RESUMEN
Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it may also be linked to viral pathogenesis. Intending to shed light on the viral determinants for severe dengue pathogenesis, we previously analyzed the DENV-2 intrahost genetic diversity in 68 patients clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18), performing viral whole-genome deep sequencing from clinical samples with an amplicon-free approach. From it, we identified a set of 141 relevant mutations distributed throughout the viral genome that deserved further attention. Therefore, we employed molecular modeling to recreate three-dimensional models of the viral proteins and secondary RNA structures to map the mutations and assess their potential effects. Results showed that, in general lines, disruptive variants were identified primarily among dengue fever cases. In contrast, potential immune-escape variants were associated mainly with warning signs and severe cases, in line with the latter's longer intrahost evolution times. Furthermore, several mutations were located on protein-surface regions, with no associated function. They could represent sites of further investigation, as the interaction of viral and host proteins is critical for both host immunomodulation and virus hijacking of the cellular machinery. The present analysis provides new information about the implications of the intrahost genetic diversity of DENV-2, contributing to the knowledge about the viral factors possibly involved in its pathogenesis within the human host. Strengthening our results with functional studies could allow many of these variants to be considered in the design of therapeutic or prophylactic compounds and the improvement of diagnostic assays. IMPORTANCE Previous evidence showed that intrahost genetic diversity in arboviruses may be linked to viral pathogenesis and that one or a few amino acid replacements within a single protein are enough to modify a biological feature of an RNA virus. To assess dengue virus serotype 2 determinants potentially involved in pathogenesis, we previously analyzed the intrahost genetic diversity of the virus in patients with different clinical outcomes and identified a set of 141 mutations that deserved further study. Thus, through a molecular modeling approach, we showed that disruptive variants were identified primarily among cases with mild dengue fever, while potential immune-escape variants were mainly associated with cases of greater severity. We believe that some of the variants pointed out in this study were attractive enough to be potentially considered in future intelligent designs of therapeutic or prophylactic compounds or the improvement of diagnostic tools. The present analysis provides new information about DENV-2 viral factors possibly involved in its pathogenesis within the human host.
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Adaptación Fisiológica/genética , Virus del Dengue/genética , Dengue/patología , Variación Genética/genética , Índice de Severidad de la Enfermedad , Virus del Dengue/inmunología , Virus del Dengue/aislamiento & purificación , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , ARN Viral/genética , Serogrupo , Regiones no Traducidas/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Secuenciación Completa del GenomaRESUMEN
We analyzed the association of polymorphisms from the 3' untranslated region of the HLA-G gene in 70 neuromyelitis optica spectrum disorder (NMOSD) patients and 162 healthy controls. No associations were found between the polymorphisms in NMOSD when compared to healthy controls, serology of the anti-AQP4 NMOSD biomarker and Expanded Disability Status Scale (EDSS). In conclusion, the 3' untranslated region 14 bp Ins/Del and +3142C/G polymorphisms seem not to be associated with NMOSD susceptibility, autoantibody production, nor a neurological deficit in patients.
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Acuaporina 4/genética , Autoanticuerpos/genética , Personas con Discapacidad , Antígenos HLA-G/genética , Neuromielitis Óptica/genética , Polimorfismo de Nucleótido Simple/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Acuaporina 4/sangre , Autoanticuerpos/sangre , Brasil/epidemiología , Femenino , Antígenos HLA-G/sangre , Humanos , Masculino , Persona de Mediana Edad , Neuromielitis Óptica/sangre , Neuromielitis Óptica/epidemiología , Regiones no Traducidas/genética , Adulto JovenRESUMEN
The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.
El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.
