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Untranslated Region in Chinese Hamster Ovary Cells
Manuscript received 26 November 1996. Initial reviews completed 10 January 1997. Revision accepted 11 March 1997.
Departments of Biochemistry and Nutritional Sciences, University of Missouri, Columbia, MO 65211
Classical glutathione peroxidase (GPX) mRNA levels fall dramatically in selenium (Se)-deficient animals, but it is not known whether this mechanism is related to the mRNA 3
untranslated region (3UTR) sequences that have been shown to direct Se incorporation. In this study, we used recombinant GPX constructs to investigate the role of the GPX 3UTR in Se regulation of GPX mRNA levels in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with GPX (pRc/GPX), GPX lacking the 3UTR (pRc/
3UTR) or the pRc/CMV vector alone, and GPX activity and GPX mRNA levels were determined in stable transfectants grown in low Se basal medium with a range of added Se concentrations. We identified two pRc/GPX transfectants with significantly elevated GPX activity levels compared with pRc/CMV transfectants. The elevated GPX expression did not dramatically shift the amount of Se that was sufficient for GPX activity to reach the Se-adequate plateau level (100 nmol/L added Se). As expected, GPX activity was not significantly different when pRc/3UTR transfectants were compared with pRc/CMV control transfectants. Among the wild type and transfected CHO cells, Se-deficient GPX activity levels averaged 35 ± 5% of Se-adequate levels. Selenium-deficient levels of endogenous GPX mRNA as well as recombinant pRc/GPX mRNA averaged 54-58% of Se-adequate levels; 3-4 nmol/L added Se was sufficient for maximal GPX mRNA levels. In contrast, pRc/3UTR mRNA levels in the unsupplemented cells remained at Se-adequate levels and showed no distinct Se regulation. These studies demonstrate that the GPX 3UTR is necessary for Se regulation of GPX mRNA levels in addition to its role in Se incorporation.
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