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1.
Nat Commun ; 12(1): 236, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431896

RESUMEN

Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)n repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarifies the mode of binding that recognizes four guanines in the UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)n repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSB-mediated splicing events. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates the disease phenotype induced by toxic r(UGGAA)n repeat RNA. These studies demonstrate that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.


Asunto(s)
Drosophila/genética , ARN/genética , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Células HeLa , Humanos , Intrones/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Fenotipo , Fosforilación , Proteínas de Unión al ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Temperatura
2.
PLoS One ; 16(1): e0243271, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33428634

RESUMEN

In an outbreak, effective detection of the aetiological agent(s) involved using molecular techniques is key to efficient diagnosis, early prevention and management of the spread. However, sequencing is necessary for mutation monitoring and tracking of clusters of transmission, development of diagnostics and for vaccines and drug development. Many sequencing methods are fast evolving to reduce test turn-around-time and to increase through-put compared to Sanger sequencing method; however, Sanger sequencing remains the gold standard for clinical research sequencing with its 99.99% accuracy This study sought to generate sequence data of SARS-CoV-2 using Sanger sequencing method and to characterize them for possible site(s) of mutations. About 30 pairs of primers were designed, synthesized, and optimized using endpoint PCR to generate amplicons for the full length of the virus. Cycle sequencing using BigDye Terminator v.3.1 and capillary gel electrophoresis on ABI 3130xl genetic analyser were performed according to the manufacturers' instructions. The sequence data generated were assembled and analysed for variations using DNASTAR Lasergene 17 SeqMan Ultra. Total length of 29,760bp of SARS-CoV-2 was assembled from the sample analysed and deposited in GenBank with accession number: MT576584. Blast result of the sequence assembly shows a 99.97% identity with the reference sequence. Variations were noticed at positions: nt201, nt2997, nt14368, nt16535, nt20334, and nt28841-28843, which caused amino acid alterations at the S (aa614) and N (aa203-204) regions. The mutations observed at S and N-gene in this study may be indicative of a gradual changes in the genetic coding of the virus hence, the need for active surveillance of the viral genome.


Asunto(s)
/virología , /genética , Secuencia de Bases , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Nigeria/epidemiología , Filogenia , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
3.
Nat Commun ; 12(1): 256, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431871

RESUMEN

In humans, inactivating mutations in MLL4, which encodes a histone H3-lysine 4-methyltransferase, lead to Kabuki syndrome (KS). While dwarfism is a cardinal feature of KS, the underlying etiology remains unclear. Here we report that Mll4 regulates the development of growth hormone-releasing hormone (GHRH)-producing neurons in the mouse hypothalamus. Our two Mll4 mutant mouse models exhibit dwarfism phenotype and impairment of the developmental programs for GHRH-neurons. Our ChIP-seq analysis reveals that, in the developing mouse hypothalamus, Mll4 interacts with the transcription factor Nrf1 to trigger the expression of GHRH-neuronal genes. Interestingly, the deficiency of Mll4 results in a marked reduction of histone marks of active transcription, while treatment with the histone deacetylase inhibitor AR-42 rescues the histone mark signature and restores GHRH-neuronal production in Mll4 mutant mice. Our results suggest that the developmental dysregulation of Mll4-directed epigenetic control of transcription plays a role in the development of GHRH-neurons and dwarfism phenotype in mice.


Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/biosíntesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Hipotálamo/citología , Neuronas/metabolismo , Animales , Secuencia de Bases , Enanismo/metabolismo , Embrión de Mamíferos/metabolismo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Hipotálamo/embriología , Masculino , Ratones Noqueados , Modelos Biológicos , Factor Nuclear 1 de Respiración/metabolismo , Fenilbutiratos/farmacología , Factores de Transcripción/metabolismo
4.
Nat Commun ; 12(1): 328, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436566

RESUMEN

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.