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Productos Biológicos , Proteínas Recombinantes/biosíntesis , Regiones no Traducidas , Proteínas Fluorescentes Verdes/metabolismo , Virus de la Encefalomiocarditis/metabolismo , Biotecnología , Genoma Viral , Técnicas de Cultivo de Célula , Estabilidad del ARN , Virus de la Encefalomiocarditis/genéticaRESUMEN
BACKGROUND: Oculo-auriculo-vertebral spectrum (OAVS) is a craniofacial developmental disorder that affects structures derived from the first and second pharyngeal arches. The clinically heterogeneous phenotype involves mandibular, oral, and ear development anomalies. Etiology is complex and poorly understood. Genetic factors have been associated, evidenced by chromosomal abnormalities affecting different genomic regions and genes. However, known pathogenic single-nucleotide variants (SNVs) have only been identified in MYT1 in a restricted number of patients. Therefore, investigations of SNVs on candidate genes may reveal other pathogenic mechanisms. METHODS: In a cohort of 73 patients, coding and untranslated regions (UTR) of 10 candidate genes (CRKL, YPEL1, MAPK1, NKX3-2, HMX1, MYT1, OTX2, GSC, PUF60, HOXA2) were sequenced. Rare SNVs were selected and in silico predictions were performed to ascertain pathogenicity. Likely pathogenic variants were validated by Sanger sequencing and heritability was assessed when possible. RESULTS: Four likely pathogenic variants in heterozygous state were identified in different patients. Two SNVs were located in the 5'UTR of YPEL1; one in the 3'UTR of CRKL and one in the 3'UTR of OTX2. CONCLUSION: Our work described variants in candidate genes for OAVS and supported the genetic heterogeneity of the spectrum.
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Discapacidades del Desarrollo/patología , Polimorfismo de Nucleótido Simple , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Niño , Preescolar , Discapacidades del Desarrollo/genética , Femenino , Humanos , Lactante , Masculino , Proteínas Nucleares/genética , Factores de Transcripción Otx/genética , Regiones no TraducidasRESUMEN
Human astrovirus (HAstV) constitutes a major cause of acute gastroenteritis in children. The viral 5' and 3' untranslated regions (UTR) have been involved in the regulation of several molecular mechanisms. However, in astrovirues have been less characterized. Here, we analyzed the secondary structures of the 5' and 3' UTR of HAstV, as well as their putative target sites that might be recognized by cellular factors. To our knowledge, this is the first bioinformatic analysis that predicts the HAstV 5' UTR secondary structure. The analysis showed that both the UTR sequence and secondary structure are highly conserved in all HAstVs analyzed, suggesting their regulatory role of viral activities. Notably, the UTRs of HAstVs contain putative binding sites for the serine/arginine-rich factors SRSF2, SRSF5, SRSF6, SRSF3, and the multifunctional hnRNPE2 protein. More importantly, putative binding sites for PTB were localized in single-stranded RNA sequences, while hnRNPE2 sites were localized in double-stranded sequence of the HAstV 5' and 3' UTR structures. These analyses suggest that the combination of SRSF proteins, hnRNPE2 and PTB described here could be involved in the maintenance of the secondary structure of the HAstVs, possibly allowing the recruitment of the replication complex that selects and recruits viral RNA replication templates.
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Simulación por Computador , Mamastrovirus/genética , Proteínas/metabolismo , Regiones no Traducidas/genética , Secuencia de Bases , Sitios de Unión , Conformación de Ácido NucleicoRESUMEN
Abstract In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of the mRNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYTl and kLYTl, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5' and 3' UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5' UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T. cruzi DTU. In addition, for the first time, was unscovered the existence of two types of LYT1 mRNAs transcripts, differing in length by 116 nts, that are generated by alternative polyadenylation. Furthermore, an in-silico analysis of the experimentally obtained UTRs, and ten additional LYT1 sequences retrieved from TritrypDB and GenBank databases, together with a thoroughly search of structural motifs, showed a remarkable conservation of relevant structural motifs previously associated with RNA metabolism in the different UTRs; these elements might be involved in the differential stage-specific expression of each LYT1 isoform.
