TISSUE SPECIFIC EXPRESSION OF CYTOCHROME C OXIDASE

这个项目的目标是确定基因组 组织和基因调控序列控制 细胞色素C氧化酶亚基的核基因表达 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， 暗示存在一个以上的表达基因 这些亚单位。具体的目标是确定模式 的组织特异性表达和基因组组织 亚基IV基因。这些目标将通过以下方式实现 骨骼肌和胎儿cDNA的分离和测序 心脏文库，并将这些序列与 人肝cDNA.亚基IV的表达模式 肝脏、心脏、肌肉和肾脏中的基因将由 转录本的Northern印迹分析，S1-核酸酶 作图和引物延伸实验。基因组 组织将通过以下方式确定： 基因组克隆测序。调控序列将是 通过与猫基因的基因融合进行分析。这些 融合将被引入到几个不同的人类和 啮齿动物基因(S)将被定位到人类染色体上 体细胞杂交和人类DNA将被分析 限制性片段长度聚合体。最后，cDNA3 其他氧化酶亚基的探针将通过以下方式分离 牛心表达文库的构建及筛选 抗牛肉心脏氧化酶抗体和人工合成的 寡核苷酸探针。 这些研究对我们理解监管是至关重要的 参与控制这一必需基因表达的信号 酶复合体和在理解遗传基础方面 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.