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We located and characterized a downstream transcriptional regulatory element in the human gastrin gene by transferring the gastrin gene 3' fragment, from which the polyadenylation signal sequence was deleted, into the shuttle vector pSCAT10 at a site located immediately downstream from the chloramphenicol acetyltransferase (CAT) gene and upstream from the simian virus 40 polyadenylation region. Study of CAT RNA derived from the hybrid plasmids, indicated regulation of transcription on the gastrin gene fragment. Analysis of deletion mutants generated from the 5' region of the fragment by CAT assay and by S1 nuclease mapping of mRNAs indicated the possible involvement of an oligothymidylate-rich sequence in transcription regulation. Mapping of gastrin gene RNA 3' ends to the 5' side proximal to the oligothymidylate-rich sequence clearly demonstrated that this sequence is a transcriptional terminator element. This unique sequence, interspersed with one or two adenines, which also functions in an orientation-dependent manner, is located 192 nucleotides downstream from the gastrin gene polyadenylation site, and serves as a transcriptional termination signal.