摘要
目的:应用酵母双杂交和哺乳动物双杂交系统研究野生型双链RNA腺苷酸脱氨基酶ADAR1蛋白(ADAR1WT)与其突变体R916W(ADAR1R916W)之间的相互作用,从而探讨ADAR1基因错义突变在遗传性对称性色素异常症中的分子致病机制。方法:通过RT-PCR方法从患者外周血白细胞中获得含ADAR1基因R916W突变的全长cDNA,将野生型和突变cDNA分别克隆到相关质粒载体上,利用MatchmakerTMGAL4酵母双杂交系统3和CheckMateTM哺乳动物双杂交系统检测蛋白单体间的相互作用。结果:在酵母细胞中未检测到野生型-野生型ADAR1蛋白及野生型-突变体ADAR1蛋白间的相互作用;与对照细胞相比,pBIND-ADAR1WT和pACT-ADAR1WT共转染的HeLa细胞中荧光素相对活性显著升高(P<0.05);与共转染pBIND-ADAR1WT和pACT-ADAR1WT的HeLa细胞比较,pBIND-ADAR1R916W和pACT-ADAR1WT共转染的细胞中荧光素相对活性明显降低(P<0.05),为前者的66%。结论:在哺乳动物细胞中ADAR1WT自身相互作用,ADAR1WT和ADAR1R916W突变体蛋白相互作用依然存在但作用明显减弱。
Objective： Mutations in the ADAR1 gene cause dyschromatosis symmetrica hereditaria （DSH）, a rare autosomal dominant skin disorder. We have shown that nonsense and frame-shift mutations in the ADAR1 gene result in nonsense-mediated mRNA decay thereby leading to ADAR1 haploinsufficiency. The present study was designed to detect protein-protein interaction between the wild type ADAR1 （ADAR1^WT） and its R916W mutant （ADAR1^R916W） using yeast and mammalian two-hybrid systems, and to gain some insight into the molecular mechanism by which the missense mutations in the ADAR1 gene cause DSH. Methods： The full length normal and mutant ADAR1 cDNA clones were obtained from the peripheral lymphocytes from a DSH patient with R916W missense mutation by RT-PCR. The cDNA fragments were cloned into the bait and prey vectors in the Matchmaker^TM GAL4 yeast two-hybrid system 3 and the CheckMate^TM mammalian two-hybrid system, respectively. The two-hybrid experiments were performed by following the manufacturer＇s protocols. Results： Yeast cells co-transformed with plasmids pGADT7-ADAR1^WT and pGBKT7-ADAR1^WT or plasmids pGADT7-ADAR1^WTand pGBKT7-ADAR1^R9l6W failed to form colonies on SD/-Trp/-Leu/-His/-Ade plates, indicating a lack of protein-protein interaction. HeLa cells co-transfected with plasmids pACT-ADAR1^WT, pBIND-ADAR1^WT and pG5luc showed a significant increase in lueiferase activity （ P 〈 0.05）. Whereas cells co-transfected with pACT- ADAR1^WT, pBIND-ADAR1^R916W and pG5luc exhibited a considerable decrease in lueiferase activity （ P 〈 0.05）, retainhag 66% of the activity compared with the wild-type counterpart. Conclusion： ADAR1^R916W displayed decreased interaction with ADAR1^WT in mammalian cells, as compared with the ADAR1^WT-ADAR1^WT interaction.
出处
《山东大学学报（医学版）》
CAS
北大核心
2007年第5期450-454,共5页
Journal of Shandong University:Health Sciences
基金
国家杰出青年科学基金资助课题(30125017)
