摘要目的 检测人类免疫缺陷病毒转录反式激活因子( TAT)对DNA依赖蛋白激酶催化亚单位( DN A-PKcs)基因启动子活性的影响.方法 用PCR方法克隆DNA-PKcs基因启动子的系列截短体,通过DNA重组构建pGL3-basic-DN A-PKcs肩动子报告质粒载体,通过双荧光素酶报告基因检测DNA-PKcs基因的肩动子活性,采用基于乳糖抑制子和乳糖操纵子并结合绿色荧光蛋白分子荧光的大规模染色质松弛报告系统观察染色质的重构活性,并研究了电离辐射对TAT参与染色体重构的影响.结果构建了DNA-PKcs启动子(全长区域为-939 bp～-1 bp)的系列截短体的报告质粒载体,鉴定出其核心区域为-64 bp～-1 bp.TAT能够抑制DNA-PKcs基因启动子的转录活性.TAT具有大规模的染色体松弛活性,电离辐射能抑制TAT参与染色体重构的作用.结论 TAT能抑制DNA修复基因DNA-PKcs的启动子活性,电离辐射能抑制TAT参与染色体重构的作用.

abstractsObjective To explore the influence of human immunodeficiency virus transactivator of transcription(TAT) on the promoter activity of DNA dependent protein kinase catalytic subunit (DNA-PKcs).Methods The truncated promoters of DNA-PKcs were cloned by PCR from the template DNA from HeLa genomic DNA,and the pGL3-basic-DNA-PKcs promoter reporter plasmids were constructed.The activity of DNA-PKcs promoters was detected by dual-luciferase reporter assay system.A Lac-repressor and Lacoperator based green fluorescent protein imaging system was used to assay the chromatin remodeling activity.Results A series of reporter plasmids harboring the truncated promoters of DNA-PKcs from -939 bp to -1 bp were constructed.The sequence of -64 bp to-1 bp was identified as a critical element for the activity of DNA-PKes promoter.TAT can suppress the activity of DNA-PKcs promoter.TAT participates in the regulation of the large scale chromatin relaxation.Ionizing radiation attenuates the activity of TAT played in the chromatin remodeling.Conclusion TAT represses the promoter activity of DNA repair protein DNA-PKcs,and also play a role of large scale chromatin remodeling which can te attenuated by ionizing radiation.