We investigated whether the two variant elements of CRE(TGcCGTCA[5'CRE], TGACcTCA[3'CRE] in the 5'flanking region of the rat TRH gene, which are different from the CRE consensus sequence(5'-TGACGTCA-3') by one base pair, are responsive to cAMP
responsiveness of the gene. When CA 77 cells were stimulated with forskolin and isobutylmethylxanthine for 4 hours, the level of TRH mRNA was increased by only two fold. The transient gene expression study using serial 5' deletion of the TRH gene
in
PC12 cells showed that the region between-113 and-77, which includes 5'CRE, was crucial for the Camp resonsiveness. When the plasmid, which contains the 30 bp oligonucleotide including either 5'CRE or 3' CRE ligated to the enhancerless RSV
promoter, was
transfected into PC12 cells, it did not significantly affect not only the basal transcription but cAMP responsiveness. The 65 bp oligonucleotide including both 5'CRE and 3'CRE, however, increased both of the basal transcription and
cAMP-stimulat-ed
transcription by 2-3 fold. When the sequence of 5'CRE was converted to that of the CRE consensus by replacing one base pair, the cAMP responsiveness was increased by two fold although the basal transcription was not increased. The one base pair
mutant
of 3'CRE increased both of the basal and CAMP-stimulated transcription by 3-4 fold. These results suggest that there are the two variant CREs in rat TRH gene, which are relatively weak CRE compared to the CREs of other neuropeptide genes and
cooperative
for the activation of both the basal and cAMP-stimulated transcription. The one base pair difference of the variant CREs from the CRE consensus sequence is responsible for the weak responsivenss to cAMP. (J Kor Soc Endocrinol 9: 190-199, 1994)
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