Novel baculovirus DNA elements strongly stimulate activities of exogenous and endogenous promoters.
A DNA sequence upstream from the polyhedrin gene of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) was found to activate strongly the expression of full or minimal promoters derived from AcMNPV and other sources. Promoters tested included the minimal CMV (CMVm) promoter from human cytomegalovirus, the full heat shock 70 promoter from Drosophila, and the minimal p35 promoter from baculovirus. Deletion and mutagenesis analyses showed that this functional polyhedrin upstream (pu) activator sequence contains three open reading frames (ORFs), ORF4, ORF5, and lef2. In plasmid transfection assays, the pu sequence was able to confer high level luciferase expression driven by all of these full or minimal promoters in insect Sf21 cells. A known baculovirus enhancer, the homologous region (hr) of AcMNPV, further enhanced the expression of these promoters. Experiments showed that although multiple hr sequences function in an additive manner, pu and hr together function synergistically, resulting in as much as 18,000-fold promoter activation. Furthermore, a modified CMVm promoter containing pu and/or hr was inserted into the baculovirus genome to drive the luciferase coding region. The CMVm promoter expressed luciferase much earlier, and although it expressed a bit less than did the p10 promoter, the CMVm promoter gave rise to greater luciferase activity. Therefore, we have uncovered a cryptic viral sequence capable of activating a diverse group of promoters. Finally, these experiments demonstrate that synthetic sequences containing pu, hr, and different full or minimal promoters can generate a set of essentially unlimited novel promoters for weak to very strong expression of foreign proteins using baculovirus.
Lo
Huei-Ru
HR
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China.
Chou
Cheng-Chung
CC
Wu
Tzong-Yuan
TY
Yuen
Joyce Pui-Yee
JP
Chao
Yu-Chan
YC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D004252:DNA Mutational Analysis; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D007313:Insecta; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014162:Transfection
D018840:HSP70 Heat-Shock Proteins; D004247:DNA; D008156:Luciferases