Identification and characterization of new mutants affecting Notch trafficking

该项目致力于对影响Notch贩运的新突变体进行遗传筛选，以及潜在的其他突变体&amp；#947；-分泌酶底物，通过细胞膜隔室。尽管对影响细胞增殖、极性和模式的突变体的筛选已经确定了影响内体Notch运输的基因，但对直接改变Notch细胞分布的突变的系统筛选尚未进行。基于我们之前对Ca2+- atp酶突变对Notch分泌通路运输的影响的分析，果蝇盘细胞的高度极化结构，以及在发育中的果蝇组织中产生致命突变克隆的遗传技术，我们认为果蝇是尝试这种类型的细胞生物学筛选的合适模型。为了进行筛选，我们对雄性果蝇进行诱变，建立平衡种群，诱导单个诱变染色体臂的纯合组织克隆，并分析突变克隆中Notch蛋白相对于邻近野生型组织的分布。目前，我们正在完成第二条染色体的筛选。筛选了大约4000个突变染色体臂，恢复了91个突变，这些突变定义了20多个独立的基因（每个基因由2-8个突变等位基因代表）。不同的突变体可以根据其对Notch转运和受影响的细胞膜腔室的影响进行分类。对这些新突变体的进一步表征，包括图谱绘制和分子克隆，目前正在进行中。

This project is devoted to forward genetic screens for new mutants that affect trafficking of Notch, and potentially other γ-secretase substrates, through intracellular membrane compartments. Although screens for mutants affecting cell proliferation, polarity, and patterning have led to the identification of genes influencing endosomal Notch trafficking, systematic screens for mutations that directly alter the cellular distribution of Notch have not been performed. Based on our previous analysis of the effects of Ca2+-ATPase mutations on the trafficking of Notch through the secretory pathway, the highly polarized structure of Drosophila disc cells, and the genetic techniques available for producing clones of lethal mutations in developing fly tissues, we reason that Drosophila is a suitable model for attempting this type of cell biological screen. To perform the screen, we mutagenize male flies, establish balanced stocks, induce tissue clones that are homozygous for a single mutagenized chromosome arm, and analyze the distribution of Notch protein in the mutant clone relative to adjacent wildtype tissue. At present, we are completing our screen of the second chromosome. Approximately 4000 mutagenized chromosome arms have been screened, resulting in the recovery of 91 mutations, which define over 20 separate genes (each represented by 2-8 mutant alleles). The different mutants can be classified with respect to their effects on Notch trafficking and the intracellular membrane compartment affected. Further characterization of several of these new mutants, including mapping and molecular cloning, is now underway.