摘要
以ｐＵＣ１９－δｇｌｏｂｉｎ为模板，用ＰＣＲ定点突变技术将δ珠蛋白基因启动子区－６４位的Ｃ突变为Ｔ，即将ＣＣＡＡＣ盒改变为ＣＣＡＡＴ，并获得野生及突变的δ珠蛋白基因启动子区约３００ｂｐ的两个片段。将此两片段分别克隆到以虫荧光素酶为报告基因的表达载体ＰＭＧ３中，转染人ＨｅＬａ细胞，以瞬时表达分析突变的ＣＣＡＡＣ盒的启动子功能。结果表明突变组发动荧光素酶表达而产生的发光强度为野生型的５倍，说明将δ珠蛋白基因启动子区－６４位的ＣＣＡＡＣ盒改变为ＣＣＡＡＴ能够增强该启动子的功能，从而证实了δ基因启动子区ＣＣＡＡＣ盒的结构特点是该基因表达水平低的主要原因之一。
With PCR point mutation technique,the CCAAC box in the promoter region of δ-globingene was changed into CCAAT which is required in the normal transcription of β-globin geneat the corresponding site.The normal and mutant segments of the δ-globin promoter regionobtained with each segment of about 300 bp(-280/+54 bp),a fragment sufficient for therequirement of its normal promoter function,were separately cloned into pUC19/Hinc Ⅱ.After sequencing,it showed that the CCAAC box had been successfully changed intoCCAAT without other mutations. The two segments were released by Nco Ⅰ and BamH Ⅰ,and were cloned into the corresponding site of plasmid pMG3 which has the firefly luciferaseas its reporter gene.The positive clones were selected by Dot Blot and were comfirmed withrestriction mapping. Then the two recombinant plasmids were transfected into HeLa cell linerespectively and the promoter activities of the two fragn1ents were identified by comparingthe light intensity generated by the cell extracts when added with luciferase assay substrate.The results demonstrated that the luminescence of the mutant group is on average five timeshigher than that of the normal group,which indicate that the C-T mutation of the CCAACbox of δ-globin promoter region could increase its promoter activity. Our results have confirmed the hypothesis that the sequence of CCAAC box of human δ-globin gene is one of themain reasons which accounts for its low expression level.The author is now studying thespecific DNA binding protein of the upstream promoter region of human δ-globin gene.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
1996年第6期429-434,共6页
Acta Academiae Medicinae Sinicae
基金
中国医学科学院基金
国家自然科学基金
关键词
δ
珠蛋白
基因
启动子
点突变
δ-globin gene
promoter region
point mutation
