STAGE-SPECIFIC GENES IN LEISHMANIA ENRIETTII

利什曼原虫 ENRIETTII 的阶段特异性基因

• 批准号: 3139608
• 负责人: Scott M Landfear
• 金额: $ 17.51万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139608
• 关键词: Leishmania; autoradiography; developmental genetics; electroporation; environmental adaptation; gene expression; genetic promoter element; genetic regulation; genetic transcription; guinea pigs; histones; immunoelectron microscopy; immunofluorescence technique; laboratory rabbit; life cycle; membrane proteins; microorganism genetics; molecular cloning; molecular genetics; protein sequence; protein structure function; radiotracer; restriction mapping; scintillation spectrometry; southern blotting; transfection; transport proteins; western blottings

Leishmania are parasitic protozoa that cause a major, life-threatening disease which afflicts millions of people living in tropical regions of the globe. The major objective of this proposal is to study several genes in Leishmania parasites whose expression is restricted to the stage of the life cycle that inhabits the insect vector. These genes code for membrane transport proteins which are apparently involved in the parasite's adaption to the environment of the insect gut, probably by allowing the parasite to utilize nutrients which are available in the insect but not in the mammalian host. One of these genes, designated Pro-1, codes for a protein related in sequence known glucose transport proteins from mammals, yeast and bacteria and which probably transports either glucose or another sugar. The ligand that is transported by the Pro-1 protein will be determined by transfecting Leishmania parasites with this cloned gene under conditions which will allow accumulation of many copies of the transformed gene. These transfected parasites will then be assayed for increased ability to transport various radiolabeled sugar ligands, compared to untransfected parasites. In addition, the location of the Pro-1 protein within the parasite will be investigated by immunoelectron microscopy to determine whether it is located in the plasma membrane and whether it is uniformly distributed or localized to a specific region of the membrane. Finally, the transcription of the Pro-1 gene will be investigated to determine where its promoter is located. Understanding transcription of the Pro-1 gene may help reveal how this gene is regulated during the parasite life cycle, being expressed in the insect from parasites but not in the form that infects the mammalian host. Two other genes that code for related membrane transport proteins, designated D1 and D2, will also be studied in detail. Complete genomic copies of D1 and D2 will be isolated and sequenced to determine the deduced amino acid sequence of each protein. The ligands which are transported by D1 and D2 will be determined, either by transfecting parasites with many copies of each gene and assaying for increased transport activity or be disrupting each gene and assaying for loss of specific transport function. Together these studies will advance our understanding of how genes are regulated during the parasite life cycle, allowing the parasite to adapt both its host and insect vector, and of how membrane transport proteins function to allow the parasite to survive in its changing environment.

利什曼原虫是一种寄生原虫， 这种疾病折磨着生活在热带地区的数百万人， 地球仪。 这项计划的主要目的是研究几个基因， 利什曼原虫属寄生虫，其表达限于 寄生在昆虫载体上的生命周期。 这些基因编码膜 显然与寄生虫的适应有关的运输蛋白 昆虫肠道的环境，可能是通过允许寄生虫 利用昆虫体内的营养物质， 哺乳动物宿主 其中一个基因，命名为Pro-1，编码一种蛋白质， 哺乳动物、酵母和细菌中已知的葡萄糖转运蛋白的序列 它可能运输葡萄糖或其他糖。 配体 由Pro-1蛋白质转运的蛋白质将由 在一定条件下用该克隆基因对利什曼原虫进行免疫 这将允许转化基因的许多拷贝的积累。 然后将测定这些转染的寄生虫增加的能力， 与未转染的相比， 寄生虫 此外，Pro-1蛋白在细胞内的位置也是确定的。 寄生虫将通过免疫电子显微镜进行研究，以确定 它是否位于质膜上，以及它是否均匀地 分布或定位于膜的特定区域。 最后， 将研究Pro-1基因的转录，以确定 其启动子位于。 了解Pro-1基因的转录 可能有助于揭示这种基因在寄生虫生命周期中是如何调节的， 在昆虫体内由寄生虫表达， 感染哺乳动物宿主 另外两个编码相关膜转运蛋白的基因， 指定为D1和D2，也将进行详细研究。 全基因组 将分离D1和D2的拷贝并测序，以确定推导的 每种蛋白质的氨基酸序列。 被转运的配体 D1和D2将通过用许多 每个基因的拷贝，并测定增加的转运活性或 破坏每个基因并测定特定运输功能的丧失。 这些研究将共同推进我们对基因是如何 在寄生虫的生命周期中进行调节，使寄生虫能够适应 它的宿主和昆虫载体，以及膜转运蛋白如何 使寄生虫能够在不断变化的环境中生存。