REGULATION OF MITOCHONDRIAL BIOGENESIS IN ANIMAL CELLS

动物细胞线粒体生物发生的调控

• 批准号: 3276490
• 负责人: NARAYAN G AVADHANI
• 金额: $ 13.52万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1991-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276490
• 关键词: DNA; DNA directed RNA polymerase; Ehrlich's tumor; HeLa cells; adenosinetriphosphatase; affinity chromatography; biological transport; cell nucleus; complementary DNA; cytochrome oxidase; electron microscopy; embryo /fetus; gene expression; genetic manipulation; genetic mapping; genetic regulation; genetic transcription; genetic translation; genome; liver cells; messenger RNA; mitochondria; molecular cloning; plasmids; polysomes; radiotracer; synthetic peptide; tissue /cell culture

Mitochondrial biogenesis in animal cells involves the coordinate expression of a large number of nuclear genes coding for varied tissue specific and generally occurring mitochondrial proteins and a limited number of genes of the 16 kbp mitochondrial genome. Despite intensive efforts, however, the molecular mechanisms involved in the coordinate expression of the two genetic systems have not yet been elucidated. Recent results in our laboratory showed the possibility that three of the mitochondrial genome coded proteins are transported to the nucleus as possible regulatory signals. Based on this, the objective of this grant proposal is to undertake a detailed characterization of the three proteins transported to the nucleus with respect to their nuclear DNA binding characteristics, mode of regulation of nuclear genes. It is proposed to continue the present line of studies on the characterization of observed induction of nuclear genes coding for varied mitochondrial proteins like cytochrome oxidase, ATPase, RNA polymerase, etc. by long-term exposure of cells to mitochondrial translation inhibitors. One of the objectives is to characterize the high affinity binding sites for the three mitochondrial proteins on the nuclear chromosomal DNA and determine their physical relationship or proximity to genes coding for the known mitochondrial proteins using cDNA cloning, in situ hybridization to G-banded chromosomes and mapping of genomic clones. Finally, the precise regulatory roles of the three proteins and the nature of gene(s) or gene clusters under their influence will be assessed using in vitro transcription in isolated nuclei and in HeLa cell extracts with added DNA templates. The genomic areas showing high specificity for the proteins will be sequenced to gain insight on the mode of regulation by the three mitochondrial proteins.

动物细胞中的线粒体生物发生涉及坐标表达 编码各种组织特异性的大量核基因和 通常发生的线粒体蛋白和有限数量的基因 16 kbp线粒体基因组。 尽管有密集的努力，但是 参与两者的坐标表达的分子机制 遗传系统尚未阐明。 我们的最新结果 实验室表明了三个线粒体基因组的可能性 将编码的蛋白质作为可能的调节转运到核中 信号。 基于此，该赠款提案的目的是 对运输到的三种蛋白质进行详细表征 核相对于其核DNA结合特性，模式 核基因的调节。 建议继续现在 关于观察到的核诱导表征表征的研究线 编码多种线粒体蛋白（如细胞色素氧化酶）的基因， 通过长期暴露于细胞，ATPase，RNA聚合酶等 线粒体翻译抑制剂。 目标之一是 表征三个线粒体的高亲和力结合位点 核染色体DNA上的蛋白质并确定其物理 与已知线粒体编码的基因的关系或接近性 使用cDNA克隆的蛋白质，原位与G带染色体的原位杂交 和基因组克隆的映射。 最后，确切的监管角色 三种蛋白质以及基因或基因簇的性质 将在分离核中使用体外转录评估影响 并在带有添加的DNA模板的HeLa细胞提取物中。 基因组区域 对蛋白质显示高特异性将被测序以获得洞察力 在三个线粒体蛋白调节的模式下。