LEISHMANIA ATPASES--REGULATION OF TRANSCRIPT ABUNDANCE

利什曼原虫ATP酶——转录丰度的调节

• 批准号: 2068591
• 负责人: JOHN CHRISTOPHER MEADE
• 金额: $ 12.01万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2068591
• 关键词: DNA binding protein; DNA footprinting; Leishmania; RNA biosynthesis; RNase protection assay; adenosinetriphosphatase; denaturing gradient gel electrophoresis; developmental genetics; electroporation; enzyme structure; gene expression; genetic mapping; genetic regulatory element; genetic transcription; messenger RNA; microorganism culture; microorganism genetics; microorganism growth; molecular cloning; northern blottings; nuclear runoff assay; nucleic acid sequence; polymerase chain reaction; reporter genes; southern blotting; transfection

Leishmania parasites exist as flagellated promastigote forms in the sandfly vector and as obligate intracellular amastigotes within the acidic environment of host phagolysosomal vacuoles. The environmental habitats of these two developmental forms are extremely different and adaptation to the shift in environment requires a dramatic morphological and physiological transformation. The molecular basis for regulation of this event as well as the molecular process of gene transcription in Leishmania is poorly understood. This proposal examines the control of transcript abundance in a tandemly linked pair of developmentally regulated ion transporting ATPase genes as a model for Leishmania gene transcription and molecular regulation during transformation. Two in vitro culture systems for Leishmania differentiation are used; the genesis of an infective promastigote form in late stationary phase cultures of L. donovani, and the transformation of a xenically grown L. m. pifanoi amastigotes to promastigotes. Initial work will focus on the organization, sequence and expression of the ATPase locus in both species. The 5' and 3' ends of ATPase transcripts will be mapped and sequenced using RNase protection, primer extension and PCR techniques. The effect of mRNA stability on transcript abundance will be measured by mRNA half life determinations (pulse chase) and the size of the ATPase transcription unit estimated by nuclear run-on and UV inactivation assays. ATPase sequences implicated in transcript control will be joined to reporter genes, transfected into Leishmania, and reporter gene expression assayed during transformation. Successive deletion of the ATPase sequences and measurement of the effects on reporter gene expression in differentiating cultures will define regions involved in control of transcription in Leishmania developmental forms. This data will be confirmed with gel retardation experiments and DNA footprinting which can localize DNA binding proteins to specific ATPase sequences. Leishmania is a significant human pathogen. Better understanding of the mechanisms controlling gene transcription during parasite development will potentiate new strategies for the design of anti-leishmanial drugs.

利什曼原虫作为鞭毛前鞭毛体形式存在于 白蛉载体和作为专性细胞内无鞭毛体内 宿主吞噬溶酶体空泡的酸性环境。 环境 这两种发展形式的栖息地是非常不同的， 为了适应环境的变化， 和生理转变。 调节的分子基础 这一事件以及基因转录的分子过程， 对利什曼原虫的了解很少。 本建议探讨管制 发育上串联的一对转录本丰度 作为利什曼原虫基因模型的调节离子转运ATP酶基因 转化过程中的转录和分子调控。 两 使用用于利什曼原虫分化的体外培养系统; 稳定期晚期感染性前鞭毛体的发生 L.的培养物donovani，以及对异源生长的L. M. pifanoi无鞭毛体到前鞭毛体。 初期工作将侧重于 组织，序列和表达的ATP酶基因座在这两个 物种 ATP酶转录物的5'和3'末端将被定位， 使用RNA酶保护、引物延伸和PCR技术测序。 mRNA稳定性对转录本丰度的影响将通过以下方法测量： mRNA半衰期测定（脉冲追踪）和ATP酶的大小 通过核运行和UV灭活估计转录单位 分析。 参与转录控制的ATP酶序列将被连接到 报告基因转染利什曼原虫， 在转化期间测定表达。 连续删除 ATP酶序列及其对报告基因影响的测定 在分化的文化中的表达将定义涉及 利什曼原虫发育形式的转录控制。 该数据 将通过凝胶阻滞实验和DNA足迹来证实 其可以将DNA结合蛋白定位于特定的ATP酶序列。 利什曼原虫是一种重要的人类病原体。 更好地理解 寄生虫发育过程中控制基因转录的机制 将为抗利什曼原虫药物的设计提供新的策略。