Identification and characterization of two critical sequences in SV40PolyA that activate the green fluorescent protein reporter gene

Honggang Wang,Xiufang Wang,Wuzhuang Sun,Zhanjun Lv,Zhu Li

doi:10.1590/s1415-47572011005000018

Honggang Wang, Xiufang Wang + Show 3 more

Open Access

https://doi.org/10.1590/s1415-47572011005000018

Copy DOI

Abstract

Alu repeats or Line-1-ORF2 (ORF2) inhibit expression of the green fluorescent protein (GFP) gene when inserted downstream of this gene in the vector pEGFP-C1. In this work, we studied cis-acting elements that eliminated the repression of GFP gene expression induced by Alu and ORF2 and sequence characteristics of these elements. We found that sense and antisense PolyA of simian virus 40 (SV40PolyA, 240 bp) eliminated the repression of GFP gene expression when inserted between the GFP gene and the Alu (283 bp) repeats or ORF2 (3825 bp) in pAlu14 (14 tandem Alu repeats were inserted downstream of the GFP gene in the vector pEGFP-C1) or pORF2. Antisense SV40PolyA (PolyAas) induced stronger gene expression than its sense orientation (PolyA). Of four 60-bp segments of PolyAas (1F1R, 2F2R, 3F3R and 4F4R) inserted independently into pAlu14, only two (2F2R and 3F3R) eliminated the inhibition of GFP gene expression induced by Alu repeats. Deletion analysis revealed that a 17 nucleotide AT repeat (17ntAT; 5′-AAAAAAATGCTTTATTT-3′) in 2F2R and the fragment 3F38d9 (5′-ATAAACAAGTTAACAACA ACAATTGCATT-3′) in 3F3R were critical sequences for activating the GFP gene. Sequence and structural analyses showed that 17ntAT and 3F38d9 included imperfect palindromes and may form a variety of unstable stem-loops. We suggest that the presence of imperfect palindromes and unstable stem-loops in DNA enhancer elements plays an important role in GFP gene activation.

Highlights

Considerable attention has been given to proteins and their encoding genes
We have shown that Alu tandem repeats and Line-1-ORF2 (ORF2) inhibited green fluorescent protein (GFP) gene expression when inserted downstream of this gene in the pEGFP-C1 vector (Wang et al, 2009a,b)
We examined the ability of sense and antisense SV40PolyA to eliminate the repression of GFP gene expression when inserted between the GFP gene and Alu repeats or ORF2 in pAlu14 or pORF2

Summary

Introduction

Considerable attention has been given to proteins and their encoding genes. With completion of the human and mouse genomes and a better understanding of eukaryotic gene expression, the noncoding sequences of genes have attracted increasing attention. Alu and Line-1 repeat elements represent about 10% and 17% of the whole human genome, respectively, and are the most important noncoding sequences (Belgnaoui et al, 2006; Polak and Domany, 2006). We have shown that Alu tandem repeats and Line-1-ORF2 (ORF2) inhibited green fluorescent protein (GFP) gene expression when inserted downstream of this gene in the pEGFP-C1 vector (Wang et al, 2009a,b). Downstream noncoding gene sequences are highly structured and contain important regulatory elements such as 3' UTRs, transcription termination signals (Andreassi and Riccio, 2009) and enhancers (Mao et al, 2010)

Methods

Results

Conclusion