RESUMEN
As the COVID-19 pandemic winds down, it leaves behind the serious concern that future, even more disruptive pandemics may eventually surface. One of the crucial steps in handling the SARS-CoV-2 pandemic was being able to detect the presence of the virus in an accurate and timely manner, to then develop policies counteracting the spread. Nevertheless, as the pandemic evolved, new variants with potentially dangerous mutations appeared. Faced by these developments, it becomes clear that there is a need for fast and reliable techniques to create highly specific molecular tests, able to uniquely identify VOCs. Using an automated pipeline built around evolutionary algorithms, we designed primer sets for SARS-CoV-2 (main lineage) and for VOC, B.1.1.7 (Alpha) and B.1.1.529 (Omicron). Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for the main lineage and each variant in a matter of hours. Preliminary in-silico validation showed that the sequences in the primer sets featured high accuracy. A pilot test in a laboratory setting confirmed the results: the developed primers were favorably compared against existing commercial versions for the main lineage, and the specific versions for the VOCs B.1.1.7 and B.1.1.529 were clinically tested successfully.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Pandemias , Inteligencia ArtificialRESUMEN
Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-off between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the gene's ability to read positional information along the embryo's anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica/genética , Modelos Genéticos , Factores de Transcripción , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Larva , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression.
Asunto(s)
Drosophila melanogaster/embriología , Proteínas de Homeodominio/fisiología , Morfogénesis/fisiología , Transactivadores/fisiología , Animales , Sitios de Unión/genética , Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Imagen Óptica/métodos , Regiones Promotoras Genéticas/genética , Transactivadores/genética , Factores de Transcripción/genética , Cigoto/metabolismoRESUMEN
Live imaging has been used in recent years for the understanding of dynamic processes in biology, such as embryo development. This was made possible by a combination of advancements in microscopy, leading to improved signal-to-noise ratios and better spatial and temporal resolutions, and by the development of new fluorescence markers, allowing for the quantification of protein expression and transcriptional dynamics in vivo. Here we describe a general protocol, which can be used in standard confocal microscopes to image early Drosophila melanogaster embryos, in order to learn about the transcriptional dynamics of a fluorescently labeled RNA.
Asunto(s)
Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , ARN Mensajero/genética , Transcripción Genética , Animales , Drosophila melanogaster/embriología , Drosophila melanogaster/ultraestructura , Femenino , Masculino , ARN Mensajero/biosíntesisRESUMEN
We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nuclei-labeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semiautomatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Programas Informáticos , Transcripción Genética , Animales , Núcleo Celular/genética , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Microscopía Confocal/métodosRESUMEN
Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response.
