CLONING OF PLASMODIUM REFRACTORINESS GENES IN A GAMBIAE

每年导致200万至300万人死亡的疟疾是 世界上第二重要的病原体致死原因 在肺结核之后的今天。大多数疟疾死亡病例发生在婴儿或年轻人中。 非洲的儿童，那里的冈比亚按蚊是主要的 向量。由于广泛出现的抗药性菌株 疟疾寄生虫和抗药性菌株的媒介，新 迫切需要疟疾控制战略。这导致了一种 人们对蚊子-寄生虫相互作用的研究重新产生了兴趣 可能的知识来源，有助于新形式的 控制力。在这个长期目标的框架内，我已经 选择了一种能够包裹并杀死冈比亚按蚊的菌株 这种蚊子通常寄生的大多数疟疾寄生虫 易受影响。白纹伊蚊易感性的微卫星初步定位 猴子寄生虫食蟹猴疟原虫B已经将一种主要的和两种 这种表型的次要基因。该项目总体目标是 克隆与这种包膜表型有关的基因，并 确定这些基因产物赋予耐热性的方式 疟疾寄生虫。这项工作将首先集中在专业上 封装基因。为了实现这一目标，我将谈到4 具体目标：(1)使难治冈比亚按蚊的基因 包裹并杀死食蟹猴寄生虫的菌株B将是 利用微卫星标记将遗传图谱定位于 基因组DNA不超过几百个DNA碱基或丢失 (我的目标是小于300kb)；(2)一组重叠的邻接 跨越定义主基因的遗传区间的克隆(重叠群) 将被识别；(3)这些间隔内的基因将 通过基因组测序和筛选cDNA相结合的方法进行鉴定 从感染食蟹猴B的食蟹猴分离的mRNA中产生文库， 当时的成年雌性难治性和易感性品系蚊子 当包被发生时；(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 用定性RT-PCR方法对分离自沙门氏菌的mRNA进行检测 食蟹猴B感染耐药和敏感品系蚊子和 两株病毒的cDNA序列比较.如果一种技术 冈比亚按蚊种系转化在时间内成为可能 这个项目的框架，这项技术将被用来确定 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.