[The effects of SV40 PolyA sequence and its AATAAA signal on upstream GFP gene expression and transcription termination].
SV40 PolyA (Simian virus 40 PolyA, also called PolyA) sequence is DNA sequence (240 bp) that possesses the activity of transcription termination and can add PolyA tail to mRNA. PolyA contains AATAAA hexanucleotide polyadenylation signal. Fourteen copies of Alu in sense orientation (Alu14) were inserted downstream of GFP in pEGFP-C1 to construct pAlu14 plasmid, and then HeLa cells were transiently transfected with pAlu14. Northern blot and fluorescence microscope were used to observe GFP RNA and protein expressions. Our results found that Alu tandem sequence inhibited remarkably GFP gene expression, but produced higher-molecular-mass GFP fusion RNA. PolyA and its sequence that was deleted AATAAA signal in sense or antisense orientation were inserted between GFP and Alu tandem sequence in pAlu14. The results showed that all the inserted PolyA sequences partly eliminated the inhibition induced by Alu14. PolyA sequences without AATAAA signal in sense or antisense orientation still induced transcription termination. Antisense PolyA (PolyAas) was divided into four fragments that all are 60 bp long and the middle two fragments were named 2F2R and 3F3R. 2F2R or 3F3R was inserted upstream of Alu tandem sequence in pAlu14. The molecular mass of GFP fusion RNA increased when the copy number of 2F2R increased. 2F2R can support transcription elongation when 2F2R is located upstream of other 2F2R. Nevertheless, 2F2R located upstream of Alu tandem sequence can induce transcription termination. Inserting one copy or 64 copies of 3F3R in upstream of Alu tandem sequence caused the production of lower-molecular-mass GFP RNA.
Department of Genetics, Hebei Medical University, Shijiazhuang, China. lilishuping01@163.com
Shu-Ping
SP
Li
Jing-Jing
JJ
Feng
Hong-Gang
HG
Wang
Xiu-Fang
XF
Wang
Zhan-Jun
ZJ
Lv
D020087:Alu Elements; D018388:Codon, Terminator; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D018388:Codon, Terminator; D049452:Green Fluorescent Proteins; D011061:Poly A