Hebei Key Lab of Laboratory Animal, Department of Genetics, Hebei Medical University, Shijiazhuang, 050017, China.
Sci China Life Sci. 2010 May;53(5):606-12. doi: 10.1007/s11427-010-0110-8. Epub 2010 May 23.
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. p2F2R4-Alu28, p3F3R4-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.
Alu 重复序列的存在会下调绿色荧光蛋白 (GFP) 基因的表达。我们发现，SV40PolyA（PolyA，240bp），无论其取向如何，当它放置在 GFP 基因和 Alu 重复序列之间时，都可以消除 Alu 重复序列对 GFP 基因表达的抑制作用。在本研究中，通过 PCR 从反义 PolyA（PolyAas）扩增了 4 个不同的片段（每个 60bp），并插入到 pAlu14 质粒中的 Alu14 上游（载体 pEGFP-C1 中的 14 个 Alu 重复序列以头尾串联的方式插入 GFP 基因下游）。片段 1F1R（PolyAas 5'端的第一个 60bp 片段）和 4F4R（PolyAas 5'端的第四个 60bp 片段）不能激活 GFP 基因的表达，而 2F2R 和 3F3R（中间两个片段）则可以（通过 Northern blot 分析和荧光显微镜检测）。不同数量的 2F2R 和 3F3R 片段以头尾串联的方式插入到 pAlu14 中的 GFP 基因下游。p2F2R4-Alu28、p3F3R4-Alu18 和 p3F3R*4-Alu28 被用作长度对照，以验证 GFP 表达的降低不是由于插入表达载体中的插入片段的长度增加所致。我们发现，2 个和 4 个拷贝的 2F2R 或 3F3R 比 1 个拷贝更能强烈地激活 GFP 基因。然而，超过 8 个拷贝的 2F2R 或 3F3R 会降低 GFP 基因的激活。我们得出结论，SV40PolyAas 至少包含两个基因激活元件（2F2R 和 3F3R），并且 2 到 4 个拷贝的 2F2R 或 3F3R 是 GFP 基因表达的最佳选择。
