In vitro transcription from baculovirus late gene promoters: accurate mRNA initiation by nuclear extracts prepared from infected Spodoptera frugiperda cells.

AUTOR(ES)

Glocker, B

RESUMO

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.

Documentos Relacionados

• In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells.
• In vitro transactivation of baculovirus early genes by nuclear extracts from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells.
• Differential transcription of baculovirus late and very late promoters: fractionation of nuclear extracts by phosphocellulose chromatography.
• Control of protein synthesis in extracts from poliovirus-infected cells. I. mRNA discrimination by crude initiation factors.
• Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells.