PHYSIOLOGICAL REGULATORS OF CYTOCHROME OXIDASE GENES

细胞色素氧化酶，线粒体电子的末端氧化酶 转运链是调节线粒体的主要位点， 氧化功能 来自高等脊椎动物的酶复合物含有 3个线粒体和10个核编码亚基。 与长期 目的是了解分子之间相互作用的基础， 核和线粒体基因，我们已经表征了核编码 小鼠考克斯IV和Vb基因相对于基础转录启动子 元素 初步结果表明，一种新的负增强子 该地区参与了各种各样的水平（超过10倍） 考克斯Vb在不同组织中的表达。 结果还显示， 亚基Vb含量与催化活性的相关性 来自肝脏、肾脏和心脏的考克斯提示其可能的作用 调节酶活性或组装的亚基。 的目的 格兰特是确定细胞色素氧化酶的核基因的作用， 功能;核和线粒体考克斯基因如何响应 细胞的氧化需求，以及它们的调节如何在 病理状况如缺氧、局部缺血和心肌病。 拟进行以下具体实验：1） 考克斯IV和Vb的启动子/增强子元件的进一步表征 基因和肌肉特异性考克斯VIII基因，以确定 各种积极的作用元素，如E盒，CArG和GTG基序。 考克斯Vb基因负调控区在决定乳腺癌预后中的作用 其在肝脏中低表达，在肌肉和肾脏中高表达 将使用体内和体外转录和蛋白质 约束力研究 同样，诱导考克斯表达的机制 肌生成过程中的Vb和考克斯VIII基因将使用 骨骼肌细胞和心肌细胞2）不同亚基Vb的作用 将研究肝脏、心脏和肾脏酶中的VIII水平 使用生物化学和免疫化学方法。 这些研究将 包括心脏组织的不同隔室， 和一个心肌病心脏模型 细胞核编码亚基在 酶的功能将通过负显性表达来研究， 在培养的肝细胞和肌肉细胞中， 转基因小鼠系统中的靶向表达。3)的影响 生理调节剂如血红素生物合成的抑制剂， 氯化血红素、缺氧、缺血等对考克斯Vb和VIII mRNA的逆转将 被研究。 最后，血红素/O2依赖性调节的机制， 核编码的Vb和线粒体基因将被研究， 确定推定的顺式DNA元件和反式作用因子 在转录调控中。

Cytochrome oxidase, the terminal oxidase of the mitochondrial electron transport chain is a major site for the regulation of mitochondrial oxidative function. The enzyme complex from higher vertebrates contain 3 mitochondrial- and 10 nuclear-encoded subunits. With the long term objective of understanding the molecular basis of interaction between the nuclear and mitochondrial genes, we have characterized nuclear encoded murine COX IV and Vb genes with respect to basal transcription promoter elements. Preliminary results indicate that a novel negative enhancer region is involved in the vastly varying levels (over 10 fold) of expression of COX Vb in different tissue. The results also show a correlation between the subunit Vb content and catalytic activities of COX from liver, kidney and heart suggesting a possible role for this subunit in modulating enzyme activity or assembly. The objective of this grant is to determine the role of nuclear genes in cytochrome oxidase function; how the nuclear and mitochondrial COX genes respond to the oxidative demand of cells and how their regulation is altered during pathological conditions like hypoxia, ischemia, and cardiomyopathies. It is proposed to carry out the following specific experiments: 1) Further characterization of promoter/enhancer elements of COX IV, and Vb genes and the muscle specific COX VIII gene to determine the roles of various positive acting elements like the E-box, CArG and GTG motifs. The role of the negative regulatory region of COX Vb gene in determining its low expression in liver, and high expression in muscle and kidney will be investigated using in vivo and in vitro transcription and protein binding studies. Similarly, the mechanisms of induced expression of COX Vb and COX VIII genes during myogenesis will be investigated using skeletal and also cardiac muscle cells 2) The role of varying subunit Vb and VIII levels in the liver, heart and kidney enzymes will be studied using biochemical and immunochemical approaches. These studies will include different compartments of the heart tissue, and also an ischemic and a myopathic heart models. The role of nuclear encoded subunits in enzyme function will be investigated by negative dominant expression of different subunits in cultured hepatic and muscle cells also by a targeted expression in the transgenic mouse system. 3) The effects of physiological regulators such as inhibitors of heme biosynthesis, reversal by hemin, hypoxia, ischemia, etc. on COX Vb and VIII mRNAs will be studied. Finally, mechanisms of heme/O2 dependent regulation of the nuclear encoded Vb and also mitochondrial genes will be investigated to identify the putative cis DNA elements and trans acting factors involved in transcription regulation.