INVITRO TRANSCRIPTION FROM BACULOVIRUS LATE GENE PROMOTERS - ACCURATEMESSENGER-RNA INITIATION BY NUCLEAR EXTRACTS PREPARED FROM INFECTED SPODOPTERA-FRUGIPERDA CELLS

Extracts prepared from nuclei of Autographa californica nuclear polyhe drosis virus-infected Spodoptera frugiperda cells were shown to suppor t in vitro transcription from baculovirus late gene promoters. In vitr o transcription was optimized for the late promoter of the 39K gene. T he Mg2+ concentration was critical; concentrations higher than 1 to 2 mM did not support late transcription. Additional conditions included template (40 mug/ml), extract (2.5 mg/ml), and incubation time (25 min ). Using a combination of runoff assays and high-resolution primer ext ension analyses, this system was shown to accurately initiate transcri ption from a variety of baculovirus late gene promoters, including tho se from the 39K and p39/capsid late genes and the hyperexpressed p10 a nd polyhedrin very late genes. In vitro transcription from the 39K lat e promoter was resistant to high concentrations of both alpha-amanitin (100 mug/ml) and tagetitoxin (4,000 U/ml), suggesting that neither RN A polymerase II nor III is responsible for the transcription of baculo virus late genes.