REGULATION OF TRYPANOSOME DNA REPLICATION

有鞭毛的原生动物属于动植体目，含有单一的 线粒体称为动质体体。两个不同的双胞胎种群 单链环状DNA，即上环和小环，是局部化的 在动粒体内形成单一的高度连接的网络。这些 生物体通常是寄生的，并对各种 人类和动物疾病，包括利什曼病，非洲睡眠 疾病和恰加斯病。我们将确定生化机制 参与锥虫DNA微环的复制 束状克里迪亚。这些研究应该为我们提供关于 真核复制机制的一般和可能揭示独特的特征 在发育过程中可以作为靶点的运动基质复制的 化疗药物。 我们将分离并鉴定微环复制中间体 在分离的动质粒中合成，并将开发出一种 小环的复制和分区的机制。复制 可溶提取物中微环的数量将作为一种手段进行研究 确定复制过程中的特定酶步骤和 开发纯化所需的个别酶的分析方法 用来复制小圆圈。在体外生产的复制产品将是 以物理和生化方法为特征的，以阐明 参与整个复制的各种酶反应 机械装置。针对特定成分制备的抗体 复制机制将用于调查以下方面的要求 处于不同复制阶段的各个组件，以确定 它们的细胞定位并为快速检测提供试剂 免疫亲和纯化各种蛋白质。

Flagellated protozoa of the Order Kinetoplastida contain a single mitochondrion termed a kinetoplast. Two distinct populations of double stranded circular DNA, the maxicircles and the minicircles, are localized within the kinetoplast as a single highly catenated network. These organisms are generally parasitic and are responsible for a variety of human and animal diseases including leishmaniasis, African sleeping sickness and Chagas disease. We will determine the biochemical mechanisms involved in the replication of the DNA minicircles of the trypanosomatid Crithidia fasciculata. These studies should provide insight into eukaryotic replication mechanisms in general and may reveal unique features of kinetoplast replication that can be targeted in the development of chemotherapeutic agents. We will isolate and characterize minicircle replication intermediates synthesized in isolated kinetoplasts and will develop a model of the mechanics of the replication and partitioning of minicircles. Replication of minicircles in soluble extracts will be investigated as a means of identifying specific enzymatic steps in the replication process and developing assays for the purification of the individual enzymes required for minicircle duplication. Replication products produced in vitro will be characterized by physical and biochemical methods in order to elucidate the various enzymatic reactions involved in the overall replication mechanisms. Antibodies prepared against specific components of the replication machinery will be used to investigate the requirements for individual components in different stages of replication, to determine their cellular localization and to provide reagents for rapid immunoaffinity purification of the various proteins.