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Our previous studies have shown that tandem Alu repeats  inhibit green fluorescent protein (GFP) gene expression when inserted  downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence  (5'-GTGTGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation  signal) of simian virus 40, eliminated repression of GFP gene expression when  inserted between the GFP gene and the Alu repeats. The 22R sequence  contains an imperfect palindrome; based on RNA structure software prediction,  it forms an unstable stem-loop structure, including a loop, a first stem, a  bulge, and a second stem. Analysis of mutations of the loop length of the 22R  sequence showed that the three-nucleotide loop (wild-type, 22R) induced much  stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T,  A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge  and in the second stem in the 22R sequence, induced stronger GFP gene  expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete  complementation of the stem, caused weaker GFP gene expression. Sequences  without a palindrome (7pieA, 5'-GTGTG CGT-3', 7pieT,  5'-GTGGCGT-3') did not activate GFP gene expression. We conclude  that an imperfect palindrome affects and can increase GFP gene expression.

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression