Features of the RAP1A and RAP1B core promoters: recombinant approaches to testing regulatory element function in expression control (946.10)

Abstract

The RAP1 genes, closely related to the RAS oncogenes, have been shown to have numerous functions, yet little is known about the expression regulation of these genes in human cells. Our lab has constructed plasmids containing putative promoter regions of human RAP1A and RAP1B, driving a gene for green fluorescent protein (GFP). We have transiently transfected these plasmids into a wide‐range of human cell types, and analyzed GFP expression via fluorescence microscopy, qRT‐PCR, and western blotting. By comparing the efficacy of each putative core promoter segment, some distinct regulatory regions have been revealed. Additionally, comparing promoter‐driven expression to the actual native expression of the RAP1B genes in specific cell types has suggested an important regulatory role for enhancer elements not included in the core promoter regions. Various sub‐cloning techniques, including inverse fusion PCR cloning (IFPC), have been employed to add putative regulatory regions to existing GFP core promoter plasmids. These should allow us to further characterize the expression control of the RAP1 genes, and to understand the cell‐type differences observed in native RAP1 expression.