Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been ascribed to this element. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. Several years ago, we proposed that the health benefits of selenium are due to the presence of selenium in selenoproteins as selenocysteine (Sec), which is the amino acid that contains selenium. We previously reported the biosynthetic pathway of Sec, the 21st amino acid in the genetic code, in eukaryotes and Archaea. Our more recent attention has focused on the two Sec tRNA isoforms that we have shown are responsible for the synthesis of the two subclasses of selenoproteins designated housekeeping and stress-related selenoproteins. In addition, we previously worked on the methylase, designated Um34 methylase, that synthesizes the methyl group at the 2'-O-postion on the ribosyl moiety at nucleotide 34 of Sec tRNA. We provided strong evidence that addition of Um34 to the isoform, 5-methylcarobxymethyl-uridine (mcmU), to form 5-methylcarboxymethyl, 2'-O-methyluridine (mcmUm) requires that mcmU is aminoacylated with Sec, i.e., that the substrate for the methylase (designated Um34 methylase) which carries out this reaction is selenocysteyl-tRNA. In the past year, we have purified these two isoforms from bovine liver and characterized them by mass spectrophometric analysis. The mcmU isoform was totally intact, but the mcmUm isoform existed in both the fully intact isoform and unexpectedly as an isoform without the CCA terminus. We are examining how these two isoforms are synthesized and are turned over during selenoprotein synthesis in HL60 cells. HL60 cells are highly enriched in Sec tRNA. In cells grown in the presence of selenium, stress-related selenoproteins are highly synthesized and the mcmUm isoform is highly enriched in these cells, while in the absence of selenium, housekeeping selenoproteins are primarily synthesized and the mcmU isoform is enriched. Mass spectrophotometric analysis of the two isoforms, when HL60 cells are grown in the presence and absence of selenium, should provide further insight into how the two isoforms are synthesized and turned over. In addition, and in collaboration with Dr. Vadim Gladyshev and his group, the genome of Euplotes crassus and Euplotes focardii were examined by bioinformatics and a high percentage of the open reading frames of genes were found to be translated by ribosomal frameshifting. Dr. Gladyshev's laboratory had previously sequenced the genomes of both of these species of organisms. Furthermore, and in an additional collaboration with Dr. Gladyshev and his group, the selenium content of selenoprotein P was examined in relation to diet. The amount of selenium in selenoprotein P varied with the amounts of selenium in the diet of mice and the lower the levels of dietary selenium, the greater the replacement of selenocysteine with cysteine in selenoprotein P.