Salivary gland nuclei of Drosophila hydei, isolated by a modification of the procedure described by Boyd et al. (9), retain their normal morphology during the isolation and subsequent incubation procedure. RNA synthesis was studied in isolated nuclei by biochemical and cytological techniques. In radioautographs 70% of the nuclei displayed a distribution of labeled RNA over the nuclear constituents similar to the distribution obtained after in vivo incorporation of radioactive precursor. Chromosome puffs and the nucleoli were specifically labeled. The remaining 30% of the nuclei showed a weak to very weak incorporation of radioactive precursor. In these nuclei most of the radioautographic grains were concentrated over the nucleolus, and a few grains were randomly distributed over the chromosomes. Actinomycin D and the absence of ATP, GTP, and CTP in the medium inhibited incorporation of radioactive precursor. The radioactive product was sensitive to combined pronase and RNase digestion. Addition of E. coli RNA polymerase to the incubation medium enhanced the specific labeling over the puffed regions. The sedimentation behavior of the RNA synthesized in isolated nuclei was different from that of RNA synthesized during a 20 min pulse of radioactive precursor administered to whole glands in vivo and in vitro. Neither the steroid ecdysterone nor a temperature treatment was effective in inducing new puffs in isolated nuclei.

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