CLONING OF PLASMODIUM REFRACTORINESS GENES IN A GAMBIAE

疟疾每年造成200万至300万人死亡， 全球第二大病原体特异性死亡原因 今天肺结核之后。大多数疟疾死亡发生在婴儿或年轻人中 在非洲，冈比亚按蚊是主要的蚊子， vector.由于抗药性菌株的广泛出现， 疟疾寄生虫和对杀虫剂有抗药性的病媒菌株， 迫切需要疟疾控制战略。这导致 重新对蚊子-寄生虫相互作用的研究产生了兴趣， 可能的知识来源，可以促进新的形式， 控制在这个长期目标的框架内，我 选择了A.冈比亚能够封装并杀死 大多数种类的疟疾寄生虫， 易受影响微卫星定位初步研究 猴子寄生虫食蟹猴疟原虫B将一个主要的和两个主要的 这种表型的次要基因。该项目的总体目标是 克隆负责这种包囊表型的基因， 确定这些基因产物赋予不应性的方式 疟疾寄生虫。这项工作最初将侧重于主要的 包囊基因为了实现这一目标，我将在4 具体目标：（1）基因，使难治性A。冈比亚按蚊 用于封装和杀死寄生虫食蟹猴疟原虫B的菌株将 使用微卫星标记进行遗传作图， 基因组DNA，不超过几百个DNA酶或损失 (my目标小于300 kb）;（2）一组重叠的连续 跨越定义主基因的遗传间隔的克隆（重叠群） 将被识别;（3）这些区间内的基因将被识别。 通过基因组测序和筛选cDNA的组合来鉴定 从O.食蟹猴B病毒感染， 成年雌性难治性和易感性蚊子 （4）表达模式（mRNA水平和 产物的大小）和这些区间中的基因序列将 通过对分离自以下的mRNA进行定性RT-PCR分析来确定 食蟹猴疟原虫B感染的难治性和敏感性品系蚊子， 比较两株菌的cDNA序列。如果一种技术， A.冈比亚将在 本项目的框架，这项技术将用于确认 候选基因的功能。

Malaria, which causes between 2 and 3 million deaths each year, is the second most important pathogen-specific cause of mortality in the world today after tuberculosis. Most malaria deaths occur in infants or young children in African, where the mosquito Anopheles gambiae is the major vector. Because of the widespread emergence of drug-resistant strains of malaria parasites and insecticide resistant strains of the vector, new strategies for malaria control are urgently needed. This has led to a renewed interest in the study of the mosquito-parasite interaction as a possible source of knowledge that could contribute to new forms of control. Within the framework of this long term objective, I have selected a strain of A. gambiae that is able to encapsulate and kill most species of malaria parasites to which this mosquito is normally susceptible. Preliminary microsatellite mapping of susceptibility to the monkey parasite Plasmodium cynomolgi B has linked one major and two minor genes to this phenotype. The broad objective of this project are to clone the genes responsible for this encapsulation phenotype and to determine the manner in which those gene products confer refractoriness to malaria parasites. The work will focus initially on the major encapsulation gene. To accomplish this objective, I will address 4 specific aims: (1) the genes that enable the refractory A. gambiae strain to encapsulate and kill the parasite P. cynomolgi B will be genetically mapped using microsatellite markers to within regions of genomic DNA that are no more than a few hundred kilobases of DNA or loss (my target is less than 300 kb); (2) a set of overlapping contiguous clones (contigs) which span the genetic interval defining the major gene will be identified; (3) the genes within these intervals will be identified by a combination of genomic sequencing and by screening cDNA libraries produced from mRNA isolated from O. cynomolgi B-infected, adult female refractory and susceptible strain mosquitos at the time when encapsulation occurs; (4) expression patterns (level of mRNA and size of the products) and sequences of the genes in these intervals will be determined by qualitative RT-PCR analysis of mRNA isolated from P.cynomolgi B-infected refractory and susceptible strain mosquitoes and comparison of sequences the cDNAs of the two strains. If a technique for germ line transformation of A. gambiae becomes available within the time frame of this project, this technique will be used to confirm the function of candidate genes.