MOLECULAR GENETICS OF LEISHMANIA

描述(改编自研究者的摘要)：利什曼病是 一种热带疾病感染了1000多万人，导致 疾病的范围从轻微到毁容到致命。这个 这项应用的长期目标是识别与 作为基本知识的寄生虫的毒力可能允许 开发更复杂的控制策略。要完成 这种，利什曼原虫的各种遗传方法被开发出来， 在之前的授权期内申请。 这些进展包括表达载体、同源基因替换 和基因“敲除”的产生，以及功能性遗传 互补性。后一种方法表明，以前未知的 在利什曼原虫中可以从基因上鉴定出毒力基因，如下 在原核生物发病机制研究中建立的经典范式。 因此，易感鼠感染了一群 无毒的大青霉，以前用粘粒文库 从毒力很强的利什曼原虫基因组DNA中。与无毒控制不同的是， 感染是从转基因群体中获得的。宇宙星是 从这些寄生虫中恢复，随后的测试确认了10 不同的宇宙体能够增加一种 完全无毒的菌株。目前的数据表明，这些基因会影响 (S)在哺乳动物感染周期中的某一阶段 与人类疾病最相关的。 这些VIR宇宙星将被更详细地描述，并补充 对毒力基因进行筛选，重点放在 侵染循环，并使用其他无毒品系。具体目标是： 首先确定受每个VIR影响的生命周期的步骤 粘粒使用特定的分析方法。第二，每个基因中的活性基因 COSMID将被绘制和排序；它的位置和作用机制 都会被追捕。第三，VIR基因作用的遗传基础将是 探索以确定它们是通过互补还是通过互补发挥作用 压制。VIR基因将为零突变体将由基因获得 替换，结合了获得零突变的新策略 基本基因座。将研究发育调节，并 寻求不同基因之间的相互作用。四、生态文明建设基础研究 利什曼原虫纯合子突变的频率、类型及诱因 将会被启动。这一信息将导致改进的协议 恢复突变，这对功能基因筛选是必不可少的。

DESCRIPTION (adapted from the investigator's abstract): Leishmaniasis is a tropical disease infecting more than 10 million people, causing a spectrum of disease ranging from mild to disfiguring to fatal. The long-term goal of this application is to identify genes responsible for virulence of the parasite as the basic knowledge may permit the development of more sophisticated control strategies. To accomplish this, a variety of genetic methods in Leishmania were developed and applied in the prior grant period. These advances included expression vectors, homologous gene replacement and the creation of gene `knockouts', and functional genetic complementation. This latter method suggested that previously unknown virulence genes could be genetically identified in Leishmania, following the classic paradigm established in prokaryotic pathogenesis studies. Accordingly, susceptible mice were infected with a population of avirulent L. major, previously transfected with a cosmid library made from virulent Leishmania genomic DNA. Unlike the avirulent control, infections were obtained from the transfectant population. Cosmids were recovered from these parasites, and subsequent tests confirmed 10 different cosmids were capable of increasing the infectivity of a completely avirulent line. Current data suggest that these genes affect some step(s) in the mammalian part of the infectious cycle, the stage most relevant to human disease. These VIR cosmids will be characterized in more detail, and additional screens for virulence genes performed, focusing on specific steps of the infectious cycle and using other avirulent lines. Specific aims are: first to determine the step of the life cycle affected by each VIR cosmid using specific assays. Second, the active genes within each cosmid will be mapped and sequenced; its site and mechanisms of action will be sought. Third the genetic basis for VIR gene action will be probed to determine whether they function by complementation or suppression. VIR gene will null mutants will be obtained by gene replacement, incorporating new strategies for obtaining null mutants in essential loci. Developmental regulation will be examined and interactions amongst different genes sought. Fourth, basic studies of the frequency, type and induction of homozygous mutations in Leishmania will be initiated. This information will lead to improved protocols for recovering mutants, which is essential for functional genetic screens.