Sense Or Antisense SV40 PolyA Sequence And AATAAA Signal Influence Upstream GFP Report Gene Expression

Objective: In the human genome, 98% DNA sequence belongs to the non-coding protein sequences. Nearly a half non-coding protein sequences are the repetitive sequences. L1(long interspersed nuclear elements 1, LINE-1) and Alu elements are two main repetitive sequence families.Polyadenylation signal,that is PolyA signal,is needed for most eukaryotic mRNAs to form their 3'ends and AAUAAA (AATAAA for genomic sequence) is its main form. Many kinds of biologic phenomenon are associated with Alu elements and the PolyA signal. Alu insertion or the deletion may cause the tumor occurrence, the chromosome reorganization and so on while a large class of hemoglobinopathies leading to thalassemias is due to point mutations in the AAUAAA sequence. We have designed a series of recombinant plasmids, which were loaded with Alu14(14 Alu elements syntropic series) and sense or antisense SV40 PolyA(Simian Virus 40 Early Polyadenylation Signal)sequence or the sequence lack of AATAAA.Then Hela cells were transfected with these plasmids. The GFP report gene expression changed with different inserted sequences, so we can conclude the role of Alu14, SV40 PolyA as well as AATAAA to the upstream sequence expression and support experimental foundation for further Alu and the PolyA signal mechanism research.Methods: 1 Construction and identification of p-A-Alu14 and p-Aas-Alu14 SV40 PolyA sequence from pEGFP-C1 was amplified by PCR. The PCR product was digested by HindⅢenzyme,and then ligated with p-Alu14.After identified by enzyme digestion analysis, PCR amplification analysis, and DNA sequencing, recombinant plasmid was named p-A-Alu14,in which PolyA sequence was inserted in sense orientation between GFP geng and Alu14 sequence or p-Aas-Alu14 in which PolyA sequence inserted in antisense oritation.2 Construction and identification of p-?A-Alu14 The sense PolyA sequence has two AATAAA sequence.To remove them, PCR amplified the first AATAAA upstream sequence PolyAa and the second AATAAA downstream sequence PolyAb; PolyAa and the PolyAb DNA fragments were fused by PCR recombination. Sense PolyA without AATAAA was formed.The following steps are similar with the construction of p-A-Alu14.The plasmid constructed was named p-?A-Alu14.3 Construction and identification of p-?Aas-Alu14 The antisense PolyA sequence has only one AATAAA. To remove it, PCR amplified AATAAA upstream sequence PolyAasa and the downstream sequence PolyAasb; then PolyAasa and the PolyAasb DNA fragments were fused by PCR recombination and antisense PolyA without AATAAA was formed. The following steps are similar as above. The plasmid constructed named p-ΔAas-Alu14.4 Transfection and cell count pEGFP-C1, p-Alu14 and all recombinant plasmids were transiently transfected into Hela cells by liposome.After 24 hours, positive cells which showed GFP expression were counted with fluorescence microscope.Results: 1 The recombinant plasmids p-A-Alu14 and p-Aas-Alu14 were proved to be correct by the restriction enzyme digestion analysis, PCR analysis, and DNA sequencing.2 p-ΔA-Alu14 and p-ΔAas-Alu14 were identified to be correct by DNA sequencing.3 The GFP expression percent of Hela cells transfected: p-Alu14 (0.10±0.02608)% p-A-Alu14 (4.05±0.32094)% p-Aas-Alu14 (5.08±0.30605)% p-ΔA-Alu14 (2.08±0.19918)% p-ΔAas-Alu14 (4.03±0.24221)% pEGFP-C1 (26.5±1.01587)%Conclusion: 1 Recombinant plasmids p-A-Alu14 and p-Aas-Alu14 were successfully constructed.2 Recombinant plasmids p-ΔA-Alu14 and p-ΔAas-Alu14 without PolyA signal AATAAA were successfully constructed.3 Alu14 sequence declined upstream GFP gene expression contrasted with pEGFP-C1 vector.4 SV40 PolyA insertion in both sense and antisense orientation can increase upstream GPF gene expression. 5 Sense or antisense SV40 PolyA without AATAAA can still increase upstream GPF gene expression.6 The increased GFP gene expression may be caused by other factors besides AATAAA.