The pathogenesis of anthrax, a severe disease of humans and numerous livestock species, involves host sensitivity to various exotoxins secreted by the causative agent of anthrax, Bacillus anthracis. One of these toxins, called Lethal Factor (LF), is a mononuclear phagocyte specific toxin, causing cytolysis of these very important immune system cells. The overall goal of this project is to identify genes in the mouse that play a role in the poorly understood LF intoxication process. Monocytic cells isolated from various inbred mouse strains exhibit differential susceptibility to cytolysis caused by LF. In at least one resistant/susceptible strain pair comparison, C57BL/6J X C3H/HeJ, the trait differences segregates as a single gene character that maps to chromosome 11. One of the main goals of this project is to isolate the gene on chromosome 11, called Ltx1, that is responsible for the difference in LF sensitivity. A high resolution genetic map of the Ltx1 region will be constructed using a (C57BL/6J X C3H/HeJ) X C57BL/6J cross, and regional mapped polymorphisms will be used to construct a physical map of YAC, BAC, and P1 clones. The search for Ltx1 in this physical map will consist of typical positional cloning strategies, including genomic sequencing, exon trapping, and identification of cross species conserved probes. Any genes identified will be analyzed to determine if they are involved in LF intoxication using functional complementation of the trait in tissue culture assays. Isolation of differentially expressed genes and genes whose proteins interact with LF will be used as a complement to the positional cloning strategies for isolating genes involved in LF intoxication. Specifically, differential display PCR or subtractive hybridization techniques will be used to search for genes whose transcription is altered upon LF intoxication. In addition, screens for LF interacting proteins utilizing 2-hybrid methods and co-immunoprecipitation will be undertaken.
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