Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene

Abstract

The presence of Alu repeats downregulates the expression of the green fluorescent protein (GFP) gene. We found that SV40PolyA (PolyA, 240 bp), in either orientation, eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats. In this study, 4 different segments (each 60 bp) were amplified from antisense PolyA (PolyAas) by PCR, and inserted upstream of Alu14 in pAlu14 plasmid (14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in a head-tail tandem manner). Segments 1F1R (the first 60 bp segment at the 5' end of PolyAas) and 4F4R (the fourth 60 bp segment from the 5' end of PolyAas) did not activate GFP gene expression, whereas 2F2R and 3F3R (the middle two segments) did (as detected by Northern blot analysis and fluorescent microscopy). Different copy numbers of 2F2R and 3F3R segments, in a head and tail tandem manner, were inserted downstream of the GFP gene in pAlu14. p2F2R*4-Alu28, p3F3R*4-Alu18 and p3F3R*4-Alu28 were used as length controls to verify that the decrease in the expression of GFP was not due to the increased length of the inserted segment in the expression vectors. We found that 2 and 4 copies of 2F2R or 3F3R activated the GFP gene more strongly than one copy of them. However, more than 8 copies of 2F2R or 3F3R reduced the activation of the GFP gene. We concluded that SV40PolyAas contained at least two gene-activating elements (2F2R and 3F3R) and 2-4 copies of 2F2R or 3F3R were optimal for the expression of the GFP gene.