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The 5'-sequences flanking the human MUC1 gene have been analyzed for their ability to direct expression of a reporter gene (the chloramphenicol acetyltransferase gene (CAT)) in cell lines that normally express or do not express the MUC1 gene. A construct containing 2.9 kilobase pairs of MUC1 5'-flanking sequence sequence showed expression of CAT in breast and pancreatic cell lines but not in the non-epithelial cell lines HT-1080, SK23, and HTB96. Deletion analysis showed that maximum expression was obtained in ZR-75 (breast cancer line) and HPAF (pancreatic cancer line) with only 743 base pairs of 5'-flanking sequence. Sequences within 1.6 kilobase pairs of the transcriptional start site showed enhancing activity in a vector carrying an enhancerless SV40 promoter. Analysis of proximal 5'-sequences in a promoterless CAT vector carrying the SV40 enhancer showed that sequences between -60 and …