STAGE-SPECIFIC GENES IN LEISHMANIA ENRIETTII

利什曼原虫 ENRIETTII 的阶段特异性基因

• 批准号: 3139605
• 负责人: Scott M Landfear
• 金额: $ 16.66万
• 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 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139605
• 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; 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生动物，它会引起一种严重的、危及生命的 这种疾病折磨着生活在热带地区的数百万人 环球网。这项建议的主要目标是研究人类的几个基因 利什曼原虫，其表达仅限于 生活在昆虫媒介中的生命周期。这些基因编码细胞膜 明显参与寄生虫适应的运输蛋白 到昆虫肠道的环境，可能是通过允许寄生虫 利用昆虫体内可获得但昆虫体内没有的营养物质 哺乳动物宿主。 其中一个基因被命名为Pro-1，编码一种与 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另一种糖。配基 由Pro-1蛋白运输的蛋白质将由 用该克隆基因在一定条件下转染利什曼原虫 这将使转化基因的许多拷贝积累起来。 然后将对这些转基因寄生虫进行检测，以确定它们是否具有增强的 运输各种放射性标记的糖配体，与未转染组比较 寄生虫。此外，Pro-1蛋白在细胞内的位置 寄生虫将通过免疫电子显微镜进行研究，以确定 它是否位于质膜中，是否均匀 分布或定位于膜的特定区域。最后， 将研究Pro-1基因的转录，以确定在哪里 它的启动子被定位。理解Pro-1基因的转录 可能有助于揭示该基因在寄生虫生命周期中是如何受到调控的， 由寄生虫在昆虫体内表达的，但不是以 感染哺乳动物的宿主。 另外两个编码相关膜运输蛋白的基因， 此外，我们亦会详细研究编号为d1和d2的。全基因组 对d1和d2的拷贝进行分离和测序，以确定推导出的 每种蛋白质的氨基酸序列。由其运输的配体 D1和d2将被确定，要么通过将许多寄生虫 每个基因的拷贝并分析增加的转运活性或BE 扰乱每个基因，并分析特定运输功能的丧失。 总之，这些研究将促进我们对基因是如何 在寄生虫生命周期中进行调节，允许寄生虫适应 寄主和昆虫媒介，以及膜如何运输蛋白质 功能，使寄生虫能够在不断变化的环境中生存。