LEISHMANIA ATPASES--REGULATION OF TRANSCRIPT ABUNDANCE

利什曼原虫ATP酶——转录丰度的调节

• 批准号: 3456282
• 负责人: JOHN CHRISTOPHER MEADE
• 金额: $ 6.93万
• 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/3456282
• 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.

利什曼原虫寄生虫以有鞭毛的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 宿主吞噬溶酶体液泡的酸性环境。 环境 这两种发育形式的栖息地截然不同 适应环境的变化需要戏剧性的形态变化 和生理转变。 调控的分子基础 这一事件以及基因转录的分子过程 人们对利什曼原虫知之甚少。 该提案审查了控制 串联连接的一对发育中的转录丰度 调节离子转运 ATP 酶基因作为利什曼原虫基因模型 转化过程中的转录和分子调控。 二进 使用体外培养系统进行利什曼原虫分化；这 稳定期晚期感染性前鞭毛体形式的起源 L. donovani 的培养物，以及异种生长的 L. donovani 的转化。 米。 pifanoi 无鞭毛体到前鞭毛体。 初步工作将集中于 ATP酶基因座的组织、序列和表达 物种。 ATPase 转录物的 5' 和 3' 末端将被映射并 使用 RNase 保护、引物延伸和 PCR 技术进行测序。 mRNA 稳定性对转录本丰度的影响将通过以下方式测量 mRNA 半衰期测定（脉冲追踪）和 ATP 酶的大小 通过核连续和紫外线灭活估计转录单位 化验。 参与转录控制的 ATP 酶序列将被连接 转染至利什曼原虫和报告基因 在转化过程中测定表达。 连续删除 ATP酶序列和对报告基因影响的测量 不同文化中的表达将定义涉及的区域 利什曼原虫发育形式的转录控制。 这个数据 将通过凝胶阻滞实验和 DNA 足迹来证实 它可以将 DNA 结合蛋白定位到特定的 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 将加强抗利什曼病药物设计的新策略。