EXPRESSION OF THE LEISHMANIA TARENTOLAE MINI EXON GENE

动质体原生动物如利什曼原虫属，布氏锥虫和 克氏锥虫引起人类和牲畜的多种疾病。 寄生虫通过添加一个共同的 39-编码蛋白质的核苷酸序列（称为小外显子） 前体RNA在反式剪接反应中。 反式剪接还没有被 在这些寄生虫的哺乳动物宿主中鉴定，因此， 该过程是寄生虫特异性的，并且是抗寄生虫的选择性靶标。 攻击 这项研究的长期目标是了解 基因的表达。 因为小外显子 在所有这些细胞中，作为短的、离散的前体（称为medRNA）合成， 物种，这项建议的研究旨在了解 合成medRNA（在模式生物利什曼原虫tarentolae中）。 将定义小外显子基因的必需启动子元件。 的 一个克隆的小外显子基因的上游区域，它已被标记为 40-bp标签，将进行1）接头扫描诱变和2） -67/-58区域的点突变分析。 突变基因包含在 将稳定的转染载体pX导入L.塔伦托拉埃比 电穿孔 附加体基因的转录将通过 北方印迹、核酸酶保护、引物延伸和新生RNA 分析。 在凝胶中，突变基因也将被用作模板， 迁移率变化分析，以确定蛋白质结合的位点。 与必需的-67/-58区域结合的蛋白质也将被 以两种互补的方法为特征。 DNA结合蛋白序列 将从已经通过以下方法分离的cDNA克隆推断： 它们的结合是“-67/58”双链寡核苷酸。 原生 还将通过柱从核提取物中纯化蛋白质 层析 蛋白质的纯化将由 凝胶迁移率变动分析。 所提议的实验将确定必需的上游启动子 利什曼原虫小外显子基因的元件。 它还将描述 与必需元件相关的潜在转录因子 这个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.