Analysis of the Drosophila Gene Rhomboid

本研究计划“The Drosophila gene rhomboid Analysis”的目的是了解Drosophila rhomboid （rho）基因如何通过egf -受体（EGF-R）途径放大信号。菱形体编码一种完整膜蛋白（Rho），该蛋白集中在顶端细胞表面独特的斑块状结构中。rho和Star基因的产物可能促进EGF- r信号传导，因为rho、Star、EGF- r和编码EGF/TGF-a型生长因子（spitz）的基因突变导致极其相似的胚胎表型。rho以空间限制性模式表达，与需要EGF-R通路过度活跃的细胞相对应，而编码EGF-R、Star和Spitz （Spi）配体的基因则普遍表达。这种rho的局部表达对于限制EGF-R信号向适当细胞的增强至关重要。以下是该提案的具体研究目标：I. Bier博士将通过生成Rho蛋白的表位标记版本，并使用针对表位标签的特征抗体来免疫沉淀活性标记的Rho蛋白以及其他潜在的相互作用蛋白（如Star或EGF-R），来确定Rho是否与其他蛋白质发生物理相互作用。他还将确定Rho蛋白和Star蛋白是否在斑块中共定位。2。他将通过体外诱变和随机体内诱变产生一组有限的靶向Rho突变体来识别Rho蛋白的重要功能区域，以恢复携带混合表达Rho转基因的苍蝇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的潜在功能亚域，以及突变的rho蛋白是否可以形成斑块。3。他将比较翼静脉发育过程中rho、Star和spi的遗传需求，并利用flip-out技术对rho进行克隆混合表达，进一步研究rho的局部非自主作用。他还将通过马赛克分析来确定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.