Asunto(s)
Sistema de Lectura Ribosómico/genética , Genoma Bacteriano , ARN de Transferencia/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , Aminoacilación , Anticodón/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Codón/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Hidrólisis , Metilación , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismo
5.
Ecotoxicol Environ Saf ; 208: 111731, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33396062

RESUMEN

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.


Asunto(s)
Cadmio/toxicidad , Catalasa/metabolismo , Clonación Molecular , Sustancias Peligrosas/toxicidad , Metalotioneína/metabolismo , Salamandridae/fisiología , Testículo/efectos de los fármacos , Animales , Secuencia de Bases , Humanos , Hígado/metabolismo , Masculino , ARN Mensajero/metabolismo , Salamandridae/genética , Salamandridae/metabolismo , Células de Sertoli/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo
6.
Nat Commun ; 12(1): 33, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397927

RESUMEN

The Origin Recognition Complex (ORC) is an evolutionarily conserved six-subunit protein complex that binds specific sites at many locations to coordinately replicate the entire eukaryote genome. Though highly conserved in structure, ORC's selectivity for replication origins has diverged tremendously between yeasts and humans to adapt to vastly different life cycles. In this work, we demonstrate that the selectivity determinant of ORC for DNA binding lies in a 19-amino acid insertion helix in the Orc4 subunit, which is present in yeast but absent in human. Removal of this motif from Orc4 transforms the yeast ORC, which selects origins based on base-specific binding at defined locations, into one whose selectivity is dictated by chromatin landscape and afforded with plasticity, as reported for human. Notably, the altered yeast ORC has acquired an affinity for regions near transcriptional start sites (TSSs), which the human ORC also favors.


Asunto(s)
Complejo de Reconocimiento del Origen/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN de Hongos/metabolismo , Fase G2/genética , Genoma Fúngico , Humanos , Modelos Genéticos , Mutación/genética , Nucleosomas/metabolismo , Motivos de Nucleótidos/genética , Complejo de Reconocimiento del Origen/química , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Procesos Estocásticos , Sitio de Iniciación de la Transcripción
7.
Nucleic Acids Res ; 49(1): 547-567, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33330920

RESUMEN

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.


Asunto(s)
Bacteroidetes/genética , Iniciación de la Cadena Peptídica Traduccional , Secuencias Reguladoras de Ácido Ribonucleico , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Codón Iniciador , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coli/genética , Flavobacterium/genética , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Puromicina/farmacología , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , ARN Ribosómico 5S/genética , Ribosomas/ultraestructura , Alineación de Secuencia , Homología de Secuencia , Especificidad de la Especie
8.
Nucleic Acids Res ; 49(1): 479-490, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33330934

RESUMEN

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.


Asunto(s)
Empalme Alternativo/genética , Aminoaciltransferasas/genética , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , Pliegue del ARN , Precursores del ARN/genética , ARN Mensajero/genética , Células A549 , Secuencia de Bases , Línea Celular Tumoral , Secuencia Conservada , Exones/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Intrones/genética , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/genética , Oligonucleótidos/genética , Oligonucleótidos Antisentido/genética , Especificidad de Órganos , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Elongación de la Transcripción Genética
9.
Nucleic Acids Res ; 49(1): 568-583, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33332555

RESUMEN

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.


Asunto(s)
Proteínas Protozoarias/química , Estabilidad del ARN/fisiología , ARN Guia/metabolismo , ARN Protozoario/metabolismo , Ribonucleasa III/química , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , Regulación de la Expresión Génica , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Dominios Proteicos , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleasa III/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Dedos de Zinc
10.
Gene ; 764: 145078, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32858175

