GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

本研究的长期目标是详细了解 转录和转录后的分子机制 人类寄生线虫Brugia的RNA加工基因调控 马来丝虫，一种淋巴丝虫病的病原体，以及蛔虫 蛔虫是最常见的人体肠道寄生虫 初步 实验表明，B.马来语和 蛔虫含有一个共同的反式剪接前导外显子。 在这两种生物中， 前导外显子来源于短的非多聚腺苷酸化RNA（SL-RNA） 从5S rRNA基因座内转录。 SL-RNA是 由RNA聚合酶II转录并具有三甲基鸟苷帽 snRNAs的结构特征。 B.马来语和 蛔虫在无细胞提取物中准确转录， 蛔虫胚胎发育 SL-RNA基因的体外转录物是 通过帽结构的三甲基化进行转录后修饰。 为了确定B中的基因。马来人和蛔虫， 其是反式剪接的，将构建cDNA文库， 链合成由对应于所述寡核苷酸的寡核苷酸引发。 拼接前导 来自反式剪接的cDNA克隆的表征 mRNA将指示是否有相似的特定mRNA谱， 在B中反式剪接。马来人和蛔虫。 使用删除分析和 定点突变，指导起始的序列元件 和B中SL-RNA合成的终止。马来人和蛔虫 定义了 突变分析也将用于确定哪些特征 SL-RNA的三甲基化作用是指导SL-RNA帽的三甲基化。 构建体 将使用含有蛋白质编码基因的5 ′侧翼区的 也作为体外转录的底物。 如果这些结构是 转录，适当的突变技术将用于定义 编码反式剪接mRNA的基因的转录控制元件， 编码非反式剪接的mRNA的基因。 最后，直接RNA 序列分析将用于完全表征B的snRNA。 马来人和蛔虫。 这项研究计划可以提供有关信息 寄生线虫基因表达的基本机制。 等 信息是理解复杂性的必要前提 关于寄生的分子基础的问题，并可能提出新的 控制寄生线虫感染的治疗策略。

The long term goal of this research is to understand, in detail, the molecular mechanisms of transcriptional and post-transcriptional (RNA-processing) gene regulation in the human parasitic nematodes, Brugia malayi, a causative agent of lymphatic filariasis, and Ascaris lumbricoides, the most common human intestinal parasite. In preliminary experiments it has been shown that a subset of mRNAs in B. malayi and Ascaris contain a common trans-spliced leader exon. In both organisms, the leader exon is derived from a short non-polyadenylated RNA (SL-RNA) transcribed from within the 5S rRNA gene locus. The SL-RNAs are transcribed by RNA polymerase II and possess the trimethyl-guanosine cap structure characteristic of snRNAs. The SL-RNA genes of both B. malayi and Ascaris are accurately transcribed in a cell-free extract derived from developing Ascaris embryos. In vitro transcripts of SL-RNA genes are post-transcriptionally modified by trimethylation of their cap structures. To define the genes in B. malayi and Ascaris which give rise to transcripts which are trans-spliced, cDNA libraries will be constructed where second strand synthesis is primed by an oligonucleotide corresponding to the spliced leader. Characterization of cDNA clones derived from trans-spliced mRNAs will indicate whether a similar spectrum of specific mRNAs are trans-spliced in B. malayi and Ascaris. Using deletional analysis and site-directed mutagenesis, the sequence elements which direct initiation and termination of SL-RNA synthesis in B. malayi and Ascaris will be defined. Mutational analysis will also be used to determine which features of the SL-RNAs serve to direct trimethylation of SL-RNA caps. Constructs containing the 5' flanking regions of protein coding genes will be used also as substrates for in vitro transcription. If these constructs are transcribed, appropriate mutational techniques will be used to define transcriptional control elements of genes encoding trans-spliced mRNAs and genes encoding mRNAs which are not trans-spliced. Finally, direct RNA sequence analysis will be used to completely characterize the snRNAs of B. malayi and Ascaris. This research program may provide information relevant to the basic mechanisms of gene expression in parasitic nematodes. Such information is a necessary prerequisite to the understanding of complex questions regarding the molecular basis of parasitism and may suggest novel therapeutic strategies for control of parasite nematode infection.