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Previous analysis of the bovine SLC11A1 complementary DNA (cDNA) failed to identify any nucleotide variations other than a microsatellite length variation within the 3' untranslated region functionally associated with bovine brucellosis. In this study I set out to identify mutations in the genomic complement of the gene that may be associated with resistance or susceptibility to bovine brucellosis, and to determine if the microsatellite length polymorphism in the 3'UTR of bovine SLC11A1 modulates gene expression and subsequent disease resistance in a phase dependent manner. The results of this study demonstrate that there are seventy-five total single nucleotide polymorphic (SNP) sites (excluding indels) located within the bovine genomic SLC11A1 sequence of a Brucella abortus resistant bull and a susceptible cow. Twenty of these SNPs segregated between resistant and susceptible populations, with 3 non-synonymous SNPs significantly associating with resistance or susceptibility to B. abortus infection. An A695G within exon 2 resulted in a histidine (resistant allele) to arginine (susceptible allele) amino acid substitution and was in significant linkage disequilibrium with the previously described 3' untranslated region (UTR) microsatellite length variation associated with brucellosis resistance. A transcriptional element search in the 3' UTR revealed a ETS-domain PU.1 site, an IFN-γ activation site (GAS), an Interferon Consensus Sequence Binding Protein site (ICSBP) and several Initiation Response sites (Inr), suggesting a possible function for this region in regulation of the expression of SLC11A1. A mobility shift assay confirmed sequence-specific DNA-protein interaction within this region. A luciferase reporter assay indicated that the 3'UTR of SLC11A1 could act as a downstream enhancer for expression. Macrophage killing assays with RAW264.7 cells expressing bovine SLC11A1 demonstrated that the microsatellite repeat is functionally associated with the macrophage killing efficiency, but not in a phase-dependent manner, suggesting that these length polymorphisms do not affect the angular orientation between cooperatively binding transcription factors, and leaves the possibility that the 3'UTR microsatellites regulate SLC11A1 transcription through some alternate mechanism, possibly mRNA stability.