Characterization of the Giardia lamblia Transcriptome

贾第鞭毛虫转录组的表征

• 批准号: 6632604
• 负责人: ANDREW G MCARTHUR
• 金额: $ 23.52万
• 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/6632604
• 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.

这是一个建议，以确定整个生命周期的重要环境传播的人类病原体贾第鞭毛虫的全基因组基因表达模式。贾第虫病是造成全球人类肠道疾病巨大负担的主要原因，但这种寄生虫的基本生物学尚不清楚。 没有贾第鞭毛虫毒力因子是已知的，这种原生生物在不同的，恶劣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的大小变化做出反应，以便在其生命周期中适当地选择关键事件的时间。 全基因组的基因表达模式的检查将提供一个连贯的图片激活和灭活的生物途径在贾第虫的生活周期。 由于贾第虫的基因组序列将很快被知道，因为我们能够在体外复制贾第虫的生命周期，我们建议利用基因表达系列分析（SAGE）来监测整个贾第虫的生命周期的信使RNA（mRNA）表达的全基因组水平。 我们将进行SAGE蓝氏贾第鞭毛虫产生约12,2000个15 bp的核苷酸序列标签从mRNA的14个阶段的生命周期，在体外模拟。 我们将检测基因的上调和下调，这些基因与囊外感染（脱囊）、致病性（滋养体）、传播（包囊化）和在环境中存活（包囊）有关。 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。 我们将使用半定量北方印迹和逆转录酶聚合酶链反应（RT-PCR）确认SAGE预测的表达水平变化最大的基因的mRNA表达水平。 这项研究将提供一个全面的了解giardiac基因表达的变化，响应重要的宿主生理信号，并将作为一个有价值的模型，研究其他寄生虫和复杂的真核生物，如酵母和动物。