EXPRESSION OF CDNA FOR HUMAN ORNITHINE TRANSCARBAMYLASE

鸟氨酸转氨甲酰酶（OTC）是一种线粒体酶， 周期，编码在X染色体上，其遗传缺陷导致 严重的，往往是致命的氨中毒，在受影响的人。 的 建议的研究是针对1）了解系统的 线粒体区室化，由此线粒体蛋白质前体， 在细胞核中编码并在细胞质多聚核糖体上合成， 由线粒体识别后，跨一个或多个线粒体易位， 两种膜，并蛋白水解加工成其活性形式;和2） 发展肝酶缺乏症的基因疗法。 在这两个领域 所采取的实验方法将涉及 操作克隆的编码人的生物活性cDNA序列 OTC。 对线粒体输入的研究主要集中在以下几个方面： 存在于细胞质合成的OTC前体中的序列， 是其进口所必需的;研究假定的细胞质因子， 参与OTC识别的外膜受体分子 前体线粒体;和澄清是否一个或两个 需要NH 2-末端蛋白水解加工步骤来产生 成熟OTC亚基。 NH 2-末端前导序列的编码序列 部分，编程用于在真核细胞中表达，将与 一种细胞质酶，以确定是否单独的领导人是 足以引导线粒体输入。 的编码序列 整个OTC前体，编程表达，将受到 缺失和点突变改变，以及对导入的影响 测定了 前体编码序列也将被编程用于高表达。 在真核和原核细胞中水平表达， 分离前体用作结合中的底物， 蛋白水解研究。 基因替代实验的目的是提供 足够的额外肝脏OTC活性，由引入的 顺序，以允许纠正缺乏氮催化剂， OTC缺陷小鼠。 OTC cDNA序列将被编程用于 在克隆的逆转录病毒基因组中表达，该基因组将被包装到 逆转录病毒颗粒 这些颗粒将用于感染缺乏OTC的 分析小鼠胚胎和这些动物的肝脏中是否存在 引入的序列，和酶活性人OTC。

Ornithine transcarbamylase (OTC) is a mitochondrial enzyme of the urea cycle, encoded on the X chromosome, whose inherited deficiency leads to severe, often lethal, ammonia intoxication in affected humans. The proposed studies are directed 1) to understanding the system of mitochondrial compartmentation, whereby mitochondrial protein precursors, encoded in the nucleus and synthesized on cytoplasmic polyribosomes, are posttranslationally recognized by mitochondria, translocated across one or both membranes, and proteolytically processed to their active forms; and 2) to developing gene therapy for hepatic enzyme deficiencies. In both areas of investigation the experimental approaches taken will involve manipulation of the cloned biologically active cDNA sequence encoding human OTC. The studies of mitochondrial import are aimed at: definition of the sequences present in the cytoplasmically-synthesized precursor of OTC that are required for its import; study of putative cytoplasmic factors and outer membrane receptor molecules involved with recognition of the OTC precursor by mitochondria; and clarification of whether one or two NH2-terminal proteolytic processing steps are required to produce the mature OTC subunit. The coding sequence of the NH2-terminal leader portion, programmed for expression in eukaryotic cells, will be joined with that of a cytoplasmic enzyme to determine whether the leader alone is sufficient to direct mitochondrial import. The coding sequence of the entire OTC precursor, programmed for expression, will be subjected to deletion and point mutational alteration, and the effects upon import measured. The precursor coding sequence will also be programmed for high level expression in both eukaryotic and prokaryotic cells, enabling isolation of the precursor for use as a substrate in binding and proteolysis studies. Gene replacement experiments will aim to provide sufficient additional hepatic OTC activity, expressed from an introduced sequence, to permit correction of deficient nitrogen catabolism in OTC-deficient mice. The OTC cDNA sequence will be programmed for expression in a cloned retroviral genome that will be packaged into retroviral particles. The particles will be used to infect OTC-deficient mouse embryos and the liver of these animals analyzed for presence of the introduced sequence, and for enzymatically active human OTC.