RESUMEN
The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements or enhancers. Here, we present a rapid and highly efficient system for the large-scale analysis of enhancer elements, site-specifically integrated into the Drosophila genome. This system, which is scalable for either small projects or high-throughput approaches, makes use of the Gateway cloning technology and the PhiC31 site-specific integration system, which allows the insertion of constructs at predetermined genomic locations. Thus, this system allows not only a fast and easy analysis of reporter gene expression in live animals, but also the simultaneous analysis of different regulatory outputs on a cellular resolution by recombining in the same animal distinct enhancer elements fused to different fluorescent proteins.
Asunto(s)
Clonación Molecular/métodos , Drosophila melanogaster/genética , Genes Reporteros , Vectores Genéticos , Ensayos Analíticos de Alto Rendimiento/métodos , Integrasas/genética , Animales , Animales Modificados Genéticamente , Sitios de Unión , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación GenéticaRESUMEN
The hydrophobic patch of cyclins interacts with cyclin-dependent kinase (Cdk) substrates and p27-type Cdk inhibitors. Although this interaction is assumed to contribute to the specificity of different Cdk-Cyclin complexes, its role in specific steps of the cell cycle has not been demonstrated. Here, we show that in Drosophila the mitotic inhibitor Frühstart (Frs) binds specifically and with high affinity to the hydrophobic patch of cyclins. In contrast to p27-type Cdk inhibitors, Frs does not form a stable interaction with the catalytic centre of Cdk and allows phosphorylation of generic model substrates, such as histone H1. Consistent with a 2.5 times stronger binding to CycA than to CycE in vitro, ectopic expression of frs induces endocycles, in a manner similar to that reported previously for downregulation of CycA or Cdk1. We propose that binding of Frs to cyclins blocks the hydrophobic patch to interfere with Cdk1 substrate recognition.