Resumen En los trypanosomátidos, la expresión génica se regula principalmente en el nivel post-transcripcional mediante mecanismos basados en la interacción entre las proteínas de unión del ARN [RBP] y las figuras presentes en las regiones no traducidas [UTR] de las ARN, que en conjunto forman complejos ribonucleoproteicos [RNP] que definen el destino de la ARN. El pre-ARN derivado del gen LYT1 del Trypanosoma cruzi es procesado por trans-empalme alternativo, dando como resultado diferentes ARN que codifican las isoformas mLYTl y kLYTl, proteínas con expresión diferencial, localización celular y función. El objetivo de este estudio fue caracterizar los 5' y 3' UTR de las ARN LYT1 como el paso inicial hacia la identificación de los RPB responsables de la expresión diferencial. Se confirmó la presencia de los dos tipos de 5' UTR en dos aislantes del T. cruzi pertenecientes al DTU I; de esta forma también se comprobó la ocurrencia del trans-empalme alternativo en el gen LYT1 de este T. cruzi DTU. Además, por primera vez, se pudo demostrar la existencia de dos tipos de transcripciones de ARN LYT1, que difieren en longitud por 116 nts, y son generadas por poliadenilación alternativa. Adicionalmente, se realizó un análisis in-silico de la UTR obtenida experimentalmente, y otras diez secuencias LYT1 recuperadas de las bases de datos TritrypDB y GenBank, junto con una búsqueda exhaustiva de figuras estructuradas, mostrando una notable conservación de los figuras estructurales asociadas con el metabolismo del ARN en los diferentes UTR; estos elementos podrían estar implicados en la expresión diferenciada de la etapa específica de cada isoforma LYT1.
Resumo Nos tripanossomatídeos, a expressão génica é regulada principalmente a nível pós-transcricional mediante mecanismos baseados na interação entre as proteínas de união do RNA [RBPs] e as fugiras presentes nas regiões não-traduzidas [UTRs] do RNA. O pré-RNA derivado do gene LYT1 do Trypanosoma cruzí é processado por uma junção trans-alternativa, resultando em diferentes RNA que codificam as isoformas mLYTl e kLYTl, proteínas com expressão, localização celular e função diferenciadas. O objetivo de este estudo foi caracterizar as 5' e 3' UTRs dos RNAs LYT1 como sendo o passo inicial na identificação das RBPs responsáveis pela expressão diferenciada. A presença dos dois tipos de 5' UTRs foi confirmada em dois isolados de T. cruzí pertencentes ao DTU I; corroborando assim com a ocorrência da junção trans-alternativa no gene LYT1 de este T. crují DTU. Adicionalmente, se demonstrou pela primeira vez a existência de dois tipos de transcrições de RNA LYT1, que se diferenciam em comprimento por 116 nts, e são geradas por poliadenização alternativa. Além disso, realizou-se uma análise in-sílico da UTR obtida experimentalmente e outras dez sequencias LYT1 recuperadas das bases de dados TritrypDB e GenBank, junto com uma busca exaustiva de figuras estruturadas, mostrando uma notável conservação das figuras estruturais associadas com o metabolismo do RNA nas diferentes UTRs. Estes elementos poderiam estar envolvidos na expressão estágio-específica diferenciada de cada isoforma LYT1.
Asunto(s)
Humanos , Trypanosoma cruzi , Regulación de la Expresión Génica , Proteínas de Unión al ARN , Regiones no TraducidasRESUMEN
PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Genes BRCA1 , Genes BRCA2 , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Adulto , Anciano , Estudios de Cohortes , Biología Computacional , Femenino , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Intrones/genética , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Regiones no Traducidas/genéticaRESUMEN
BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.
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Humanos , Femenino , Neoplasias de la Mama/genética , Adenosina Desaminasa/genética , Proteínas de Unión al ARN/genética , Edición de ARN/genética , Regiones no Traducidas/genética , Estabilidad del ARN/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Adenosina Desaminasa/metabolismo , Proteínas de Unión al ARN/metabolismo , Perfilación de la Expresión Génica , Estabilidad del ARN/fisiología , Línea Celular TumoralRESUMEN
ABSTRACT Objective: miR-483-5p has been identified as a miRNA oncogene in certain cancers. However, its role in prostate cancer has not been sufficiently investigated. In this study, we investigated the role of miR-483-5p in prostate cancer and examined RBM5 regulation by miR-483-5p. Material and methods: Expression levels of miR-483-5p were determined by quantitative real-time PCR. The effect of miR-483-5p on proliferation was evaluated by MTT assay, cell invasion was evaluated by trans-well invasion assays, and target protein expression was determined by western blotting in LNCaP, DU-145, and PC-3 cells. Luciferase reporter plasmids were constructed to confirm the action of miR-483-5p on downstream target gene RBM5 in HEK-293T cells. Results: we observed that miR-483-5p was upregulated in prostate cancer cell lines and tissues. A miR-483-5p inhibitor inhibited prostate cancer cell growth and invasion in DU-145 and PC-3 cells. miR-483-5p directly bound to the 3' untranslated region (3'UTR) of RBM5 in HEK-293T cells. RBM5 overexpression inhibited prostate cancer cell growth and invasion in LNCaP cells. Enforced RBM5 expression alleviated miR-483-5p promotion of prostate cancer cell growth and invasion in LNCaP cells. Conclusion: The present study describes a potential mechanism underlying a miR-483-5p/RBM5 link that contributes to prostate cancer development.
