Cloning and characterization of the 5′ flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells

Autor: Shari A. PRICE-SCHIAVI, Aymee PEREZ, Roy BARCO, Kermit L. CARRAWAY
Rok vydání: 2000
Předmět:
Zdroj: Biochemical Journal. 349:641-649
ISSN: 1470-8728
0264-6021
DOI: 10.1042/bj3490641
Popis: Sialomucin complex (SMC/Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ascites sialoglycoprotein-1 (ASGP-1) and a transmembrane subunit (ASGP-2), which is aberrantly expressed on the surfaces of a variety of tumour cells. SMC is transcribed from a single gene, translated into a large polypeptide precursor, and further processed to yield the mature ASGP-1/ASGP-2 complex. SMC has complex spatial and temporal expression patterns in the normal rat, suggesting that it has complex regulatory mechanisms. A crude exon/intron map of the 5ʹ regions of the SMC/Muc4 gene generated from clones isolated from a normal rat liver genomic DNA library reveals that this gene has a small first exon comprising the 5ʹ untranslated region and signal peptide, followed by a large intron. The second exon appears to be large, comprising the 5ʹ unique region and a large part (probably all) of the tandem repeat domain. This structure is strikingly similar to that reported for the human MUC4 gene. Using PCR-based DNA walking, 2.4 kb of the 5ʹ-flanking region of the SMC/Muc4 gene was cloned and characterized. Promoter-pattern searches yielded multiple motifs commonly found in tissue-specific promoters. Reporter constructs generated from this 2.4 kb fragment demonstrate promoter activity in primary rat mammary epithelial cells (MEC), the human colon tumour cell line HCT-116, and the human lung carcinoma cell line NCI-H292, but not in COS-7 cells, suggesting epithelial cell specificity. Deletion constructs of this sequence transfected into rat MEC or HCT-116 cells demonstrate greatly varying levels of activity, suggesting that there are positive and negative, as well as tissue-specific, regulatory elements in this sequence. Taken together, these data suggest that the rat SMC/Muc4 promoter has been identified, that it is tissue- (epithelial cell-) specific, and that there are both positive and negative, as well as tissue-specific, regulatory elements in the sequence.
Databáze: OpenAIRE
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