The goal of this research proposal is to investigate the molecular mechanisms by which thyroid hormone and nutritional factors act to regulate the expression of specific hepatic genes. In this regard, we are currently focusing our attention on the gene designated spot 14. The hepatic concentration of spot14 mRNA increases very rapidly (less than 15 min) and dramatically (greater than 10-fold) following administration of thyroid hormone or carbohydrate feeding. Thus, this response may represent a primary effect in the liver to these stimuli. Our current working hypotheses is that this gene is regulated at two distinct sites by either effector; a minor change occurs in the rate of gene transcription, but the major change is due to a post-transcriptional increase in the stability of the nuclear precursor to spot 14 mRNA. To further study the regulation of spot 14 gene expression, the following studies will be performed. First, the induction of spot 14 gene expression will be examined in the presence of inhibitors of protein synthesis to test whether this response is truly a primary effect to hormone and diet. Second, the regulation will be examined in primary hepatocyte culture in which the cellular site of action of hormonal and nutritional stimuli will be confirmed. Third, the transcriptional activity of the spot 14 gene will be examined during fetal and neonatal development to determine when the expression of this gene is activated. Fourth, the expression of several other hepatic genes which are regulated by thyroid hormone and carbohydrate feeding will be determined to test whether post-transcriptional control is an unusual feature for the spot 14 gene or a common feature for regulated genes. Further studies will explore the association of the spot 14 mRNA precursor with the nuclear matrix, the presumed site of RNA processing, during hormonal, dietary or developmental changes. In addition, the post-transcriptional modification of the primary spot 14 transcript will be examined in these states. We hope that these studies may elucidate the mechanism of regulation. Finally, a cell culture system will be developed for testing the function of the isolated spot 14 gene by DNA-mediated gene transfer. Subsequently, the extent and nature of DNA sequences essential for regulation will be defined by 'in vitro' mutagenesis. This system should provide an excellent model system for exploring the basis of hormonal and dietary gene regulation and may provide clues regarding the nuclear processing of RNA and how this process can be regulated.
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