Figure 8 | Scientific Reports

Figure 8

From: Pharmacogenomics of poor drug metabolism in Greyhounds: Cytochrome P450 (CYP) 2B11 genetic variation, breed distribution, and functional characterization

Figure 8

Effect of CYP2B11 3′-UTR polymorphisms on transcript length. (a) PCR primers were designed to amplify CYP2B11 3′-UTR cDNA upstream (5′) of any polymorphism (Region 1) and downstream (3′) adjacent to Region 1 overlapping two polymorphisms (c.2137 TG/CA and c.2166 G/A) that demonstrated significant allelic imbalance (Regions 2 and 3). (b) PCR was then conducted with each primer set using reverse transcribed RNA extracted from MDCK cells transfected with luciferase reporter constructs containing each of the CYP2B11 3′-UTR haplotypes (H1, H2 or H3), untransfected cells (C), or no input RNA (−). PCR primers for the housekeeping gene, GAPDH, were included to exclude an effect of differences in RNA extraction and reverse transcription efficiency. DNA bands of the appropriate size were identified by agarose gel electrophoresis with Sybr Green staining. Supplementary Figure S1 contains full length gels. (c) Sequence analysis of the CYP2B11 3′-UTR identified two canonical consensus polyadenylation signal (AAUAAA) sites. Their locations are indicated relative to the predicted 3′ ends of each 3′-UTR haplotype. (?) indicates the region likely to contain the 3′-end of the mRNA based on RT-PCR and RNA-seq data.

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