Analysis of the Drosophila Gene Rhomboid

Bier 9603801 本研究项目“果蝇基因rhomboid分析”的目的是了解果蝇rhomboid（rho）基因如何通过EGF受体（EGF-R）途径放大信号传导。rhomboid编码一种整合膜蛋白（Rho），其集中在顶端细胞表面的独特斑块样结构中。rho和星星基因的产物可能促进EGF-R信号传导，因为rho、星星、Egf-r和编码EGF/TGF-α型生长因子（spitz）的基因的突变导致极其相似的胚胎表型。rho以对应于需要EGF-R途径过度活性的细胞的空间受限模式表达，而编码EGF-R、星星和Spitz（Spi）配体的基因普遍表达。rho的这种局部表达对于将增强的EGF-R信号传导限制到适当的细胞至关重要。以下是本建议的具体研究目标：一。Bier博士将通过产生Rho蛋白的表位标记版本并使用针对表位标记的特征抗体来免疫沉淀活性标记的Rho蛋白沿着其他潜在相互作用蛋白（如星星或EGF-R），确定Rho是否与其他蛋白发生物理相互作用。他还将确定Rho和星星蛋白是否在斑块中共定位。二.他将通过使用体外诱变和随机体内诱变产生一组有限的靶向rho突变体来鉴定Rho蛋白的功能重要区域，以恢复携带混合表达rho转基因的蝇种的显性异位静脉表型。他将对恢复的rho突变体进行测序，以确定它们是否聚集在Rho的潜在功能亚结构域中，以及突变的Rho蛋白是否可以形成斑块。 三.他将比较翅静脉发育过程中rho、星星和spi的遗传要求，并使用翻转技术克隆混合表达rho，以进一步研究rho的局部非自主作用。他还将使用镶嵌分析来确定spi在翅膀发育中的作用。

Bier 9603801 The goal of this research proposal "Analysis of the Drosophila gene rhomboid" is to understand how the Drosophila rhomboid (rho) gene amplifies signaling through the EGF-Receptor (EGF-R) pathway. rhomboid encodes an integral membrane protein (Rho) which is concentrated in unique plaque-like structures at the apical cell surface. The products of the rho and Star genes are likely to promote EGF-R signaling since mutations in rho, Star, Egf-r, and a gene encoding an EGF/TGF-a type of growth factor (spitz) lead to extremely similar embryonic phenotypes. rho is expressed in a spatially restricted pattern corresponding to cells requiring hyperactivity of the EGF-R pathway, while genes encoding EGF-R, Star, and the Spitz (Spi) ligand are ubiquitously expressed. This localized expression of rho is critical for limiting augmented EGF-R signaling to appropriate cells. The following are specific research goals for this proposal: I. Dr. Bier will determine whether Rho interacts physically with other proteins by generating epitope tagged versions of the Rho protein and using characterized antibodies against the epitope tags to immune-precipitate active tagged Rho proteins along with other potential interacting proteins such as Star or EGF-R. He will also determine whether Rho and Star proteins co-localize in plaques. II. He will identify functionally important regions of the Rho protein by generating a limited set of targeted rho mutants using in-vitro mutagenesis and random in vivo mutagenesis to revert the dominant ectopic vein phenotype of a fly stock carrying a mix-expressed rho transgene. He will sequence recovered rho mutants to determine whether they cluster in potential functional sub-domains of Rho and whether mutant Rho proteins can form plaques. III. He will compare the genetic requirements for rho, Star, and spi during wing vein development and mix-express rho clonally using the flip-out technique to further investigate the locally non-autonomous action Of rho. He also will us e mosaic analysis to determine the role of spi in wing development.