RESUMEN

In maize, eat rot and stalk rot caused by Fusarium verticillioides and Fusarium graminearum lead to contamination of moldy grains to produce mycotoxins. Identification of resistance genes against these pathogens for maize breeding is an effective way for disease control. Several 2-oxoglutarate-dependent dioxygenase (2OGD) proteins have been found to confer resistance to different pathogens in diverse plant species. However, little is known about the 2OGD superfamily in maize. Here, we identified 103 putative 2OGD genes in maize from a genome-wide analysis, and divided them into three classes - DOXA, DOXB, and DOXC. We further comprehensively investigated their gene structure, chromosome distribution, phylogenetic tree, gene-function enrichment, and expression profiles among different tissues. The genes encoding three 2OGD proteins, ACO, F3H, and NCS involved in ethylene biosynthesis, flavonoids biosynthesis, and alkaloids biosynthesis pathways, respectively, were identified to be induced by F. verticillioides and F. graminearum. The promoters of the three genes contain the binding sites for the transcription factor ZmDOF and ZmHSF, which are also induced by the two pathogens. The results imply that the three 2OGDs and the two transcription factors might be involved in the resistance to the two pathogens. This study provided a comprehensive understanding of the 2OGD superfamily in maize and laid the foundation for the further functional analysis of their roles in maize resistance to eat rot and stalk rot.


Asunto(s)
Dioxigenasas/genética , Fusarium/inmunología , Proteínas de Plantas/genética , Zea mays/fisiología , Secuencia de Bases/genética , Sitios de Unión/genética , Cromosomas de las Plantas/genética , Coenzimas/metabolismo , Secuencia Conservada/genética , Dioxigenasas/inmunología , Dioxigenasas/metabolismo , Resistencia a la Enfermedad/genética , Evolución Molecular , Fusarium/patogenicidad , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/fisiología , Estudio de Asociación del Genoma Completo , Ácidos Cetoglutáricos/metabolismo , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Tallos de la Planta/enzimología , Tallos de la Planta/crecimiento & desarrollo , Tallos de la Planta/microbiología , Regiones Promotoras Genéticas/genética , RNA-Seq , Factores de Transcripción/metabolismo , Zea mays/microbiología
11.
Gene ; 766: 145144, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-32916248

RESUMEN

The elongases of very long-chain fatty acids (Elovls) are involved in the rate-limiting of the carbon chain elongation reaction in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovls family, Elovl4, has been regarded as a critical enzyme involved in the biosynthesis pathway of polyunsaturated fatty acids (PUFAs). To explore the role of Elovl4 in PUFA synthesis in Trachinotus ovatus, the cDNA of the Elovl4b gene is cloned from T. ovatus (ToElovl4b). The ORF of ToElovl4b was 918 bp and encoded 305 amino acid (aa) protein sequences. Sequence alignment showed that the deduced amino acids contained significant structural features of the Elovl4 family, such as a histidine box motif (HXXHH), multiple transmembrane domains and an endoplasmic reticulum (ER) retention signal. Moreover, phylogenetic analysis revealed that ToElovl4b was highly conserved with that of Rachycentron canadum Elovl4b. Moreover, heterologous expression in yeast demonstrated that ToElovl4b could efficiently elongate 18:2n-6, 18:3n-6 and 20:5n-3 FAs up to 20:2n-6, 20:3n-6 and 22:5n-3, respectively. Furthermore, the tissue expression profile indicated that mRNA expression of ToElovl4b was higher in the gonads and brain than in other tissues. Additionally, nutritional regulation suggested the highest mRNA levels of ToElovl4b in liver and brain were under feeding with 1:1 FO-SO (fish oil, FO; soybean oil, SO) and 1:1 FO-CO (corn oil, CO)), respectively. These new insights were useful for understanding the molecular basis and regulation of LC-PUFA biosynthesis in fish.


Asunto(s)
Proteínas de Peces/genética , Peces/genética , Peces/metabolismo , Distribución Tisular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Elongasas de Ácidos Grasos/genética , Ácidos Grasos Insaturados/genética , Femenino , Hígado/metabolismo , Masculino , Filogenia , ARN Mensajero/genética , Alineación de Secuencia
12.
Gene ; 767: 145186, 2021 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-32998045

RESUMEN

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from 'Macronuclear Destined Sequences' that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3' regions. Instead, its 5' region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome.


