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Originally published In Press as doi:10.1074/jbc.M108895200 on December 7, 2001
J. Biol. Chem., Vol. 277, Issue 7, 5256-5264, February 15, 2002
Novel Baculovirus DNA Elements Strongly Stimulate Activities of
Exogenous and Endogenous Promoters*
Huei-Ru
Lo,
Cheng-Chung
Chou ,
Tzong-Yuan
Wu§,
Joyce Pui-Yee
Yuen, and
Yu-Chan
Chao¶
From the Institute of Molecular Biology, Academia Sinica, Nankang,
Taipei 115, Taiwan, Republic of China
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.
*
This work was supported by a grant from the Academia Sinica
and Grant NSC 89-2313-B-001-003 from National Science Council, Taiwan,
Republic of China.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Institute of Biomedical Sciences, Academia
Sinica, Nankang, Taipei 115, Taiwan, ROC.
§
Present address: Institute of Biotechnology, National Chiayi
University, Chiayi 600, Taiwan, ROC.
¶
To whom correspondence should be addressed. Tel.:
886-2-2788-2697; Fax: 886-2-2788-2697 or 886-2-2782-6085; E-mail:
mbycchao@ ccvax.sinica.edu.tw.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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