Growth hormone is a single chain polypeptide hormone which plays a major role in somatic growth and in the control of protein, lipid and carbohydrate metabolism. By analyzing the DNA structure of the rat growth hormone gene (rGH), we hope to elucidate the molecular mechanisms controlling the production of growth hormone. The goals of this proposal are to characterize the DNA secondary structures which form in vitro in the rGH 5' flanking region at the nucleotide level and to investigate whether Z-DNA formation in particular plays a biological role in the regulation of rGH gene expression within transfected GH3 rat pituitary cells. The results of these studies may have broader implications for the role of DNA structure in gene regulation in general.
In Aims #1 and #2 a chemical (diethylpyrocarbonate, DEPC) footprinting technique will be used to define the exact nucleotides within the first 1751 bp of rGH 5' flanking DNA (urGH1751) that undergo Z-DNA and cruciform DNA structures. We will test the hypothesis that not all possible regions of DNA secondary structure in urGH1751 exist simultaneously. This is because there is not enough free energy (deltaG) of plasmid supercoiling to simultaneously stabilize them all. We propose that stabilization or de-stabilization of certain DNA secondary structures may provide a previously uncharacterized mechanism for regulating rGH gene expression.
In Aim #3 formation of Z-DNA within plasmids bearing wild-type mutant rGH promoters will be assayed after electroporation into GH3 cells by in vivo footprinting with OS04 and DEPC. Extension of these techniques by ligation-mediated PCR will allow us to assay for Z-DNA formation in the genomic rGH gene in GH3 cells. The GH3 cell line has been used extensively to examine the genetic regulation of rGH production.
In Aim #4 we will use both deletion and point mutations within the rGH upstream region to test for effect(s) of DNA secondary structure on rGH promoter activity. Appropriately mutated sequences will be cloned in front of the firefly luciferase reporter gene and introduced by electroporation into GH3 pituitary cells. Promoter activity will be monitored in cell extracts by light production.
