Alu And L1-ORF2 Segment Inhibit The Report Gene Expression And SV40PolyA Relieves The Suppression

Objective: The repeat sequences of Alu element and long interspersed nuclear element 1 (L1) represent 27% of the whole human genome mass, and mechanism research of gene expression regulation on repeat sequences has a significance for understanding biology function of non-coding sequences and human genome structure. Laboratorial research and analysis of bioinformatics have shown that L1 has the potential function to down-regulating gene expression; LINE-1 elements of high density are around inactive genes, monoallelic genes and their flanking sequences, such as immunoglobin loci genes, TCR genes, interleukins (IL-2 and so on). Most of laboratorial studies show Alu down-regulating gene expression, but a few tests and analyses of genome reveal Alu up-regulating gene expression. In human genome, the distributions of Alu elements are single or tandem; the majority of L1 repeat sequences are un-integrity sequences, so it is important to study influence of Alu and L1 tandem repeats on gene expression. Alu and L1 tandem repeats were inserted into report gene downstream to observe the effects on report gene transcriptive levels and transcriptive termination; SV40PolyA was inserted to report gene downstream to observe effects of Alu14 and ORF2 as flanking gene sequences on report gene; and whether Alu sequences change chromatin conformation was studied.Methods: 1 Construction of recombinant plasmids 280-4 sequence (1006bp-1285bp on ORF2) from pORF2 was amplified using primers with suitable restriction sites. The amplified DNA fragment was inserted to pEGFP-C1 vector to generate pEGFP-L1-ORF2-280-4*1 (p280-4*1). 280-4 sequences were inserted again and again to generate p280-4*2, p280-4*4, p280-4*8 and p280-4*14 tandem repeat plasmids. 280-4*1, *2, *4 were inserted into pAlu14 plasmid in sense and antisense orientation between GFP downstream and Alu14 upstream to gain six plasmids.pORF2 (L1-ORF2 was inserted into pEGFP-C1 plasmid), pAlu1, pAlu2, pAlu4, pAlu8, pAlu14 (1, 2, 4, 8 or 14 Alus were inserted into GFP downstream of pEGFP-C1 plasmid), pA-Alu14 (PolyA was inserted into the upstream of Alu14 in pAlu14 plasmid.), pAas-Alu14 (Antisense PolyA was inserted into the upstream of Alu14 in pAlu14 plasmid.), pΔA-Alu14 (Sense PolyA without AATAAA was inserted into the upstream of Alu14 in pAlu14 plasmid.), pΔAas-Alu14, pA-ORF2 and pAas-ORF2 were prepared. Recombinant plasmids used in this article are showed in Table 1.2 Cell transfection and fluorescence microscopy observation The 280-4 tandem repeat plasmids were transiently transfected into HeLa cells using LipofectamineTM2000. GFP expression in HeLa cells was observed with fluorescence microscopy.3 Northern blot GFP probe marked byα-32P was prepared by PCR amplification. Total RNA of HeLa cells transfected was abstracted, electrophoresed and transferred to the nylon film. Hybridizion with GFP probe and autoradiography were performed. The nylon film was treated with removal liquid, and then was again hybridized with the probe for neo RNA as comparison.4 Assay of DNaseⅠsensitivity Integrity of template DNA is essential for PCR amplification, whereas degradation of template DNA caused by DNaseⅠdigestion results failure of PCR amplification. Open chromatin is sensitive to DNaseⅠdigestion, but DNA packed tightly is resistant to DNaseⅠdigestion, so after DNaseⅠdigestion, we can estimate chromatin packing state by PCR amplification of some chromatin segments. The nucleus transfected with pAlu1, pAlu14 were digested using DNaseⅠfor 1 or 3 min. 4 pairs of primers were used to amplify GFP gene and GFP-Alu fusion gene.Results: 1 Identification of recombinant plasmids The recombinant plasmids were proved to be correct by the restriction digestion analysis, PCR analysis, and DNA sequencing. Fig.2 is electrophoresis pattern of the 280-4 tandem repeat plasmids digested with HindⅢand KpnⅠ. Fig.3 is p280-4*2 sequencing map and sequence. Fig.4 shows restriction digestion and PCR electrophoresis pattern for 280-4*1, *2, *4 