SV40PolyA顺式活化基因元件中不完整茎环结构的发现和序列研究

研究室的前期工作发现,Alu串连序列插入pEGFP-C1质粒的GFP基因下游,瞬时转染HeLa细胞抑制GFP基因表达,2F2R(来自SV40PolyA反序5′端的第2个60 bp)插入GFP和Alu串连序列之间可以解除Alu序列对GFP基因的抑制作用。文章通过删减2F2R发现,45R(2F2R 5′端的45 bp)、30R和22R可以活化基因,且二串连体活化基因作用高于单体。Secloop(2F2R近中部的22 bp)和Poly4(2F2R 3′端的30 bp)不能活化基因。30R与Poly4用9碱基连接形成30R-Poly4,其活化基因作用低于2F2R,两个22R之间连接碱基数对活化GFP基因作用没有明显的影响。22R(5′-GTGAAAAAAATGCTTTATTTGT-3′)含有不完整的回文序列,可以形成不完整的茎环结构,包括一个3碱基loop、3 bp第一茎、2碱基泡和3 bp第二茎。改变22R茎环结构的碱基突变明显影响其活化GFP基因的作用,过多互补和过少互补的茎环结构均不利于活化基因,提示适当的不完整茎环结构与活化基因有关。

Detection and sequence analysis of an imperfect stem-loop structure in cis activating gene element from SV40PolyA

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.