Identification and characterization of genes encoding proteins with polar expression in different Drosophila cells

不同果蝇细胞中极性表达蛋白编码基因的鉴定和表征

• 批准号: 5410028
• 负责人: Professor Dr. Christian Klämbt
• 金额: --
• 依托单位: Europäisches Laboratorium für Molekularbiologie (EMBL)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2003
• 资助国家: 德国
• 起止时间: 2002-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5410028?language=en
• 关键词: Identification; characterization; genes; encoding; proteins

In all organisms cell polarity crucially depends on the interplay of often-polar localized determinants and a polarized organization of the cytoskeleton. In the past, genetic approaches have identified several key players organizing polar cells such as Inscuteable, Crumbs, Bazooka and Stardust. Subsequently, the use of antibodies generated against the encoded proteins revealed their polar localization in fixed material. These tools turned out to be extremely valuable in analyzing cell behaviour in the context of a developing organism. Howver, to fully understand the generation and maintenance of polarized cells it will be necessary to use GFP-tagged proteins which will allow in vivo analyses of the dynamic regulation of cell polarity. In addition, we have to seek additional genes that encode proteins with a polar distribution. One way to identify such proteins is to use a random GFPtagging approach (Morin et al., 2001). Here, A GFP containing exon, which is inserted in a transposon based vector, is introduced by chance into an intron of an endogenous fly gene. Following splicing a fusion protein is generated (depending on the fusion frame) that is generally transported to its normal subcellular localization. Once a specific subcellular expression pattern is defined, mutations in the gene can be easily generated by imprecise excision of the transposon. This powerful combination of genetics and cell biology will provide new insights into the mechanisms that regulate cell polarity in a broad range of tissues.

在所有生物体中，细胞极性关键取决于通常极性的局部决定因素和细胞骨架的极化组织的相互作用。过去，基因方法已经确定了组织极地细胞的几个关键参与者，例如 Inscuteable、Crumbs、Bazooka 和 Stardust。随后，使用针对编码蛋白质产生的抗体揭示了它们在固定材料中的极性定位。事实证明，这些工具对于分析发育中的有机体中的细胞行为非常有价值。然而，为了充分了解极化细胞的产生和维持，有必要使用 GFP 标记的蛋白质，这将允许对细胞极性的动态调节进行体内分析。此外，我们必须寻找编码具有极性分布的蛋白质的额外基因。识别此类蛋白质的一种方法是使用随机 GFP 标记方法（Morin 等，2001）。在此，含有外显子的 GFP，插入基于转座子的载体中，偶然被引入内源果蝇基因的内含子中。剪接后生成融合蛋白（取决于融合框架），该融合蛋白通常被转运至其正常的亚细胞定位。一旦确定了特定的亚细胞表达模式，通过不精确的转座子切除就可以很容易地产生基因突变。遗传学和细胞生物学的这种强大结合将为调节广泛组织中细胞极性的机制提供新的见解。