inserting into pAlu14 plasmid in sense and antisense orientation between GFP downstream and Alu14 upstream. Results of restriction digestion, PCR and sequencing were consistent with desire and consensus sequences.2 The effects of Alu and 280-4 tandem repeats on GFP expression The percentages of GFP fluorescent positive cells in transfected HeLa cells of p280-4*1, p280-4*2, p280-4*4, p280-4*8, p280-4*14 and pEGFP-C1 were (4.7±1.30)%, (2.5±0.30)%, (1.8±0.30)%, (0.5±0.30)%, (0.1±0.30)% and (35.3±2.66)% respectively. Along with increase of inserted segment of L1-ORF2-280-4, fluorescence positive cells reduced. The percentages of fluorescence positive cells transfected with p280-4*1 was 47 times higher than that with p280-4*14(Fig.5). The percentages of GFP fluorescent positive cells in transfected HeLa cells of pAlu1, pAlu2, pAlu4, pAlu8 and pAlu14 were reported in another article.Total RNAs of HeLa cells transfected with Alu and 280-4 tandem repeats and pEGFP-C1 plasmids were extracted. Northern blot showed that along with Alu length increasing, GFP transcript levels decreased and transcript lengths increased. Ratios of the relative transcript level of pEGFP-C1/pAlu1, pAlu1/pAlu2, pAlu2/pAlu4, pAlu4/pAlu8 and pAlu8/pAlu14 were 1.18, 1.14, 1.39, 1.58 and 55.5 respectively, which showed that stronger GFP inhibition took place from pAlu8 to pAlu14. Northern results of 280-4 insertion indicated that lengths of 280-4 segments increased and GFP transcript levels decreased. 280-4 insertion inhibition level exceeded the Alu insertion (Fig.6).3 Relieving the suppression by SV40PolyA Insertions of PolyA, PolyAas,ΔpolyA (deleted the AATAAA core sequence) andΔpolyAas into Alu14 upstream of pAlu14 plasmid, which made Alu14 as flanking sequence, relieved GFP gene expression suppression by Alu14. The relieving suppression abilities of PolyAas were higher than that of PolyA. The relieving suppression abilities ofΔpolyA (ΔpolyAas) were similar to PolyA and PolyAas. Premature transcriptions took place in all PolyA recombinant plasmids;Insertions of PolyA and PolyAas into pORF2 also relieved the GFP repression by ORF2 (Fig.7).4 The effects of mixed array on GFP gene expression Insertions of 280-4*1, *2, *4 in sense and antisense orientation between GFP downstream and Alu14 upstream of pAlu14 plasmid, along with length increasing, inhibited GFP transcript levels stronger. The relieving suppression abilities of 280-4 antisense were higher than that of 280-4 sense sequences. GFP gene transcription of 280-4 tandem sequences inserted to pEGFP-C1 in antisense orientation was higher than that in sense orientation, which was similar to 280-4 inserting into Alu14 upstream of pAlu14 (Fig.8).5 DNaseⅠdigestion sensitility The nucleus of transfected HeLa cells with pAlu1, pAlu14 were digested using DNaseⅠ, and 4 pairs of primers were used for PCR amplification. The lengths of amplified fragments were GFP5F/GFP4R (215bp), GFP2/GFP3 (311bp), 1GFP1F/Alu2 (460bp) and ECor-xba/Alu2 (340bp). DNA fragments were detected from nucleus of cells transfcted with pAlu14 using DNaseⅠtreatment for 1 min, whereas not detected when treated for 3 min. DNA fragments were not detected from nucleus of cells transfcted with pAlu1 digested for 1 or 3 min. These results suggest that GFP gene of pAlu14 or fusing gene have more tightness chromatin construction (Fig.9).Conclusion: 1 The longer the Alu sequence is, the stronger the gene inhibitory action is.2 The Alu element belongs to elongating sequences for transcription and has not terminators on it.3 The L1-ORF2-280-4 is also belonging to elongating sequences, but the activity of transcription is weaker than Alu element.4 Insertions of PolyA, PolyAas,ΔpolyA andΔpolyAas into pAlu14 or pORF2, making Alu14 and ORF2 as gene flanking sequences, relieve the GFP repression, which are not influenced by PolyA orientation and deleted AATAAA hexamer.5 Mixed array of Alu14 and 280-4 shows that along with 280-4 length increasing, GFP transcript levels decrease.6 GFP gene expression inhibition by Alu14 tandem sequence is related to chromatin construction.