Molecular Genetics of Mammalian Cytochrome c Oxidase

细胞色素c氧化酶是电子传递链的末端蛋白，在哺乳动物中由13个亚基组成，其中3个亚基由线粒体DNA编码，其余的由核DNA编码。除了对所有10个核编码亚基的功能未知之外，发现至少有3个，可能更多，甚至可能所有这些核亚基以一种以上的细胞形式(异构体)出现，这引入了第二层次的复杂性。我们将(1)集中于细胞色素C氧化酶的两个亚基，VIIa和VIc，它们以异构体的形式存在，以便(A)分离和鉴定每个异构体的cDNA，以及(B)通过检查它们的转录本和它们在基因组中编码区的组织来确定这些异构体产生的机制；(2)通过检测其转录产物在不同成年牛组织中的分布，以及在不同发育阶段的组织中的分布，寻求对这些异构体的作用的洞察；以及(3)通过在培养的牛细胞中观察它们的化学计量学，特别是在反义转录干扰特定亚基的合成之后，来研究这些和我们有探针的其他细胞色素c氧化酶亚基的调节。像细胞色素c氧化酶这样的复杂蛋白质是如何正确组装的还不清楚。更神秘的是基因表达的同步调控机制，特别是当一些基因位于核基因组，另一些位于细胞器基因组时，从而产生适当的亚基组装比例。这项研究的结果不会让我们对这个复杂的过程有更多的了解。

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. In addition to the fact that the function is unknown for all ten of the nuclear-encoded subunits, a second level of complexity has been introduced by the finding that at least three, probably more, and possibly even all of these nuclear subunits occur in more than one cellular form (isoforms). We will (1) focus on two subunits of cytochrome c oxidase, VIIa and VIIc, that are present as isoforms in order to (a) isolate and characterize cDNAs for each isoform, and (b) determine the mechanism by which these isoforms arise by examining their transcripts and the organization of their coding regions in the genome; (2) seek insights into the role of these isoforms by examining the distribution of their transcripts in various adult bovine tissues, and in tissues taken from several different developmental stages; and (3) investigate the regulation of these and other cytochrome c oxidase subunits for which we have probes by observing their stoichiometry in cultured bovine cells, particularly after perturbation of the synthesis of specific subunits with antisense transcripts. How a complex protein such as cytochrome c oxidase is correctly assembled is obscure. Even more mysterious are the mechanisms involved in the synchronous regulation of gene expression, particularly when some are located on the nuclear genome and others on the organelle genome, so that the proper ration of subunits to be assembled results. The results of this study will give us little more insight into that complex process.