TISSUE SPECIFIC EXPRESSION OF CYTOCHROME C OXIDASE

这个项目的目标是确定 组织和基因调控序列控制 细胞色素c氧化酶亚基核基因的表达 在人类身上。 哺乳动物细胞色素氧化酶至少由 十二个亚基，它们是两个独立的 基因组 三种最大的多肽（I-III）是 线粒体基因产物，而较小的多肽 （IV-VIII）是核基因产物。 此外，许多 核编码的氧化酶亚基具有组织特异性形式， 这意味着存在一个以上的表达基因， 这些亚单位。 具体目标是确定模式 组织特异性表达和基因组组织的 亚基IV基因。 这些目标将通过以下方式实现： 分离和测序骨骼肌和胎儿的cDNA 心脏文库，并将这些序列与 人肝cDNA。 亚单位IV的表达模式 肝脏、心脏、肌肉和肾脏中的基因将由 通过北方印迹分析、S1-核酸酶 作图和引物延伸实验。 基因组 组织将通过表征和 测序基因组克隆。 调节序列将是 通过与CAT基因的基因融合进行分析。 这些 融合体将被引入到几种不同的人类和 啮齿类动物的基因将通过 将分析体细胞杂交体和人类DNA， 限制性片段长度聚合。 最后，cDNA 其它氧化酶亚基的探针将通过 构建并筛选牛心表达文库， 牛心氧化酶抗体和合成的 寡核苷酸探针。 这些研究对于我们理解调控机制至关重要。 信号参与控制这种重要的 酶复合物和理解的遗传基础， 遗传性细胞色素氧化酶缺乏症

The goals of this project are to determine the genome organization and genetic regulatory sequences controlling expression of nuclear genes for cytochromoe c oxidase subunits in humans. Mammalian cytochrome oxidase consist of at least twelve subunits, which are the products of two separate genomes. The three largest polypeptides (I-III) are mitochondrial gene products, whereas the smaller polypeptides (IV-VIII) are nuclear gene products. Futhermore, many of the nuclear encoded oxidase subunits have tissue-specific forms, implying the existence of more than one expressed gene for these subunits. The specific aims are to determine the pattern of tissue-specific expression and the genome organization of subunit IV genes. These goals will be accomplished by isolating and sequencing cDNAs for skeletal muscle and fetal heart libraries and comparing these sequences with that of human liver cDNA. The pattern of expression of subunit IV genes in liver, heart, muscle and kidney will be determined by analyzing transcripts by Northern blot analysis, S1-nuclease mapping and primer extension experiments. The genome organization will be determined by characterizing and sequencing genomic clones. Regulatory sequences will be analyzed by means of gene fusions with the CAT gene. These fusions will be introduced into several different human and rodent gene(s) will be mapped to human chromosomes by means of somatic celll hybrids and human DNA will be analyzed for restriction fragment length polymerphisms. Finally, cDNA probes for other oxidase subunits will be isolated by constructing and screening a beef heart expression library with antibody to beef heart oxidase and with synthetic oligonucleotide probes. These studies are crucial to our understanding of regulatory signals involved in controlling expression of this essential enzyme complex and in understanding the genetic basis of inherited cytochrome oxidase deficiencies.