EXPRESSION OF THE LEISHMANIA TARENTOLAE MINI EXON GENE

动体原生动物，如利什曼原虫、布氏锥虫和 克氏锥虫会引起人和牲畜的多种疾病。 寄生虫产生成熟的信使RNA是通过添加共同的 编码蛋白质的39个核苷酸序列(称为微型外显子) 反式剪接反应中的前体RNA。反式剪接还没有被 在这些寄生虫的哺乳动物宿主中发现，因此 该过程是特定于寄生虫的，是抗寄生虫的选择性目标 袭击。 本研究的长期目标是了解心力衰竭的共同机制 这些致病原虫的基因表达。因为迷你外显子是 作为一种短的、离散的前体(称为MedRNA)在所有这些 物种，这项建议中的研究旨在理解 MedRNA的合成(在模式生物塔氏利什曼原虫中)。 将定义微小外显子基因的基本启动子元件(S)。这个 克隆的迷你外显子基因的上游区域，该区域已被标记为 40个碱基的标签，将受到1)接头扫描突变和2) -67/-58区点突变分析。包含的突变基因 将稳定的转导载体px通过以下途径导入狼尾藻 电穿孔。上体基因的转录将通过以下方式进行监测 Northern杂交、核酸酶保护、引物延伸和新生RNA 分析。突变的基因也将被用作凝胶中的模板 迁移率改变分析，以确定蛋白质结合的位置。 与Essential-67/-58区域结合的蛋白质也将是 以两种互补的方法为特点。DNA结合蛋白序列 将从已分离的cdna克隆中推断出 它们结合了一个“-67/58”双链寡核苷酸。原生的 蛋白质(S)也将通过柱层析从核提取液中纯化 层析法。蛋白质(S)的纯化将由 凝胶迁移率-位移分析。 拟议中的实验将定义重要的上游启动子 利什曼原虫迷你外显子基因的元件。它还将表现出 与必需元件相关的潜在转录因子 这种重要的寄生虫特异性基因。

Kinetoplastid protozoa such as Leishmania spp., Trypanosoma brucei and Trypanosoma cruzi cause a variety of diseases in humans and livestock. The parasites generate mature messenger RNA by the addition of a common 39-nucleotide sequence (termed the mini-exon) to protein-encoding precursor RNAs in a trans-splicing reaction. Trans-splicing has not been identified in the mammalian hosts of these parasites, therefore the process is parasite-specific and a selective target for anti-parasite attacks. The long term aim of this research is to understand common mechanisms of gene expression in these pathogenic protozoa. Because the mini-exon is synthesized as a short, discrete precursor (termed medRNA) in all these species, the research in this proposal is directed to understanding the synthesis of medRNA (in the model organism Leishmania tarentolae). Essential promoter element(s) of the mini-exon gene will be defined. The upstream region of a cloned mini-exon gene, which has been marked with a 40-bp tag, will be subjected 1) to linker-scanning mutagenesis and 2) point mutation analysis of the -67/-58 region. Mutated genes contained on the stable transfection vector pX will be introduced into L. tarentolae by electroporation. Transcription of the episomal genes will be monitored by Northern blotting, nuclease protection, primer extension and nascent RNA analyses. Mutated genes will also be used as templates, in the gel mobility-shift assay, to define the sites of protein binding. Proteins that bind to the essential -67/-58 region will also be characterized by two complementary methods. DNA-binding protein sequence will be inferred from cDNA clones that have been isolated by virtue of their binding a "-67/58" double-stranded oligonucleotide. Native protein(s) will also be purified from nuclear extracts by column chromatography. Purification of the protein(s) will be monitored by the gel mobility-shift assay. The proposed experiments will define the essential upstream promoter element of the Leishmania mini-exon gene. It will also characterize potential transcription factors that associate with the essential element of this important, parasite-specific gene.