Microarray analysis of gene expresion in T. cruzi

描述（由申请方提供）：大约30%的克氏锥虫感染导致潜在致命的慢性病理学，称为恰加斯病。我们的长期目标是确定南美锥虫病预防和干预的可能目标。实现这一目标的前提是发现和鉴定T.克鲁兹最近完成的T. cruzi基因组为确定疫苗和治疗干预的新靶点提供了前所未有的机会。我们假设：1）T. cruzi生活史中的每一个都有独特的基因表达标记，2）确定这些标记对于阶段转换中涉及的机制和信号传导途径的性质将是有指导意义的，和3）在T. cruzi生命周期将包括许多基因，这些基因将成为疾病预防和干预的特别有用的靶点。本项目的目标是建立一个高质量的、注释的T.克氏锥鞭毛体（TRP）、无鞭毛体（AMA）、上鞭毛体（EPI）和亚环锥鞭毛体（MET）。我们将使用来自每个生命周期阶段的单个时间点的RNA来确定基因表达特征，以询问含有与新测序的T中的每个注释基因互补的长寡核苷酸的DNA微阵列。cruzi基因组为了研究mRNA稳定性和翻译效率对T.克鲁兹这些分析的结果将与我们实验室产生的蛋白质组学数据进行比较，以确定使用DNA微阵列的分析是否准确反映蛋白质的存在和丰度。我们还将进行一个时间过程的研究TRP- AMA阶段转换，以检查在寄生虫发育的这一关键过程中的基因表达的波动。在T. cruzi生命周期中，TRP到AMA是最适合于时间过程实验，特别是相关的宿主-寄生虫在T。克鲁兹感染的宿主此外，委员会认为， 使用多聚核糖体RNA和延长时间进程将建立在我们以前对这一阶段过渡的研究基础上。这些研究的数据和分析，沿着蛋白质组数据，将以符合MIAME的格式存放在TcruziDB中，供研究界使用。本研究的完成将为T. cruzi，特别是许多是“假设的”，并为阐明这些基因的功能提供了一个起点。

DESCRIPTION (provided by the applicant): Roughly 30% of Trypanosoma cruzi infections result in potentially fatal chronic pathology known as Chagas disease. Our long-term goal is to identify likely targets for Chagas disease prevention and intervention. Prerequisite to this goal is the discovery and characterization of genes and their pattern of expression in T. cruzi. The recent completion of sequencing and annotation of the T. cruzi genome provides an unprecedented opportunity to identify new targets for vaccine and therapeutic interventions. We hypothesize that: 1) stages in the T. cruzi life cycle each have unique signatures of gene expression, 2) determining these signatures will be instructive as to the nature of the mechanisms and signaling pathways involved in stage conversion, and 3) that genes expressed non-constitutively during the T. cruzi lifecycle will include many genes that will be particularly useful targets for disease prevention and intervention. The aims of this project are to initiate the development of a high quality, annotated gene expression database of T. cruzi trypomastigotes (TRP), amastigotes (AMA), epimastigotes (EPI), and metacyclic trypomastigotes (MET). We will determine gene expression signatures using RNAs from a single time point in each life-cycle stage to interrogate DNA micro-arrays containing long oligonucleotides complementary to every annotated gene in the newly sequenced T. cruzi genome. Both total KNA and polysomal RNAs will be used in this analysis in order to investigate the relative contributions of mRNA stability and translational efficiency to control of gene expression in T. cruzi. The results of these analyses will be compared with proteomic data generated in our laboratory to determine if profiling using DNA micro-arrays accurately reflects protein presence and abundance. We will also perform a time-course study of TRP-to- AMA stage conversion to examine the fluctuations hi gene expression during this critical process in parasite development. Of the five stage transitions in the T. cruzi life cycle, the TRP-to-AMA is the most amenable to time course experimentation and is particularly relevant to the host-parasite relationship in T. cruzi-infected hosts. Moreover, using polysomal RNA and extending the time course will build upon our previous study of this stage transition. The data and analysis of these studies, along with proteome data, will be made available to the research community by deposition in TcruziDB in a MIAME compliant format. Completion of this study will provide valuable data on the expression of genes in T. cruzi, especially the many that are "hypothetical," and provide a starting point for elucidating the functions of these genes.