Characterization of the Giardia lamblia Transcriptome

贾第鞭毛虫转录组的表征

• 批准号: 6365064
• 负责人: ANDREW G MCARTHUR
• 金额: $ 23.61万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6365064
• 关键词: Giardia; bacteriophage M13; complementary DNA; computer program /software; functional /structural genomics; gene expression; genetic library; genetic regulation; genetic transcription; giardiasis; host organism interaction; intracellular parasitism; life cycle; messenger RNA; molecular cloning; northern blottings; nucleic acid sequence; open reading frames; polymerase chain reaction; protozoal genetics; serial analysis of gene expression; small intestines; virulence

This is a proposal to determine patterns of genome-wide gene expression throughout the life cycle of the important environmentally transmitted human pathogen Giardia lamblia. Giardiasis is a major contributor to the enormous worldwide burden of human diarrheal diseases, yet the basic biology of this parasite is not well understood. No giardial virulence factor is known and this protist's ability to survive in diverse, hostile environments may be a key to its pathophysiology, as Giardia must be able to respond to large and small changes in its external environment for appropriate timing of crucial events in its life cycle. Examination of genome-wide gene expression patterns will provide a coherent picture of activation and inactivation of biological pathways throughout Giardia's life cycle. As the genome sequence of Giardia will soon be known and because we are able to reproduce Giardia's life cycle in vitro, we propose to utilize Serial Analysis of Gene Expression (SAGE) to monitor genome-wide levels of messenger RNA (mRNA) expression throughout Giardia's life cycle. We will perform SAGE for Giardia lamblia by generating approximately 12,2000 15 bp nucleotide sequence tags from the mRNA of 14 stages of its life cycle, modeled in vitro. We will detect up- and down-regulation of genes related to giardial infection (excystation), pathogenicity (trophozoites), transmission (encystation), and survival in the environment (cysts). We will additionally predict which genes and biochemical pathways are constitutively expressed in Giardia lamblia. We will confirm mRNA expression levels of genes with the most variable expression levels predicted by SAGE using semi- quantitative Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR). This research will provide a comprehensive understanding of changes in giardial gene expression in response to important host physiological signals and will serve as a valuable model for study of other parasites and complex eukaryotes, such as yeast and animals.

这是一项建议，以确定重要环保人类病原体Lamblia的整个生命周期中全基因组基因表达的模式。贾第鞭毛病是人类腹泻疾病的巨大负担的主要因素，但这种寄生虫的基本生物学尚不清楚。 没有贾dial毒力因素是众所周知的，该原生物在多种多样的环境中生存的能力可能是其病理生理学的关键，因为贾第鞭毛虫必须能够对其外部环境中的大小变化做出反应，以在其生命周期中适当的关键事件时间。 整个基因组基因表达模式的检查将提供整个贾第鞭毛虫生命周期中生物途径的激活和失活的连贯图。 由于贾第鞭毛虫的基因组序列很快就会被众所周知，并且由于我们能够在体外重现贾第鞭毛虫的生命周期，因此我们建议利用基因表达（SAGE）的序列分析来监测整个贾第酵母生命周期的整个基因组范围的Messenger RNA（mRNA）表达。 我们将通过从其生命周期的14个阶段的mRNA产生大约12,000 15 bp核苷酸序列标签来对贾第鞭毛虫进行鼠尾草的鼠尾草。 我们将检测到与贾第鞭毛虫感染（ExcyStation），致病性（滋养体），传播（encystation）和环境中生存相关的基因的上调和下调（囊肿）。 我们还将预测哪些基因和生化途径在贾迪亚兰布利亚（Giardia lamblia）中构成表达。 我们将使用半定量北印迹和逆转录酶聚合酶链反应（RT-PCR）确认具有大量可变表达水平的基因的mRNA表达水平。 这项研究将对重要的宿主生理信号的吉他基因表达的变化提供全面的理解，并将作为研究其他寄生虫和复杂真核生物（例如酵母和动物）的宝贵模型。