SV40 PolyA(猴空泡病毒PolyA, 简称PolyA)序列是有转录终止作用和使转录的mRNA添加PolyA尾的DNA序列(240 bp), 含有AATAAA六核苷酸多腺苷化信号(Polyadenylation signal)。在pEGFP-C1质粒的GFP基因下游插入14个同向串联的Alu序列(Alu14), 构建pAlu14质粒, 瞬时转染HeLa细胞, 用Northern blot检测和荧光显微镜观察GFP RNA和GFP蛋白表达, 发现Alu串联序列强烈抑制GFP基因表达, 该序列没有转录终止作用产生高分子量GFP融合RNA。又在pAlu14质粒GFP基因和Alu串联序列之间按正、反方向插入 PolyA序列及去除AATAAA信号的PolyA序列, 插入的这些PolyA序列均能部分解除Alu14对GFP基因的抑制作用; 去除AATAAA信号的PolyA正、反序列仍然引起转录终止。将PolyA反序(PolyAas)分为4段每段60 bp, 中间的2段分别称为2F2R和3F3R, 将2F2R或3F3R插在pAlu14质粒的Alu串联序列的上游, 随着插入2F2R片段拷贝数的增加转录的GFP融合RNA的分子量增加; 2F2R的下游如果依然是2F2R那么2F2R可以支持转录延伸, 如果2F2R下游是Alu串联序列则2F2R导致转录终止。无论插入一个3F3R或插入64个3F3R, 均产生低分子量GFP RNA。
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 tran-scription 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.
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