Transcription of the major Drosophila heat-shock gene in vitro

Abstract

Active eukaryotic genes are more accessible to some proteins than bind DNA than are inactive genes. In order to probe the accessibility of the Drosophila heat-shock genes we have isolated nuclei from Drosophila tissue culture cells and have used these nuclei as templates for Escherichia coli RNA polymerase. With nuclei isolated from cells that had not been heat shocked, the synthesis of heat-shock RNA was not detected by hybridization to a DNA clone containing sequences from the major heat-shock region. In contrast, approximately 0.22% of the RNA synthesized in nuclei isolated from cells that had been previously heat shocked hybridized to the heat-shock clone. The synthesis of heat-shock RNA was DNA dependent, was sensitive to rifampicin and to actinomycin D, and represented a 70-fold enrichment over random transcription of the Drosophila genome. Transcription showed an extraordinary preference for a region 5' distal to the structural gene. These results demonstrate that preferential transcription by the bacterial RNA polymerase is indicative of the active state of Drosophila genes.