SELENOCYSTEINE TRNA([SER]SEC) LEVELS AND SELENIUM-DEPENDENT GLUTATHIONE-PEROXIDASE ACTIVITY IN MOUSE EMBRYONIC STEM-CELLS HETEROZYGOUS FOR A TARGETED MUTATION IN THE TRNA[(SER]SEC) GENE

To investigate the effect of a reduced level of selenocysteine (Sec) t RNA([Ser]Sec) in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA([Ser]Sec) gene, The presence o f a single functional gene in ES cells afforded us an opportunity to d etermine directly in the cell line the effect of reduced gene dosage o n (1) the levels of the Sec tRNA([Ser]Sec) population, (2) the distrib utions of the isoacceptors within the Sec tRNA population, and (3) sel enoprotein biosynthesis. We therefore determined the amounts and distr ibutions of the two major tRNA([Ser]Sec) isoacceptors, designated mcm( 5)U and mcm(5)Um, within the Sec tRNA population and determined the ac tivity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium, The level of the Sec tRNA([Ser]Sec) popul ation in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the m cmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1, In the presence of media supplemented with selenium, the Sec tRNA([Ser]Sec) population increase d about 20% in wild type cells and virtually not all in heterozygous c ells, and the level of the Sec tRNA([Ser]Sec) population was, therefor e, approximately 50% of that of wild type cells, GPx activity was indi stinguishable among these cell lines in either selenium-supplemented o r unsupplemented media, indicating that the resultant changes in tRNA( [Ser]Sec) levels did not have a measurable effect on GPx biosynthesis.