Identification of genomic promoter sequences and translational activity of mRNA variants for bovine pyruvate carboxylase
Abstract
The objectives of this research were to identify the promoter regions of bovine pyruvate carboxylase (PC), determine the translational efficiency of the PC 5' UTR variants and develop a quantitative PCR (qPCR) assay to screen expression of the PC 5' UTR variants. Genomic DNA sequence analysis indicates an exon arrangement from 5' to 3' of 178 (exon 1A and 1B), 48 (exon II), 41 (exon III) and 185 (exon IV) bp for bovine PC 5' UTR variants. Pyruvate carboxylase has three promoter regions located within 42.6 kb of the translation start site, and these promoters sites, P1, P2 and P3 are located upstream of exon I, II and III, respectively. Although P2 and P3 contain glucocorticoid response elements, neither of these promoters are responsive to glucocorticoids in vitro. Based on our model of promoter location, P1 is responsible for the transcription of PC 5' UTR A, PC 5' UTR B, PC 5' UTR C and PC 5' UTR F. Promoter 2 is responsible for transcription of PC 5' UTR D, and P3 is responsible for transcription of PC 5' UTR E. The PC 5' UTR variants display differential translational efficiencies. Pyruvate carboxylase 5' UTR D exhibits the highest translational efficiency of the bovine PC UTR variants, whereas PC 5' UTR C and PC 5' UTR E exhibited the lowest translational efficiencies. However, qPCR analysis of liver RNA from mid-lactation dairy cows revealed that PC 5' UTR E mRNA expression is higher than PC 5' UTR D. These data on the promoter regions and 5' UTR exon arrangement of bovine PC provide essential information about transcriptional regulation of bovine PC 5' UTR variants. The qPCR protocol for PC 5' UTR D, PC 5' UTR E and total PC will aid in rapid screening of PC expression under a variety of physiological and developmental scenarios and when used in conjunction with the translational efficiency data obtained from this work, will facilitate studies that correlate PC mRNA expression to PC protein abundance.
Degree
Ph.D.
Advisors
Donkin, Purdue University.
Subject Area
Anatomy & physiology|Animals|Veterinary services|Livestock
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