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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-1874 |
| Date | 02 June 2009 |
| Creators | Schutta, Christopher John |
| Contributors | Templeton, Joe W. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
Page generated in 0.0022 seconds