Transcriptional Regulators of the Malarial var Multigene Family

描述（由申请人提供）：本研究项目的目标是开发一种结合染色质免疫沉淀、串联亲和纯化和质谱的新方法，以鉴定编码疟疾毒力因子、恶性疟原虫红细胞膜蛋白1 （PfEMP1）的var多基因家族可能的激活因子和/或抑制因子。在它们的红细胞发育过程中，疟疾寄生虫转换表达这种变体表面蛋白的替代形式以逃避宿主的免疫反应。每个恶性疟原虫的基因组包含大约60个变异基因，分布在不同的染色体上。然而，每个寄生虫只有一个var基因以互斥的方式表达。var基因的启动子区域在这一调控中起着重要作用，据信这一机制可能涉及特异性抑制因子（PfVPR）和/或激活因子（PfVPA）分子。本提案的具体目标包括实验设计和方法，其中一些是我们实验室新开发的恶性疟原虫的串联亲和纯化和荧光原位杂交（FISH）。目的：利用染色质免疫沉淀、串联亲和纯化和质谱技术鉴定PfVPA和PfVPR基因表达调控因子；和Aim II。启动恶性疟原虫PfVPA和PfVPR蛋白的功能分析。

DESCRIPTION (provided by applicant): The goal of this research project is to develop a new approach that combines chromatin immunoprecipitation, tandem affinity purification and mass spectrometry to identify possible activators and/or repressors of the var multigene family encoding the malaria virulence factor, Plasmodium falciparum erythrocyte membrane protein 1, PfEMP1. During their intraerythrocytic development, malaria parasites switch expression to alternative forms of this variant surface protein to evade the host immune response. The genome of each P. falciparum parasite contains approximately 60 var genes distributed on different chromosomes. However, only one var gene is expressed by each parasite in a mutually exclusive manner. The promoter region of var genes plays an important role in this regulation and it is believed that this mechanism might involve specialized repressor (PfVPR) and/or activator (PfVPA) molecules. The specific aims of the current proposal include experimental designs and methods, some newly developed in our laboratories for P. falciparum such as tandem affinity purification and fluorescent in situ hybridization (FISH). They are: Aim I. To identify PfVPA and PfVPR regulators of var gene expression by chromatin immunoprecipitation, tandem affinity purification and mass spectrometry; and Aim II. To initiate a functional analysis of the PfVPA and PfVPR proteins in P. falciparum. PUBLIC HEALTH RELEVANCE: Malaria parasites invade and ultimately destroy circulating red blood cells of their host, often leading to severe clinical illness or death. During their intraerythrocytic development, malaria parasites switch expression to alternative forms of a variant surface protein PfEMP1 encoded by the var genes to evade the host immune response. The goal of this research collaboration is to combine tools and techniques from genetics, molecular biology, and immunochemistry, and the expertise of two laboratories to unravel the molecular determinants of antigenic variation during malaria infection.