Stage-specific Flagellar genes of Leishmania

本提案描述了一种探索利什曼原虫基因调控新机制的方法，利什曼原虫是一种锥虫原虫，也是最古老的真核生物谱系之一。许多证据表明，转录起始可能不是利什曼原虫基因表达的主要调控点。一个有吸引力的替代模型可能是基因表达是由反式剪接调节的。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进行的，在这个过程中，一个三甲基g盖帽的小外显子连接到位于多顺反子前体RNA中每个编码序列5'端附近的剪接受体位点。因此，是剪接受体位点而不是启动子决定了成熟mrna的5'端。反式剪接将是一个理想的基因调控点，其表达仅限于利什曼原虫生命周期的一个阶段。编码鞭毛利什曼原虫特异性成分的基因将被分离出来。通过序列分析鉴定Promastigote特异性基因，并分离基因组克隆。将鉴定负责鞭毛基因的promastigote特异性表达的Cis元件，并确定其作用模式。负责阶段特异性表达模式的序列将通过转染嵌合构建体来确定，嵌合构建体将分离的阶段特异性基因侧翼的潜在调控序列附加到报告基因上，形成无尾线虫和promastigote形式。将构建缺乏鞭毛的寄生虫突变系，以测试克隆基因的功能重要性。对突变体的分析将结合超微结构和荧光显微镜检查，以了解缺失基因的功能。利什曼原虫在其生命周期中有两个不同的阶段，有不同的宿主，并已进化出一种形态上不同的形式，专门用于在每个阶段生存。无尾鞭毛虫最适合在哺乳动物巨噬细胞的溶酶体腔室中生长，而原鞭毛虫则在白蛉载体的肠道细胞外繁殖。每个发育阶段的独特特性必然反映了发育阶段特异性基因的表达，但迄今为止，人们对这些基因的表达如何受到调控知之甚少。这项研究的结果应该让我们更深入地了解这种基因表达的阶段特异性调控。

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.