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Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Ciclo Celular/metabolismo , Regiones no Traducidas/genética , Proteínas Supresoras de Tumor/metabolismo , MicroARNs/fisiología , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias de la Próstata/mortalidad , Regulación hacia Abajo , Regulación hacia Arriba , Proteínas de Unión al ARN/metabolismo , MicroARNs/antagonistas & inhibidores , Línea Celular Tumoral , Invasividad NeoplásicaRESUMEN
Non-coding RNAs (ncRNAs) are regulatory elements present in a wide range of organisms, including trypanosomatids. ncRNAs transcribed from the untranslated regions (UTRs) of coding genes have been described in the transcriptomes of several eukaryotes, including Trypanosoma brucei. To uncover novel putative ncRNAs in two Leishmania species, we examined a L. major cDNA library and a L. donovani non-polysomal RNA library. Using a combination of computational analysis and experimental approaches, we classified 26 putative ncRNA in L. major, of these, 5 arising from intergenic regions and 21 from untranslated regions. In L. donovani, we classified 37 putative ncRNAs, of these, 7 arising from intergenic regions, and 30 from UTRs. Our results suggest, for the first time, that UTR-transcripts may be a common feature in the eukaryote Leishmania similarly to those previously shown in T. brucei and other eukaryotes.
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Leishmania donovani/genética , Leishmania major/genética , ARN no Traducido/análisis , ARN no Traducido/genética , Regiones no Traducidas , Biología Computacional , Perfilación de la Expresión Génica , Biblioteca de GenesRESUMEN
BACKGROUND Dengue is considered one of the world’s most important mosquito-borne diseases. MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that play an important role in the regulation of gene expression in eukaryotes. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in Dengue virus (DENV) replication remains poorly understood. OBJECTIVE To determine the role of miR-484 and miR-744 in DENV infection and to examine whether DENV infection alters the expression of both miRNAs. METHODS We used bioinformatics tools to explore the relationship between DENV and cellular miRNAs. We then overexpressed miR-484 or miR-744 in Vero cells to examine their role in DENV replication using flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and western blotting. FINDINGS We found several cellular miRNAs that target a conserved region within the 3′ untranslated region (3′ UTR) of the genome of the four DENV serotypes and found that overexpression of miR-484 or miR-744 inhibits infection by DENV-1 to DENV-4. Furthermore, we observed that DENV RNA might be involved in the downregulation of endogenous miR-484 and miR-744. CONCLUSION Our study identifies miR-484 and miR-744 as two possible restriction host factors against DENV infection. However, further studies are needed to directly verify whether miR-484 and miR-744 both have an anti-DENV effect in vivo.
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Animales , Replicación Viral/fisiología , Replicación Viral/genética , Chlorocebus aethiops , Regulación de la Expresión Génica/genética , Western Blotting , Reacción en Cadena de la Polimerasa , Biología Computacional , Regiones no Traducidas , Regiones no Traducidas/fisiología , Virus del Dengue/fisiología , Virus del Dengue/genética , MicroARNs/metabolismo , Citometría de FlujoRESUMEN
DRA encodes the alpha chain of the DR heterodimer, is closely linked to DRB and is considered almost monomorphic in major histocompatibility complex region. In this study, we identified the exon 2 of DRA to evaluate the immunogenetic diversity of Chinese south indigenous goat. Two single nucleotide polymorphisms in an untranslated region and one synonymous substitution in coding region were identified. These data suggest that high immunodiversity in native Chinese population.