Asunto(s)
Quimera/genética , Euplotes/genética , Feromonas/genética , Secuencia de Aminoácidos/genética , Secuencia de Bases/genética , Cilióforos/genética , ADN/genética , Reordenamiento Génico/genética , Intrones/genética , ARN/genética , Empalme del ARN/genética
13.
Biosens Bioelectron ; 172: 112766, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33126177

RESUMEN

The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.


Asunto(s)
/métodos , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /genética , Secuencia de Bases , /instrumentación , Humanos , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/estadística & datos numéricos , Pandemias , ARN Viral/genética , Sensibilidad y Especificidad
14.
Viruses ; 12(12)2020 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-33371200

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.


Asunto(s)
Regiones no Traducidas 3'/genética , Conformación de Ácido Nucleico , Pandemias , ARN Viral/genética , /genética , Animales , Secuencia de Bases , Línea Celular , Secuencia Conservada , Cricetinae , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mesocricetus , Modelos Moleculares , Plásmidos , Mutación Puntual , Genética Inversa/métodos , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Ésteres del Ácido Sulfúrico , Transcripción Genética , Virión/genética , Virión/fisiología
15.
J Vis Exp ; (166)2020 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-33346188

RESUMEN

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).


Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Genes Reporteros , Luciferasas/metabolismo , Mamíferos/metabolismo , Plásmidos/genética , Animales , Secuencia de Bases , Línea Celular , ADN/metabolismo , Reparación del ADN , Vectores Genéticos/metabolismo , Luciferasas/genética , Oligonucleótidos/metabolismo , ARN Guia/genética , Reproducibilidad de los Resultados , Ovinos , Transformación Genética
16.
J Vis Exp ; (166)2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33369608

RESUMEN

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing in less than two weeks.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colágeno/genética , Pruebas Genéticas , Ribonucleoproteínas/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN/metabolismo , Gónadas/metabolismo , Homocigoto , Microinyecciones , Edición de ARN/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Mapeo Restrictivo , Ribonucleoproteínas/genética , Streptococcus pyogenes/genética
17.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(6): 584-590, 2020 Nov 25.
Artículo en Chino | MEDLINE | ID: mdl-33325192

RESUMEN

OBJECTIVE: To characterize Torso-like (tsl) gene and investigate its expression characteristics in Anopheles dirus, so as to provide a theoretical basis for subsequent functional studies of the tsl gene. METHODS: According to the coding sequences of Drosophila melanogaster and An. gambiae tsl genes, the complete genome of An. dirus was retrieved and the An. dirus tsl gene was characterized. Specific primers were designed and the target gene was amplified using PCR and reverse-transcription PCR assays. The physicochemical properties, signal peptide, transmembrane structure, secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools, and a phylogenetic analysis was performed. In addition, the specific expression of the tls gene was detected in various tissues of An. dirus using a quantitative real-time PCR assay. RESULTS: The An. dirus tsl gene was 16 751 bp in length with a CDS region of 1 134 bp, encoding 377 amino acids, and the encoded TSL protein was a stably hydrophilic protein. The TSL protein was predicted to be a secretory protein that was located in extra-membrane regions containing signal peptides. The secondary structure of the TSL protein contained α-helix (51.72%), extended strand (12.20%), ß-bridge (4.78%) and random coil (31.30%) in the secondary structure, and a 3D homology model was generated using 5cj9.1.A as a template. Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An. dirus and An. farauti. In addition, quantitative real-time PCR assay detected the tsl gene expression in the head, chest, abdomen and foot of An. dirus, with the highest expression in the head and low expression in the foot. CONCLUSIONS: The tsl gene is characterized in An. dirus at a genomic level, and the prediction of the TSL protein structure and the elucidation of the tissue-specific tsl gene expression in An. dirus provide a basis for the further studies on the gene functions.


