Genetic Analysis of Bacterial Toxins in Drosophia

果蝇是与人类关系最密切的无脊椎动物遗传模式系统，正在成为分析人类疾病基因功能的有力工具。在果蝇和人类之间高度保守的基因是那些编码细菌毒素的靶基因，如那些引起炭疽、霍乱和破伤风的基因。令人严重关切的是，包括炭疽LF和霍乱毒素CTX-A1在内的几种细菌毒素也可用于生物武器。 这些毒素进入宿主细胞，可造成不可逆的损伤或 死亡，甚至在病原菌被消灭后。对抗这种毒素的发病机制的一个重要贡献将是开发抑制其在宿主细胞内的活性的试剂。我们最近在果蝇中开发了一种遗传学方法，称为“新的OVerexpression等位基因（NOVA）”筛选，该方法允许有效分离感兴趣基因中的显性等位基因。这项探索性/发展性R21资助的一个重要内容是在我的实验室启动一项新的研究，我们使用NOVA方法产生果蝇中表达的细菌毒素基因的显性阴性（DN）等位基因。这些DN等位基因与野生型毒素功能相关，可用于开发治疗暴露于细菌毒素的患者的试剂，并可用作进一步确定毒素作用机制的工具。根据R21供资机制的另一个关键目标，我们 希望获得必要的初步数据，以便提交RO 1资助，继续和扩展这些研究。该补助金具有以下具体目标，以产生和表征DN-毒素等位基因： 目标1.细菌毒素基因A型亚单位在果蝇中的表达 1-1：将细菌毒素基因亚克隆到pUASW 2表达载体中 1-2：通过在果蝇细胞的特定亚群中表达毒素来产生表型 1-3：表征由pUASW 2-毒素转基因表达产生的表型 目标2.果蝇细菌毒素基因显性负等位基因的筛选 2-1：诱变果蝇pUASW 2-毒素转基因并使用NOVA筛选DN等位基因 2-2：表征DNA-毒素NOVA等位基因中的分子损伤 2-3：确认DN-毒素等位基因干扰野生型毒素基因功能 2-4：确定DN-毒素等位基因是否在哺乳动物细胞中起作用

Drosophila is the invertebrate genetic model system most closely related to humans and is emerging as a powerful tool for analyzing the function of human disease genes. Among genes highly conserved between Drosophila and humans are those encoding targets for bacterial toxins such as those causing anthrax, cholera, and tetanus. There is significant concern that several bacterial toxins, including anthrax LF and cholera toxin CTX-A1, could also be used in biological weapons. These toxins enter host cells and can cause irreversible damage or death, even after the pathogenic bacteria have been eliminated. An important contribution to fighting the pathogenesis of such toxins would be to develop agents inhibiting their activity within the host cell. We have recently developed a genetic method in Drosophila called "Novel OVerexpression Allele (NOVA)" screening, that permits the efficient isolation of dominant alleles in a gene of interest. An important element of this exploratory/developmental R21 grant is to initiate a new line of research in my laboratory in which we use the NOVA method to generate dominant negative (DN) alleles of bacterial toxin genes expressed in Drosophila. These DN alleles, which interefere with wild-type toxin function, may be useful in developing reagents to treat patients exposed to bacterial toxins and may serve as tools to further define the mechanism of toxin action. In line with another key objective of the R21 funding mechanism, we hope to obtain the necessary preliminary data for submitting an RO1 grant to continue and extend these studies. This grant has the following specific aims to generate and characterize DN-toxin alleles: Aim 1. Express A-type subunits of bacterial toxin genes in Drosophila 1-1: Subclone bacterial toxin genes into the pUASW2 expression vector 1-2: Generate phenotypes by expressing toxins in specific subsets of Drosophila cells 1-3: Characterize phenotypes resulting from expression of pUASW2-toxin transgenes Aim 2. Screen for dominant negative (DN) alleles of bacterial toxin genes in Drosophila 2-1: Mutagenize flies pUASW2-toxin transgenes and use NOVA to screen for DN alleles 2-2: Characterize molecular lesion in DN-toxin NOVA alleles 2-3: Confirm that DN-toxin alleles interfere with wild-type toxin gene function 2-4: Determine whether DN-toxin alleles function in mammalian cells