Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells

Abstract

A simple method is presented for the preparation of nuclear extracts from suspension cultures of rice, wheat and tobacco cells. These extracts are shown to be capable of RNA Polymerase II-dependent transcription from two plant promoters in vitro; a 250 bp fragment of a wheat gliadin promoter containing sequences from -167 bp to +83 relative to the in vivo transcriptional initiation site and two fragments of the CaMV 35S promoter, containing sequences from -419 to +17, and from -90 to +17. Using the rice extract, transcription is shown to be extract-dependent, DNA-dependent, alpha-amanatin-sensitive, promoter-dependent, and accurate with respect to initiation site selection on the gliadin promoter and the -90 to +17 35S promoter, but not accurate on the -419 to +17 35S promoter.