MOLECULAR BASIS FOR SELENIUM DIETARY REQUIREMENT

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The broad, long-term objective of this work is to establish the molecular basis for selenium (Se) nutrient requirement. Selenium is an essential trace element in the diets of man and animals. However, the mechanisms for many of its biological effects remain unexplained. Unfortunately there is no single, comprehensive experiment that can be designed to answer the question "What other physiological processes depend on Se intake?" or "What other enzymes depend on Se for activity?". However, using the techniques of modern biology, it is possible to ask and answer the questions "What genes are regulated by Se?" and "How are they regulated?". By identifying such genes, the products for which they code, and the mechanisms of their regulation, additional functions for Se will be revealed. Mechanisms for previously observed biological effects of Se will be suggested. New markers for Se nutritional status may be discovered. In addition, the foundation will be established to explore the genetic basis for variability in Se nutrient requirement. Specific Aim 1 in this project is to isolate rat cDNAs for genes whose expression is varied by changes in dietary Se intake. Specific Aim 2 is to determine the pretranslational reaction(s) in the expression of these genes most dependent on dietary intake of Se. Rats will be fed diets inadequate, adequate and high but nontoxic in Se. The techniques of differential display of mRNA will be used to identify and clone cDNAs for genes expressed at different levels in tissues of rat consuming different levels of Se. The sequences of these differentially expressed clones will be compared to those entered into GenBank and other data bases to determine if they represented previously characterized or newly discovered genes, whose expression is regulated by Se intake. These experiments will accomplish Specific Aim 1. For each of these genes, run-on transcription assays and Northern blot analyses will show whether gene transcription or mRNA stabilization is the step in pretranslational gene expression most sensitive to Se intake. Comparison among sequences in the UTRs of cDNAs for Se-regulated genes will show sequences conserved among these different species which may serve as Se-responsive elements. These experiments will accomplish Specific Aim 2.