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Cabras/genética , Cadenas alfa de HLA-DR/genética , Polimorfismo Genético , Animales , Animales Domésticos , China , Exones , Cabras/clasificación , Cabras/inmunología , Cadenas alfa de HLA-DR/inmunología , Intrones , Sistemas de Lectura Abierta , Regiones no TraducidasRESUMEN
New sequencing studies of the nonsegmented dsRNA genome of penaeid shrimp infectious myonecrosis virus (IMNV), a tentatively assigned member of the family Totiviridae, identified previously unread sequences at both genome termini in three previously analyzed IMNV strains, one from Brazil (the prototype strain of IMNV) and two from Indonesia. The new sequence determinations add >600 nt to the 5' end of the genomic plus strand of each strain, increasing the length of the 5' nontranslated region to at least 469-472 nt and the length of the upstream open reading frame (ORF1) translation product by at least 48 aa. These new findings are similar to recent ones for two other IMNV strains (GenBank KF836757.1 and KJ556923.1) and thereby corroborate important amendments to the full-length IMNV genome sequence.
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Penaeidae/virología , Totiviridae/genética , Animales , Secuencia de Bases , Brasil/epidemiología , Genoma Viral/genética , Indonesia/epidemiología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Regiones no Traducidas/genéticaRESUMEN
Carassius auratus var. pingxiangnensis is a natural triploid crucian carp mutant. In order to understand its placement and genetic background at the gene level, the characteristics of mitochondrial DNA sequences and phylogenetic relationship were examined. The results showed that the mitochondrial DNA is a circular double-stranded DNA molecule that is 16,576 bp in length with 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a non-coding control region. Mitochondrial genes overlapped by a total of 40 bp in 11 different locations from 1 to 14 bp. The base composition of the C. auratus mitogenome was estimated to be 29.70% A, 26.74% C, 15.35% G, and 28.21% T. The central conserved blocks and the conserved blocks were compared and were similar among C. auratus var. pingxiangnensis and six other cyprinids with different ploidies. The origin of light strand replication was similar to that of other vertebrates; it was 33 bp, but the characteristic sequence motif 5ê-GCCGG-3ê at the base of the stem within tRNA(Cys) was mutated to 5ê-GGCGG- 3ê. Our phylogenetic analysis based on whole mitogenome sequences indicated that C. auratus var. pingxiangnensis was clustered with C. auratus and then sister-grouped with Carassius gibelio. The systemic developmental tree of crucian carp with different chromosome ploidies showed that diploid C. auratus auratus was clustered with triploid C. auratus auratus, sister-grouped with tetraploid C. auratus auratus, and clustered with other diploid, triploid, and tetraploid C. auratus.
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Carpas/clasificación , Carpas/genética , Genoma Mitocondrial , Carpa Dorada/genética , Filogenia , Triploidía , Animales , Composición de Base , Secuencia de Bases , Codón , Orden Génico , Genes Mitocondriales , Genes de ARNr , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Ploidias , Poliploidía , ARN de Transferencia , Regiones no TraducidasRESUMEN
Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure
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Humanos , ARN Viral/sangre , Hepatitis C/diagnóstico , Hepacivirus/aislamiento & purificación , Regiones no Traducidas/genética , ARN Viral/genética , Hepatitis C/virología , Hepacivirus/genética , Carga Viral/métodosRESUMEN
In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.
Asunto(s)
Repeticiones de Dinucleótido/fisiología , Regulación de la Expresión Génica/fisiología , Poli A/genética , Poli C/genética , ARN Protozoario/química , Trypanosoma cruzi/metabolismo , Análisis por Conglomerados , Biología Computacional , Repeticiones de Dinucleótido/genética , Regulación de la Expresión Génica/genética , Genes Reporteros , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN/fisiología , Trypanosoma cruzi/genética , Regiones no Traducidas/genética , Regiones no Traducidas/fisiologíaRESUMEN
The aims of this study were to evaluate torque teno sus virus (TTSuV) infection in healthy slaughter-age pigs and to compare the similarities of the untranslated region (UTR) nucleotide (nt) sequences obtained from different biological samples of the same animals. Fifty-eight pigs were evaluated by PCR assay for the presence of TTSuV in paired samples of liver and serum (Group 1, n = 27) and lung and serum (Group 2, n = 31). All the pigs were positive for TTSuV infection in the organs sampled and 94.8 % (n = 55) presented with viraemia. The nt sequence similarities between the Groups 1 and 2 varied from 91.7 % to 96.6 % (TTSuV1) and 91 % to 95 % (TTSuV2). In Group 1, the nt sequence similarities were 93 % (TTSuV1) and 95.4 % (TTSuV2). In Group 2, the nt sequence similarities were 95 % (TTSuV1) and 91 % (TTSuV2). These results revealed the simultaneous infection with distinct strains of TTSuV1 and 2 in healthy pigs at slaughter age.
