MOLECULAR GENETICS OF C OXIDASE

细胞色素c氧化酶，电子传递的终末蛋白 链，在哺乳动物中由13个亚基组成，其中3个由 线粒体DNA，其余的由核DNA决定。至少有三个 这些核亚基以一种以上的分子形式、亚型、 不同基因的产物。拟议的实验将提供 对这些异构体的细胞功能的洞察，这可能是 与个体发育和细胞生理学有关。在……里面 此外，人类神经肌肉的异构体表达也被发现。 疾病。该项目将重点关注COX VIIA和COX VIIC两个子单元， 由三个基因编码。COX7a显示心脏和肌肉特有的 已分离的同型基因COX7a-H和一个同型基因 COX7a-L在所有组织中均有表达。第三个基因，COX7c，似乎有 一种在所有组织中表达的单一形式。具体目标是(1) 分离和鉴定COX7a-L和COX7c基因，并比较 COX7a-H的调控区域；(2)制作报告结构 每个基因通过缺失和缺失来定义顺式作用的调控元件 突变途径；(3)研究生理学效应。 异构体表达调控条件；(4)分离与研究 反式调节因素。第三个目的，研究法律法规 异构体的表达将通过两种方式进行：通过原位 异构体特异性探针与心脏区域的杂交 在非常不同的氧化压力下工作，并通过共转染 两种异构体基因在可区分的报告结构中进入细胞 处于各种生理条件下。这个项目很适合 更长期的目标是推断核的作用-- 编码的亚基和用于调节氧化功能的机制， 并将这一知识应用于核基因的研究 线粒体疾病。

Cytochrome c oxidase, the terminal protein of the electron transport chain, is composed in mammals of thirteen subunits, three encoded by mitochondrial DNA and the remainder by nuclear DNA. At least three of these nuclear subunits occur in more than one molecular form, isoforms, the products of separate genes. The proposed experiments will provide insights into the cellular function of these isoforms, which may be related to both ontogenetic development and cellular physiology. In addition, isoform expression has been implicated in human neuromuscular disease. This project will focus on two subunits, COX VIIa and COX VIIc, encoded by three genes. COX7a displays a heart- and muscle-specific isoform gene, COX7a-H, which is already isolated, and an isoform gene expressed in all tissues, COX7a-L. A third gene, COX7c, appears to have a single expressed form in all tissues. The specific aims are to (1) isolate and characterize the COX7a-L and COX7c genes, and compare the regulatory regions to those of COX7a-H; (2) make reporter constructs of each gene to define cis-acting regulatory elements by deletional and mutational approaches; (3) investigate the effect of physiological conditions on regulation of isoform expression; and (4) isolate and study trans-acting regulatory factors. The third aim, studying regulation of isoform expression, will be pursued in two ways: by in situ hybridization of isoform-specific probes to regions of the heart that operate under very different oxidative stresses, and by cotransfection of both isoform genes in distinguishable reporter constructs into cells placed under a variety of physiological conditions. This project fits into the longer term objectives of deducing the role of the nuclear- encoded subunits and the mechanisms used to regulate oxidative function, and applying this knowledge to the study of nuclear genes in mitochondrial disease.