摘要
为研究生肌素在染色质重建过程中的作用 ,采用生肌素真核表达载体转染C2C12肌细胞 ,细胞核经微球菌核酸酶消化后 ,提取DNA进行SDS PAGE分析 .生肌素转染的细胞 ,其核小体 (染色质 )在Mg2 + 溶液中的溶解性明显增加 ,提示染色质组蛋白乙酰化程度提高 .组蛋白经TAU SDS(2 D)双相凝胶电泳分析 ,发现在转染生肌素真核表达载体 2 4h后 ,组蛋白H4的乙酰化修饰程度最高 .采用抗乙酰化组蛋白H3和H4抗体进行的Western印迹分析进一步证明了乙酰化的发生 .上述变化与染色质的活跃程度相关 .RT PCR结果显示 ,生肌素的靶基因烟碱样乙酰胆碱受体 (nAChR)α亚基和肌酸激酶 (MCK)基因在转染后表达水平提高 .结果提示 ,强制性表达外源性生肌素可引起肌细胞核染色质重建 。
Summary After myogenin transfection into C2C12 muscle cells, the Mg 2+ containing buffer soluble fractionation of chromatin from hyperacetylated nuclei digested with micrococcal nuclease resulted in a substantial increase compared with that of control nuclei. Histones extracted from transfected cells were resolved by TAU SDS (2 D) gel electrophoresis, which showed that the acetylated species of histone H4 were apparently increased in 24 hours posttransfection. Western blot analyses did confirm these results. These findings indicate that the changes of histone hyperacetylation and chromatin higher solubility are related to the active chromatin. The results from RT PCR showed that the expression levels of the nicotinic acetylcholine receptor (nAChR) α subunit and muscle creatine kinase (MCK), the myogenin target genes, were enhanced. From the results of solubility, 2 D gel electrophoresis and RT PCR experiments, it is concluded that the forced expression of myogenin can stimulate chromatin remodeling, followed the activation of myogenin target genes.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2003年第1期92-95,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金 (3 9870 3 94)
北京大学 985计划资助~~
