GH regulatory mutations may explain at least three forms of human pituitary dwarfism in which the GH structural genes are intact, and also contribute in more subtle ways to other metabolic syndromes. Availability of cloned DNA probes to GH regulatory genes will enable rapid screening of human DNA for mutations in these loci. The long-term objective of this research plan is the preparation of such molecular probes. Pieces of cloned DNAs encoding GH regulatory elements will also be used to isolate full length cDNAs which can be transferred into bacterial production vectors. This way, large amounts of GH regulatory elements can be isolated for studies of their mechanism of action in reconstituted systems.
The first aim i s to identify rat GH gene flanking DNA regions critical to proper GH gene expression. This will be accomplished through the construction of fusion genes between GH genes 5' and 3' flanking DNA and bacterial genes encoding easily assayable gene products. GH-bacterial fusion gene and endogenous rat GH gene expression will be compared after transfer into GH-expressiong GH3 rat pituitary tissue culture cells. Within these putative GH regulatory regions, sequences specifically required for dexamethasone-induced gene expression, and effects of 5-methylcytosine, particularly in regions of potential Z-DNA formation, will be investigated. DNA probes specifically designed to test the integrity of these regions could then be isolated and used to screen cells for potential defects in GH expression. Somatic cell hybridization of GH3 rat pituitary cells with mouse fibroblasts results in extinction of GH RNA expression.
AIM 2 is to test for fibroblast-derived or -induced repressors which mediate GH extinction via an interaction with GH flanking DNA. Transdominant, diffusible, and titratible, reversible or non-reversible repressors should be detected by looking for GH gene reactivation in hybrid cells in the presence of transferred copies of GH regulatory DNA. Potentially, these experiments provide the basis for using hybrid cells to obtain cDNA clones of molecules with GH gene repressor activity.
