Nucleic Acids Research, Vol 24, Issue 15 3010-3016, Copyright © 1996 by Oxford University Press
H Kawasaki, J Ohkawa, N Tanishige, K Yoshinari, T Murata, KK Yokoyama and K Taira
The cellular 300 kDa protein known as p300 is a target for the adenoviral
E1A oncoprotein and it is thought to participate in prevention of the G0/G1
transition during the cell cycle, in activation of certain enhancers and in
the stimulation of differentiation pathways. In order to determine the
exact function of p300, as a first step we constructed a simple assay
system for the selection of a potential target site of a hammerhead
ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used
a fusion gene (p300- luc) that consisted of the sequence encoding the
N-terminal region of p300 and the gene for luciferase, as the reporter
gene. We were also interested in the correlation of the GUX rule, for the
triplet adjacent to the cleavage site, with ribozyme activity in vivo.
Therefore, we selected five target sites that all included GUX The rank
order of activities in vitro indeed followed the GUX rule; with respect to
the kcat, a C residue as the third 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-driven ribozyme, targeted to a GUA located
upstream of the initiation codon, had the highest inhibitory effect (96%)
in HeLa S3 cells when the 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 not conform to the GUX rule, it is unlikely that the
rate limiting step for 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.
ARTICLES
Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase
National Institute of Bioscience and Human Technology, MITI, Japan.
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