Identification and characterization of new mutants affecting Notch trafficking

影响Notch贩运的新突变体的鉴定和表征

• 批准号: 7733328
• 负责人: Mark E Fortini
• 金额: $ 47.13万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733328
• 关键词: Acute T Cell Leukemia; Affect; Alleles; Alzheimer' s Disease; Area; Aspartic Endopeptidases; Biological; Biology; Ca(2+)-Transporting ATPase; Cell Proliferation; Cell physiology; Cell surface; Cells; Chromosome Arm; Chromosomes; Classification; Cleaved cell; Complex; Development; Disease; Drosophila genus; Equilibrium; Eukaryota; Eukaryotic Cell; Event; Genes; Genetic Screening; Genetic Techniques; Goals; Human; Integral Membrane Protein; Intracellular Membranes; Lead; Malignant Neoplasms; Maps; Membrane; Membrane Proteins; Modeling; Molecular Cloning; Mutation; Nerve Degeneration; Numbers; Pathway interactions; Pattern; Process; Receptor Signaling; Recovery; Relative (related person); Research; Screening Result; Signal Pathway; Signal Transduction; Structure; Therapeutic; Thinking; Tissues; amyloid peptide; amyloid precursor protein processing; base; cell type; cellular targeting; fly; functional genomics; human disease; male; mutant; notch protein; secretase; trafficking; tumorigenesis

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.

这个项目致力于对新的突变体进行遗传筛选， Notch和潜在的其他分泌酶底物的运输，通过 细胞内膜区室。虽然筛选影响细胞的突变体 增殖、极性和模式化导致了对基因的鉴定， 内体Notch运输，系统筛选直接改变细胞的突变， Notch的分布尚未执行。根据我们之前对 Ca 2 +-ATP酶突变对Notch通过分泌途径运输的影响， 果蝇盘细胞的极化结构，以及可用于 在发育中的果蝇组织中产生致命突变的克隆，我们推断果蝇是一种 用于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 诱变雄性果蝇，建立平衡的种群，诱导组织克隆， 单个诱变的染色体臂，并分析Notch蛋白在 相对于相邻野生型组织的突变克隆。目前，我们正在完成我们的屏幕 第二条染色体已经筛选了大约4000个诱变的染色体臂， 导致91个突变的恢复，这些突变定义了20多个独立的基因（每个基因 由2-8个突变等位基因表示）。不同的突变体可以根据 它们对Notch运输和受影响的细胞内膜区室的影响。 这些新突变体中的几个的进一步表征，包括作图和分子标记。 克隆正在进行中。