人β珠蛋白基因5’旁侧－1.5Kb处新沉默子的结构功能研究

A novel silencer fragment (310bp) was recently identified to locate between -2132～-1822bp upstream from the cap site of β-globin gene. DNaseⅠfootprinting assays were performed based on this discovery, and the results showed that there were two nuclear protein binding sites in this silencer, which are the -2017～-2011bp sequence"CTTCCGC" and the -2006～-1997bp sequence"CACTTTATTT". Subsequently, directed-mutagenesis assays and competitive gel retardation assays were performed, and the results showed that the two binding sites of nuclear proteins are involved or associated with a potential DNA-DNA interaction. Furthermore, two specific DNA-binding proteins were purified from the nuclear extract of Hela cells by affinity chromatography. By SDS -PAGE assays, the molecular weight of each protein was determined to be 37kDa and 81kDa, respectively. Moreover, a series of recombinant luciferase reporter gene vectors were constructed, which contain the promoter of different globin gene. These reporter gene vectors can be used to study the potential function of this silencer in the regulatory network of globin gene. Based on the accumulation of long-term scientific research and the internal and external research actuality, we bring forward an innovative viewpoint or hypothesis that is β-globin gene and even other eukaryotic genes should be controlled by complex network, which is composed of numerous regulatory elements, rather than few regulatory elements.

我们新0近发现并报道人β珠蛋白基因上游远端存在一沉默子活性功能区，内含众多潜在转嫉骺卦═REs）。拟采用凝胶阻滞、DNA足纹分析、Southwestern印迹杂交、核蛋白亲和层析和部分氨基酸序列分析以及荧光素酶报告基因检测等技术深入TREs及相关核蛋白的结构和功能，为进一步阐明β珠蛋白基因簇时空表达调控机制和β地贫的基因治疗提供新理论依据。..