Figure 2. Structure and Responsiveness of Target HREs for T3 and RA.
Target HREs for the TR and RAR were synthesized as double-stranded oligonucleotides with an overhanging tetranucleotide (5’-agct-3’) at both ends. A single copy of these oligonucleotides was cloned at the unique Hindlll site present in a basal promoter CAT construct, ΔSV-CAT (see Experimental Procedures). Capitalized portions in the nucleotide sequences correspond to those found in the natural promoters except TREp and rGH21, which are synthetic. Bold letters and arrows indicate the AGGTCA motif and “X” denotes a nucleotide substitution from this motif. Numbers between the arrows are the size of the spacer and those in columns represent fold inductions of the CAT enzyme activity stimulated by the hormones in either the TR8groducing (± T3 at 100 nM) or RARa-producing (± RA at 1 μM) CV-1 cells. Inductions observed on the basal construct ΔSV-CAT by T3 and RA are 0.8 and 1 4-fold, respectively.
(a) TREp is an optimized palindromic rGH TRE (Glass et al., 1988). which stands also as an efficient RARE (Umesono et al., 1988). MHC-L encodes a TRE localized at positions between −154 and −122 from a transcription start site in the MHC gene promoter (shown as an antisense; Glass et al., 1989). MHC-S and -D contain deletion(s) (indicated by brackets) from the MHC-L.
(b) MHC-N encodes a core sequence of the wild-type MHC TRE, whereas Ml through M3 contain specific nucleotide substitutions (shaded letters and “X” in the arrow if they are located in the AGGTCA motif).
(c) Malic enzyme TRE corresponds to −281 to −261 from a transcription start site (Petty et al., 1990). A TRE found in the MLV LTR was taken from Sap et al. (1989) and rGH21 is a mutant of the rGH gene TRE (Brent et al., 1989a). βRARE corresponds to 8RE2 reported in Sucov et al. (1990).