of the sea anemone Nematostella vectensis. Loss of Hox-Gbx activity also elicits marked defects in tentacle patterning along the directive (orthogonal) axis of primary polyps. On the basis of our results, we propose that an axial Hox code may have controlled body patterning and tissue segmentation before the evolution of the bilaterian A-P axis.
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Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/fisiología , Anémonas de Mar/crecimiento & desarrollo , Factores de Transcripción/fisiología , Animales , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Endodermo/citología , Endodermo/crecimiento & desarrollo , Endonucleasas , Técnicas de Silenciamiento del Gen/métodos , Genes Homeobox/genética , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Mutagénesis , ARN Interferente Pequeño/genética , Anémonas de Mar/citología , Anémonas de Mar/genética , Factores de Transcripción/genéticaRESUMEN
Canonical Wnt signaling coordinates many critical aspects of embryonic development, while dysregulated Wnt signaling contributes to common diseases, including congenital malformations and cancer. The nuclear localization of ß-catenin is the defining step in pathway activation. However, despite intensive investigation, the mechanisms regulating ß-catenin nuclear transport remain undefined. In a patient with congenital heart disease and heterotaxy, a disorder of left-right patterning, we previously identified the guanine nucleotide exchange factor, RAPGEF5. Here, we demonstrate that RAPGEF5 regulates left-right patterning via Wnt signaling. In particular, RAPGEF5 regulates the nuclear translocation of ß-catenin independently of both ß-catenin cytoplasmic stabilization and the importin ß1/Ran-mediated transport system. We propose a model whereby RAPGEF5 activates the nuclear GTPases, Rap1a/b, to facilitate the nuclear transport of ß-catenin, defining a parallel nuclear transport pathway to Ran. Our results suggest new targets for modulating Wnt signaling in disease states.
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Tipificación del Cuerpo , Núcleo Celular/metabolismo , Vía de Señalización Wnt , Proteínas de Xenopus/fisiología , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Animales , Factores de Intercambio de Guanina Nucleótido/fisiología , XenopusRESUMEN
Nucleoporins (nups) compose the structure of the nuclear pore complex (NPC) of all cells, but several studies have illuminated nucleoporins' additional roles in development and the cell cycle. However, a comprehensive study of nup expression in embryonic development has not yet been reported. We synthesized antisense probes for all nup genes and used whole-mount in situ hybridization techniques to determine the expression pattern of all members of the nup family of genes at three different developmental stages in Xenopus tropicalis. We found that the expression of nups was not ubiquitous in embryos, but was localized to specific and distinguishable anatomical structures at all three stages tested. We also found that the expression patterns for nups within the same subcomplexes were not necessarily identical. Thus, nup expression is subject to a significant level of regulation during development. These results provide new information for functional studies of nups to unravel their roles in embryonic development.
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Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Complejo Poro Nuclear/genética , Poro Nuclear/genética , Xenopus/genética , Animales , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Xenopus/embriología , Xenopus/metabolismoRESUMEN
Human genomics is identifying candidate genes for congenital heart disease (CHD), but discovering the underlying mechanisms remains challenging. In a patient with CHD and heterotaxy (Htx), a disorder of left-right patterning, we previously identified a duplication in Nup188. However, a mechanism to explain how a component of the nuclear pore complex (NPC) could cause Htx/CHD was undefined. Here, we show that knockdown of Nup188 or its binding partner Nup93 leads to a loss of cilia during embryonic development while leaving NPC function largely intact. Many data, including the localization of endogenous Nup188/93 at cilia bases, support their direct role at cilia. Super-resolution imaging of Nup188 shows two barrel-like structures with dimensions and organization incompatible with an NPC-like ring, arguing against a proposed "ciliary pore complex." We suggest that the nanoscale organization and function of nucleoporins are context dependent in a way that is required for the structure of the heart.
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Cardiopatías Congénitas/metabolismo , Síndrome de Heterotaxia/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Xenopus/genética , Animales , Cilios/genética , Cilios/patología , Técnicas de Silenciamiento del Gen , Genoma Humano , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Síndrome de Heterotaxia/patología , Humanos , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Conformación Proteica , Mapas de Interacción de Proteínas/genética , Transporte de Proteínas/genética , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismoRESUMEN
A spectacular advantage of Xenopus tropicalis is the ease with which diploid embryos can be generated year round. By the simple administration of human chorionic gonadotropin, an investigator can generate many hundreds of synchronized embryos by in vitro fertilization or thousands of embryos from a mating pair. The ability to induce ovulations when desired facilitates many different experiments such as experimental embryology, molecular manipulation of gene products, and genetics.
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Diploidia , Embrión no Mamífero/fisiología , Xenopus/genética , Crianza de Animales Domésticos , Animales , Tampones (Química) , Gonadotropina Coriónica/administración & dosificación , Copulación , Femenino , Fertilización In Vitro/métodos , Masculino , Ovulación , Sustancias para el Control de la Reproducción/administración & dosificaciónRESUMEN
We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.
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Adaptación Fisiológica , Bromus/enzimología , Congelación , Hexosiltransferasas/metabolismo , Nicotiana/genética , Pichia/genética , Secuencia de Bases , Cromatografía por Intercambio Iónico , Cartilla de ADN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The AtEm1 and AtEm6 gene products accumulate exclusively in embryos during Arabidopsis seed maturation. The transcription factor ABI3 and the phytohormone abscisic acid are required for normal expression of both genes. However, the expression of these genes occurs in extremely small embryos limiting the availability of tissue to directly study DNA-protein interactions. We generated callus lines derived from embryos to determine if the regulation of Em expression was similar to wild type embryos. Expression of AtEm1 and AtEm6 was strongly induced by abscisic acid in callus derived from wild type embryos, but not in embryo callus derived from ABI3 mutant embryos (abi3-6). Epitopes to 14-3-3 proteins were found in complexes with the AtEm1 promoter in mobility shift experiments using nuclear extracts derived from both wild type and abi3-6 calli. Using phosphorylated peptides that bind to 14-3-3 proteins, we show that 14-3-3 proteins are required for the maintenance of the transcriptional complex generated in nuclear extracts. Chromatin immunoprecipitation experiments using a 14-3-3 antibody display the expected 241-bp band from the AtEm1 promoter. Hence, 14-3-3 proteins are physically present in the AtEm1 transcriptional complex in vivo and are required for the maintenance of the transcriptional complex in vitro.