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

凝集素是一种独特的牛血浆蛋白，可介导致敏红细胞固相iC3b（凝集）的凝集。该蛋白被鉴定为一种Ca^<2+>依赖性动物凝集素，对n-乙酰氨基葡萄糖具有特异性，并且是具有胶原样结构域的蛋白质（集合素，胶原样凝集素）的成员。本研究通过对凝集素基因5'调控区序列和表达调控的研究，从基因结构上阐明了凝集素的生物学功能。1)粘连素基因5'-上游区域的表征。利用载体（盒式）连接介导的PCR技术，克隆了凝集素基因的5′侧区，并对其进行了基因行走测序。在该基因的启动子区（700bp），鉴定了几个可能参与粘连素表达的假定共识序列。2)用报告基因法分析转录调控序列。利用荧光素酶作为报告基因，对con . More glutinin基因（700bp） 5'侧区启动子活性进行了检测。为了确定粘连蛋白启动子负责转录的区域，在荧光素酶质粒中构建了一系列5'-逐步缺失和位点定向突变。这些质粒启动子DNA构建体被转染到人肝癌细胞系HepG2中。荧光素酶启动子分析显示，在200bp附近存在未知的正向控制顺式作用元件。这个未知的顺式元件与AP-1序列在158bp- 154bp处协同作用。3)转录调控位点的Gel-shift实验和DNase I足迹分析。凝胶迁移转移实验证实了未知的顺式作用元件（约-200bp）和AP-1（-158—154bp）与HepG2细胞和牛肝细胞的核蛋白提取物的结合。DNase I足迹分析在核蛋白约180bp处发现了未知的顺式作用元件与反式作用元件的结合位点。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