Genetic inactivation of the Drosophila P450 system

果蝇 P450 系统的基因失活

• 批准号: 7011238
• 负责人: KEVIN Andrew EDWARDS
• 金额: $ 6.84万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011238
• 关键词: Drosophilidae; NADPH cytochrome c2 reductase; arthropod genetics; cytochrome P450; developmental genetics; drug metabolism; drug resistance; embryo /fetus; endoplasmic reticulum; enzyme activity; gene environment interaction; gene expression; gene induction /repression; gene mutation; larva; method development; phenotype; technology /technique development; toxin; transfection

DESCRIPTION (provided by applicant): Cytochrome P450 monooxygenases are heme-containing enzymes that insert oxygen into a wide range of xenobiotics, such as pollutants, pesticides, and drugs, promoting their clearance from the body. Although they provide a vital defense system against environmental toxins, P450s may also catalyze the formation of products that are more toxic or carcinogenic than their precursors. P450s perform essential roles in development and homeostasis as well, including steroid hormone synthesis. It is unclear how these defensive and developmental roles of the P450 system are coordinated and regulated; to address this problem in vivo, a genetic analysis was initiated in the model organism Drosophila melanogaster. P450s that reside on the endoplasmic reticulum (ER) generally require a redox partner, Cytochrome P450 Reductase (CPR). Most animals have dozens of P450 genes, but CPR is often encoded by a single gene, providing a unique "Achilles' heel" for genetically inactivating the ER P450 system. The principal investigator's lab generated an allelic series of mutations in the Drosophila CPR gene (Cpr), including 6 novel lethal alleles. Flies were constructed which conditionally express a Cpr cDNA; this transgene can rescue the lethal mutations, and it causes visible phenotypes when overexpressed. These genetic tools provide the means to elucidate the numerous functions of the highly pleiotropic Cpr gene. 3 aims are proposed: I) Characterize embryonic and larval phenotypes associated with Cpr, to identify its developmental functions. II) Develop an assay system to determine the degree to which metabolism of a compound depends on CPR. The sensitivity (or resistance) of the mutants to several xenobiotics and mixtures will be tested. A method for generating CPR-deficient adults will be established, to facilitate compound testing and analysis of adult Cpr phenotypes. Ill) Test for feedback induction of the P450s. Toxin and drug interactions are often caused by P450 induction. It was recently hypothesized that vertebrates have a large-scale system to overexpress P450s when CPR is compromised. P450 mRNAs will be quantified in Cpr mutants to determine if this system exists and can be investigated in Drosophila.

描述（由申请人提供）：细胞色素 P450 单加氧酶是含血红素的酶，可将氧气插入多种异生物质（例如污染物、杀虫剂和药物）中，促进其从体内清除。尽管它们提供了针对环境毒素的重要防御系统，但 P450 也可能催化比其前体更具毒性或致癌性的产物的形成。 P450 在发育和体内平衡中也发挥着重要作用，包括类固醇激素的合成。目前尚不清楚 P450 系统的这些防御和发育作用是如何协调和调节的；为了在体内解决这个问题，在模式生物果蝇中启动了遗传分析。驻留在内质网 (ER) 上的 P450 通常需要氧化还原伙伴，即细胞色素 P450 还原酶 (CPR)。大多数动物都有数十个 P450 基因，但 CPR 通常由单个基因编码，为从基因上灭活 ER P450 系统提供了独特的“致命弱点”。首席研究员的实验室在果蝇 CPR 基因 (Cpr) 中产生了一系列等位基因突变，其中包括 6 个新的致死等位基因。构建了条件表达 Cpr cDNA 的果蝇；这种转基因可以挽救致命突变，并且在过度表达时会导致可见的表型。这些遗传工具提供了阐明高度多效性 Cpr 基因的众多功能的方法。提出了 3 个目标： I) 表征与 Cpr 相关的胚胎和幼虫表型，以确定其发育功能。 II) 开发一个测定系统来确定化合物代谢对 CPR 的依赖程度。将测试突变体对几种外源物和混合物的敏感性（或抗性）。将建立一种产生 CPR 缺陷成人的方法，以促进成人 CPR 表型的复合测试和分析。 Ill) 测试 P450 的反馈感应。毒素和药物相互作用通常是由 P450 诱导引起的。最近有人假设，当心肺复苏受到损害时，脊椎动物有一个大规模的系统来过度表达 P450。将在 Cpr 突变体中对 P450 mRNA 进行定量，以确定该系统是否存在并可以在果蝇中进行研究。