Characterization of the Giardia lamblia Transcriptome

这是一项确定重要的环境传播人类病原体贾第鞭毛虫整个生命周期的全基因组基因表达模式的建议。贾第虫病是造成世界范围内人类腹泻疾病巨大负担的一个主要原因，但这种寄生虫的基本生物学尚未得到很好的了解。贾第鞭毛虫必须能够对其外部环境的大大小小的变化作出反应，以便在其生命周期的关键事件的适当时机作出适当的反应，因此贾第鞭毛虫在各种敌对环境中生存的能力可能是其病理生理学的关键。对全基因组基因表达模式的检查将为贾第鞭毛虫整个生命周期中生物途径的激活和失活提供一个连贯的画面。由于贾第鞭毛虫的基因组序列很快就会被知道，而且我们能够在体外复制贾第鞭毛虫的生命周期，我们建议利用基因表达序列分析（SAGE）来监测贾第鞭毛虫整个生命周期中信使RNA （mRNA）的全基因组表达水平。我们将对贾第鞭毛虫进行SAGE，从其体外建模的生命周期的14个阶段的mRNA中产生大约12,2000个15 bp的核苷酸序列标签。我们将检测与贲门感染（囊胞）、致病性（滋养体）、传播性（囊胞）和在环境中生存（囊肿）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贾第鞭毛虫中组成性表达。我们将使用半定量Northern blot和逆转录聚合酶链反应（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。该研究将提供对重要宿主生理信号响应的gi贲门基因表达变化的全面理解，并将为其他寄生虫和复杂真核生物（如酵母和动物）的研究提供有价值的模型。

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.