Genetic Analysis of Bacterial Toxins in Drosophia

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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