PHYSIOLOGICAL REGULATORS OF CYTOCHROME OXIDASE GENES

线粒体电子的末端氧化酶--细胞色素氧化酶 传输链是线粒体调控的主要部位 氧化功能。高等脊椎动物的酶复合体含有 3个线粒体亚基和10个核编码亚基。从长远来看 目的：了解细胞间相互作用的分子基础。 核和线粒体基因，我们已经确定了核编码的 小鼠环氧合酶IV和VB基因与基础转录启动子的关系 元素。初步结果表明，一种新型的负性增强剂 地区涉及的水平差异很大(超过10倍) COX-VB在不同组织中的表达结果还显示， 维生素B亚基含量与其催化活性的相关性 来自肝脏、肾脏和心脏的COX暗示这可能在其中起作用 调节酶活性或组装的亚基。这样做的目的是 格兰特将确定核基因在细胞色素氧化酶中的作用 功能；核和线粒体COX基因如何对 细胞的氧化需求及其调控是如何改变的 病理状态，如缺氧、缺血和心肌病。 拟开展以下具体实验：1) COX IV和VB启动子/增强子元件的进一步鉴定 基因和肌肉特异性环氧合酶VIII基因的作用 各种积极的行动元素，如E-box，Carg和GTG主题。 环氧合酶VB基因负调控区在决定人类免疫缺陷中的作用 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进行研究 约束性研究。同样，诱导COX表达的机制 肌肉发生过程中的VB和COX VIII基因将用 骨骼肌细胞和心肌细胞2)不同亚基VB的作用 将研究肝脏、心脏和肾脏酶的viii水平。 使用生化和免疫化学方法。这些研究将 包括心脏组织的不同隔室，也包括缺血 和一个肌病心脏模型。核编码亚基在人类免疫系统中的作用 酶的功能将通过负显性表达来研究 培养的肝细胞和肌肉细胞中的不同亚单位也通过 转基因小鼠系统中的靶向表达。3)网络的影响 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COX Vb和VIIImRNAs的逆转作用 被研究。最后，血红素/氧依赖的调节机制 核编码的VB以及线粒体基因将被研究 鉴定可能涉及的顺式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.