Cloning and characterization of the 5' flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells.
Cloning and characterization of the 5' flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells.
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DOI：
10.1042/0264-6021:3490641
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发表时间：
2000-07
期刊：
影响因子：
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通讯作者：
S. Price‐Schiavi;Aymee Perez;Roy Barco;K. L. Carraway
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文献类型：
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作者：
S. Price‐Schiavi;Aymee Perez;Roy Barco;K. L. Carraway
唾液粘蛋白复合物（SMC/Muc 4）是由粘蛋白亚基腹水唾液糖蛋白-1（ASGP-1）和跨膜亚基（ASGP-2）组成的异源二聚体糖蛋白复合物，在多种肿瘤细胞表面异常表达。SMC从单个基因转录，翻译成大的多肽前体，并进一步加工以产生成熟的ASGP-1/ASGP-2复合物。正常大鼠SMC具有复杂的时空表达模式，提示其具有复杂的调控机制。从正常大鼠肝基因组DNA文库分离的克隆产生的SMC/Muc 4基因5'区的粗外显子/内含子图谱显示，该基因具有包含5'非翻译区和信号肽的小的第一外显子，随后是大的内含子。第二个外显子似乎很大，包括5'独特区域和大部分（可能是全部）串联重复结构域。这种结构与人类MUC 4基因的报道惊人地相似。采用PCR-DNA步移法，克隆了SMC/Muc 4基因5 ′-侧翼区的2.4kb片段，并进行了表征。启动子模式搜索产生了多个图案中常见的组织特异性启动子。从该2.4kb片段产生的报告构建体在原代大鼠乳腺上皮细胞（MEC）、人结肠肿瘤细胞系HCT-116和人肺癌细胞系NCI-H292中显示启动子活性，但在COS-7细胞中不显示，表明上皮细胞特异性。转染到大鼠MEC或HCT-116细胞中的该序列的缺失构建体表现出极大变化的活性水平，表明该序列中存在阳性和阴性以及组织特异性的调控元件。总之，这些数据表明，大鼠SMC/Muc 4启动子已被确定，它是组织（上皮细胞）特异性的，并有积极和消极的，以及组织特异性，在序列中的调节元件。
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.