GENE EXPRESSION IN HUMAN PARASITIC NEMATODES

人类寄生线虫的基因表达

• 批准号: 6626482
• 负责人: TIMOTHY W. NILSEN
• 金额: $ 42.35万
• 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 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626482
• 关键词: Ascaris; DNA footprinting; RNA biosynthesis; RNA splicing; X ray crystallography; autoradiography; crosslink; electron microscopy; gene expression; genetic promoter element; messenger RNA; microorganism genetics; nucleic acid hybridization; nucleic acid methylation; nucleic acid sequence; parasitic diseases; posttranscriptional RNA processing; precursor mRNA; site directed mutagenesis; small nuclear RNA; spliceosomes; transcription factor

The long term goal of the proposed research is to achieve a detailed understanding of the mechanism of nuclear pre-mRNA splicing (both cis- and trans-) using cell free extracts derived from embryos of the parasitic nematode, Ascaris. Trans-splicing is used as a mechanism of pre-mRNA processing in a variety of medically relevant parasites, including nematodes, trypanosomatid protaozoa and trematodes; the Ascaris system permits biochemical analysis of this reaction. Four Specific Aims are proposed: 1. Trans-spliceosome assembly involves independent recognition of the 3' splice site on the trans-splice acceptor and the 5' splice site on the SL RNA (the trans-splice donor). A combination of biochemical fractionation and site-specific crosslinking approaches will be used to characterize factors required for 3' splice site recognition. In addition, the hypothesis that the SL RNA is recruited to the acceptor as part of a pre-formed quadruple snRNA will be directly tested.; 2. Accurate maturation of nematode pre-mRNAs requires that the SL be excluded from internal (cis)- 3' splice acceptor sites. Evidence suggests that the cis- 5' splice site and the 5' splice site of the SL RNA are both present and may compete within the cis-spliceosome. Site-directed mutagenesis of both 5' splice sites will be used to determine how a particular 5' splice site is selected for the first transesterification reaction; 3. Tethered (site-specific) hydroxyl radical cleavage will be used to probe the RNA environments of the 5' splice site, the branch point, and the 3' splice site in staged cis- and trans-spliceosomes.; 4. Large (mg) quantities of staged spliceosomes will be purified for the purpose of biophysical analyses; electron cryomicroscopy and X-ray crystallography. In combination, the proposed biochemical and biophysical studies should provide new insight into the mechanism of trans-splicing in particular, and the mechanism of pre-mRNA splicing in general. Finally, a thorough understanding of trans-splicing may suggest possible avenues for therapeutic intervention in diseases caused by parasitic organisms that employ this pre-mRNA processing pathway.

拟议的研究的长期目标是使用源自寄生线虫胚胎胚胎的胚胎的无细胞提取物，对核前mRNA剪接（顺式和反式）的机理进行详细的了解。跨剪接用作多种医学相关寄生虫的MRNA加工机制，包括线虫，锥虫protaozoa和Trematodes； Ascaris系统允许对该反应的生化分析。提出了四个具体目的：1。Trans-Spliceomssbly涉及对变式式受体上3'剪接位点的独立识别，以及SL RNA上的5'剪接位点（thrans-Splice供体）。生化分馏和特定地点的交联方法的组合将用于表征3'剪接站点识别所需的因素。此外，将直接测试将SL RNA作为预先形成的四倍SNRNA的一部分募集到受体的假设。 2。线虫预先mRNA的准确成熟要求将SL从内部（CIS）-3'剪接受体位点排除。有证据表明，CIS-5'剪接位点和SL RNA的5'剪接位点既存在，又可能在顺板中竞争。两个5'剪接位点的位置定向诱变将用于确定如何选择特定的5'剪接位点作为第一个式式反应； 3。将使用束缚（特定于位点的）羟基自由基裂解来探测5'剪接位点，分支点和3'剪接位点的RNA环境。 4。将纯化大量（mg）分级剪接体，以进行生物物理分析；电子冷冻显微镜和X射线晶体学。结合起来，提出的生化和生物物理研究应特别了解尤其是变速器的机制，以及一般的mRNA剪接机制。最后，对跨性变化的透彻理解可能表明，可能会导致寄生生物引起的疾病治疗干预，这些寄生虫采用了这种MRNA加工途径。