Genetic Analysis of Hypoxic Cell Death in C. elegans

描述（由申请人提供）：以中风和心肌梗死形式出现的低分化细胞死亡是美国最大的单一死亡原因。缺氧细胞死亡的基本分子机制尚未完全了解。对秀丽隐杆线虫和果蝇等遗传学上易处理的模式生物的研究在细胞死亡、缺氧感知和适应等领域取得了重大进展。 然而，在C.在果蝇中的结果是有限的。这项工作将利用C. elegans来鉴定缺氧细胞死亡的决定因素。具体目标是：1）鉴定C.优雅这将通过筛选耐缺氧死亡的突变动物来实现。在中试诱变筛选中，已经鉴定了几种耐缺氧的突变体。其中一个突变基因，daf-2，编码胰岛素/IGF受体，以前显示在C中调节寿命。优雅这项工作也将类似地确定其他调节缺氧死亡的基因。2)定义所识别的基因调节缺氧死亡的机制。一旦发现了一些缺氧死亡基因，问题将变成它们如何执行这一功能。基因产物的序列将可能提示基因发挥功能的潜在机制和途径。将确定保护免于细胞死亡的细胞类型和表达基因的细胞类型。对于每一个确定的基因，具体的缺氧保护机制将进行测试，包括：超氧化物水平的降低，氧消耗的改变，缺氧预处理的增强，以及细胞凋亡或坏死性死亡的调节。3)使用DNA微阵列鉴定控制缺氧反应/存活的基因产物。缺氧死亡基因可能直接或间接地控制许多其他基因的表达水平，这些基因介导它们的功能。为了找到这些“下游”基因，我们检测了整个C.将使用DNA微阵列在正常和缺氧条件下测量突变型和野生型动物中的线虫基因组。通过RNA介导的干扰和过表达实验，将通过减少和增加它们的功能来测试微阵列在控制缺氧细胞死亡中所涉及的优秀候选物的作用。将遗传学和基因组学工具相结合，在C.秀丽隐杆线虫应该为脊椎动物研究提供新的方向，旨在改善缺氧损伤。

DESCRIPTION (provided by applicant): Hypoxic cell death in the form of stroke and myocardial infarction is the largest single cause of death in the United States. The fundamental molecular mechanisms of hypoxic cell death are incompletely understood. Studies in genetically tractable model organisms such as Caenorhabditis elegans and Drosophila have made major advances in the fields of cell death, hypoxia sensing, and adaptation. However, direct screens for mutations that alter hypoxia-induced cell death have not been reported in C. elegans and results in Drosophila have been limited. The proposed work will utilize the powerful genetic tools in C. elegans to identify determinants of hypoxic cell death. The specific aims are: 1) Identify genes regulating hypoxic death in C. elegans. This will be accomplished by screening for mutant animals that are resistant to hypoxic death. In pilot mutagenesis screens, several mutants have been identified that are hypoxia resistant. One of the mutant genes, daf-2, codes for an insulin/IGF receptor, previously shown to regulate lifespan in C. elegans. The proposed work will similarly identify other genes regulating hypoxic death. 2) Define the mechanisms whereby the identified genes regulate hypoxic death. Once a number of hypoxic death genes are found, the question will become how do they perform this function. The sequence of the gene product will likely suggest potential mechanisms and pathways through which the genes function. The cell types protected from cell death and the cell types in which the genes are expressed will be determined. For each identified gene, specific hypoxic protective mechanisms will be tested including: reduction in superoxide levels, alterations in oxygen consumption, enhancement of hypoxic preconditioning, and regulation of apoptotic or necrotic death. 3) Use DNA microarrays to identify gene products controlling hypoxic response/survival. The hypoxic death genes will likely control the expression levels, either directly or indirectly, of a number of other genes mediating their function. To find these "downstream" genes, the RNA expression levels of the whole C. elegans genome will be measured in mutant and wild type animals in normal and hypoxic conditions using DNA microarrays. The role of outstanding candidates implicated by the microarrays in the control of hypoxic cell death will be tested by reducing and increasing their function through RNA-mediated interference and over-expression experiments. The combination of genetic, and genomic tools in C. elegans should provide novel directions for vertebrate studies aimed at ameliorating damage from hypoxic insults.