Asunto(s)
Hígado/virología , Pulmón/virología , Porcinos/virología , Torque teno virus/aislamiento & purificación , Animales , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Análisis de Secuencia de ADN , Porcinos/sangre , Torque teno virus/genética , Regiones no TraducidasRESUMEN
One of the common forms of multidrug resistance (MDR) is caused by activation of the mdr1 (ABCB1) gene, resulting in overexpression of P-glycoprotein (P-gp) and conferring cancer cell resistance to a broad range of chemotherapeutics. Recently, P-gp-mediated MDR has been associated with aberrant expression of microRNAs (miRNAs) in several types of cancer. miRNAs are small noncoding RNAs that regulate gene expression in a posttranscriptional manner through partial or total hybridization with specific sequences in the 3'-UTR of target mRNAs. Interestingly, there are at least two reports that suggest an additional regulation by miRNAs at the mdr1 promoter level. Here, we critically analyzed some of the miRNAs that regulate P-gp expression at two different levels: posttranscriptional and transcriptional. We proposed that the latter may occur through two possible scenarios: (1) direct miRNA hybridization with an active promoter and (2) triplex structure formation (double-stranded DNA/RNA) stabilized by Argonaute 2. Also, we classified transcriptional gene silencing (1) by homology, represented by small interfering RNAs directed to viral promoters, and (2) by complementarity (Watson-Crick/Hoogsteen base pairing), mediated by miRNAs. Transcriptional regulation could represent a new avenue of knowledge applicable to the modulation of other genes mediated by these noncoding RNAs.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos , Regulación de la Expresión Génica , MicroARNs/genética , Regiones Promotoras Genéticas , Regiones no Traducidas , Animales , HumanosRESUMEN
Physalis peruviana, commonly known as Cape gooseberry, is an Andean Solanaceae fruit with high nutritional value and interesting medicinal properties. In the present study we report the development and characterization of microsatellite loci from a P. peruviana commercial Colombian genotype. We identified 932 imperfect and 201 perfect Simple Sequence Repeats (SSR) loci in untranslated regions (UTRs) and 304 imperfect and 83 perfect SSR loci in coding regions from the assembled Physalis peruviana leaf transcriptome. The UTR SSR loci were used for the development of 162 primers for amplification. The efficiency of these primers was tested via PCR in a panel of seven P. peruviana accessions including Colombia, Kenya and Ecuador ecotypes and one closely related species Physalis floridana. We obtained an amplification rate of 83% and a polymorphic rate of 22%. Here we report the first P. peruviana specific microsatellite set, a valuable tool for a wide variety of applications, including functional diversity, conservation and improvement of the species.
Asunto(s)
Marcadores Genéticos , Repeticiones de Microsatélite/genética , Ribes/genética , Alelos , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Transcriptoma , Regiones no TraducidasRESUMEN
Dengue virus (DENV) genome amplification is a process that involves the viral RNA, cellular and viral proteins, and a complex architecture of cellular membranes. The viral RNA is not a passive template during this process; it plays an active role providing RNA signals that act as promoters, enhancers and/or silencers of the replication process. RNA elements that modulate RNA replication were found at the 5' and 3' UTRs and within the viral coding sequence. The promoter for DENV RNA synthesis is a large stem loop structure located at the 5' end of the genome. This structure specifically interacts with the viral polymerase NS5 and promotes RNA synthesis at the 3' end of a circularized genome. The circular conformation of the viral genome is mediated by long range RNA-RNA interactions that span thousands of nucleotides. Recent studies have provided new information about the requirement of alternative, mutually exclusive, structures in the viral RNA, highlighting the idea that the viral genome is flexible and exists in different conformations. In this article, we describe elements in the promoter SLA and other RNA signals involved in NS5 polymerase binding and activity, and provide new ideas of how dynamic secondary and tertiary structures of the viral RNA participate in the viral life cycle.