IDENTIFICATION OF A NOVEL SELD HOMOLOG FROM EUKARYOTES, BACTERIA, ANDARCHAEA - IS THERE AN AUTOREGULATORY MECHANISM IN SELENOCYSTEINE METABOLISM

Escherichia coli selenophosphate synthetase (SPS, the selD gene produc t) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tR NAs. We report the molecular cloning of human kind mouse homologs of t he selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site, These sequ ences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus j annaschii, which had been previously misinterpreted due to their in-fr ame TGA codon. Sps2 mRNA levels are elevated in organs previously impl icated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mou se chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression tha t results in the insertion of a FLAG Lag sequence at the N terminus of the SPS2 protein, This strategy allowed us to document: the readthrou gh of the in-frame TGA codon and the incorporation of Se-75 into SPS2. These results suggest the existence of an autoregulatory mechanism in volving the incorporation of Sec into SPS2 that might be relevant to b lood cell biology. This mechanism is likely to have been present in an cient life forms and conserved in a variety of living organisms from a ll domains of life.