title: Cloning and characterization of the 5' flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells.
abstract: 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 5' 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 5' untranslated region and signal peptide, followed by a large intron. The second exon appears to be large, comprising the 5' 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 5'-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.
authors.affiliation: Department of Cell Biology and Anatomy (R-124), University of Miami School of Medicine, PO Box 016960, Miami, FL 33101, U.S.A.
authors.forename: S A
authors.initials: SA
authors.lastname: Price-Schiavi
authors.forename: A
authors.initials: A
authors.lastname: Perez
authors.forename: R
authors.initials: R
authors.lastname: Barco
authors.forename: K L
authors.initials: KL
authors.lastname: Carraway
mesh_terms: D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D005786:Gene Expression Regulation; D016678:Genome; D006801:Humans; D008969:Molecular Sequence Data; D055264:Mucin-4; D009077:Mucins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
chemical_list: C524658:MUC4 protein, human; C524660:Muc4 protein, rat; D055264:Mucin-4; D009077:Mucins; D004247:DNA