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

Abstract

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.