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

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.

AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice

“…The discrepancy between vector-mediated protein and mRNA desmin expression is impressive and probably due to the SV40 poly(A) domain added in the 3′UTR, which is known to further stabilize mRNA and enhance translation. 18 , 19 …”

Proteomic Analysis of Apis cerana and Apis mellifera Larvae Fed with Heterospecific Royal Jelly and by CSBV Challenge

“…In recent years, heterospecific RJ breeding between Ac and Am has been concerned in the bee industry [39]. This technology was used between different species of western honeybees in 1957.…”

“…For the up-regulated proteins, pORF2 is a retrovirus reverse transcriptase with RNA-directed DNA polymerase activity [39]. Yellow-e2 responses to fungus/G + /G − , and functions in caste determination, and is influenced by environmental factors [51].…”

“…Heterospecific RJ breeding means during the growth or reproduction period of the colony or individual, changing the comb from Ac to Am or from Am to Ac or feeding RJ artificially. In recent years, heterospecific RJ breeding between Ac and Am has been concerned in the bee industry [39] . This technology was used between different species of western honeybees in 1957.…”

“…Correspondingly, 17 differentially expressed proteins from 3-day old larvae of Am fed with RJC included 6 up-regulated (pORF2, yellow-e2, MDH, Oat, RpS12, one unknown protein), and 11 down-regulated proteins (Hsc70-5, Tpx-1, Ef2b, FAH, invA, Cct5, Rps12, Hsp60,Gapdh1, Hex110, one unknown protein). For the up-regulated proteins, pORF2 is a retrovirus reverse transcriptase with RNA-directed DNA polymerase activity [39] . Yellow-e2 responses to fungus/G + /G − , and functions in caste determination, and is influenced by environmental factors [51] .…”