In vitro transcription from baculovirus late gene promoters: accurate mRNA initiation by nuclear extracts prepared from infected Spodoptera frugiperda cells.
Extracts prepared from nuclei of Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells were shown to support in vitro transcription from baculovirus late gene promoters. In vitro transcription was optimized for the late promoter of the 39K gene. The Mg2+ concentration was critical; concentrations higher than 1 to 2 mM did not support late transcription. Additional conditions included template (40 micrograms/ml), extract (2.5 mg/ml), and incubation time (25 min). Using a combination of runoff assays and high-resolution primer extension analyses, this system was shown to accurately initiate transcription from a variety of baculovirus late gene promoters, including those from the 39K and p39/capsid late genes and the hyperexpressed p10 and polyhedrin very late genes. In vitro transcription from the 39K late promoter was resistant to high concentrations of both alpha-amanitin (100 micrograms/ml) and tagetitoxin (4,000 U/ml), suggesting that neither RNA polymerase II nor III is responsible for the transcription of baculovirus late genes.
Glocker
B
B
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301.
Hoopes
R R
RR
Hodges
L
L
Rohrmann
G F
GF
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D000077183:Occlusion Body Matrix Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D000077183:Occlusion Body Matrix Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D012319:RNA Polymerase II; D012320:RNA Polymerase III