KINETOPLAST DNA ASSOCIATED PROTEINS IN KINETOPLASTID PROTOZOA

动质体原生动物中动质体 DNA 相关蛋白

• 批准号: 6107057
• 负责人: MANUEL SEVERO VALENZUELA
• 金额: $ 7.35万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107057
• 关键词: DNA binding protein; DNA replication; Leishmania donovani; SDS polyacrylamide gel electrophoresis; Trypanosoma brucei rhodesiense; Trypanosoma cruzi; antibody; circular DNA; laboratory rabbit; mitochondrial DNA; molecular cloning; organelles; protein structure function; protozoa

Members of the order Kinetoplastida, which include trypanosomes and leishmania, share one of the most unusual mitochondrial DNA structures found in nature. The single mitochondria present in these organisms contains a relatively large proportion of the cellular DNA (kinetoplast DNA, kDNA) organized in a disk-shaped structure and made up of a population of two types of topologically linked circular molecules. In spite of the recent advances made in our understanding of the mechanism of replication of the major DNA component of the networks, some outstanding questions about the structure and function of kDNA networks still remain unanswered. For example, it is not clear what mitochondrial function the network structure serves, nor how it is partitioned at the end of replication. The maintenance and possibly the function of kDNA may depend on some proteins components of the networks. Our preliminary studies with Trypanosoma cruzi indicate that several proteins remain associated with kDNA networks after exposure of trypanosome cells to a mild detergent in presence of 2M NaCl. Rabbit antibodies raised against these T.cruzi kDNA-protein complexes recognize some protein(s) that appear to be bound to the supercoiled form of T.cruzi minicircle DNA. More interestingly, using both immunofluorescence and immunoelectron microscopy, we have observed that the same antibody preparation cross-reacts with kinetoplast DNA present in related kinetoplastid protozoa. Based on these observations, the present research proposal is designed to unravel some basic features about the structure and function of kinetoplast DNA networks, by investigating the function of some of the proteins associated with kDNA. We propose first, to identify and characterize proteins bound to T.cruzi minicircles; second, to identify common kDNA-associated proteins (KAPs) present in kDNA network preparations from other kinetoplastid protozoa; and third, to isolate and clone the genes encoding for selected KAPs. By understanding the structure and function of kDNA-associated proteins we may learn a great deal about how kDNA networks are maintained and propagated. This information may also provide us with a more rational approach toward the complete eradication of these parasites, some of which are the causative agents of several important human diseases.

动质体目的成员，包括锥虫和 利什曼原虫，共享最不寻常的线粒体DNA结构之一， 在自然界中发现的。存在于这些生物体中的单个线粒体 含有相对大比例的细胞DNA（动基体 DNA，kDNA）以盘状结构组织，由 两种拓扑连接的环状分子的群体。 尽管最近我们在理解上取得了进展， 网络的主要DNA成分的复制机制， 关于kDNA结构和功能的一些悬而未决的问题 网络仍然没有答案。例如，不清楚什么是 线粒体功能的网络结构服务，也不知道它是如何 在复制结束时进行分区。维护，可能还有 kDNA的功能可能取决于某些蛋白质成分的 网络.我们对克氏锥虫的初步研究表明， 几种蛋白质在暴露后仍然与kDNA网络相关联 在2 M NaCl存在下，将锥虫细胞溶解于温和的洗涤剂中。 针对这些克氏锥虫kDNA-蛋白复合物产生的兔抗体 识别一些似乎与超螺旋结合的蛋白质 克氏锥虫小环DNA更有意思的是， 免疫荧光和免疫电子显微镜，我们已经观察到， 相同抗体制剂与存在的动基体DNA交叉反应 在相关的动质体原生动物中。根据这些观察， 目前的研究计划旨在揭示一些基本特征 关于动质体DNA网络的结构和功能， 研究一些蛋白质的功能， kDNA 我们建议首先识别和表征与 克氏锥虫小环;第二，识别常见的kDNA相关的 蛋白质（KAP）存在于来自其他的kDNA网络制备物中 第三，分离和克隆基因 为选定的KAP进行编码。 通过了解kDNA相关蛋白的结构和功能， 我们可能会学到很多关于kDNA网络是如何维持的， 传播。这些信息也可能为我们提供一个更合理的 为了彻底根除这些寄生虫， 它们是几种重要的人类疾病的病原体。