Copy Number Variation in Malaria Parasites

描述（由申请人提供）：基因拷贝数变异（CNV）在真核生物基因组中普遍存在，由此产生的剂量变化在决定表型中起重要作用。我们的目的是确定疟原虫基因组（恶性疟原虫）中CNV的范围、进化动力学和功能。为了实现这一目标，我们将使用比较基因组杂交（cGH），它为分析CNV和单核苷酸多态性（SNP）提供了一种有效的工具，在单倍体基因组中尤其有效。初步数据显示，恶性疟原虫基因组中存在丰富的CNV（缺失和扩增），并证明了对snp的准确预测。该项目将分为三个部分。首先，我们将在全球寄生虫收集中识别所有CNV并编目侧翼snp。抽样的人群将包括来自泰国、巴布亚新几内亚、哥伦比亚和冈比亚的新分离的单克隆感染。我们将测量基因组重排的大小、基因含量、排列和种群频率。我们的第二个目标将是评估显示CNV的基因组区域的适应性进化证据，以突出可能受到近期进化压力（如药物选择）影响的基因组片段。我们将通过检查连锁不平衡模式和侧链SNP多态性的单倍型结构，频率的地理变异，以及通过检查cnv的独立起源数量来做到这一点。最后，我们将重点研究一个含有GTP环水解酶位点的CNV，这可能具有重要的功能。我们将验证这一假设，即该基因的扩增弥补了叶酸途径下游酶代谢效率的降低，这些酶携带的突变赋予了抗叶酸化合物的抗性。这将通过选择和转染实验，结合耐药性、适应性和叶酸途径通量的测定来完成。本研究与公共卫生的相关性。基因拷贝数的变化对寄生虫的生物学有很大的影响。我们建议系统地描述疟疾寄生虫基因组中的DNA重排，并评估它们在这一重要病原体中发挥的作用。

DESCRIPTION (provided by applicant): Gene copy number variation (CNV) is ubiquitous in eukaryotic genomes and the resultant dosage changes can play an important role in determining phenotype. We aim to define the extent, evolutionary dynamics and functionality of CNV in the malaria parasite genome (Plasmodium falciparum). To achieve this we will use comparative genomic hybridization (cGH) which provides an efficient tool for assaying both CNV and single nucleotide polymorphism (SNP) and is particularly effective in haploid genomes. Preliminary data reveal abundant CNV (both deletions and amplifications) in the P. falciparum genome and demonstrate accurate prediction of SNPs. The project will be divided into three parts. First, we will identify all CNV and catalogue flanking SNPs in a worldwide parasite collection. The populations sampled will comprise freshly isolated single-clone infections from Thailand, Papua New Guinea, Colombia and Gambia. We will measure the size, gene content, and arrangement and population frequency of genome rearrangements. Our second aim will be to evaluate evidence for adaptive evolution of genome regions showing CNV to highlight genome segments that could be influenced by recent evolutionary pressures such as selection by drugs. We will do this by examining patterns of linkage disequilibrium and haplotype structure in flanking SNP polymorphism, geographical variation in frequency, and by examination of numbers of independent origins of CNVs. Finally, we will conduct focused investigations of one CNV - containing the GTP cyclohydrolase locus - that is likely to be of functional importance. We will test the hypothesis that amplification of this gene compensates for reduced metabolic efficiency of enzymes further down the folate pathway carrying mutations that confer resistance to anti-folate compounds. This will be done using selection and transfection experiments in combination with assays of drug resistance, fitness, and flux through the folate pathway. Relevance of this research to public health. Variation in the number of gene copies can have a large influence on parasite biology. We propose to systematically describe DNA rearrangements in the malaria parasite genome, and evaluate the role that they play in this important pathogen.