Microarray analysis of gene expresion in T. cruzi

描述（由申请人提供）：大约30%的克氏锥虫感染导致潜在致命的慢性病理，称为恰加斯病。我们的长期目标是确定预防和干预南美锥虫病的可能目标。实现这一目标的先决条件是发现和表征克氏锥虫的基因及其表达模式。最近完成的克氏锥虫基因组测序和注释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)克氏锥虫生命周期的每个阶段都有独特的基因表达特征，2)确定这些特征将对涉及阶段转换的机制和信号通路的性质具有指导意义，以及3)在克氏锥虫生命周期中非组成性表达的基因将包括许多基因，这些基因将成为疾病预防和干预的特别有用的靶标。本项目旨在建立一个高质量、带注释的克氏锥虫（T. cruzi trypmasastigotes， TRP）、amastigotes （AMA）、epimastigotes （EPI）和metacyclic trypmasastigotes （MET）基因表达数据库。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. cruzi基因组中每个注释基因互补的长寡核苷酸的DNA微阵列。为了研究克氏锥虫基因表达控制中mRNA稳定性和翻译效率的相对贡献，本研究将使用总KNA和多体rna进行分析。这些分析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微阵列分析是否准确反映了蛋白质的存在和丰度。我们还将进行trp到AMA阶段转化的时间过程研究，以检查在寄生虫发育的这一关键过程中hi基因表达的波动。在克氏锥虫生命周期的五个阶段转变中，TRP-to-AMA是最适合时间过程实验的，并且与克氏锥虫感染宿主的宿主-寄生虫关系特别相关。此外,

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.