β珠蛋白基因5′远端新沉默子克隆及定点诱变

Subcloning and Site-directed Mutation of a Novel Silencer Fragment in 5′ Flanking Distal Region of Human β Globin Gene

通过凝胶滞留分析和荧光素酶报告基因检测系统 ,鉴定出人类 β珠蛋白基因 5′旁侧远端帽位上游 - 2 1 32～ - 1 82 2 bp间存在一个活性沉默子片段 (31 0 bp) ,将其亚克隆至p UC- T载体中 ,然后采用人工设计的突变引物 ,将经 DNA足纹分析确定的两个结合位点的核心基序 (β珠蛋白基因帽位上游 - 2 0 1 7～ - 2 0 1 1 bp间的“CTTCCGC”序列和 - 2 0 0 6～ -1 997bp间的“CACTTTATTT”序列 )分别定点诱变为“CTTAAGC”和“CACTTAAGTT”两个突变序列 ,从而构建成两种突变型 31 0 bp片段 ,可用于对活性沉默子位点的结构与功能及β珠蛋白基因表达调控机制的深入研究 .

A novel silencer fragment(310 bp), which was recently discovered and identified by gel retardation assay and luciferase reporter gene expression analysis to locate between -2 132 bp and -1 822 bp upstream from the cap site of β globin gene, was subcloned \{into a pUC T\} vector. Then by using the DNA footprinting assays, it showed that there were two Hela cell nuclear protein binding sites in this fragment, one of them located at the sequence of -2 017～ -2 011 bp( i.e. “CTTCCGC”) and the other at -2 006～ -1 997 bp( i.e. “CACTTTATTT”). These two sequences were further sustained the site directed mutagenesis and accurately turned into the sequences of “CTTAAGC” and “CACTTAAGTT” respectively by two mutagenic primer in order to reconstruct mutated types of 310 bp fragment. They could be used to study the structure and function of the silencer as well as the mechanism of its regulation on gene transcription.