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KMID : 0381120140360040455
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Genes and Genomics
2014 Volume.36 No. 4 p.455 ~ p.464
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The effects of a short sequence enhancer (5′-GTGAAATAAATGCAAATAAAGT) and its derived sequences on green fluorescent protein expression
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Ma Zhihong
Jing Xiangyang
Cheng Jianjun
Wang Xiufang
Lv Zhanjun
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Abstract
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Cis-regulatory elements are regions of DNA that regulate the expression of genes located on that same molecule of DNA. Though these elements are important for gene expression regulation, the functions of cis elements remain largely unknown. To explore the mechanisms of gene activation by short sequence enhancers, we examined the enhancer activity of short sequence DNA and its derived sequences using a GFP expression system. We found that AA sequence (5′-GTGAAATAAATGCAAATAAAGT) induced strong GFP gene expression, while 7pieA (5′-GTGAAAAAAATGCAAAAAAAGT) did not. We mutated the five T bases of the AA sequence to A, C or G. Our findings indicated that sequences retaining the 7th and/or 17th Ts possessed strong enhancer activity. RT-PCR and RNA synthesis inhibition analysis using actinomycin D revealed that the enhanced GFP gene expression induced by the AA sequence occurred at the transcriptional level. To determine whether the AA sequence formed a secondary structure via atypical complementation, PAGE method was used, and the results showed that the AA sequence formed a secondary structure. Our results support previous evidence that AATAAA is an important composition of cis elements (enhancer/promoter), and suggest that the formation of an unstable stem-loop structure via atypical complementation may be a new mechanism of enhancer activity.
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KEYWORD
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GFP, Enhancer, DNA structure, PAGE
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