Thyroid hormone produces a coordinate increase in the levels of all five enzymes of the urea cycle in liver of the bullfrog tadpole (Rana catesbeiana). Preliminary experiments demonstrate that for at least two of the urea cycle enzymes in tadpole liver -- argininosuccinate synthetase and carbamyl phosphate synthetase I -- the increase in enzyme levels reflects a several-fold increase in the corresponding mRNA levels. The proposed research is directed toward elucidating the molecular mechanisms whereby thyroid hormone regulates the levels of the mRNAs for the urea cycle enzymes. I plan to construct a cDNA library for bullfrog liver and isolate cDNA clones for argininosuccinate synthetase and carbamyl phosphate synthetase I. Using RNA-DNA hybridization assays, I plan to analyze in detail the response of the mRNA levels for these enzymes to thyroid hormone. A major objective of the research is to determine whether regulation of the specific mRNA levels occurs primarily at the transcriptional or post-transcriptional level. Experiments using primary cultures of tadpole hepatocytes will determine whether thyroid hormone alone or in combination with other hormones acts directly to elicit the increases in mRNA levels for the urea cycle enzymes observed in the intact animal. Although the relative synthesis rates and/or mRNA levels for a number of proteins have been shown to change following administration of thyroid hormone to intact animals, a direct effect of thyroid hormone alone on specific mRNA levels in isolated cells in culture has to date been demonstrated for only two identified proteins: avian malic enzyme and rat growth hormone. Neither of these has been shown to be coordinatley regulated with other proteins of known function by thyroid hormone alone. The tadpole liver is potentially useful in providing an experimental system for studying the molecular mechanisms of thyroid hormone action in early development, especially for coordinate regulation of the different enzymes of an important metabolic pathway.
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