Induction of ecdysterone-stimulated chromosomal puffs in permeabilized Drosophila salivary glands: A new method for assaying the gene-regulating activity of cytoplasm

Abstract

A simple assay system for gene regulation using chromosomal puffing as an index of gene activity was established. Salivary glands of Drosophila melanogaster treated with a mild detergent, digitonin, were permeable to high molecular substances, including β-galactosidase (MW 465,000). The permeabilized salivary glands retained the ability to form puffs at the ecdysterone-stimulated loci (74EF and 75B) in response to the hormone. Incubation of the permeabilized salivary glands at puff stage 1 (PS1) for 2 hr in a medium containing both ecdysterone and a homogenate of intact salivary glands at puff stage 8–9 (PS8–9) induced a puff at 78C, where puffing occurs only at puff stages 6–11 in vivo. The puff at 78C was not induced when the permeabilized PS1 glands were incubated with the combination of ecdysterone and a homogenate of the PS1 salivary glands. Likewise, the 78C puff was not induced in intact PS1 salivary glands by a 2-hr incubation with ecdysterone and PS8–9 gland homogenate. These results indicate that a factor(s) required for 78C puff formation is present in PS8–9 but not in PS1 salivary glands and that factor(s) can permeate digitonin-treated salivary glands but not intact glands. The effectiveness of the permeabilized salivary glands as an assay system for gene-regulating factors is discussed.