GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

这项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 淋巴丝虫病的病原体马来和蛔虫 蛔虫，人类最常见的肠道寄生虫。在预赛中 实验表明，马来丝虫和马来丝虫中的mRNAs的一个子集 蛔虫含有一个常见的反式剪接的前导外显子。在这两种生物中， 前导外显子来源于一个短的非多聚腺苷RNA(SL-RNA) 从5S rRNA基因座转录而来。SL-RNAs是 由RNA聚合酶II转录，并具有三甲基鸟苷帽子 小核糖核酸的结构特征。马来丝虫和马来丝虫的SL-RNA基因 蛔虫在一种无细胞提取物中准确转录，这种提取物来源于 发育中的蛔虫胚胎。SL-RNA基因的体外转录产物是 通过其帽子结构的三甲基化进行转录后修饰。 确定马来丝虫和蛔虫中产生转录本的基因 这些都是反式剪接的，将在第二个 链的合成是由对应于 拼接引线。反式剪接获得的cDNA克隆的鉴定 MRNAs将指示类似谱的特定mRNAs是否 在马来丝虫和蛔虫中反式剪接。使用缺失分析和 定点突变，即直接启动的序列元件 并终止马来丝虫和蛔虫中SL-RNA的合成 已定义。突变分析也将用于确定哪些特征 的SL-RNA用于直接SL-RNA帽的三甲基化。构造 将使用包含蛋白质编码基因5‘侧翼区的 也可作为体外转录的底物。如果这些构造是 转录后，将使用适当的突变技术来定义 转录调控元件编码反式剪接的mRNAs和 编码非反式剪接的mRNA的基因。最后，直接RNA 序列分析将被用来完整地描述B。 马来和蛔虫。该研究计划可能会提供相关信息 寄生线虫基因表达的基本机制。是这样的 信息是理解复杂事物的必要前提 关于寄生的分子基础的问题，可能会提出新的 控制寄生虫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.