Genetic Analysis of Hypoxic Cell Death in C. elegans

描述（由申请人提供）：中风和心肌梗死形式的缺氧细胞死亡是美国最大的单一死亡原因。细胞缺氧死亡的基本分子机制尚不完全清楚。对秀丽隐杆线虫（Caenorhabditis elegans）和果蝇（Drosophila）等遗传易感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，对改变缺氧诱导细胞死亡的突变的直接筛选尚未在秀丽隐杆线虫中报道，在果蝇中的结果也有限。提出的工作将利用秀丽隐杆线虫强大的遗传工具来确定缺氧细胞死亡的决定因素。具体目的是：1)鉴定线虫缺氧死亡调控基因。这将通过筛选抗缺氧死亡的突变动物来完成。在先导诱变筛选中，已经确定了几种抗缺氧突变体。其中一个突变基因daf-2编码胰岛素/IGF受体，该受体先前被证明可以调节秀丽隐杆线虫的寿命。拟议的工作将类似地确定其他调节缺氧死亡的基因。2)明确已鉴定基因调控缺氧死亡的机制。一旦发现了一些缺氧死亡基因，问题就变成了它们是如何发挥这一功能的。基因产物的序列可能会提示基因发挥作用的潜在机制和途径。将确定保护免受细胞死亡的细胞类型以及表达基因的细胞类型。对于每个已确定的基因，将测试特定的缺氧保护机制，包括：超氧化物水平的降低，氧气消耗的改变，缺氧预处理的增强，以及凋亡或坏死死亡的调节。3)利用DNA微阵列技术鉴定控制缺氧反应/生存的基因产物。缺氧死亡基因可能会直接或间接地控制其他一些介导其功能的基因的表达水平。为了找到这些“下游”基因，将使用DNA微阵列技术测量正常和缺氧条件下突变型和野生型秀丽隐杆线虫整个基因组的RNA表达水平。通过rna介导的干扰和过表达实验，将通过降低和增加其功能来测试微阵列在控制缺氧细胞死亡中的作用。遗传和基因组工具在秀丽隐杆线虫中的结合应该为旨在改善缺氧损伤的脊椎动物研究提供新的方向。

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.