RESUMEN
Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.
Asunto(s)
ADN/genética , Análisis por Micromatrices/métodos , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Animales , ADN/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Marcadores Genéticos , Humanos , Meiosis , Análisis por Micromatrices/instrumentación , Plásmidos/química , Plásmidos/metabolismo , Ploidias , Populus/citología , Unión Proteica , Protoplastos/citología , Protoplastos/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Yeast one-hybrid (Y1H) is a common technique for identifying DNA-protein interactions, and robotic platforms have been developed for high-throughput analyses to unravel the gene regulatory networks in many organisms. Use of these high-throughput techniques has led to the generation of increasingly large datasets, and several software packages have been developed to analyze such data. We previously established the currently most efficient Y1H system, meiosis-directed Y1H; however, the available software tools were not designed for processing the additional parameters suggested by meiosis-directed Y1H to avoid false positives and required programming skills for operation. RESULTS: We developed a new tool named GateMultiplex with high computing performance using C++. GateMultiplex incorporated a graphical user interface (GUI), which allows the operation without any programming skills. Flexible parameter options were designed for multiple experimental purposes to enable the application of GateMultiplex even beyond Y1H platforms. We further demonstrated the data analysis from other three fields using GateMultiplex, the identification of lead compounds in preclinical cancer drug discovery, the crop line selection in precision agriculture, and the ocean pollution detection from deep-sea fishery. CONCLUSIONS: The user-friendly GUI, fast C++ computing speed, flexible parameter setting, and applicability of GateMultiplex facilitate the feasibility of large-scale data analysis in life science fields.
Asunto(s)
Saccharomyces cerevisiae , Análisis de Datos , Redes Reguladoras de Genes , Robótica , Saccharomyces cerevisiae/genética , Programas InformáticosRESUMEN
Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.