A problem of major interest in current biology centers not only on the question of the mechanisms whereby hormones regulate the expression of specific genes, but also on how coordinate expression of genes -- e.g., for individual enzymes within a metabolic pathway -- is achieved. Expression of the genes encoding enzymes of the urea cycle constitutes an excellent model for investigating both aspects of this problem. The urea cycle is comprised of 2 mitochondrial and 3 cytosolic enzymes, all encoded by nuclear genes which are coordinately expressed to a significant extent only in liver of ureotelic animals. Levels of the urea cycle enzymes in mammals are responsive to changes in dietary protein content, glucocorticoids, glucagon, insulin, and thyroid hormone. Results from this laboratory, as well as from others, have demonstrated that a primary component of these changes involves altered cellular concentrations of the mRNAs for these enzymes. Studies in this laboratory have also demonstrated that the transcription rate of these genes in rat liver is increased in response to cyclic AMP and that glucocorticoids and dibutyryl cyclic AMP are each necessary and sufficient to elevate mRNA levels for urea cycle enzymes in rat hepatocytes cultured in serum-free medium. This grant proposes to isolate and characterize genomic DNA clones encoding two of the urea cycle enzymes -- carbamyl phosphate synthetase I and argininosuccinate synthetase. mRNAs for these two enzymes exhibit distinctive patterns of expression in cultured rat hepatocytes. Recombinant DNA constructs incorporating genomic DNA sequences for these enzymes and a reporter gene will be used in cell transfection experiments to identify and characterize cis-acting DNA sequences involved in the response to hormones. Preliminary experiments will also be undertaken to identify DNA-binding proteins with sequence- specific affinity for regions of DNA shown by transfection experiments to be important for expression.
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