用酵母单杂交系统筛选与人ε-珠蛋白基因表达相关的基因  

Screening of Genes Related to the Expression of Human ε-globin Gene by Using Yeast One hybrid System

作　　者：黄建[1] 侯春晖[1] 钱若兰[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2001年第2期246-250,共5页

基　　金：国家自然科学基金资助项目 !(No .3989332 0;No .39870 378)&&

摘　　要：人体胚胎型ε 珠蛋白基因 5′旁侧一个沉默子 (- 392～ - 177bp)对关闭该基因在胎儿期的表达起重要作用。DNaseI足迹法分析已表明在沉默子区内存在着一个明显的蛋白质因子结合位点 (- 2 78～ - 2 35bp)。为了筛选与沉默子DNA特异结合的蛋白质因子 ,将该保护区域的DNA序列作为靶序列 ,采用酵母单杂交系统对人胎儿肝cDNA表达文库进行筛选 ,并对获得的克隆进行序列分析。初步的结果表明 ,已分离到了人核糖体大亚基蛋白3(ribosomalproteinL3 ,RPL3)的全长cDNA克隆。体外凝胶电泳阻抑分析及竞争实验亦证明该蛋白质与沉默子DNA存在相互作用。提示RPL3可能通过与沉默子DNA的结合参与调节εThe developmental stage specific silencing of the human ε globin gene during embryonic period is controlled, in part, by a silencer (from -392 to -177 bp) upstream of this gene. In our previous work, by using DNase I footprinting assay, a major protected region from -278 to -235 bp within this silencer DNA was identified. In order to isolate genes encoding proteins that bind to this target element, a human fetal liver cDNA library was screened by using yeast one hybrid system. The candidate clones were sequenced, and the ribosomal protein L3 (RPL3) was preliminarily isolated. Electrophoretic mobility shift assay (EMSA) and competition tests also demonstrated its binding activity to the target element. These imply that RPL3 might play an important role in silencing the human embryonic ε globin gene expression through the interaction with this silencer.

关 键 词：人ε-珠蛋白基因 酵母单杂交系统 沉默子 人核糖体大亚基蛋白3 表达 

分 类 号：Q786[生物学—分子生物学]