Li Shu-Ping, Feng Jing-Jing, Wang Hong-Gang, Wang Xiu-Fang, Lv Zhan-Jun
Department of Genetics, Hebei Medical University, Shijiazhuang, China.
Yi Chuan. 2012 Jan;34(1):113-9. doi: 10.3724/sp.j.1005.2012.00113.
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.
SV40多聚腺苷酸（猴病毒40多聚腺苷酸，也称为多聚腺苷酸）序列是一种DNA序列（240碱基对），具有转录终止活性，可在mRNA上添加多聚腺苷酸尾巴。多聚腺苷酸包含AATAAA六核苷酸聚腺苷酸化信号。将14个正向的Alu拷贝（Alu14）插入pEGFP-C1中GFP的下游，构建pAlu14质粒，然后用pAlu14瞬时转染HeLa细胞。使用Northern印迹法和荧光显微镜观察GFP RNA和蛋白质表达。我们的结果发现，Alu串联序列显著抑制GFP基因表达，但产生了更高分子量的GFP融合RNA。将多聚腺苷酸及其在正向或反向缺失AATAAA信号的序列插入pAlu14中GFP和Alu串联序列之间。结果表明，所有插入的多聚腺苷酸序列都部分消除了Alu14诱导的抑制作用。正向或反向无AATAAA信号的多聚腺苷酸序列仍可诱导转录终止。反义多聚腺苷酸（PolyAas）被分成四个长度均为60碱基对的片段，中间的两个片段分别命名为2F2R和3F3R。将2F2R或3F3R插入pAlu14中Alu串联序列的上游。当2F2R的拷贝数增加时，GFP融合RNA的分子量增加。当2F2R位于其他2F2R的上游时，2F2R可支持转录延伸。然而，位于Alu串联序列上游的2F2R可诱导转录终止。在Alu串联序列上游插入一个拷贝或64个拷贝的3F3R会导致产生较低分子量的GFP RNA。
