GENES FOR T CRUZI KINETOPLAST DNA ASSOCIATED PROTEINS

T Cruzi 动质体 DNA 相关蛋白的基因

• 批准号: 2665075
• 负责人: MANUEL SEVERO VALENZUELA
• 金额: $ 10.88万
• 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: 1998-09-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2665075
• 关键词: DNA binding protein; Trypanosoma cruzi; animal tissue; gene expression; genetic library; immunoelectron microscopy; immunofluorescence technique; nucleic acid sequence; restriction mapping; western blottings

Members of the order Kinetoplastida which include Trypanosoma and Leishmania are responsible for major tropical diseases. These flagellated protozoa 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 recent advances made in our understanding of kDNA replication, outstanding questions remain about this complex DNA structure. The major goal of our research is to contribute to a better understanding about the structure and function of kDNA networks. We wish to argue that specific protein-kDNA interactions play a key role in the maintenance and segregation of these structures. Therefore, the identification and characterization of kDNA-associated proteins (KAPS) may provide some information about the function of these unusual DNA structures. We have focused our attention on a set of non-histone-like proteins that remain associated to Trypanosoma cruzi kDNA after a 2M NaCl extraction of kDNA in the presence of a mild detergent. Using immuno-electron microscopy and fluorescent microscopy we have observed that rabbit antisera raised again these proteins, recognize epitopes localized in the kinetoplast of T. cruzi cells. More interestingly, the same antibody preparation cross-reacts with similarly localized epitopes in Leishmania amazonensis and Trypanosoma brucei. Based on these observations, the present research proposal attempts to identify and characterize T. cruzi KAPS. We propose first, to screen a T. cruzi cDNA expression library with the antibody preparation described above and second, to select and characterize clones encoding putative T. cruzi KAPs. By understanding the structure and function of T. cruzi KAPs 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 T. cruzi the causative agent of American trypanosomiasis.

包括锥虫和 利什曼尼亚（Leishmania）负责主要的热带疾病。 这些 鞭毛原生动物共享最不寻常的线粒体DNA之一 自然界中发现的结构。 其中存在的单个线粒体 生物体包含相对较大的细胞DNA， 动力学DNA（kDNA），以圆盘形结构组织，并制成 在两种类型的拓扑连接的圆形的人群中 分子。 尽管我们对我们的理解取得了最新的进步 kDNA复制，关于这种复杂DNA仍然存在的出色问题 结构。我们研究的主要目标是为更好 了解KDNA网络的结构和功能。我们希望 认为特定的蛋白质-KDNA相互作用在 这些结构的维护和隔离。因此， KDNA相关蛋白（KAP）的鉴定和表征 可能会提供有关这些不寻常DNA功能的一些信息 结构。 我们将注意力集中在一组类似于内的人上 在2M之后与Cruzi kDNA保持联系的蛋白质 在温和清洁剂存在下kDNA的NaCl提取。 使用 免疫电子显微镜和荧光显微镜我们已经观察到 兔抗血清再次提高了这些蛋白质，识别表位 位于克鲁齐细胞的动力学中。 更有趣的是， 相同的抗体制备与类似局部表位的交叉反应 在利什曼尼亚亚马逊菌和布鲁氏锥虫。 基于这些 观察结果，本研究建议试图识别和 特征T. Cruzi Kaps。 我们首先建议筛选T. cruzi cDNA 具有上述抗体制备的表达式库， 第二，选择和表征编码推定的T. cruzi的克隆 卡普斯。通过了解T. cruzi Kaps的结构和功能我们 可能会了解有关如何维护kDNA网络的大量知识和 传播。 这些信息也可能为我们提供更合理的 彻底根除克鲁兹的方法 美国锥虫病。