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...
The polytene chromosomes of Drosophila strains that differ in the synthesis of the major salivary gland glue protein sgs-4 were examined by indirect immunofluorescence using antisera to several nonhistone chromosomal proteins. The Oregon-R X chromosome, which produces sgs-4 messenger RNA, showed a strong fluorescent band at locus 3C11-12 when stained with anti-RNA polymerase II, whereas the null mutant Berkeley 1 failed to exhibit fluorescence at that locus. The presence of another antigen (Band 2), normally associated with developmentally active loci, was clearly evident at locus 3C11-12 of both transcriptionally competent and null strains, indicating that the association of Band 2 antigen with the chromatin is an event independent of RNA polymerase II binding . Antibodies directed against Drosophila topoisomerase I stained 3C11-12 in the Sgs-4+ (wild-type) strain brightly, but gave significantly less staining in the null strain . This indicates that the high concentrations of topoisomerase I seen at active loci are closely associated with the transcriptional event . In some of these analyses, we have made use of flies heterozygous for the wild-type and null alleles in order to make internally controlled comparisons. The results suggest that this type of analysis will enable conclusions to be drawn concerning the interdependence and order of action of chromosomal proteins involved in developmental gene activation .The indirect immunofluorescence technique (1, 23) allows a visual assay for the presence or absence of protein molecules on polytene chromosomes, and can therefore be used to examine macromolecular differences between active and inactive chromatin from cytological preparations . The study of the association of nonhistone chromosomal (NHC)`proteins with active chromatin and their temporal binding order should eventually lead to a hierarchical picture of events in gene activation. Even better than the use of progressively staged larvae in exploring the course of such biochemical changes is the use ofwell-characterized variants . Such variants might show altered programs of macromolecular association, detectable by the immunofluorescent assay, leading to alteration ofgene expression .The Sgs-4 locus at 3C11-12 in Drosophila melanogaster encodes a peptide component of the larval glue which is secreted by the third instar larvae at the beginning of pupariation (3,8). Chromatin structure analyses of the wild-type and several well-characterized variant alleles have revealed a set of tissue-specific deoxyribonuclease (DNase) I hypersensi-'Abbreviations used in this paper: BER-1, Berkeley 1 variant line; DNase, deoxyribonuclease ; NHC, nonhistone chromosomal; SSC, saline-sodium citrate ; w", ln(1)w`chromosome.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.