Figure 1. Introduction of restriction sites in the GRU does not affect GRU activity.
To facilitate cloning, restriction sites were introduced into the GRU. (A) Nucleotide sequence of the CPS GRU and the REs therein (construct a), and the modified GRU upon the introduction of restriction sites (construct b). The triangles indicate the positions of the point mutations. The GRU constructs were cloned upstream of the minimal promoter and proximal enhancer of the CPS gene. These reporter-gene constructs were transiently transfected into FTO-2B hepatoma cells and induced with glucocorticoids for 24 h. The reporter-gene activities, measured in the resulting lysates, are presented as means±S.E.M. luciferase values for at least four experiments and the fold induction. The asterisk indicates significantly different results relative to the wild-type construct a. The data show that the combination of proximal enhancer and minimal promoter does not result in a marked increase in activity when induced by glucocorticoids relative to basal activity (B, construct c). In contrast, both the parent and the modified GRUs exhibited a similarly high level of activity when cultured in presence of glucocorticoids (B, constructs a and b). Dex, dexamethasone.