Our goal is to create a system that allows the identification and study of repressor sensitive (rs) mutations in the fruit fly Drosophila melanogaster. Repressor sensitive mutations are a new class of conditional lethal mutation. To make rs mutations, the transposon P[UAS] (a P element that contains multiple UAS binding sites of the Gal4 protein) will be inserted at various locations throughout the Drosophila genome. A Gal4-repressor fusion protein will be used to inhibit the transcription of genes adjacent to the P[UAS] insert. A schematic diagram of a rs mutation is shown below. Panel A shows wild type alleles of a gene being transcribed. Panel B shows a rs allele of the gene. Adjacent to the gene is a P element insert carrying UAS binding sites. The P element insertion by itself does not cause a mutant phenotype. Panel C shows a fly homozygous for a rs mutation and expressing a Gal4-repressor fusion protein. The protein will bind to the UAS sites and repress the expression of the adjacent gene, resulting in a mutant phenotype. By controlling the expression of the Gal4-repressor protein you can regulate when and where the mutant phenotype is expressed. The reason this system is needed is because many genes act at multiple time points throughout development. If a gene has a lethal phenotype, then it is much easier to study the gene the first time it is required, rather than at a later developmental time point. For labs studying post-embryonic development, this is a severe hindrance, forcing the use of cumbersome approaches like mosaic analysis. An easier approach is required. Repressor sensitive mutations will allow the direct analysis of lethal mutations at any time during development. We will initially make a rs mutation in the gene hedgehog. We will then test a number of repressor proteins to determine which one has the strongest repressor activity. This repressor will then be used in a screen for rs mutations in Drosophila.
