Summary
The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein.
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Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 74:5350–5354
Anderson DJ, Blobel G (1983) Immunoprecipitation of protein from cell free translation. Methods Enzymol 96:111–120
Benson M, Pirrotta V (1987) The product of the Drosophila zeste gene binds to specific DNA sequences in white and Ubx. EMBO J 6:1387–1392
Bittner M, Kupferer P, Morris CF (1980) Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal Biochem 102:459–471
Bonner WM, Laskey RA (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88
Bustos MM, Luckow VA, Griffing LR, Summers MD, Hall TC (1988) Expression, glycosylation and secretion of phaseolin in a baculovirus system. Plant Mol Biol 10:475–488
Doerfler W (1986) Expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells: Homologous viral and heterologous vertebrate genes — The baculovirus system. Curr Top Microbiol Immunol 131:51–67
Döring HP, Starlinger P (1986) Molecular genetics of transposable elements in plants. Annu Rev Genet 20:175–200
Gardiner GR, Stockdale H (1975) Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J Invertebr Pathol 25:363–370
Klenow H, Overgaard-Hansen K, Patkar SA (1971) Proteolytic cleavage of native DNA polymerase into two different catalytic fragments. Influence of assay conditions on the change of exonuclease activity and polymerase activity accompanying cleavage. Eur J Biochem 22:371–381
Kunze R, Stochaj U, Laufs J, Starlinger P (1987) Transcription of transposable element Activator (Ac) of Zea mays L. EMBO J 6:1555–1563
Kuroda K, Hauser C, Rott R, Klenk H-D, Doerfler W (1986) Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO J 5:1359–1365
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lübbert H, Doerfler W (1984a) Mapping of early and late transcripts encoded by the Autographa californica nuclear polyhedrosis virus genome: Is viral RNA spliced? J Virol 50:497–506
Lübbert H, Doerfler W (1984b) Transcription of overlapping sets of RNAs from the genome of Autographa californica nuclear polyhedrosis virus: A novel method for mapping RNAs. J Virol 52:255–265
Luckow VA, Summers MD (1988) Trends in the development of baculovirus expression vectors. Bio Technology 6:47–55
Maizel JV Jr, White DO, Scharff MD (1968) The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36:115–125
McClintock B (1948) Mutable loci in maize. Carnegie Inst Wash Year Book 47:155–169
McClintock B (1951) Chromosome organization and genic expression. Cold Spring Harbor Symp Quant Biol 16:13–47
McClintock B (1964) Aspects of gene regulation in maize. Carnegie Inst Wash Year Book 63:592–602
Miyamoto C, Smith GE, Farrell-Towt J, Chizzonite R, Summers MD, Ju G (1985) Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol Cell Biol 5:2860–2865
Müller-Neumann M (1985) Molekulare Isolierung und Charakterisierung des transponierbaren Elements Activator am Waxy-Locus von Zea mays L. Thesis, Universität zu Köln
Ollo R, Maniatis T (1987) Drosophila Krueppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA. Proc Natl Acad Sci USA 84:5700–5704
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schirm S, Doerfler W (1981) Expression of viral DNA in adenovirus type 12-transformed cells, in tumor cells, and in revertants. J Virol 39:694–702
Scott MRD, Westphal K-H, Rigby PWJ (1983) Activation of mouse genes in transformed cells. Cell 34:557–567
Smith GE, Summers MD, Fraser MJ (1983) Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol 3:2156–2165
Tjia ST, Carstens EB, Doerfler W (1979) Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus. II. The viral DNA and the kinetics of its replication. Virology 99:399–409
Tjia ST, Meyer zu Altenschildesche G, Doerfler W (1983) Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells. Virology 125:107–117
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Hauser, C., Fusswinkel, H., Li, J. et al. Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector. Mol Gen Genet 214, 373–378 (1988). https://doi.org/10.1007/BF00330469
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DOI: https://doi.org/10.1007/BF00330469