Analysis of the Drosophila Gene Rhomboid

Bier 9603801这项名为“果蝇菱形基因分析”的研究计划的目的是了解果蝇菱形基因(Rho)是如何通过表皮生长因子受体(EGF-R)途径放大信号的。菱形蛋白编码一个完整的膜蛋白(Rho)，它集中在细胞顶端独特的斑状结构中。Rho和Star基因的产物可能促进EGF-R信号转导，因为Rho、Star、EGF-r和一个编码EGF/TGF-a类型的生长因子(Spitz)的基因突变导致非常相似的胚胎表型。Rho以空间受限的模式表达，与需要EGF-R途径高活性的细胞相对应，而编码EGF-R、Star和Spitz(Spi)配体的基因则普遍表达。这种Rho的局部表达对于将增强的EGF-R信号限制到适当的细胞是至关重要的。这项提议的具体研究目标如下：I.比尔博士将通过产生Rho蛋白的表位标记版本，并使用针对表位标记的特征性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，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和Star蛋白是否在斑块中共定位。他将通过使用体外突变和体内随机突变来产生一组有限的靶向Rho突变，以逆转携带混合表达的Rho转基因的苍蝇种群的主要异位静脉表型，从而识别Rho蛋白的功能重要区域。他将对恢复的Rho突变进行测序，以确定它们是否聚集在Rho的潜在功能亚域中，以及突变的Rho蛋白是否可以形成斑块。他将比较Rho、Star和Spi在翼脉发育过程中的遗传需求，并使用Flip-Out技术克隆混合表达Rho，以进一步研究Rho的局部非自主作用。他还将使用电子马赛克分析来确定SPI在机翼发育中的作用。

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.