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.

该提案旨在确定重要的环境传播人类病原体兰氏贾第鞭毛虫整个生命周期的全基因组基因表达模式。贾第鞭毛虫病是造成全球人类腹泻疾病巨大负担的主要原因，但这种寄生虫的基本生物学原理尚不清楚。 贾第虫毒力因子尚不清楚，这种原生生物在多样化、恶劣环境中生存的能力可能是其病理生理学的关键，因为贾第虫必须能够对其外部环境的大大小小的变化做出反应，以便在其生命周期中的关键事件的适当时机发生。 对全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激活和失活的连贯图景。 由于贾第虫的基因组序列很快就会为人所知，并且我们能够在体外重现贾第虫的生命周期，因此我们建议利用基因表达系列分析 (SAGE) 来监测整个贾第虫生命周期的全基因组信使 RNA (mRNA) 表达水平。 我们将通过在体外模拟贾第鞭毛虫生命周期 14 个阶段的 mRNA 生成约 12,2000 个 15 bp 核苷酸序列标签，对兰氏贾第鞭毛虫进行 SAGE。 我们将检测与贾第鞭毛虫感染（脱囊）、致病性（滋养体）、传播（包囊）和环境生存（包囊）相关的基因的上调和下调。 我们还将预测哪些基因和生化途径在蓝氏贾第鞭毛虫中组成型表达。 我们将使用半定量 Northern blot 和逆转录聚合酶链式反应 (RT-PCR) 来确认 SAGE 预测的表达水平变化最大的基因的 mRNA 表达水平。 这项研究将全面了解贾第鞭毛虫基因表达响应重要宿主生理信号的变化，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）的有价值的模型。