In vitro transcription with extracts of nuclei of Drosophila embryos

Abstract

An in vitro transcription system has been developed from 0.3M NaCl extracts of nuclei of Drosophila embryos. Optimal transcription in the Drosophila embryo extract (DEX) was at 5mM MgCl2, 70mM KCl, 25 degrees C and with promoter concentrations of 0,75-1.0 pmol/assay. In vitro transcription from the Adenovirus-2 major late and the Drosophila histone gene promoters was studied in particular. S1-nuclease protection experiments showed that in vitro transcription from these promoters was accurate. In vitro transcription from the Adenovirus-2 major late promoter was less efficient than from histone gene H3 and H4 promoters in DEX. Vicecersa, in vitro transcription from Adenovirus-2 major late promoter was more efficient in HeLa whole cell extracts. The efficiencies of transcription from histone gene promoters decreased in DEX in the order H4 greater than or equal to H3 greater than H2a. Transcription from H2b and H1 promoters was not detected in DEX. The transcription from the Adenovirus-2 major late promoter was completely inhibited by histone H3 and H4 promoters. Preincubation of DEX with the adenoviral template, however, did not inhibit transcription from histone H3 and H4 promoters. The transcription start sites of histone genes H3 and H4 are separated by 160 base pairs. The H3 and H4 transcription start sites were subcloned separately. Now, a competition of transcription from the H3/H4 promoters with the Adenovirus-2 major late promoter was observed. The competition studies suggest that preincubation of DEX with the adenoviral template inhibited transcription from the H3 promoter more strongly than from the H4 promoter.