GP46 GENES AND PROTEIN EXPRESSION IN LEISHMANIA CHAGASI

恰加斯利什曼原虫中 GP46 基因和蛋白质表达

• 批准号: 6373830
• 负责人: John E Donelson
• 金额: $ 20.24万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6373830
• 关键词: Leishmania; RNA binding protein; RNase protection assay; SDS polyacrylamide gel electrophoresis; affinity chromatography; developmental genetics; gel mobility shift assay; gene expression; glycoproteins; hamsters; intracellular parasitism; leishmaniasis; life cycle; messenger RNA; microorganism immunology; molecular cloning; molecular pathology; nuclear runoff assay; posttranscriptional RNA processing; posttranslational modifications; protein structure function; regulatory gene; southern blotting; transcription factor; virulence; western blottings

Most Leishmania species, including Leishmania chagasi, possess on their surface an abundant, highly conserved, glycoprotein called GP46 or PSA-2. Different molecular weights for GP46 have been reported within and among different Leishmania species. The function of GP46 is unknown, but GP46 has been shown to confer partial protection of mice against challenge with Leishmania amazonensis. In those Leishmania species where it has been studied, the GP46 gene organization is complex and consists of multiple, non-identical, genes arranged in clusters. In Preliminary studies and as described in reference 1, we found that in L. chagasi the abundance of 44 and 66 kDa versions of GP46 increases 30-fold as virulent promastigotes develop from less infectious to highly infectious forms, but in attenuated promastigotes the GP46 abundance does not increase during development. Two species of GP46 RNA exist (2.8 and 4.8 kb), and both also increase 30-fold during virulent promastigote development. However, whereas the abundances of the 44 and 66 kDa GP46 are within 2-fold of each other, the abundance of the 2.8 kb RNA is 40-fold more than that of the 4.8 kb RNA. Nuclear run-on experiments indicate that the GP46 mRNA levels are regulated post- transcriptionally. Experiments using promastigotes stably transfected with plasmids containing various regions of one of the L. chagasi GP46 genes showed that its 3' UTR confers a developmentally regulated abundance to a reporter gene/RNA. Thus, the specific aims of this project are to: (1) determine the relationships between the GP46 genes and the abundances of the different GP46 mRNAs and proteins, (2) determine the molecular basis for the loss of regulated GP46 expression in attenuated promastigotes, (3) identify and characterize cis- and trans-acting elements that participate in the developmental regulation of GP46 mRNA abundance, and (4) determine whether pre-processing, co- processing or post-processing of the RNA mediates the increased abundance of GP46 mRNA in the infectious form. These studies are intended to identify the molecular processes that regulate GP46 expression, fundamental processes that we expect will require proteins which will be targets for future research aimed at new treatments and prevention of visceral leishmaniasis.

大多数利什曼原虫物种，包括利什曼原虫Chagasi，都拥有 表面一种称为gp46或的丰富，高度保守的糖蛋白 PSA-2。 据报道，GP46的分子量不同 在不同的利什曼原虫物种中。 GP46的功能是 未知，但GP46已显示出对小鼠的部分保护 反对利什曼尼亚亚马逊人的挑战。 在那些利什曼尼亚 研究了该物种，GP46基因组织很复杂 并由簇中排列的多个，非相同的基因组成。 在初步研究中，如参考文献1所述，我们发现 在L. Chagasi中，GP46的44和66 kDa版本的丰度增加 30倍，因为有毒的前寄生物从不太感染到高度发展 传染性形式，但在衰减的前毒体中gp46丰度 在开发过程中不会增加。 存在两种GP46 RNA （2.8和4.8 kb），在有毒期间都增加了30倍 Promastigote的发展。 但是，尽管44和 66 kDa GP46彼此2倍以内，2.8的丰度 KB RNA比4.8 kb RNA的RNA多40倍。 核跑步 实验表明，GP46 mRNA水平在 - 转录。 使用前寄生虫稳定转染的实验 存在的质粒含有Chagasi L. gp46的各个区域 基因表明，其3'UTR赋予了一个受发展的监管 对记者基因/RNA的丰度。 因此，这是特定目标 项目是：（1）确定GP46基因之间的关系 以及不同的GP46 mRNA和蛋白质的丰度，（2） 确定丢失受调节GP46表达的分子基础 在衰减的前差，（3）识别和表征顺式和表征 参与发育法规的跨作用元素 gp46 mRNA丰度，（4）确定是否进行预处理 RNA的处理或后处理介导了增加 传染性形式的GP46 mRNA丰度。 这些研究是 旨在确定调节GP46的分子过程 表达，我们期望需要蛋白质的基本过程 这将是针对新疗法的未来研究的目标 预防内脏利什曼病。