The distribution of DNA topoisomerase I within Drosophila polytene chromosomes was observed by immunofluorescent staining with affinity-purified antibodies. The enzyme is preferentially associated with active loci, as shown by prominent staining of puffs. The heat shock loci 87A-87C are stained after, but not before, heat shock induction. A detailed comparison of the distribution of topoisomerase I with that of RNA polymerase II reveals a similar, al, though not identical, pattern of association. Topoisomerase I is also found in association with the nucleolus, the site of transcription by RNA polymerase I.The polytene chromosomes of the Drosophila salivary gland provide an excellent system for investigating the distribution of specific chromosomal proteins. Although highly organized, these giant chromosomes behave like diploid interphase chromatin in many assays of function and fine structure (1-6). They can, however, be easily observed under the light microscope; some substructure, such as transcriptionally active sites, can be recognized. It is possible to localize proteins in these chromosomes and thereby obtain some information as to the biological processes in which the given proteins might be involved. In order to visualize the chromosomal proteins, the method of indirect immunofluorescence was developed several years ago (7,8). The distribution patterns of a number of chromosomal proteins of unknown function have been determined by this approach (8-11). The method has allowed identification of a subclass of nonhistone chromosomal proteins that are prominently associated with loci that are active or inducible at some time in the salivary glands of the third instar larvae and prepupae (10,12,13).In addition, distribution patterns of proteins of known function have been analyzed-e.g., RNA polymerase 11 (14-17) and ribonucleoproteins (18). These proteins are preferentially associated with the transcriptionally active regions of the genome. In this paper we report the distribution pattern of DNA topoisomerase I. This enzyme is well characterized at the molecular level, but the range of its biological functions in eukaryotes has yet to be established (for reviews, see refs. 19-25). The ability of eukaryotic topoisomerase I to relax either negatively or positively supercoiled DNA hints that the enzyme might play a role in gene activation, either effecting structural changes in chromatin as it assumes a more "open" conformation (e.g., as detected by the appearance of puffs) and/or facilitating transcription per se (26, 27). There is some evidence in favor of a role of topoisomerase in transcription. For example, topoisomerase has been shown to be associated with ribosomal gene chromatin actively expressing rRNA (28); topoisomerase I is recovered with nucleosomes in a fraction enriched for transcriptionally active genes (29). Complex formation between eukaryotic DNA topoisomerase I and chromosomal high mobility group proteins and histone H1 has also been reported (30). However, there has been no direct evi...

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Photoactivation of the a,/?-unsaturated ketones of natural and synthetic steroid molecules by light of 2 2 330 nm allows their covalent attachment to steroid-binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid-binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non-radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive pcptidcs, demonstrating that covalent attachment takcs placc at specific binding sites.

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