eQTL analysis of Toxoplasma development

描述（由申请人提供）：最近对寄生虫毒力的遗传分析证实，寄生虫负荷增加与艾滋病病原体弓形虫引起的疾病之间存在重要联系。控制寄生虫负担的因素尚不清楚，但很明显，速殖子复制和切换到持久性组织包囊之间的平衡对宿主中的寄生虫数量至关重要。基因转录是控制弓形虫发育的一个重要机制，这一点可以通过与导致组织包囊的寄生虫阶段转换相关的转录中的大的坐标变化来证明。我们估计，在中间的生命周期发育控制的基因的数量是在这种寄生虫中检测到的总基因表达的20%。基因表达在遗传多样性菌株之间也不同，这些差异是寄生虫细胞内复制、组织迁移和侵袭、毒力和长期持久性建立的表型差异的基础。在这个建议中，我们将调查的转换机制，伴随着在组织囊肿中发现的缓殖子阶段的形成的差异的遗传基础。在目的1中，我们将构建代表三种典型谱系（I型- GT-1、II型-Me 49 B7和III型-CTG）的早期传代系中速殖子至缓殖子mRNA表达的综合谱。将来自所有三种体外诱导分化菌株的详细动力学系列的微阵列数据与脑囊肿衍生的缓殖子的谱进行比较，以构建速殖子至缓殖子调节的mRNA的高质量谱。这些RNA谱也将与来自几种缓殖子突变体和来自替代诱导方法和宿主细胞类型的数据进行比较，以提高这些数据的总体分辨率。该项目产生的所有杂交结果将以其他研究者可访问的格式下载到ToxoDB上。在目标2中，我们将研究从I-GT-1型和III-CTG型之间的遗传杂交获得的一组独立后代中发育变异的遗传基础。我们将利用微阵列数据，以产生全球mRNA的遗传后代的条件下，诱导缓殖子特异性基因表达。将通过标准方法鉴定差异表达的mRNA，然后将来自这些分析的定量表达值和比率用作QTL分析中的性状，以定义控制特定基因表达的染色体区域及其相互作用。这些研究有可能鉴定出降低I型菌株发育效率的基因，同时揭示出在更发育活跃的菌株中积极调节缓殖子转换的基因组。最近对寄生虫毒力的遗传分析证实，寄生虫负担增加与艾滋病病原体弓形虫引起的疾病之间存在重要联系。控制寄生虫负担的因素尚不清楚，但很明显，速殖子复制和切换到持久性组织包囊之间的平衡对宿主中的寄生虫数量至关重要。在这项建议中，我们将调查的转换机制，伴随着形成的缓殖子阶段的组织囊肿，这是负责慢性弓形虫病的差异的遗传基础。

DESCRIPTION (provided by applicant): Recent genetic analysis of parasite virulence confirms that there is an important link between increased parasite burden and disease caused by the AIDS pathogen, Toxoplasma gondii. The factors that control parasite burden are not understood, but it is clear that the balance between tachyzoite replication and switching to the persistence tissue cyst is critical to parasite numbers in the host. Gene transcription is an important mechanism in the control of Toxoplasma gondii development as evidenced by the large coordinate changes in transcription associated with parasite stage transitions that lead to the tissue cyst. We estimate that the number of genes under developmental control in the intermediate life cycle is ~20% of total gene expression detected in this parasite. Gene expression also varies between genetically diverse strains and these differences underlie phenotypic differences in parasite intracellular replication, tissue migration and invasion, virulence, and the establishment of long-term persistence. In this proposal, we will investigate the genetic basis for differences in the switching mechanism that accompanies formation of the bradyzoite stage found in the tissue cyst. In Aim 1, we will construct a comprehensive profile of tachyzoite- to-bradyzoite mRNA expression in early passage lines representing the three canonical lineages (Type I- GT-1, Type II-Me49B7, and Type III-CTG). Microarray data from a detailed kinetic series of all three strains induced to differentiate in vitro will be compared to profiles of brain cyst-derived bradyzoites in order to construct a high quality profile of tachyzoite-to-bradyzoite regulated mRNAs. These RNA profiles will also be compared with data from several bradyzoite mutants and from alternate methods of induction and host cell types in order to improve the overall resolution of these data. All hybridization results generated by this project will be downloaded onto ToxoDB in a format accessible to other investigators. In Aim 2, we will examine the genetic basis of developmental variation in a set of independent progeny obtained from a genetic cross between Type I-GT-1 and Type III-CTG. We will utilize microarray data to generate global mRNA profiles for genetic progeny under conditions that induce bradyzoite-specific gene expression. Differentially expressed mRNAs will be identified by standard methods, and then quantitative expression values and ratios from these analyses will be utilized as traits in QTL analysis to define chromosome regions and their interactions that control the expression of specific genes. These studies have the potential to identify genes that reduce developmental efficiency in Type I strains, while at the same time revealing groups of genes that positively regulate bradyzoite switching in more developmentally active strains. Recent genetic analysis of parasite virulence confirms that there is an important link between increased parasite burden and disease caused by the AIDS pathogen, Toxoplasma gondii. The factors that control parasite burden are not understood, but it is clear that the balance between tachyzoite replication and switching to the persistence tissue cyst is critical to parasite numbers in the host. In this proposal, we will investigate the genetic basis for differences in the switching mechanism that accompanies formation of the bradyzoite stage found in the tissue cyst, which is responsible for chronic toxoplasmosis.