Both Of SV40PolyA And PolyAas Can Decrease The Inhibition Of GFP Expression Caused By L1, Alu Insertion

Objectives: 98% of DNA sequence in human genome belongs to non-coding sequence. Half of them are comprised of the repeat sequence. The main types of the repeat sequence include long interspersed nuclear elements (LINEs),short interspersed nuclear elements (SINEs) and long terminal repeat (LTR), etc. LINE is composed by L1 and L2, while SINES is primarily constituted of Alu family. L1 and Alu which account for 16.9% and 9.9% of human genome sequence respectively are the most important repeat sequences. Some important biologic heredity and differentiation information are hiding in the L1 sequence. L1 is in preference to exist in allelic exclusion genes or the flanking of these genes, such as BCR (B lymphatic cell receptor) gene, TCR (T lymphatic cell receptor) gene, IL2 (interleukin2, IL2) gene, ect. Data show that L1 is also associated with the regulation of gene expression, cancer occurence, individual development and biologic evolution, ect.The Alu family is a set of dispersed, related sequences, each ~300 bp long, in the human genome. There are 1000,000 members in the haploid genome. It is the secondary more of the repeat sequences. Alu is related with multiple biologic events, including cancer occurence, chromosome recombination, translation regulation and so on. Researches also indicate that there is a close correlation between Alu and immunity systerm. Alu could be identified by some cis-regulatory elements such as specific enhancer in human CD8αgene of T cell. The research of Hamor proved that Alu inserted in the last intron of CD8αgene may take effect as enhancer.It is known that L1 has 10 families including L1M4, L1M3, L1M2, L1M1, L1P5,L1P4,L1P3,L1P2,L1P1 and Hs. According to their ORF2 (opened reading frame 2) and 3'untranslated region classification, these families also can be divided into 75 subfamilies. Han JS, et al found that inserting L1 ORF2 which originated from L1.2 subfamily into the downstream of EGFP (Enhanced green fluorescent protein) gene decreased the EGFP expression. However, it is still unknown whether other subfamilies have the similar function and the reason accounting for the decreased EGFP expression is not clear. To discuss the influence on GFP expression by different repeat sequences, we use a method by inserting ORF2 of another L1 subfamily member L1PA3 in the sense and antisense orientation and 14 tandem-repeated Alu into the pEGFP-C1. Considering the multiple mechanisms may result in the inhibition, we further constructed six new recombinant plasmids by inserting simian virus 40 polyadenylation signal (SV40PolyA , PolyA as brief) and PolyAas (PolyA antisense) into the above three reconstructed plasmids in purpose of exploring the underlying mechanism of L1/Alu in regulating the gene expression. After being transiently transfected 24 hours with the nine reconstructed plasmids and pEGFP as a contrast, HeLa cells are observed with fluorescence microscopy to examain the GFP expression.Methods:1 Construction of p-ORF2,p-ORF2as,p-Alu14ORF2 and ORF2as of L1PA3 on RP11 clone were amplified by PCR, and primers were designed with suitable restriction enzymes site, PCR production was digested with the restriction enzymes. p-ORF2, p-ORF2as were constructed by inserting into MCS (multiple clone site) downstream of GFP on pEGFP-C1.Alu on RP11 was amplified by PCR, and primers were designed with suitable restriction enzymes site. p-Alu14 was constructed by inserting 14 Alus into pEGFP-C1.2 Constructing recombinant plasmids which are inserting PolyA.PolyA was amplified by PCR and primers were designed with suitable restriction enzymes site. p-A-ORF2, p-A-ORF2as, p-A-Alu14 were constructed by inserting PolyA into the site between EGFP and ORF2/ORF2as/Alu14, while p-Aas-ORF2, p-Aas-ORF2as, p-Aas-Alu14 were constructed by inserting PolyAas into the site between EGFP and ORF2/ORF2as/Alu14. 3 Cell transfection and fluorescence microscopy observationThe nine plasmids were transiently transfected into HeLa cells using LipofectamineTM2000 with pEGFP-C1 as contrast. GFP expression in HeLa cells was observed with fluorescence microscopy. The total cell number was counted (at least 500 cells) with the light microscopy and the GFP positive cell number was counted with the fluorescence microscopy in the same scope. Take pictures respectively.Results:1 GFP expression was inhibited differently by inserting various repeat sequencesThe GFP expression percentages of HeLa cells transfected with p-ORF2,p-ORF2as,p-Alu14 and pEGFP-C1 after 24 hours show that all of them can decrease the GFP expression: (0.47±0.10)%,(3.72±0.58)%,(0.03±0.05)%. All are much lower than pEGFP-C1(35.93±1.81)%. The plasmid inserted by Alu inhibit the GFP expression mostly, then the ORF2, and the ORF2as inhibition ability is the least in the three.2 Both of PolyA and PolyAas can increase the inhibited GFP expression:The GFP expression percentages of HeLa cells transfected with p-A-ORF2,p-A-ORF2as,p-A-Alu14 after 24 hours are (7.69±0.55)%, (11.2±1.14)%,(3.94±0.26)%. All of the plasmids inserted by PolyA can partly highlight the expression compared with their carrier reconstructed plasmids respectively.The GFP expression percentages of HeLa cells transfected with p-Aas-ORF2,p-Aas-ORF2as,p-Aas-Alu14 after 24hours show that p-Aas-ORF2 is (9.15±0.77)%,p-Aas-ORF2as is (15.24±0.88)%,and p-Aas-Alu14 is (5.67±0.59)%。All of the plasmids inserted by PolyAas can partly highlight the expression compared to their carrier plasmids.Conclusion:1 Compared to pEGFP-C1, all of the reconstructed plasmids inserted by ORF2, ORF2as or Alu14 can supress GFP expression.2 The decreased GFP expression is in a different extent according to various sequences. The plasmid inserted by Alu inhibits the GFP expression mostly, then the ORF2, and the ORF2as inhibition ability is the least in the three.3 Both of PolyA and PolyAas can increase the inhibited GFP expression partly, and have no relation with the inserting direction.