Var gene regulation and antigenic variation in malaria

说明(由申请人提供)：疟疾仍然是发展中国家发病率和死亡率的主要原因之一。这种疾病是由原生动物寄生虫入侵并最终破坏宿主的循环红细胞引起的，导致严重贫血和经常致命的脑型疟疾综合征。这些寄生虫进化出一种复杂的免疫逃避机制，在感染过程中，出现具有改变抗原表型的小亚群寄生虫，从而避开宿主的抗体反应。这一抗原变异的过程是该病持续存在的原因，也是在感染恶性疟原虫的个体中经常观察到的寄生虫血症波动的原因。抗原表型的差异是多拷贝var基因家族成员之间表达切换的结果。这个家族由大约40-50个基因组成，编码主要的抗原决定簇，一种被称为PIEMPI的蛋白质。任何给定的寄生虫一次只表达一个var基因，从而确定受感染细胞的抗原类型。Var基因表达的变化和由此产生的抗原变异似乎是在转录水平上控制的。这一建议的目的是确定维持除单个var基因外的所有基因处于转录沉默状态的分子机制。需要检验的假设是，var启动子通过一种浓缩的异染色态的组装而沉默，染色质的修饰和重塑调节了var基因的表达和恶性疟原虫的抗原变异。实验设计利用了最近的发现，即var启动子上游的元件与在所有var基因中发现的保守内含子以协同方式起作用，以沉默除单个基因外所有基因的转录。这种转录沉默的状态可以组装在含有报告基因的转基因上，报告基因的两侧是var启动子和内含子。利用这个异构体系统，可以确定沉默和活跃的var启动子的染色质结构和亚核定位。此外，还将确定var基因沉默和激活所需的特定DNA元件，并阐明DNA元件作为沉默或激活剂的序列要求。

DESCRIPTION (provided by the applicant): Malaria remains one of the leading causes of morbidity and mortality in the developing world. The disease is caused by protozoan parasites that invade and ultimately destroy circulating red blood cells of their host, leading to severe anemia and the frequently lethal syndrome of cerebral malaria. These parasites have evolved a complex mechanism of immune evasion whereby, over the course of an infection, small sub-populations of parasites arise that have an altered antigenic phenotype, thus avoiding the antibody response of the host. This process of antigenic variation and is responsible for the persistent nature of the disease as well as the waves of parasitemia frequently observed in individuals infected by P. falciparum. The variation in antigenic phenotype is the result of switches in expression between individual members of the multicopy var gene family. This family consists of approximately 40-50 genes that encode the predominant antigenic determinant, a protein called PIEMPI. Only a single var gene is expressed at a time by any given parasite, thus determining the antigenic type of the infected cell. Changes in var gene expression and the resulting antigenic variation appear to be controlled at the level of transcription. The objective of this proposal is to determine the molecular mechanisms that maintain all but a single var gene in a transcriptionally silent state. The hypothesis to be tested is that var promoters are silenced through the assembly of a condensed, heterochromatic state and that chromatin modification and remodeling regulates expression of var genes and antigenic variation in Plasmodium falciparum. The experimental design exploits the recent discovery that elements upstream of var promoters act in a cooperative fashion with a conserved intron found in all var genes to silence transcription of all but a single gene. This transcriptionally silent state can be assembled on transfected episomes containing a reporter gene flanked by a var promoter and intron. Using this episomal system, the chromatin structure and subnuclear localization of silent and active var promoters will be determined. In addition, the specific DNA elements necessary for var gene silencing and activation will be identified, and the sequence requirements for DNA elements to functions as silencers or activators elucidated.