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.

Alu元件（约280bp）是人类基因组中的重要重复序列,串联Alu呈长度依赖性下调GFP报告基因表达,在Alu串联序列上游正向或反向插入SV40PolyA（简称PolyA,240bp）,解除Alu串联序列对GFP基因的抑制作用.PCR法扩增PolyA反序（PolyAas）不同位置的60bp片段,插入pAlu14质粒（14个Alu正向串联插入pEGFP-C1）的GFP基因和Alu14之间,瞬时转染HeLa细胞,通过荧光显微镜观察和Northern检测,1F1R（PolyAas5′端第1个60bp片段）和4F4R（自PolyAas5′端计算第4个60bp片段）不能活化基因;2F2R和3F3R（PolyAas中间的2段）可以解除Alu14对GFP基因的抑制作用.将2F2R和3F3R各自反复首尾串联,分别插入pAlu14质粒GFP基因和Alu串联序列之间,瞬时转染HeLa细胞,用插入4个2F2R的pAlu28,插入4个3F3R的pAlu18,插入4个3F3R的pAlu28作长度对照,以排除由于插入片段长度增加可能对结果造成的干扰,发现2～4个拷贝的2F2R和3F3R活化基因作用高于一个拷贝的同样片段,但是多于8拷贝以后,活化GFP基因作用减弱.本实验证明,SV40 PolyAas至少含有两段活化基因序列,活化基因序列（2F2R,3F3R）的最适活化基因条件需要合适的拷贝数.

The presence of Alu repeats downregulates the expression of the green fluorescent protein (GFP) gene. We found that SV40PolyA (PolyA, 240 bp), in either orientation, eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats. In this study, 4 different segments (each 60 bp) were amplified from antisense PolyA (PolyAas) by PCR, and inserted upstream of Alu14 in pAlu14 plasmid (14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in a head-tail tandem manner). Segments 1F1R (the first 60 bp segment at the 5' end of PolyAas) and 4F4R (the fourth 60 bp segment from the 5' end of PolyAas) did not activate GFP gene expression, whereas 2F2R and 3F3R (the middle two segments) did (as detected by Northern blot analysis and fluorescent microscopy). Different copy numbers of 2F2R and 3F3R segments, in a head and tail tandem manner, were inserted downstream of the GFP gene in pAlu14. p2F2R*4-Alu28, p3F3R*4-Alu18 and p3F3R*4-Alu28 were used as length controls to verify that the decrease in the expression of GFP was not due to the increased length of the inserted segment in the expression vectors. We found that 2 and 4 copies of 2F2R or 3F3R activated the GFP gene more strongly than one copy of them. However, more than 8 copies of 2F2R or 3F3R reduced the activation of the GFP gene. We concluded that SV40PolyAas contained at least two gene-activating elements (2F2R and 3F3R) and 2-4 copies of 2F2R or 3F3R were optimal for the expression of the GFP gene.

The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vector downstream of GFP. All of the ORF2 fragments in sense orientation inhibited GFP expression more than when in antisense orientation, which suggests that small ORF2 fragments contribute to the distinct inhibitory effects of this ORF on gene expression. These results provide the first evidence that different 280-bp fragments have distinct effects on the termination of gene transcription, and that when inserted in the antisense direction, fragment 280-9 (the 3' end fragment of ORF2) induces premature termination of transcription that is consistent with the effect of ORF2.

Alu elements belonging to the short interspersed nuclear elements (SINE) of repetitive elements are present in more than one million copies which altogether represent 10% of the whole human genome. In this study, the roles ofAlu tandem sequences in the process of GFP gene (GFP) expression and packing into chromatin of its DNA were studied. To detect the effect ofAlu repeats on gene expression, different copies ofAlus were insertedGFP downstream respectively in pEGFP-C1 vector. We found thatAlu sequences decreased the amount ofGFP transcription, the percentage of GFP positive cells and the accessibility to DNase I in length-dependent manner. InsertingAlu caused the production of higher-molecular-mass RNA, indicatingAlu sequence did not induce premature transcriptional termination. Tight packing chromatins keep silent and resist to DNase I digestion, which is a general phenomenon. We suggested that head and tail tandemAlu sequences suppressedGFP expression in length dependent manner by triggering chromatin packing.