Gene expression control of animal lectins containing a collagen-like domain.

粘附素是一种独特的牛血浆蛋白，它介导致敏的红细胞-固相IC3b的凝集(粘连)。该蛋白被鉴定为一种钙离子。依赖动物凝集素的N-乙酰氨基葡萄糖专一性凝集素是一类具有胶原样结构域的蛋白质(胶原素、胶原样凝集素)。本研究从基因结构的角度研究了凝集素基因的5‘-调控区序列及其表达调控。1)凝集素基因5’-上游区域的特征。利用基因步行载体(盒式连接)-连接介导的PCR克隆了该基因的5‘-侧翼区，并对其序列进行了测定。鉴定了几个可能参与粘连蛋白表达的共同序列。2)用报告基因分析转录调控序列。CON…的5‘侧翼区以荧光素酶为报告基因，检测了More凝集素基因(700bp)的启动子活性。为了确定负责转录的conglitinin启动子区域，将一系列5‘-步进式启动子缺失和定点突变构建到荧光素酶载体中。将这些PLALAM启动子DNA载体导入人肝癌细胞系HepG2。荧光素酶启动子分析表明，在-200bp左右存在一个未知的正调控顺式作用元件。3)凝胶漂移实验和转录调控位点的DNase I足迹分析。凝胶迁移率分析表明，该未知顺式作用元件(约-200bp)和AP-1(约-158-154bp)与HepG2细胞和牛肝细胞的核蛋白提取物结合。DNase I足迹分析确定了未知顺式作用元件与核蛋白中反式作用元件的结合位置，约为-180bp。较少

Conglutinin is a unique bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC3b (conglutination). The protein is identified as a Ca^<2+>-dependent animal lectin specific for N-acetylglucosamine and a member of proteins which have a collagen-like domain (collectins, collagen-like lectins).In this research, the 5'-regulatory region sequence and the expression control of conglutinin gene were studied to elucidate the biological function of conglutinin with respect to the gene structure.1) Characterization of 5'-upstream region of the conglutinin gene.The 5'-flanking region of the conglutinin gene was cloned and sequenced by gene walking using vector (cassette) -ligation mediated PCR.In the promoter region (700bp) of the gene, several putative consensus sequences that may be involved in the expression of conglutinin were identified.2) Analysis of the transcription regulatory sequences with the reporter gene assay.A 5'-flanking region of the con … More glutinin gene (700bp) was examined for its promoter activity using luciferase as a reporter gene. To determine the region of the conglitinin promoter responsible for the transcription, a series of 5'-stepwise promoter deletions and site-directed mutations were constructed into the luciferase plasmids. These plamid-promoter DNA constructs were transfected into the human hepatoma cell line HepG2. The luciferase promoter assays revealed that an unknown positively controlling cis-acting elements exists around-200bp. This unknown cis-element acted synergistically with the AP-1 sequence present at-158bp--154bp.3) Gel-shift assay and DNase I footprint analysis of the transcription regulatory sites.The bindings of the unknown cis-acting element (around-200bp) and AP-1 (-158--154bp) with the nuclear protein extracts of the HepG2 cell and bovine liver cells were demonstrated with gelmobility shift assays. The DNase I footprint analysis identified the binding sites of the unknown cis-acting element with a trans-acting element in the nuclear protein around-180bp. Less