REPRESSOR SENSITIVE MUTATIONS IN DROSOPHILA

果蝇的阻遏物敏感突变

• 批准号: 6087208
• 负责人: FRANK LASKI
• 金额: $ 11.48万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6087208
• 关键词: Drosophilidae; alleles; gene expression; gene induction /repression; gene mutation; genetic models; genetic transcription; lethal genes; model design /development; phenotype; technology /technique development; transcription factor; transposon /insertion element

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.

我们的目标是创建一个系统，可以识别和研究果蝇果蝇中阻遏物敏感 (rs) 突变。阻遏物敏感突变是一类新的条件致死突变。为了进行 rs 突变，转座子 P[UAS]（包含 Gal4 蛋白的多个 UAS 结合位点的 P 元件）将被插入到整个果蝇基因组的不同位置。 Gal4 阻遏物融合蛋白将用于抑制 P[UAS] 插入片段附近基因的转录。 rs突变的示意图如下所示。图 A 显示正在转录的基因的野生型等位基因。图 B 显示该基因的 rs 等位基因。与该基因相邻的是带有 UAS 结合位点的 P 元件插入片段。 P元件插入本身不会引起突变表型。图 C 显示了 rs 突变纯合果蝇并表达 Gal4 阻遏物融合蛋白。该蛋白质将与 UAS 位点结合并抑制邻近基因的表达，从而产生突变表型。通过控制 Gal4 阻遏蛋白的表达，您可以调节突变表型表达的时间和地点。需要这个系统的原因是因为许多基因在整个发育过程中的多个时间点发挥作用。如果一个基因具有致死表型，那么在第一次需要该基因时研究该基因会比在以后的发育时间点研究要容易得多。对于研究胚胎后发育的实验室来说，这是一个严重的障碍，迫使他们使用镶嵌分析等繁琐的方法。需要一种更简单的方法。阻遏物敏感突变将允许在发育过程中的任何时间直接分析致死突变。我们首先将对刺猬基因进行 rs 突变。然后，我们将测试一些阻遏蛋白，以确定哪一种具有最强的阻遏活性。然后，该阻遏蛋白将用于筛选果蝇中的 rs 突变。