title: Combined sense-antisense Alu elements activate the EGFP reporter gene when stable transfection.
abstract: Alu elements in the human genome are present in more than one million copies, accounting for 10% of the genome. However, the biological functions of most Alu repeats are unknown. In this present study, we detected the effects of Alu elements on EGFP gene expression using a plasmid system to find the roles of Alu elements in human genome. We inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences into the pEGFP-C1 vector to construct expression vectors. We altered the copy number of Alus, the orientation of the Alus, and the presence of an enhancer (4TMI) in the inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences. These expression vectors were stably transfected into HeLa cells, and EGFP reporter gene expression was determined. Our results showed that combined sense-antisense Alu elements activated the EGFP reporter gene in the presence of enhancers and stable transfection. The combined sense-antisense Alu vectors carrying four copies of Alus downstream of inserted CMV induced much stronger EGFP gene expression than two copies. Alus downstream of inserted CMV were replaced to AluJBs (having 76% homology with Alu) to construct expression vectors. We found that combined sense-antisense Alu (or antisense AluJB) vectors induced strong EGFP gene expression after stable transfection and heat shock. To further explore combined sense-antisense Alus activating EGFP gene expression, we constructed Tet-on system vectors, mini-C1-Alu-sense-sense and mini-C1-Alu-sense-antisense (EGFP gene was driven by mini-CMV). We found that combined sense-antisense Alus activated EGFP gene in the presence of reverse tetracycline repressor (rTetR) and doxycycline (Dox). Clone experiments showed that Mini-C1-Alu-sense-antisense vector had more positive cells than that of Mini-C1-Alu-sense-sense vector. The results in this paper proved that Alu repetitive sequences inhibited gene expression and combined sense-antisense Alus activated EGFP reporter gene when Alu transcribes, which suggests that Alus play roles in maintaining gene expression (silencing genes or activating genes) in human genome.
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Ma
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China.
authors.forename: Xianglong
authors.initials: X
authors.lastname: Kong
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China.
authors.forename: Shufeng
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China.
authors.forename: Shuxian
authors.initials: S
authors.lastname: Yin
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China.
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China. Zhanjun_lv@hotmail.com.
authors.forename: Zhanjun
authors.identifier: http://orcid.org/0000-0002-0580-1670
authors.initials: Z
authors.lastname: Lv
authors.affiliation: Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, Hebei Province, 050017, China. wangxiufang@live.cn.
authors.forename: Xiufang
authors.initials: X
authors.lastname: Wang
mesh_terms: D020087:Alu Elements / Q000235:genetics*; D016375:Antisense Elements (Genetics) / Q000235:genetics*; D045744:Cell Line, Tumor; D018628:Gene Dosage / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D015894:Genome, Human; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
chemical_list: D016375:Antisense Elements (Genetics); C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Activating gene; Alu RNA; Heat shock; Repetitive sequences; Tet-on