An investigation into camp regulation of the phosphoenolpyruvate carboxykinase gene

Author

Joseph Abner Williams

Date of Award

12-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Wesley D. Wicks

Committee Members

Leaf Huang, John Koontz, Stuart Riggsby

Abstract

The chloramphenicol acetyltransferase (CAT) gene was fused 3' to the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in a plasmid (pBB0.6CAT), injected into Xenopus oocytes and enzyme activity measured. CAT activity from oocytes treated with progesterone was 49% of the CAT activity from oocytes not treated with the hormone. The progesterone-induced drop in enzyme activity was consistent over all levels of expression and at 4 and 17 hr intervals between plasmid injection and oocyte harvest. Similar treatment of oocytes injected with a control plasmid, pSV2CAT (driven by the SV40 promoter) showed increased CAT activity (131%) following progesterone treatment.

Additionally, the progesterone-induced loss of pBBO.6CAT-derived was overcome by injection of the catalytic subunit of the A kinase prior to progesterone treatment. The A kinase did not affect CAT activity in the absence of progesterone.

Finally specific binding of an oocyte factor to the PEPCK cAMP regulatory element was detected by the band retardation assay, and the degree of binding from oocytes treated with progesterone for 3 hr decreased to 33% of the level of binding detected from untreated oocytes.

These data indicate that: i) progesterone can specifically regulate oocyte gene expression through a heterologous promoter, ii) the progesterone effect is mediated through activity of the A kinase, and iii) progesterone causes a loss of specific binding to the PEPCK cAMP CRE which may be responsible for the specific gene regulation.

DNA affinity chromatography was used to isolate proteins from rat liver nuclei which specifically bind to the PEPCK CRE as detected by the band retardation assay. Preliminary purification of the nuclear ex tract indicated that two specific protein/DNA complexes were detectable and separable by both DEAE and heparin agarose chromatography. Application of the purified fractions to the DNA affinity resin, however, resulted in complete loss of binding activity as detected by band retardation. UV crosslinking of proteins to the CRE indicated that several proteins bind directly to the CRE and/or its proximal bases, an observation which was supported by polyacrylamide gel separation of purified fractions. Identification, therefore, of a purified CRE-specific binding fac tor (s) was not possible with this DNA fragment.

Recommended Citation

Williams, Joseph Abner, "An investigation into camp regulation of the phosphoenolpyruvate carboxykinase gene. " PhD diss., University of Tennessee, 1988.
https://trace.tennessee.edu/utk_graddiss/11992