PHENOTYPE OF RHESUS DECIDUAL NATURAL KILLER CELLS

University Of Wisconsin Madison, Madison WI

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Abstract

OBJECTIVE To identify regions of DNA-protein interactions in the rhesus monkey growth hormone variant gene promoter. We have been unable to detect DNA-protein interactions at the elements adjacent to the Sp factor binding sites using gel mobility-shift assays. We therefore moved to establish DNAse footprinting assays in the lab to determine whether this approach would allow us to detect previously occult protein-protein interactions. We have conducted DNAse protection assays with -140/+2 promoter construct found to contain full basal and cAMP-responsive activity in transient transfections. Two protected regions were tentatively identified as spanning -140/-130, and -66/-32. These regions include the Sp binding sites as well as adjacent 3U elements. Competition with a consensus Sp binding site oligonucleotide revealed that the entire upstream footprint could be accounted for by Sp factors, and that the 5U half but not the 3U-half of the downstream footprint could be competed by the consensus site. Additional experiments with recombinant Sp1 were in agreement with competition experiments. In the absenc e of Sp factors at -65/-60, a footprint spans the region from -60/-40. Significantly, the presence of a footprint in the absence of an Sp site indicates that Sp binding is not needed for adjacent DNA-protein interactions, which points towards this region as a potential candidate for an element for yeast 1-hybrid library screening. FUTURE DIRECTIONS We will prepare bait constructs with the downstream element for cloning DNA-binding factors using a yeast 1-hybrid approach. KEY WORDS placenta, gene transcription, placental growth hormone, transcription factors FUNDING NIH R01 HD26458