Hepatic cAMP dependent DNA binding factors

Author

Hugh A. Miller

Date of Award

12-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Wesley D. Wicks

Committee Members

Leaf Huang, David Brian, John Koontz

Abstract

Intracellular levels of adenosine cyclic monophosphate (cAMP) have been determined to regulate the s3mthesis of several proteins at the transcriptional level. The presence of factors which would recognize and bind to sequences within the regulatory region of the gene for these proteins has been proposed.

The regulatory region of the gene for hepatic phosphoenolpyruvate carboxykinase (PEPCK) responsible for cAMP regulation has been determined to be within the first 621 base pairs immediately 5' of the transcribed region of the gene. Fragments produced by restriction endonuclease digestion of this region were utilized in the nitrocellulose filter binding assay in order to detect possible DNA binding factors.

Crude nuclear extracts prepared from rat liver treated with cAMP were found to specifically retain on nitrocellulose a fragment of the PEPCK gene from 71 to 111 base pairs 5' of the start site of transcription (A1 base pairs in size). This region is included in a fragment from 70 to 109 base pairs from the start site which confers cAMP regulation on a heterologous gene (Short et al., 1986, J. Biol. Chem. 261: 9721-9726).

Competition binding revealed that this factor(s) in the nuclear 12 extract demonstrated an approximate dissociation constant of 1 x 10 when tested with the specific 41 base pair fragment.

The ability of nuclear extracts from cAMP-treated rat liver to protect guanine residues within this 41 base pair region from methylation by dimethylsulfate was demonstrated. The extract from cAMP-treated liver nuclei protected guanines at positions 98, 94, 93, 92, and 91 on the 5' side of the start site. These guanines are close to the region that has been proposed to be the cAMP regulatory element.

These lines of evidence suggest that the identified factor(s) may be involved in the regulation of gene transcription by cAMP.

Recommended Citation

Miller, Hugh A., "Hepatic cAMP dependent DNA binding factors. " PhD diss., University of Tennessee, 1986.
https://trace.tennessee.edu/utk_graddiss/12295