GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

人类寄生线虫的基因表达

• 批准号: 3143366
• 负责人: TIMOTHY W. NILSEN
• 金额: $ 22.98万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3143366
• 关键词: Ascaris; DNA footprinting; Nematoda; RNA biosynthesis; RNA splicing; chromosome deletion; complementary DNA; endonuclease; filariasis; gene expression; genetic library; genetic promoter element; genetic regulation; genetic terminator element; genetic transcription; high performance liquid chromatography; immunoprecipitation; messenger RNA; microorganism genetics; molecular genetics; nucleic acid hybridization; nucleic acid methylation; nucleic acid sequence; parasitic diseases; parasitic gastrointestinal disorder; posttranscriptional RNA processing; site directed mutagenesis; small nuclear RNA; transcription factor

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.

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 （RNA处理）人类寄生线虫中的基因调节，Brugia 马来西，淋巴丝虫病的病因和阿斯卡利斯 Lumbricoides，最常见的人类肠道寄生虫。 在初步 实验已经表明，马来语中的mRNA子集和 Ascaris包含一个常见的移植领导者外显子。 在这两个生物中， 领位外显子衍生自短的非聚乙酰化RNA（SL-RNA） 从5S rRNA基因基因座中转录。 SL-RNA是 由RNA聚合酶II转录并具有三甲基 - 黄烷帽 snRNA的结构特征。 马来语和马来语的SL-RNA基因 Ascaris精确地转录在源自源自的无细胞提取物中 开发阿斯卡利斯胚胎。 SL-RNA基因的体外转录本是 转录后通过三甲基化的帽结构修饰。 定义马来芽孢杆菌和阿斯卡里斯的基因，这引起了转录本 是被拆卸的，将在第二个地方构建cDNA库 链合成由对应于 剪接的领导者。 源自移植的cDNA克隆的表征 mRNA将指示类似的特定mRNA频谱是 在Malayi和Ascaris中进行移植。 使用缺失分析和 定点诱变，直接启动的序列元素 Malayi和Ascaris中SL-RNA合成的终止将是 定义。 突变分析还将用于确定哪些功能 SL-RNA的含量可导致SL-RNA盖的三甲基化。 构造 将使用蛋白质编码基因的5'侧翼区域 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 编码移植的mRNA和 编码未移植的mRNA的基因。 最后，直接RNA 序列分析将用于完全表征B的SNRNA。 马来西和阿斯卡里斯。 该研究计划可能会提供相关的信息 寄生线虫中基因表达的基本机制。 这样的 信息是理解复杂的必要先决条件 有关寄生虫分子基础的问题，可能暗示新颖 控制寄生虫线虫感染的治疗策略。