IDENTIFICATION AND EXPRESSION ANALYSIS OF ABC GENES IN PLASMODIUM YOELII

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。疟疾是世界范围内最重要的寄生虫病之一。恶性疟原虫和间日疟原虫多药耐药株的进化和传播是一个主要的健康问题。尽管包括冈比亚按蚊、恶性疟原虫和约氏按蚊全基因组测序在内的研究取得了进展，但疟原虫耐药现象的分子基础尚不完全清楚。这项拟议研究的长期目标是阐明疟疾寄生虫对抗疟疾药物产生抗药性的机制。这项研究的结果可能会为药物设计和疾病控制带来更好的方法。我们的工作假设是，属于ATP结合盒(ABC)超家族转运蛋白的几个基因共同作用，赋予耐药性。约氏疟原虫的一系列药物敏感和耐药株将被用作体内的啮齿动物疟疾模型，以研究耐药性的机制。虽然有许多可能的耐药机制，但我们将重点介绍可能在寄生虫中介导药物渗出的ABC超家族转运蛋白的两个成员，即多药耐药相关蛋白基因(Pymrp)和ABCG同源基因。其具体目的是：-鉴定约氏疟原虫多药耐药相关蛋白基因(Pymrp)，并确定其在疟疾耐药性中的作用。-检测pymrp基因在药物敏感和耐药株中的转录水平。-确定pyMRP蛋白的亚细胞位置并测量其表达水平。-启动转基因研究，通过基因破坏来解决pyMRP蛋白的功能。-鉴定一种新的ABC基因，约氏P.yoelii ABCG同源物，并确定其在疟疾耐药性中的作用。-通过生物信息学分析，确定膜拓扑结构并确定约氏杆菌ABCG同源物中的保守基序。-分析来自药物敏感和耐药品系的约氏杆菌ABCG同源物的基因拷贝数、单核苷酸多态(SNP)、转录水平和染色体位置。-确定约氏假单胞菌ABCG蛋白的亚细胞位置和表达水平。这项研究的理由是，了解pymrp和约氏疟原虫ABCG同源基因在疟原虫耐药性中的作用可以为更好的药物设计提供基础，并有可能逆转含喹啉的抗疟疾药物的耐药性。这是一项创新性的研究，因为疟原虫MRP和ABCG同源基因的功能及其在耐药性中的潜在作用仍有待阐明。在第一年中，我们希望实现以下目标：1)测量pymrp基因在药物敏感和耐药品系中的转录水平；2)通过生物信息学分析确定膜拓扑结构并确定约氏疟原虫ABCG同源物中的保守基序；以及3)启动pyMRP重组蛋白的构建。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Malaria is one of the most important parasitic diseases worldwide. The evolution and spread of multidrug resistant strains of P. falciparum and P. vivax is a major health concern. Despite the advances in research, including the sequencing of the entire genomes of Anopheles gambiae, P. falciparum and P. yoelii, the molecular basis of the drug resistance phenomena in Plasmodia is not completely understood. The long-term goal of the proposed research is to elucidate mechanisms by which the malaria parasites become resistant to antimalarial drugs. The results of this research could lead to a better approach to drug design and disease control. Our working hypothesis is that several genes that belong to the ATP-binding cassette (ABC) superfamily of transporter proteins work in conjunction to confer drug resistance. A collection of drug sensitive and resistant lines of P. yoelii will be used as an in vivo rodent malaria model to investigate the mechanisms of drug resistance. Although a number of resistance mechanisms are possible, we will focus on two members of the ABC superfamily of transporter proteins that may mediate drug efftux in the parasite, the multidrug resistance associated protein gene (pymrp) and the ABCG homologue gene. The specific aims are: - To characterize the P. yoelii multidrug resistance associated protein gene (pymrp) and to determine its role in malaria drug resistance. - To measure transcript levels of the pymrp gene in drug sensitive and resistant lines. - To determine the sub-cellular location and measure expression levels of the pyMRP protein. - To initiate transfection studies to address the function of the pyMRP protein by gene disruption. - To characterize a novel ABC gene, the P. yoelii ABCG homologue, and determine its role in malaria drug resistance. - To determine membrane topology and identify conserved motifs in the P. yoelii ABCG homologue by performing bioinformatic analyses. - To analyze the P. yoelii ABCG homologue, from drug sensitive and resistant lines, for gene copy number, single nucleotide polymorphisms (SNP), transcript levels and chromosomal location. - To determine the sub-cellular location and measure expression levels of the P. yoelii ABCG protein. The rational for this research is that understanding the role of pymrp and the P. yoelii ABCG homologue gene in Plasmodium drug resistance could provide the basis for better drug design and potentially lead to reversal of drug resistance to the quinoline-containing antimalarial drugs. This is an innovative research because the function of the Plasmodium mrp and ABCG ortholog genes and their potential role in drug resistance remains to be elucidated. During the first year we expect to accomplish the following goals: 1) To measure transcript levels of the pymrp gene in drug sensitive and resistant lines; 2) To determine membrane topology and identify conserved motifs in the P. yoelii ABCG homologue by performing bioinformatic analyses; and 3) To initiate the construction of the pyMRP recombinant protein.