eQTL analysis of Toxoplasma development

描述(申请人提供)：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重要机制，与导致组织囊变的寄生虫阶段转换相关的转录大的坐标变化证明了这一点。我们估计在生命周期中期处于发育控制下的基因数量约占该寄生虫总基因表达的20%。基因表达在遗传不同的菌株之间也存在差异，这些差异是寄生虫细胞内复制、组织迁移和入侵、毒力以及建立长期持久性的表型差异的基础。在这项提案中，我们将调查在组织囊中发现的伴随着缓殖子阶段形成的转换机制的差异的遗传学基础。在目标1中，我们将构建一个代表三个典型谱系(类型I-GT-1、类型II-Me49B7和类型III-CTG)的早期传代系中速殖子到缓殖子mRNA表达的全面图谱。来自所有三个菌株在体外诱导分化的详细动力学序列的微阵列数据将与脑囊肿缓殖子的图谱进行比较，以构建高质量的速殖子到缓殖子调控的mRNAs图谱。这些RNA图谱还将与几个缓殖体突变体的数据以及来自不同诱导方法和宿主细胞类型的数据进行比较，以提高这些数据的整体分辨率。该项目产生的所有杂交结果将以其他研究人员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和III-CTG之间的遗传杂交获得的一组独立后代的发育变异的遗传基础。我们将利用微阵列数据，在诱导慢殖子特异基因表达的条件下，为遗传后代生成全球mRNA图谱。差异表达的mRNAs将通过标准方法进行鉴定，然后从这些分析中得到的定量表达值和比率将被用作QTL分析的性状，以确定控制特定基因表达的染色体区域及其相互作用。这些研究有可能在I型菌株中识别降低发育效率的基因，同时揭示在发育更活跃的菌株中积极调节缓染色体切换的基因组。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囊之间的平衡对宿主中的寄生虫数量至关重要。在这项提案中，我们将调查在组织囊中发现的伴随着缓殖子阶段形成的转换机制不同的遗传基础，这是导致慢性弓形虫病的原因。

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.