Selenium has a role in preventing heart disease and is rapidly becoming recognized as a chemopreventive agent against cancer. The beneficial affects of this element are due, at least in part, to its presence in selenoproteins as the amino acid, selenocysteine (Sec). Our program, therefore, focuses on the means by which Sec is incorporated into protein and the role of specific selenoproteins in human health. As Sec tRNA is regarded as the key molecule in selenoprotein biosynthesis, we have produced transgenic mice encoding as few as 2 and as many as 20 extra copies (transgenes) of the Sec tRNA gene to examine effects of its overexpression. We introduced a silent mutation in the transgene that enables us to distinguish its expression from that of the corresponding wild type gene by primer extension. The transgenes and the wild type genes contribute equally to the total Sec tRNA population, but the level of expression is not linear with gene copy number, strongly suggesting that gene expression is regulated by a feedback mechanism. Overexpression does not appear to affect selenoprotein synthesis or protein synthesis as a whole. This observation, coupled with our earlier findings of normal selenoprotein synthesis in cells containing only ? the Sec tRNA population, demonstrate that Sec tRNA is not a limiting component in selenoprotein translation. We have also sequenced the Sec tRNA gene in zebrafish and Chinese hamsters. Zebrafish contains two functional copies of this gene whereas all other higher and lower animals examined to date contain only one functional gene. Chinese hamsters contain three pseudogenes whereas all other organisms examined to date contain only one or no pseudogenes and since the Chinese hamster gene encodes two pyrimidine transtions compared to all other mammals, it seems likely that these transitions occurred by editing of a transcript and re-insertion into its genome. In our studies on examining the role of specific selenoproteins in human health, we have focused on a recently discovered selenoprotein, designated as the 15 kDa protein. We have shown that its levels are normally elevated in prostate as compared to other tissues, but are reduced substantially in prostate cancer. Since selenium is known to have a chemopreventive role in prostate cancer, it would seem that the 15 kDa protein may have a role in preventing malignancy in this tissue. We, therefore, have made the appropriate constructs encoding the 15 kDa protein to examine the affects of its overexpression in mammalian cells in culture and in transgenic mice. These studies are in progress. We have confirmed the occurrence of two polymorphisms in the 3-untranslated regions of the 15 kDa protein gene and 1 of the polymorphisms may be involved in regulating protein expression. There was statistically significant differences in allele distribution between head and neck cancer tissues and controls. In a related study on the effects of selenium on human health, a previous member of our group (V.N.G., see collaborators below), had shown earlier that the incorporation of selenium into protein is diminished in HIV infected cells. We have subsequently shown that the HIV encoded TAT protein binds to the SECIS element which is a specific stem-loop structure in all selenoprotein mRNAs required for insertion of Sec into protein; but TAT also binds to most double stranded RNAs examined and thus shows no specificity. We are presently using in vivo experiments to determine if specificity occurs in selenoprotein synthesis. We have discovered two new human selenoenzyme thioredoxin reductases and characterized the role of a previously identified thioredoxin reductase in redox regulation of cell signaling. In addition, we identified a thioredoxin reductase in C. elegans and this appears to be the only selenoprotein in this organism. Our study on examining the Sec tRNA population in Drosophila has been completed as well as our study on examining the role of hypermodified bases in the anticodon loop of specific tRNAs on ribosomal frameshifting. - Genes, Gene Targeting, Gene expression, Post-transcription regulation, Selenium, Selenocysteine, Transgenic mice, Translation, - Neither Human Subjects nor Human Tissues
Showing the most recent 10 out of 47 publications
