The ultimate goal of this project is to provide unequivocal demonstration of the existence of a nuclear factor(s) which presumably binds to and regulates the transcription of genes known to be subject to control by cAMP in eukaryotic cells. It is postulated that such a protein ought to exist that is likely to be a substrate for the cAMP-dependent protein kinase (PK) which exhibits low affinity for the relevant specific DNA sequences until it has been phosphjorylated. We have preliminary evidence that such a factor is, in fact, present in cAMP-treated but not control nuclei. A variety of approaches will be taken with the goals of clearly demonstrating the existence of such a protein and developing methods for its purification. Nitrocellulose filter binding of various specific restriction fragments isolated from two cAMP-inducible genes (PEPCK and prolactin) will be conducted with salt extracts of nuclei from control and cAMP-treated cells. PAGE analysis of possible complexes will be run+/- competng DNA and footprinting will be done with Exonuclease III. The ability of various fragments to exhibit factor binding will be compared with their ability to confer cAMP-inducibility upon a heterologous gene (CAT) after injection into Xenopus oocytes or transfecion into mammalian cells. Attempts will be made to render control extracts (nuclear and cytosolic) capable of specific DNA binding by addition of the pure C subunit of PK an ATP. Transient expression of plasmids constructed with various fragements of the two inducble genes inserted upstream of the E. coli CAT gene in a pSV2 derived plasmid which contains the SV40 enhancer will be sought by injectin into oocytes and by transfection. cAMP inducibility will be scored in oocytes after injecion of pure PK subunit, PK subunit antibodies or addition of progesterone. Deletion and reorientation mutants should allow location of the critical regulatory elements (s) and to determine if it acts as an enhancer. A combination of the in vitro binding and in vivo expression systems will be used to assist purificaion of the putative trans acting protein subject to regulation by PK. The use of two gnes from different cells and species should allow us to determine whether or not the putative protein is a ubiquitous mediator of cAMP contol of eukaroytic gene expression and to discover what is the basis of its presumed interaction with specific regions of genes under control.
