Association of a protease with polytene chromosomes of Drosophila melanogaster.

Abstract

Incubation of Drosophila salivary glands with radioactive diisopropyl fluorophosphate results in the uniform labeling of polytene chromosomes. Extensive labeling is seen only when chromosome squashes are prepared by a formaldehyde fixation procedure and not by standard acetic acid techniques . The labeling is inhibited in the presence of tosylphenylalanine chloromethyl ketone and phenylmethane sulfonylfluoride but not by tosyllysine chloromethyl ketone, suggesting that a chymotrypsin-like serine protease is associated with the chromosomes. Protease inhibitors show no apparent effect on heat-shock specific puffing.Chromatin isolated from various rat and mouse tissues contains a highly basic 25,000-dalton serine protease (3), which appears to be directly bound to the DNA (J. Cavagnaro and C.-B . Chae, manuscript submitted for publication) . The protease selectively cleaves H 1 histone in condensed but not in relaxed chromatin (11,12) and oligonucleosomes in vitro (Y.-J . Kim and C.-B . Chae, unpublished results), suggesting the possibility of involvement of the enzyme in the local decondensation of super-coiled chromatin fiber by controlled cleavage of H 1 histone in vivo . The polytene chromosomes of Drosophila melanogaster larval salivary glands are an excellent system with which to study this possibility because specific genes at certain loci can be activated by heat and this gene activation accompanies readily recognizable puffing at the heat-shock specific loci (18). Therefore, we have attempted to establish that a protease is associated with polytene chromosomes by labeling chromosomes with a radioactive active site-directed protease inhibitor, diisopropyl fluorophosphate (DFP) and have studied the possible effect of protease inhibitors on heat-shock specific puffing.We find that although a chymotrypsin-like protease appears to be uniformly distributed along the polytene chromsomes, protease inhibitors do not appear to affect the specific puffing elicited by heat shock . After incubation the glands were transferred to formaldehyde fixative for 1-2 min and then squashed in formaldehyde-acetic acid (22) onto a subbed slide . The slide was then dipped into liquid nitrogen. Immediately after dipping, the covershp was removed with a razor blade and the slide was placed in cold PBS for 30-120 min with three changes of buffer . After the last rinse in PBS, the slides were prepared for autoradiography. Autoradiography and Giemsa staining were performed as previously described (15). MATERIALS AND METHODS Labeling of Salivary Heat ShockFor heat-shock experiments the salivary glands were incubated at 37°C for 30 min in Drosophila PBS. SDS Gel Electrophoresis of ( 3 HJDFP-labeledProteins 20 salivary glands were incubated in 100 pl of Drosophila phosphate-buffered solution with or without inhibitor and incubated for 30 min át room temperature. The solution was removed with a tuberculin syringe and 100 pl of [a H]DFP in PBS (final concentration 6.15 x 10 -s M [ 3H]DFP) was applied to cover the glands . After 30-m...