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

含有胶原蛋白样结构域的动物凝集素的基因表达控制。

基本信息

批准号：
07672354
负责人：
KAWASAKI Nobuko
金额：
$ 1.41万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672354/
关键词：
Conglutinin Animal lectin Collagen-like structure Bovine serum Expression control of gene Luciferase Promoter Transcription regulatory sequence レクチン

项目摘要

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

凝集素是一种独特的牛血浆蛋白，介导致敏红细胞-固相iC 3b（凝集素）的凝集。该蛋白被鉴定为对N-乙酰葡糖胺特异的Ca^<2+>依赖性动物凝集素，并且是具有胶原样结构域的蛋白质的成员（collectins，胶原样凝集素）。在这项研究中，为了从基因结构上阐明conglomerin的生物学功能，我们对conglomerin基因的5 ′-调控区序列和表达调控进行了研究。通过基因步移法克隆并测序了该基因的5 ′-侧翼区，在该基因的启动子区（700 bp），鉴定了几个可能参与连接蛋白表达的推定共有序列。2）用报告基因分析法分析转录调控序列。 ...更多信息以荧光素酶为报告基因，检测了700 bp的Escherin基因的启动子活性。为了确定负责转录的伴格列汀启动子的区域，将一系列5 '-逐步启动子缺失和定点突变构建到荧光素酶质粒中。将这些质粒启动子DNA构建体转染到人肝癌细胞系HepG 2中。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）凝胶迁移率改变实验和DNA酶I足迹分析证实了未知顺式作用元件（约-200bp）和AP-1（约-158--154bp）与HepG 2细胞和牛肝细胞核蛋白提取物的结合。DNA酶I足迹分析确定了未知的顺式作用元件与核蛋白中约180 bp的反式作用元件的结合位点。少