Functional characterization of a single nucleotide polymorphism in the 5' UTR region of the bovine toll-like receptor 4 gene.
Toll-like receptor 4 (TLR4) is the key receptor for the LPS component of Gram-negative bacteria. We have identified a SNP within the 5' UTR region of the bovine TLR4 gene at position 226 (c.-226C>G) relative to start codon, which was found to be associated with health related traits in the Canadian Holstein population. TLR4 gene expression was measured over time intervals in samples collected from 18 cows (6 cows in each genotype) challenged in vitro with LPS (20 ng/ml) to determine if this SNP alters the gene expression. TLR4 gene expression was highest 6 h post LPS challenge, and cows with the GG genotype exhibited significantly higher expression compared to other genotypes. This was further validated by a dual luciferase reporter gene assay in which sequential progressive constructs were cloned into the pGL3 basic vector and transfected into bovine Mac-T cells. The reporter construct containing most of the 5' UTR (-456G) exhibited significantly higher luciferase activity compared to-456C, however, further inclusion of 5' upstream sequence showed a reversal phenomenon where the -913C construct exhibited significantly higher activity compared to -913G. These results indicate that the c.-226C>G SNP affects the expression of TLR4, suggesting that this SNP might be influencing the binding and interaction of transcription factors regulating gene expression.
Sharma
B S
BS
Mount
J
J
Karrow
N A
NA
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D051197:Toll-Like Receptor 4 / Q000235:genetics*
D020121:5' Untranslated Regions; D017931:DNA Primers; D051197:Toll-Like Receptor 4