Genetic Analysis of Hypoxic Cell Death in C. elegans

描述(申请人提供)：中风和心肌梗塞形式的缺氧性细胞死亡是美国最大的单一死亡原因。缺氧性细胞死亡的基本分子机制尚不完全清楚。秀丽线虫和果蝇等遗传易驯化模式生物的研究在细胞死亡、低氧感知和适应等领域取得了重大进展。然而，在线虫中还没有关于改变缺氧诱导的细胞死亡的突变的直接筛查的报道，在果蝇中的结果也是有限的。这项拟议的工作将利用线虫中强大的遗传工具来识别缺氧性细胞死亡的决定因素。具体目的是：1)鉴定线虫低氧死亡调控基因。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，已鉴定出几个耐缺氧的突变体。其中一个突变基因daf-2编码了一种胰岛素/IGF受体，此前已有研究表明，该受体可以调节线虫的寿命。这项拟议中的工作将同样识别其他调节低氧性死亡的基因。2)确定已识别的基因调节缺氧性死亡的机制。一旦发现了大量缺氧死亡基因，问题将变成它们如何执行这一功能。基因产物的序列可能会暗示基因发挥作用的潜在机制和途径。防止细胞死亡的细胞类型和表达基因的细胞类型将被确定。对于每个已识别的基因，将测试特定的低氧保护机制，包括：减少超氧化物水平，改变氧气消耗，增强低氧预适应，以及调节细胞凋亡或坏死性死亡。3)利用DNA微阵列鉴定控制低氧反应/存活的基因产物。低氧死亡基因可能会直接或间接地控制许多其他基因的表达水平，这些基因介导着它们的功能。为了找到这些下游基因，将使用DNA微阵列测量突变和野生动物在正常和低氧条件下的整个线虫基因组的RNA表达水平。微阵列所暗示的优秀候选基因在控制缺氧细胞死亡中的作用将通过RNA介导的干扰和过度表达实验来减少和增加它们的功能来测试。线虫的遗传和基因组工具的结合应该为脊椎动物研究提供新的方向，旨在改善低氧侮辱造成的损害。

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.