Doctoral Dissertations

Date of Award

8-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Wesley D. Wicks

Committee Members

John Koontz, Daniel Roberts, Jerry Weir

Abstract

Cyclic AMP (cAMP) has been shown to regulate the ex pression of a large number of genes at the level of transcription, including phosphoenolpyruvate carboxykinase (PEPCK) in rat liver cells. The regulatory region of the hepatic PEPCK gene responsive to cAMP has been determined to be within the immediate 5' portion of the gene (-109 to -68). Comparison with other genes sensitive to cAMP has led to the identification of a consensus octameric sequence which has been suggested to serve as a recognition site for a cAMP dependent trans-acting factor (CRF).

Initially, evidence for such a cAMP dependent fac tor (s) which interacts with the cAMP regulatory element (CRE) in the PEPCK gene has been obtained with extracts of nuclei from rat liver cells exposed to cAMP. The formation of an apparently single complex between a synthetic oligomer containing the region from -67 to -111 of the PEPCK gene and a factor in nuclear extracts from cAMP-treated rat liver was visualized by the band retardation method. Complex formation was both concentration and cAMP-dependent and could be prevented by excess specific but not non-specific competitor DNA.

An apparently similar factor(s) was detected in heterologous cell types which supports the possibility that CRF could be the mediator of cAMP action on other genes in other cells. The heterologous CRF activity detected was found to be dependent upon cAMP treatment. The time course of increases in CRF activity was consistent with the known kinetics of cAMP control of foreign gene expression in transfection experiments with some of these cell types.

The CRF binding site in the PEPCK promoter region was evaluated by DNase I footprinting and DMS protection experiments which revealed that sequences in and around the consensus octameric motif were protected. Data gained as a result of this study clearly demonstrates that the detected factor(s) has properties consistent with a possible role as mediator of the transcriptional control exerted by cAMP in eukaryotic cells.

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