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。这些适应主要是通过 通过基因表达的改变。缺氧诱导的一个家族 基因，包括促红细胞生成素（Epo），血管内皮生长 血管内皮生长因子（VEGF）和葡萄糖转运蛋白GLUT 1和GLUT 3， 也显示出被过渡金属钴上调， 镍和锰。对氧气知之甚少（02） 介导这种调节的传感途径。长期 该建议的目的是克隆和表征基因 其在氧传感途径中起重要作用， 哺乳动物细胞事实证明，这在二倍体中很难实现 真核细胞考虑到适应性的重要性， 对缺氧的反应，这似乎是合理的假设， 这些反应是高度保守的。我们已经证明，当 酵母，酿酒酵母，用报告基因转化 由厌氧诱导的启动子组成的构建体 酵母基因，ANB 1，附着在lacZ基因上，在缺氧条件下生长 条件下或在所述过渡金属存在下 上面，观察到一种诱导型表型，其类似于 缺氧和过渡金属诱导Epo、VBGF、GLUT 1和 GLUT 3。此外，酵母中候选O2传感器的破坏 即使在常氧条件下， 条件这些发现提供了一种新的策略，以确定 参与O2传感途径的基因。具体目标是 建议：1）鉴定S.酿酒酵母基因 对于ANB 1-lacZ报告基因构建体观察到的表型 并证实其缺氧和钴诱导，类似于 哺乳动物基因2)使用特定目标中确定的基因 在用于鉴定02相关基因的检测中排名第一 使用标准酵母遗传学的酿酒酵母中的传感途径 技术. 3)为了进一步验证最近 克隆的FHB基因编码S.啤酒。4)到 鉴定参与O2传感和信号传导的同源基因 使用互补策略在哺乳动物细胞中转导， 哺乳动物DNA文库的低严格性杂交，和 电脑搜索更好地了解 控制对缺氧应激反应的分子机制是 设计和开发小说的关键的第一步 治疗由疾病引起或恶化的人类疾病的战略 因为缺少02。可能的应用将旨在改善 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.