Asunto(s)
Anopheles , Genes de Insecto , Animales , Anopheles/genética , Secuencia de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster , Filogenia , Conformación Proteica
18.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(6): 618-622, 2020 Oct 15.
Artículo en Chino | MEDLINE | ID: mdl-33325197

RESUMEN

OBJECTIVE: To obtain the transcriptome data of Tyrophagus putrescentiae, so as to provide insights into the subsequent functional studies. METHODS: The mixture of male and female T. putrescentiae was sequenced using the Illumina HiSeqTM 2000 high-throughput sequencing platform. Unigenes were obtained after assembling the sequencing data using the Trinity software and compared with the protein sequences in the RefSeq non-redundant protein sequence (NR) database, nucleotide sequence (NT) database, Swiss-Prot database, Kyoto encyclopedia of genes and genomes (KEGG) database and clusters of orthologous groups (COG) database, and the function of the Unigenes was annotated. In addition, the coding DNA sequences (CDS) were predicted through alignment of the Unigenes in NR and Swiss-Prot protein databases. The SSR loci were identified by analysis of the Unigenes in T. putrescentiae with the MISA software, and the SNPs were detected using the SOAPsnp technique. RESULTS: A total of 4.67 GB high-quality data were obtained from raw sequencing data. A total of 51 271 Unigenes were obtained after assembling the sequencing data, with a total length of 41 848 995 nucleotide (nt) and a mean length of 816 nt. A total of 29 053 annotated Unigenes were obtained following comparisons with the public protein databases, and 27 443 CDS were predicted. In addition, there were 23 092 SSR loci and 148 027 SNPs identified. CONCLUSIONS: The database of T. putrescentiae transcriptome is created by sequencing, and a large number of T. putrescentiae transcripts are obtained, which provides a basis for the subsequent functional studies of allergy-related genes.


Asunto(s)
Acaridae/genética , Biología Computacional , Transcriptoma , Animales , Secuencia de Bases , Bases de Datos de Ácidos Nucleicos , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Repeticiones de Microsatélite , Anotación de Secuencia Molecular , Polimorfismo de Nucleótido Simple
19.
PLoS Biol ; 18(12): e3000978, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33320883

RESUMEN

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.


Asunto(s)
/métodos , Sistemas CRISPR-Cas/genética , /aislamiento & purificación , Secuencia de Bases , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
20.
Zootaxa ; 4892(1): zootaxa.4892.1.1, 2020 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-33311101

RESUMEN

Morocco is a well known hot-spot of biodiversity in the Mediterranean basin. While some taxa like vascular plants are relatively well recorded, important groups of pollinators like bees are still understudied. This article presents an updated checklist of the bee species of Morocco and includes a summary of global and regional distribution of each species. A total of 961 species belonging to six bee families and 68 genera are recorded: Andrenidae (8 genera, 217 species); Apidae (15 genera, 241 species); Colletidae (2 genera, 74 species), Halictidae (12 genera, 144 species), Megachilidae (28 genera, 271 species) and Melittidae (3 genera, 14 species). Among them, 67 species are recorded for the first time in Morocco. Around 70% of the bee fauna of Morocco consists of widespread Palaearctic species. Only 18% of Moroccan species recorded are restricted to North Africa and 8% are Moroccan single-country endemics (81 species). Afrotropical elements in the Moroccan fauna are few, with only 3% of Morocco species co-occuring in that region. This checklist is intended to stimulate new regional research on bees including their taxonomy and biogeography. As many groups of bees have been understudied, discovery of new species for science and new records for the country can be expected. Additional research including inventorying, monitoring, and integrative taxonomic studies are needed to develop a comprehensive strategy for bee conservation in Morocco.


Asunto(s)
Abejas , Himenópteros , Animales , Secuencia de Bases , Himenópteros/genética , Marruecos
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