GP46 GENES AND PROTEIN EXPRESSION IN LEISHMANIA CHAGASI

大多数利什曼原虫物种，包括恰加斯利什曼原虫，都拥有 表面丰富的、高度保守的糖蛋白，称为 GP46 或 PSA-2。 GP46 的不同分子量已在 以及不同的利什曼原虫物种之间。 GP46的功能是 未知，但 GP46 已被证明可以对小鼠提供部分保护 对抗亚马逊利什曼原虫的挑战。 在那些利什曼原虫中 在已研究的物种中，GP46 基因组织很复杂 由排列成簇的多个不相同的基因组成。 在初步研究和参考文献 1 中所述，我们发现 在恰加西 L. chagasi 中，44 和 66 kDa 版本的 GP46 丰度增加 毒性前鞭毛体从传染性较低发展为高度传染性时增加 30 倍 传染性形式，但在减毒前鞭毛体中 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），并且在强毒期间两者也会增加 30 倍 前鞭毛体发育。 然而，虽然 44 和 66 kDa GP46 彼此相差不到 2 倍，即 2.8 的丰度 kb RNA 是 4.8 kb RNA 的 40 倍。 核连续运行 实验表明 GP46 mRNA 水平在 转录地。 使用稳定转染的前鞭毛体进行的实验 质粒含有恰加西乳杆菌 GP46 之一的不同区域 基因显示其 3' UTR 赋予发育调节 报告基因/RNA 的丰度。 因此，本次活动的具体目标 项目目标是：（1）确定GP46基因之间的关系 以及不同 GP46 mRNA 和蛋白质的丰度，(2) 确定 GP46 表达调控丧失的分子基础 在减毒前鞭毛体中，(3) 识别并表征顺式和 参与发育调节的反式作用元件 GP46 mRNA 丰度，以及 (4) 确定是否进行预处理、共- RNA 的加工或后加工介导了增加的 感染形式中 GP46 mRNA 的丰度。 这些研究是 旨在识别调节 GP46 的分子过程 表达，我们期望的基本过程需要蛋白质 这将是未来研究的目标，旨在新的治疗方法和 预防内脏利什曼病。

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.