Fazio M J, O'Leary J, Kähäri V M, Chen Y Q, Saitta B, Uitto J
Department of Dermatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
J Invest Dermatol. 1991 Aug;97(2):281-5. doi: 10.1111/1523-1747.ep12480380.
Nidogen is a sulfated multifunctional glycoprotein present in basement membranes. In this study, we have cloned the 5'-flanking region of the human nidogen gene. Initially, an approximately 35-kb DNA clone (NCos4) was isolated from a human cosmid genomic library. Southern hybridization of EcoRI-digested NCos4 allowed isolation of a 3.7-kb fragment, which was shown to contain a portion of intron 1, the entire exon 1, and approximately 0.9 kb of 5'-flanking sequences of the nidogen gene. Nucleotide sequencing of the 5'-flanking DNA revealed the presence of two canonic CCAAT consensus sequences in the antisense strand and a potential variant of the TATA motif, TATTT, in the sense strand. One putative AP-2 and six putative SP1 binding sites were also present. To test the functional promoter activity of the 5'-flanking genomic DNA, two nidogen promoter/CAT reporter gene constructs, with the promoter segment spanning from -864 to -1 and from -534 to -1, respectively, were developed and analyzed in transient transfections of human and mouse cell cultures. Both constructs showed clearly detectable promoter activity, and the activity of the larger construct could be up-regulated by 12-O-tetradecanoyl phorbol 13-acetate up to 2.5 times. The results indicate that the nidogen promoter/CAT gene constructs developed in this study provide a means to examine the transcriptional regulation of nidogen gene expression in human diseases of the basement membrane zone.
巢蛋白是一种存在于基底膜中的硫酸化多功能糖蛋白。在本研究中，我们克隆了人巢蛋白基因的5'侧翼区域。最初，从人黏粒基因组文库中分离出一个约35kb的DNA克隆（NCos4）。用EcoRI消化NCos4后进行Southern杂交，分离出一个3.7kb的片段，该片段包含内含子1的一部分、整个外显子1以及巢蛋白基因约0.9kb的5'侧翼序列。对5'侧翼DNA进行核苷酸测序，发现在反义链中有两个典型的CCAAT共有序列，在有义链中有一个潜在的TATA基序变体TATTT。还存在一个假定的AP - 2结合位点和六个假定的SP1结合位点。为了测试5'侧翼基因组DNA的功能启动子活性，构建了两个巢蛋白启动子/CAT报告基因构建体，其启动子片段分别跨越-864至-1和-534至-1，并在人和小鼠细胞培养物的瞬时转染中进行分析。两个构建体均显示出明显可检测到的启动子活性，较大构建体的活性可被12 - O - 十四烷酰佛波醇13 - 乙酸酯上调至2.5倍。结果表明，本研究中构建的巢蛋白启动子/CAT基因构建体为研究基底膜区人类疾病中巢蛋白基因表达的转录调控提供了一种手段。
