RESUMO
During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating-responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contributes to determine the kinetics of transcription in a simplified cell-fate decision system.
Assuntos
Genes Fúngicos Tipo Acasalamento/genética , Feromônios/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação Fúngica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Regiões Promotoras Genéticas , Análise de Célula ÚnicaRESUMO
The tractability of the budding yeast genome has provided many insights into the fundamental mechanisms regulating cellular life. With the advent of synthetic biology and single-cell measurements, novel tools are required to manipulate the yeast genome in a more controlled manner. We present, here, a new family of yeast shuttle vectors called single integration vectors (pSIV). Upon transformation in yeast, these plasmids replace the entire deficient auxotrophy marker locus by a cassette containing an exogenous marker. As shown using flow cytometry, this complete replacement results in a unique integration of the desired DNA fragment at the marker locus. In addition, a second transcriptional unit can be inserted to achieve the simultaneous integration of two constructs. The selection marker cassettes, present in the pSIV, were also used to generate a complete set of gene tagging plasmids (pGT) encompassing a large palette of fluorescent proteins, from a cyan fluorescent protein to a near-infrared tandem dimer red fluorescent protein. These tagging cassettes are orthogonal to each other thanks to the use of different TEF promoter and terminator couples, thereby avoiding marker cassette switching and favoring integration in the desired locus. In summary, we have created two sets of robust molecular tools for the precise genetic manipulation of the budding yeast.
