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  • Title: Identification of a transcriptional regulatory region of the rat pancreatitis-associated protein I (PAP I) gene that confers tissue specificity.
    Author: Dusetti NJ, Ortiz EM, Dagorn JC, Iovanna JL.
    Journal: Biochem J; 1995 Oct 15; 311 ( Pt 2)(Pt 2):643-7. PubMed ID: 7487908.
    Abstract:
    We have previously characterized the rat pancreatitis-associated protein I (PAP I) gene by nucleotide sequencing. We describe in this paper its promoter region by analysing the regulatory functions associated with the DNA sequence comprising nt -1253 to + 10 of the gene. That sequence strongly promoted the transcription of the promotorless chloramphenicol acetyltranferase (CAT) gene in cells of pancreatic origin (AR-42J) but not in cells of non-pancreatic origin (Rat 2 and IEC 6). The influence on CAT expression of stepwise 5' deletions in the promoter sequence was monitored in the three cell lines. In pancreatic AR-42J cells, deletion down to position -926 did not affect significantly the expression of the reporter gene. Deletion to nt -685 caused about a 30% decrease in expression. Extending the deletion to nt -444 did not have any additional effect, but a further deletion to nt -180, resulted in a reduction to about 25%. Moreover, deletion from nt -180 to -118 resulted in a further reduction to about one-third of that. Finally, deletion down to nt -61 further reduced activity by a factor of 3, although it remained above background. These results suggest the presence of several positive cis-acting elements in the PAP I promoter. In non-pancreatic cells, CAT expression remained very low when the promoter was deleted down to nt -180. Yet, deletion from -180 to -118 significantly increased CAT expression, suggesting suppression of a negative cis-acting element. Further deletion down to nt -61 decreased CAT activity by a factor of 5. The region between nt -180 and -61 was subjected to footprint analysis. A similar pattern of DNase protection was obtained with AR-42J and Rat 2 nuclear extracts, the only protected region extending from nt -125 to -95. That region was further analysed by inserting the nt -180 to -81 fragment, in both orientations, upstream of thymidine kinase (TK) or simian virus 40 (SV40) promoter-CAT constructs. In all cases CAT expression was increased in pancreatic cells but reduced in Rat 2 cells. These results indicated the presence of cell-specific positive and negative elements within that region.
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