In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells

Abstract

The production and characterization of nuclear extracts from uninfected Spodoptera frugiperda cells, capable of accurately initiating transcription of baculovirus immediate early genes in vitro, are described. Optimal in vitro transcription was dependent on the presence of a TATA box promoter element and was abolished by alpha-amanitin. Nuclear extracts from the S. frugiperda cells primed with plasmid DNA containing the adenovirus major late promoter produced run-off transcripts of the size predicted for initiation from the adenovirus promoter. In addition, nuclear extracts prepared from a human cell line accurately initiated transcription from the promoter of the baculovirus immediate early gene encoding gp64. Primer extension analysis showed that transcripts derived from the gp64 gene promoter using both the S. frugiperda and human cell nuclear extracts initiated at the same nucleotide as transcripts produced in vivo.