Malaria Parasite Development, Drug Resistance, and Gemonics

在过去的一年中，我们继续研究恶性疟原虫基因组多样性，耐药性，基因调控，并使用遗传作图，微阵列，基因敲除，遗传杂交，群体遗传学和其他方法的毒力。最终目标是将遗传多态性与寄生虫生物学联系起来，并开发控制疟疾的新方法。 我们已经从185个适应培养的分离株中获得了数千个单核苷酸多态性（SNP），测试了寄生虫对7种抗疟药物的反应，在全基因组关联分析中使用基因型/表型数据，并确定了与寄生虫对甲氟喹、双氢青蒿素和其他药物反应相关的候选基因。我们升级了我们的3.5k SNP分型阵列，以包括8000个SNP，用于以更高的分辨率绘制基因，并将该阵列提供给疟疾社区。我们已经开始对这项研究中确定的一些候选基因进行功能测试。 去年，我们启动了一个项目，系统地表征寄生虫对数千种化合物的差异反应，并收集了数百种差异化学表型（DCP）。我们现在已经在70个遗传杂交后代中测试了DCP化合物，并且已经鉴定了与寄生虫对DCP的反应相关的许多位点。我们还针对大量药物筛选了>60种田间寄生虫分离株，并鉴定了可以在体外杀死氯喹抗性寄生虫的药物。 我们一直在开发约氏疟原虫的遗传图谱，以研究疾病表型。疾病表型是寄生虫和宿主相互作用的结果。使用近交系小鼠将大大减少宿主因素对表型测定的影响。我们用3对寄生虫株（亲本）进行了23个个体的遗传杂交试验，从杂交中获得了134个独立的重组后代。在用数百个微卫星标记对后代进行分型后，已经开发了遗传连锁图。 几项研究表明，同基因寄生虫对之间的基因表达差异，但没有DNA序列的变化可以确定。我们感兴趣的是寄生虫核小体的位置和基因表达在不同的寄生虫发育阶段之间的关系。我们与NIDDK的赵克基博士合作，获得了恶性疟原虫不同发育阶段的核小体位置和mRNA序列。已经发现了预测基因模型和选择性剪接基因中的错误。我们正在分析序列数据。

In the past year, we continued to study Plasmodium falciparum genome diversity, drug resistance, gene regulation, and virulence using genetic mapping, microarray, gene knockout, genetic crosses, population genetics, and other approaches. The ultimate goals are to relate genetic polymorphisms to parasite biology and to develop novel approaches to control malaria. We have obtained thousands of single nucleotide polymorphisms (SNPs) from 185 culture adapted isolates, tested the parasite responses to seven antimalarial drugs, used genotype/phenotype data in genome wide association analysis, and identified candidate genes associated with parasite responses to mefloquine, dihydroartemisinin, and other drugs. We upgraded our 3.5k SNP typing array to include 8000 SNPs for mapping genes at higher resolution and have made the array available to the malaria community. We have started functionally testing some of the candidate genes identified from this study. Last year, we initiated a project to systematically characterize parasite differential response to thousands of chemical compounds and have collected hundreds of differential chemical phenotypes (DCPs). We have now tested the DCP compounds in 70 progeny of genetic crosses and have identified many loci that are linked to parasite responses to the DCPs. We have also screened >60 field parasite isolates against a large number of drugs and identified drugs that can kill chloroquine resistant parasites in vitro. We have been developing a genetic map for Plasmodium yoelii to study disease phenotypes. Disease phenotypes are the results of parasite and host interactions. Use of inbred mice will greatly reduce the influence of host factors on phenotype measurement. We have performed 23 individual genetic cross experiments using three pairs of parasite strains (parents) and obtained 134 independent recombinant progeny from the crosses. A genetic linkage map has been developed after typing the progeny with hundreds of microsatellite markers. Several studies have shown differences in gene expression between pairs of isogenic parasites, but no changes in DNA sequences could be identified. We are interested in relationships between parasite nucleosome position and gene expression in various parasite developmental stages. In collaboration with Dr. Keji Zhao of NIDDK, we obtained nucleosome positions and mRNA sequences from different developmental stages of P. falciparum. Mistakes in predicted gene models and alternatively spliced genes have been discovered. We are in the process of analyzing the sequence data.