SELECTION OF THE BEST TARGET SITE FOR RIBOZYME-MEDIATED CLEAVAGE WITHIN A FUSION GENE FOR ADENOVIRUS E1A-ASSOCIATED 300 KDA PROTEIN (P300) AND LUCIFERASE

The cellular 300 kDa protein known as p300 is a target for the adenovi ral E1A oncoprotein and it is thought to participate in prevention of the G(0)/G(1) transition during the cell cycle, in activation of certa in enhancers and in the stimulation of differentiation pathways, In or der to determine the exact function of p300, as a first step we constr ucted a simple assay system for the selection of a potential target si te of a hammerhead ribozyme in vivo, For the detection of ribozyme-med iated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luci ferase, as the reporter gene, We were also interested in the correlati on of the GUX rule, for the triplet adjacent to the cleavage site, wit h ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX, The rank order of activities in vitro indeed fo llowed the GUX rule; with respect to the k(cat), a C residue as the th ird base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU), However, in vivo the tRNA(Val) promoter-d riven ribozyme, targeted to a GUA located upstream of the initiation c odon, had the highest inhibitory effect (96%) in HeLa S3 cells when th e molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4, Since the rank order of activities in vivo did n ot conform to the GUX rule, it is unlikely that the rate limiting step far cleavage of the p300-luc mRNA was the chemical step, This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.