OXYGEN SENSING IN EUKARYOTIC CELLS

适应低氧应激的能力对生存至关重要。 几乎所有的生命形式。这些适应在很大程度上是通过调节 通过基因表达的变化。一个低氧诱导型家族 促红细胞生成素(EPO)等基因与血管内皮细胞生长 因子(VEGF)以及葡萄糖转运蛋白GLUT1和GLUT3具有 也被证明被过渡金属钴上调， 镍和锰。人们对氧气知之甚少(02) 介导这一调控的感觉通路(S)。从长远来看 这项提议的目的是克隆和鉴定这些基因 它们在02感觉通路(S)中起着重要作用 哺乳动物细胞。事实证明，这在二倍体中很难实现。 真核细胞。鉴于自适应技术的重要性 对缺氧的反应，似乎有理由假设 这些反应是高度保守的。我们已经证明，当 酵母，酿酒酵母，被记者转化 由厌氧诱导的启动子组成的结构 酵母基因ANB1，附着于LacZ基因并在低氧条件下生长 在上述过渡金属存在的条件下或在存在的情况下 上面，观察到一种可诱导的表型，类似于 低氧和过渡金属对EPO、VBGF、GLUT1和 GLUT3.此外，酵母中候选02传感器的破坏 即使在常氧条件下也能概括这种低氧表型。 条件。这些发现提供了一个新的策略来识别 参与02感觉通路的基因。这样做的具体目的是 建议有：1)确定酿酒酵母的致病基因(S) 对于使用ANB1-LacZ报告构建观察到的表型 并确认其低氧和钴诱导性，类似于 哺乳动物的基因。2)利用特定目的鉴定的基因(S) 在旨在确定02涉及的基因的分析中排名第一 标准酵母基因在酿酒酵母中的传感途径 技巧。3)进一步检验最近 在酿酒酵母中克隆的FHB基因编码一个02感应器。4)至 确定与02传感和信号有关的同源基因 利用互补策略在哺乳动物细胞中的转导， 哺乳动物DNA文库的低严格杂交，以及 电脑搜索。更好地了解 控制对低氧应激反应的分子机制是 小说设计和开发的关键第一步 治疗由或恶化的人类状况的策略 到目前为止，还缺少02。可能的应用程序旨在改进 治疗贫血以及血管和神经疾病的能力 疾病。

The ability to adapt to hypoxic stress is crucial to the survival of virtually all life forms. These adaptations are mediated largely by changes in gene expression. One family of hypoxia-inducible genes, including erythropoietin (Epo), vascular endothelial growth factor (VEGF), and the glucose transporters GLUT1 and GLUT3, has also been shown to be upregulated by the transition metals cobalt, nickel, and manganese. Little is known about the oxygen (02) sensing pathway(s) which mediate this regulation. The long term objective of this proposal is to clone and characterize the genes which play a significant role in the 02 sensing pathway(s) of mammalian cells. This has proven difficult to accomplish in diploid eukryotic cells. Given the paramount importance of the adaptive responses to hypoxia, it seemed reasonable to hypothesize that these responses are highly conserved. We have shown that when the yeast, Saccharomyces cerevisiae, is transformed with a reporter construct consisting of the promoter of the anaerobically-induced yeast gene, ANB1, attached to the lacZ gene and grown under hypoxic conditions or in the presence of the transition metals mentioned above, an inducible phenotype is observed which is similar to the hypoxia and transition metal induction of Epo, VBGF, GLUT1, and GLUT3. Moreover, disruption of a candidate 02 sensor in yeast recapitulates this hypoxic phenotype even under normoxic conditions. These findings provide a new strategy to identify the genes involved in the 02 sensing pathway. The specific aims of this proposal are: 1) To identify the S. cerevisiae gene(s) responsible for the phenotype observed with the ANB1-lacZ reporter construct and confirm its hypoxia and cobalt inducibility, analogous to mammalian genes. 2) To use the gene(s) identified in Specific Aim #1 in assays designed to identify the genes involved in the 02 sensing pathway in & cerevisiae using standard yeast genetic techniques. 3) To further test the hypothesis that the recently cloned FHB gene encodes an 02 sensor in S. cerevisiae. 4) To identify the homologous genes involved in 02 sensing and signal transduction in mammalian cells using complementation strategies, low stringency hybridization of mammalian DNA libraries, and computer searches. Acquiring a better understanding of the molecular mechanisms governing the response to hypoxic stress is the crucial first step in the design and development of novel strategies to treat human conditions resulting from, or exacerbated by, a lack of 02. Likely applications would be aimed at improving the ability to treat anemias as well as vascular and neurological diseases.