Abstract
BackgroundEnhancers are DNA sequences that serve as binding sites for regulatory proteins, and stimulate transcriptional activity independent of their positions and orientations with respect to the transcriptional initiation site. Previous studies considered that baculovirus homologous regions (hrs) function as enhancers in cis. In our study, a plasmid containing homologous region 3 (hr3) enhancer from Bombyx mori nucleopolyhedrovirus (BmNPV) failed to enhance transcription of promoter in other plasmid in co-transfection assays, but strong stimulation occurred when cells were infected by BmNPV. ResultsThe cotransfection results of each BmNPV genomic library plasmid, hr3 plasmid and reporter plasmid showed that there were eight library plasmids stimulated the luciferase gene expression remarkably. Sequencing these plasmids revealed that each of them contained the ie-1 gene. Transfected plasmids, containing ie-1, hr3 and various origin promoter drove reporter gene showed the function was even retained. Cotransfection of hr3 functional dissected fragment and ie-1 revealed that the 30-bp imperfect palindrome destroyed fragment can't enhance reporter gene expression even though transfected with ie-1.ConclusionIE-1 was the only early factor of BmNPV that could act as a mediator for hr enhancer function in trans and the trans-function was achieved with a broad-spectrum of promoters. The 30-bp imperfect palindrome was the elementary molecular structure by which IE-1 participated in the enhancer function in trans.
Highlights
Enhancers are DNA sequences that serve as binding sites for regulatory proteins, and stimulate transcriptional activity independent of their positions and orientations with respect to the transcriptional initiation site
Function of the hr enhancer in trans via virus infection The helicase promoter of Bombyx mori nucleopolyhedrovirus (BmNPV) was rather weak in transient assays, and only just-detectable luciferase activity was found
When the transfected cells were infected with BmNPV, or co-transfected with reporter plasmid and pKS-hr3, transcription of helicase promoter was slightly augmented, that is to say, hr3 did not appear to function as an enhancer when presented in separate plasmids in insect cells
Summary
Enhancers are DNA sequences that serve as binding sites for regulatory proteins, and stimulate transcriptional activity independent of their positions and orientations with respect to the transcriptional initiation site. A plasmid containing homologous region 3 (hr3) enhancer from Bombyx mori nucleopolyhedrovirus (BmNPV) failed to enhance transcription of promoter in other plasmid in co-transfection assays, but strong stimulation occurred when cells were infected by BmNPV. The genome of baculovirus contains interspersed homologous regions (hrs) that function as transcriptional enhancers linking in cis to viral or heterologous promoters in either insect or mammalian cells [1]. Hr from BmNPV failed to enhance the expression of the luciferase gene (luc) in trans in cotransfection assays, but strong enhancement occurred when the two independent plasmids were co-transfected into silkworm cells along with BmNPV. A random BmNPV genomic library was constructed and used to screen viral factor (s) mediating hr enhancer function in trans through co-transfection with DNAs from reporter plasmid and hr enhancer-containing plasmid. According to the structural characteristics of the hr enhancer, dissection analyses with different amounts of palindromes were conducted to uncover the basic requirement for hr enhancer function in trans
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call