Stage-specific Flagellar genes of Leishmania

该提案描述了一种探索小说的方法， 利什曼原虫基因调控机制的研究 原生动物和最古老的真核生物谱系之一。 一 大量证据表明转录起始 可能不是基因表达的主要调控点， 利什曼原虫 一个有吸引力的替代模型可能是， 通过调节反式剪接来调节表达。 的 利什曼原虫核mRNA的成熟是通过一个强制性的 反式剪接事件，其中三甲基-G加帽的小外显子被 连接到位于靠近5'末端的剪接受体位点， 每个编码序列在多顺反子前体RNA中。 因此，我们认为， 剪接受体位点而不是启动子决定了 成熟mRNA。 反式剪接将是一个理想的点， 调控基因的表达被限制在一个阶段， 利什曼原虫的生命周期 编码前鞭毛体特异性 将分离利什曼原虫鞭毛的组分。 前鞭毛体 特定基因将通过序列分析表征， 将分离基因组克隆。 Cis元素负责 鞭毛基因的前鞭毛体特异性表达将是 并确定其作用方式。 序列 负责特定阶段表达模式的是 通过检测嵌合构建体来鉴定， 分离的阶段特异性基因侧翼的调节序列， 附加到报告基因上，进入无鞭毛体样和 前鞭毛体形成。 缺乏个体的突变寄生虫系 将构建鞭毛以测试功能重要性 克隆的基因。 突变寄生虫系的分析将是 结合超微结构和荧光显微镜 考试，以了解删除的 基因. %%% 利什曼原虫在其生命周期中有两个不同的阶段， 宿主并进化出一种形态上独特的 在每一个生存。 无鞭毛体的形式是最适合生长的 在哺乳动物巨噬细胞的溶酶体区室中， 前鞭毛体形式在肠道中细胞外增殖， 白蛉媒介。 每个阶段的独特属性必须反映 阶段特异性基因的表达，但到目前为止， 这些基因的表达是如何被调控的。 结果 这项研究的结果应该更深入地了解这种阶段性的 基因表达的调控。

This proposal describes an approach to exploring novel mechanisms of gene regulation in Leishmania, a Trypanosomatid protozoan and one of the most ancient of eukaryotic lineages. A number of lines of evidence suggest that transcription initiation may not be the major regulatory point for gene expression in Leishmania. An attractive alternative model might be that gene expression is modulated by regulation of trans-splicing. The maturation of Leishmania nuclear mRNA proceeds via an obligatory trans-splicing event in which a trimethyl-G capped miniexon is ligated to splice acceptor sites located close to the 5' end of each coding sequence in a polycistronic precursor RNA. Therefore, splice acceptor sites rather than promoters determine the 5' end of mature mRNAs. Trans-splicing would be an ideal point for the regulation of genes whose expression is restricted to one stage of the Leishmania lifecycle. Genes encoding promastigote-specific components of Leishmania flagella will be isolated. Promastigote specific genes will be characterized by sequence analysis and genomic clones will be isolated. Cis elements responsible for promastigote specific expression of flagellar genes will be identified and their mode of action determined. Sequences responsible for the stage-specific pattern of expression will be identified by transfecting chimeric constructs in which potential regulatory sequences flanking isolated stage-specific genes are appended to a reporter gene into both amastigote-like and promastigote forms. Mutant parasite lines lacking individual flagellar will be constructed to test for the functional importance of cloned genes. Analysis of mutant parasite lines will be coupled with ultrastructural and fluorescence microscopic examination to develop an understanding of the function of deleted genes. %%% Leishmania has two distinct stages in its life cycle with different hosts and has evolved a morphologically distinct form specialized for survival in each. The amastigote form is optimized for growth in the lysosomal compartment of mammalian macrophage and the promastigote form multiplies extracellularly in the gut of the sandfly vector. The unique properties of each stage must reflect the expression of stage-specific genes but as yet, little is known about how expression of such genes might be regulated. The results of this study should give greater insight into such stage-specific regulation of gene expression.