ANALYSIS OF THE COX4/COX4AL BIDIRECTIONAL PROMOTER

细胞色素c氧化酶是线粒体内膜的多亚基酶复合物，它将电子从细胞色素c转移到分子氧中，并以质子梯度的形式保存能量。对细胞色素氧化酶（COX4）的最大核编码亚基（亚基IV）的人类基因的表征导致鉴定出一个功能未知的密切相关的表达基因。COX4相关位点（COX4AL）与COX4呈头对头排列，因此基因间区（250-bp）可能是一个双向启动子。该项目的第一个目标是通过免疫荧光追踪24kda编码蛋白的亚细胞位置，并通过遗传和生化方法确定其与其他细胞蛋白的关联，从而确定COX4AL的可能功能。双向启动子通常编码功能相似或相关的蛋白质；因此，COX4AL和COX4一样编码线粒体蛋白的特定假设将得到验证。第二个目的是确定COX4/COX4AL基因间区是否作为双向启动子起作用。人类细胞中转染的启动子报告基因结构的遗传分析将用于鉴定每个基因表达所必需的启动子元件。第三，COX4/COX4AL基因中的保守启动子元件将被用于扫描其他协调控制的哺乳动物基因的启动子，包括其他核编码细胞色素氧化酶亚基和双向控制基因的启动子，以确定调节基因表达的新因子或建立新的控制模式。长期目标是为16号染色体上编码细胞色素氧化酶亚基IV基因的区域的人类基因组功能数据库做出贡献。了解人类疾病分子基础的未来进展取决于通过长时间序列数据获得完整的人类基因功能目录和基因间区域作为“路线图”。

Cytochrome c oxidase is the multisubunit enzyme complex of the mitochondrial inner membrane which transfers electrons from cytochrome c to molecular oxygen and conserves energy as a proton gradient. Characterization of the human gene for the largest nuclear-encoded subunit (subunit IV) of cytochrome oxidase (COX4) led to the identification of a closely linked expressed gene of unknown function. COX4-associated locus (COX4AL) is linked in a head-to-head arrangement with COX4 and thus the intergenic region (250-bp) likely functions as a bidirectional promoter. The first aim of this project is to determine a possible function of COX4AL by tracking the subcellular location of the 24-kDa encoded protein via immunofluorescence and by determining its association with other cellular proteins using genetic and biochemical approaches. Bidirectional promoters usually encode proteins of similar or related function; thus the specific hypothesis that COX4AL, like COX4, encodes a mitochondrial protein will be tested. A second aim is to ascertain whether the COX4/COX4AL intergenic region functions as a bidirectional promoter. Genetic analysis of transfected promoter-reporter gene constructs in human cells will be used to identify promoter elements essential to the expression of each gene. Third, conserved promoter elements in COX4/COX4AL genes will be used to scan the promoters of other coordinately controlled mammalian genes, including those for other nuclear-encoded cytochrome oxidase subunits and bidirectionally controlled genes, to identify new factors that regulate gene expression or to establish new patterns of control used by well-characterized factors. The long-term objective is to contribute to the functional database of the human genome for a region of chromosome 16 shown to encode the cytochrome oxidase subunit IV gene. Future progress in understanding the molecular bases for human disorders depends on having a complete catalog of the functions of human genes and intergenic regions as a "roadmap" through long stretches of sequence data.