We propose to investigate in detail the protein organization in the regulatory region of the PEPCK gene. This gene gives rise to a key gluconeogenic enzyme (phosphoenolpyruvate carboxykinase). We will analyze the protein organization in cells which do not respond to glucagon and insulin, as well as in hepatic cells in which vigorous activity of the gene can be stimulated by glucocorticoids and/or cAMP. In addition the effect on the protein organization of the profound inhibitory effect of insulin will be studied. The techniques to be employed will include a battery of nucleases acting upon chromatin in isolated cell nuclei, coupled with methods of increased sensitivity capable of detecting single copy genes even when greatly fragmented into multiple (sometimes diffuse) components. This work will provide information about the nature of rapid hormonal responses in multihormonal systems. It should indicate whether the responses consists of (a) modifying existing regulatory protein-DNA interactions, (b) creating new interaction or (c) replacing one set of interaction with another. Several of these devices may be used to regulate the complex PEPCK system.
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