Abstract
The presence of Alu repeats downregulates the expression of the green fluorescent protein (GFP) gene. We found that SV40PolyA (PolyA, 240 bp), in either orientation, eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats. In this study, 4 different segments (each 60 bp) were amplified from antisense PolyA (PolyAas) by PCR, and inserted upstream of Alu14 in pAlu14 plasmid (14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in a head-tail tandem manner). Segments 1F1R (the first 60 bp segment at the 5′ end of PolyAas) and 4F4R (the fourth 60 bp segment from the 5′ end of PolyAas) did not activate GFP gene expression, whereas 2F2R and 3F3R (the middle two segments) did (as detected by Northern blot analysis and fluorescent microscopy). Different copy numbers of 2F2R and 3F3R segments, in a head and tail tandem manner, were inserted downstream of the GFP gene in pAlu14. p2F2R*4-Alu28, p3F3R*4-Alu18 and p3F3R*4-Alu28 were used as length controls to verify that the decrease in the expression of GFP was not due to the increased length of the inserted segment in the expression vectors. We found that 2 and 4 copies of 2F2R or 3F3R activated the GFP gene more strongly than one copy of them. However, more than 8 copies of 2F2R or 3F3R reduced the activation of the GFP gene. We concluded that SV40PolyAas contained at least two gene-activating elements (2F2R and 3F3R) and 2–4 copies of 2F2R or 3F3R were optimal for the expression of the GFP gene.
Similar content being viewed by others
References
Tomilin N V. Regulation of mammalian gene expression by retroelements and non-coding tandem repeats. Bioessays, 2008, 30: 338–348 10.1002/bies.20741, 1:CAS:528:DC%2BD1cXltl2rs7s%3D, 18348251
Duan X C, Jin X, Xie Y, et al. Different effects on reporter gene expression by distinct L1-ORF2 segments. Hereditas, 2009, 31: 50–56 1:CAS:528:DC%2BD1MXht1OlsL7E, 19138901
Lee L T, Lam I P, Chow B K. A Functional variable number of tandem repeats is located at the 5′ flanking region of the human secretin gene plays a downregulatory role in expression. J Mol Neurosci, 2008, 36:125–131 10.1007/s12031-008-9083-5, 1:CAS:528:DC%2BD1cXhsVagtLjJ, 18566919
Alyssa S, Jennifer C, Winship H. Simian virus 40 revertant enhancers exhibit restricted host ranges for enhancer function. J Virology, 1988, 62: 3364–3370
Hiroshi H, Koji H, Ryoji N, et al. Tandem repeat of a transcriptional enhancer upstream of the sterol 14a-demethylase gene (CYP51) in Penicillium digitatum. Appl Environm Microbiol, 2000, 66: 3421–3426 10.1128/AEM.66.8.3421-3426.2000
Chen H, Lowrey C H, Stamatoy G. Analysis of enhancer function of the HS-40 core sequence of the human [alpha]-globin cluster. Nucleic Acid Res, 1997, 25: 2917–2922 10.1093/nar/25.14.2917, 1:CAS:528:DyaK2sXks1Khs7Y%3D, 9207043
Lander E S, Linton L M, Birren B, et al. Initial sequencing and analysis of the human genome. Nature, 2001, 409: 860–921 10.1038/35057062, 1:CAS:528:DC%2BD3MXhsFCjtLc%3D, 11237011
Polak P, Domany E. Alu elements contain many binding sites for transcription factors and may play a role in regulation of developmental processes. BMC Genomics, 2006, 7: 133 10.1186/1471-2164-7-133, 16740159, 1:CAS:528:DC%2BD28XmsFemsLk%3D
Ebihara M, Ohba H, Ohno S, et al. Genomic organization and promoter analysis of the human nicotinic acetylcholine receptor α6 subunit (CHNRA6) gene: Alu and other elements direct transcriptional repression. Gene, 2002, 298: 101–108 10.1016/S0378-1119(02)00925-3, 1:CAS:528:DC%2BD38XotFOmurs%3D, 12406580
Rund D, Dowling C, Najjar K, et al. Two mutations in the beta- globin polyadenylylation signal reveal extended transcripts and new RNA polyadenylylation sites. Proc Natl Acad Sci USA, 1992, 89: 4324–4328 10.1073/pnas.89.10.4324, 1:CAS:528:DyaK38XksVWrsL8%3D, 1374896
Mason P J, Jones M B, Elkington J A, et al. Polyadenylation of the Xenopus beta 1 globin mRNA at a downstream minor site in the absence of the major site and utilization of an AAUACA polyadenylation signal. EMBO J, 1985, 4: 205–211 1:CAS:528:DyaL2MXhtVOgurc%3D, 2862026
Han J S, Szak S T, Boeke J D. Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature, 2004, 429: 268–274 10.1038/nature02536, 1:CAS:528:DC%2BD2cXktVOqur0%3D, 15152245
Wang X F, Wang X Y, Liu J, et al. Alu tandem sequences inhibit GFP gene expression by triggering chromatin wrapping. Gen Genomics, 2009, 31: 209–215 1:CAS:528:DC%2BD1MXpslOmsr4%3D, 10.1007/BF03191192
Lechtenberg B, Schubert D, Forsbach A, et al. Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. Plant J, 2003, 34: 507–517 10.1046/j.1365-313X.2003.01746.x, 1:CAS:528:DC%2BD3sXkslOjtbY%3D, 12753589
Dhakshinamoorthy S, Sridharan S R, Li L, et al. Protein/DNA arrays identify nitric oxide-regulated cis-element and trans-factor activities some of which govern neuroblastoma cell viability. Nucleic Acid Res, 2007, 35: 5439–5451 10.1093/nar/gkm594, 1:CAS:528:DC%2BD2sXht1ygtb7F, 17702766
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yin, K., Wang, X., Ma, H. et al. Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene. Sci. China Life Sci. 53, 606–612 (2010). https://doi.org/10.1007/s11427-010-0110-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-010-0110-8