Different Tandem Segments From L1-ORF2 And Simple Repeat Sequences Modulate GFP Gene Expression

Objectives: The human genome is composed of 40-50% repetitive DNA. Long interspersed nuclear elements(LINEs)are important repeated sequences in the human genome. LINEs contain LINE-1 (L1) and LINE-2 (L2), and the former accounts for 16.9% of the human genome sequence. The majority of the L1 inserted in mammalian genomes is 5'truncated, and is in the antisense orientation within human genes. Interleukin-2 and T cell receptor genes are flanked by significantly higher densities of L1 sequences. Inserting L1-ORF2 (the second reading frame of L1) in the sense orientation to downstream of enhanced green fluorescent protein (GFP for short) gene inhibits intensively GFP gene expression, but ORF2 sequence placed in the antisense direction lead to GFP premature transcription termination. It is unclear that whole L1-ORF2 sequence or its specific segments cause this property. To study the effects of various ORF2 segments on upstream sequence, we used a method by inserting 8 tandem repeats of segments (280bp each) from different position on L1-ORF2 and 8 tandem-repeat Alu in the sense and antisense orientation into pEGFP-C1 (C1) vector, then transiently transfecting HeLa cells. Using Northern blot analysis and fluorescence microscope, we examined the effects of downstream sequence on GFP gene expression. A-rich is a sequence feature of L1-ORF2 in the sense strand, and accounts for 40.89%. To research whether A-rich simple repeat sequence has the characteristic of distinct gene expression in the sense and antisense orientation, we fused either AAACAAA or AG (736bp) repeats downstream of GFP gene in the sense and antisense orientation, and then observed the effects on GFP gene expression.Methods: 1 Plasmid construction 5 pairs of primers (Table 1) that had proper restriction endonuclease sites were designed. Five fragments (280bp each) of L1-ORF2 (L1PA3 subfamily member,) were amplified from RP11-29107 clone (position in Xq13.1) using the primers. After amplification, PCR products were subcloned respectively into C1 vector to generate five recombinant plasmids containing one segment (e.g. p280-2*1). Cohesive terminals of XbaⅠand NheⅠwere ligated by T4 DNA ligase, but XbaⅠnor NheⅠcan neither digest the ligated products. Using this principle,we constructed five kinds of 8 tandem repeat plasmids (e.g. p280-2*8). 8 tandem repeat plasmids were digested, and then short segments were separated. C1 vector and the short segment were connected. Antisense recombinant plasmids were monitored by PCR and restriction analysis. L1-ORF2 deleted 3'end sequence was 3212bp, and was amplified with specific primers (Table 1). The segment and C1 vector were connected to generate a recombinant plasmid (p280-1~8*8as). Two oligonucleotides (78bp) were synthesized, which have EcoRⅠ/XbaⅠrestriction endonuclease sites upstream, NheⅠ/KpnⅠdownstream and AAACAAA or AG simple repeats. The oligonucleotides amplified with Eco-Xba and Nhe-Kpn primers were separately inserted into C1 plasmid. Above-mentioned steps were performed again to generate recombinant plasmids.2 Cell transfection HeLa cells were split into 12-well plates and 24-well plates respectively. Cells were transfected with the recombinant plasmids using LipofectamineTM2000, with C1 as control. Cells in 12-well plates and in 24-well plates were cultured for 36 h in 37℃, 5% CO2 environment.3 Northern hybridization Using Trizol reagent total intracellular RNA was isolated at 36 h after transfection. RNA samples were subjected to electrophoresis in agarose gel containing formaldehyde, transferred to nylon membrane, and hybridized withα-32P dCTP labeled GFP probes prepared by PCR amplification. The nylon membrane was washed for radioautography. The nylon membrane was treated with removal solution at 80℃, for 1 h twice. After washing, the nylon membrane was hybridized again, withα-32P dCTP labeled Neo(neomycin resistance cassette) RNA probes prepared by PCR amplification, as control.4 Fluorescence positive cell counting Cells were fixed by 4% paraformaldehyde at 36 h after transfection. The total cell numbers were counted at white light and the GFP positive cell numbers were counted at fluorescence light in the same scope with×100. The ratios of fluorescence positive cells were calculated.Results: 1 Recombinant plasmids1.1 Recombinant plasmid construction Eighteen recombinant plasmids (Table 2) were constructed. They were identified by restriction analysis (Fig.2, Fig.4), PCR (Fig.3, Fig.4) and sequencing (Fig.5, Fig.6, Fig.7).1.2 Other recombinant plasmids p280-1*8, p280-4*8, pAlu*8, pORF2 and pORF2as were constructed in other works of our laboratory.2 Effects of L1-ORF2 tandem segments and Alu tandem sequence on GFP gene expression2.1 Northern hybridization GFP gene expression of all L1-ORF2 8 tandem segments inserted in the antisense orientation was higher than that in the sense orientation, but the result of Alu was opposite. Different tandem segments had different transcription termination positions, p280-1*8, p280-5*8, p280-9*8, p280-1*8as and p280-9*8as plasmids took place premature transcription termination (Fig.8).2.2 Counting results of the GFP positive cells HeLa cells were transfected with the six plasmids respectively, and then the results were observed (Fig.9). The mean (±SD) of positive fluorescence cells: p280-1*8(1.81±0.87)%,p280-1*8as(14±4.63)%,p280-5*8(0.51±0.17)%,p280-5*8as(12.5±3.04)%,p280-9*8(2.54±0.59)%,p280-9*8as(11.96±4.54)%. 3 The effect of L1-ORF2 segment deleted 3'end sequence on GFP gene expression3.1 Northern hybridization GFP gene inserted L1-ORF2 in the antisense orientation (pORF2as) emerged premature transcription termination. Differently from pORF2as, L1-ORF2 deleted 3'end sequence antisense segment (p280-1~8as) emerged transcription elongation instead of premature transcription termination (Fig.10).3.2 Counting results of the GFP positive cells HeLa cells were transfected with the three plasmids respectively, and then the results were observed (Fig.11). The mean (±SD) of positive fluorescence cells: pORF2(0.16±0.03)%, pORF2-1~8as (3.93±0.25)%,pORFas(5.45±1.00)%.4 Effects of simple repeat sequences on GFP gene expression4.1 Northern hybridization GFP transcription strengths of inserting AAACAAA or AG repeats in the antisense orientation were higher than their sense sequences, and both orientations of AAACAAA repeat sequence occurred premature transcription termination (Fig.12).4.2 Counting results of the GFP positive cells HeLa cells were transfected with the four plasmids and C1 respectively, and then the results were observed (Fig.13). The mean (±SD) of positive fluorescence cells: p(AAACAAA)Rep(2.86±0.25)%, p(AAACAAA)Repas(5.71±0.41)%, p(AG)Rep(2.23±0.47)%, p(AG)Repas(2.86±0.34)%, pEGFP-C1(39±3.67)%.Conclusions: 1 All of sequences inserted downstream of GFP gene inhibited GFP gene expression, although inhibition strengths and transcription termination positions were different.2 L1-ORF2 in the antisense orientation showed premature termination. Nevertheless, the antisense segment removed of L1-ORF2 3'end sequence no longer emerged premature transcription termination. Premature transcription termination caused by antisense L1-ORF2 might be due to a part of sequence from L1-ORF2 3'end.3 Short and simple repeat sequences may have some role in the characteristic formation of L1-ORF2 and in regulating GFP gene expression.