Wang Hong-Gang, Ma Huan, Li Zhu, Zhang Bin, Jing Xiang-Yang, Zhang Yuan, Lv Zhan-Jun
Department of Genetics, Hebei Key Lab of Laboratory Animal, Hebei Medical University, Shijiazhuang 050017, China.
Yi Chuan. 2011 Apr;33(4):337-46. doi: 10.3724/sp.j.1005.2011.00337.
Our previous studies showed that tandem Alu repeats inhibited GFP gene expression when they were inserted into the downstream of GFP gene in pEGFP-C1 vector and HeLa cells were then transfected transiently. The sequence named 2F2R (second 60 bp from the 5' end of SV40PolyA antisense strand) eliminated the repression of GFP gene expression induced by Alu repeats when 2F2R was inserted between GFP and Alu repeats. In this study the deletion of 2F2R DNA showed that 45R (45 bp in 2F2R 5'end), 30R (30 bp in 2F2R 5' end) and 22R (22 bp in 2F2R 5' end) activated GFP gene expression, and the activating actions of the double tandem sequences were stronger than those of their corresponding single sequences. Secloop (22 bp near the center in 2F2R) and Poly4 (30 bp in 2F2R 3' end) sequences did not activate GFP gene expression. The activating action of 30R-Poly4 sequence formed by ligating 30R with Poly4 by 9 bp was lower than that of 2F2R. The linking base number between two 22R sequences did not influence the GFP gene expression obviously. Sequence 22R (5'-GTGAAAAAAATGCTTTATTTGT-3') contains an imperfect palindrome sequence and may form an imperfect stem-loop structure including a 3nt loop, 3 bp first stem, 2nt bulge, and 3bp second stem. The mutations changing stem-loop structure of 22R influenced the GFP gene activation significantly and neither the excessively stable nor excessively unstable stem-loop structures were in favour of GFP gene activation, which suggested that the suitably imperfect stem-loop structures had something with gene activation.
我们之前的研究表明，串联Alu重复序列插入到pEGFP-C1载体中GFP基因的下游时，会抑制GFP基因的表达，随后对HeLa细胞进行瞬时转染。当将名为2F2R（从SV40PolyA反义链5'端起的第二个60 bp）的序列插入到GFP和Alu重复序列之间时，它消除了Alu重复序列对GFP基因表达的抑制作用。在本研究中，对2F2R DNA的缺失分析表明，45R（2F2R 5'端的45 bp）、30R（2F2R 5'端的30 bp）和22R（2F2R 5'端的22 bp）可激活GFP基因表达，且双串联序列的激活作用强于其相应的单序列。Secloop（2F2R中心附近的22 bp）和Poly4（2F2R 3'端的30 bp）序列不能激活GFP基因表达。通过9 bp将30R与Poly4连接形成的30R-Poly4序列的激活作用低于2F2R。两个22R序列之间的连接碱基数对GFP基因表达没有明显影响。序列22R（5'-GTGAAAAAAATGCTTTATTTGT-3'）包含一个不完全回文序列，可能形成一个不完全茎环结构，包括一个3 nt环、3 bp的第一个茎、2 nt的凸起和3 bp的第二个茎。改变22R茎环结构的突变显著影响GFP基因的激活，过度稳定或过度不稳定的茎环结构均不利于GFP基因的激活，这表明适当的不完全茎环结构与基因激活有关。
