HPV18 Intergenic Spacer Mediating Bicistronic Expression in Eukaryotes
HPV18 is a DNA virus,with E6 and E7 gene open reading frame(ORF)separated by 8 nucleotides and expressed as a bicistron in cervical cancer HeLa cells.To investigate whether this 8 nt spacer sequence plays a crucial role in the bicis-tron gene expression,two bicistron expressing plasmids were constructed using this spacer sequence and transfected into HEK293T cells to detect the expression of both upstream and downstream ORFs.RT-PCR results showed simultaneous tran-scription of two ORFs;Western blot results showed that both proteins were expressed simultaneously.The confocal micro-scopy results further demonstrated the co-expression of red fluorescence protein(RFP)and green fluorescence protein(GFP).The above results demonstrate that the HPV18 E6 and E7 gene spacer sequence can re-initiate downstream ORF translation after the completion of upstream ORF translation.This phenomenon is relatively rare in eukaryotic cells,and the molecular mechanism might be related to the reentry of ribosomes which is worthy of further exploration.
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