In situ immunofluorescent visualization of chromosomal transcripts in polytene chromosomes

The induction and distribution of chromosomal transcripts in the polytene chromosomes of D. melanogaster and D. hydei has been investigated by indirect immunofluorescence using an antiserum directed against DNA/RNA hybrids. The fluorescence was intense and occurred in most of the chromosomal subdivisions when the chromosomes were exposed to denaturing conditions and then allowed to reanneal. The extent of hybrid formation depended both on the extent of DNA denaturation and on the maintenance of RNA integrity. Fluorescence was absent from chromosomes treated with pancreatic RNase before denaturation. The velocity of the chromosomal DNA/RNA hybridization reaction and the effects of the initiation inhibitor of RNA synthesis, DRB, suggest that in order to hybridize the RNA has to be located in its transcriptional compartment. Even though overall patterns of fluorescence seem to be similar during a developmental stage, variations were observed, particularly some correlated with puff induction after ecdysone stimulation.