Features of in vitro puffing and RNA synthesis in polytene chromosomes of Chironomus

Abstract

In order to elucidate the causal relation between RNA synthesis and puffing in polytene chromosomes, salivary gland nuclei and chromosomes from Chironomus tentans and C. pallidivittatus (Diptera, Chironomidae) were isolated in the native state under controlled and optimized conditions. The behavior of the polytene chromatin in vitro as compared to that in vivo was studied morphologically and autoradiographically. It was shown that the absence of cytologically visible puffing does not necessarily indicate transcriptional quiescence. Under suitable conditions all potential puff sites incorporate the radioactive precursor simultaneously. This can be seen in vivo and even more clearly in chromosomes decondensed by 340-400 mM NaCl in vitro. Potential puff sites therefore appear to be permanently equipped with RNA-polymerases, and there is always a basic low level of transcriptive activity. Experimental shrinking of Balbiani rings and other puffs by dimethylsulfoxide (DMSO) or heat shock in vivo, leaves the genes in the collapsed puffs still transcriptionally active, although to a reduced extent. Light optically visible puffing is therefore not a necessary prerequisite for RNA synthesis, while, conversely, RNA synthesis is required for puff formation.

Chromatin structure of ribosomal genes in Chironomus thummi (Diptera: Chironomidae): tissue specificity and behaviour under drug treatment

“…Nuclei from salivary glands were prepared as described elsewhere (Sass 1980). The plate was kept on ice until the end of the psoralen crosslinking procedure.…”

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

“…Nuclei from about 100 glands were isolated (25) and subsequently extracted after resuspension and sonication in 100 ,l of 40 mM Tris hydrochloride (pH 7.6)-10 mM MgCl2-50 mM KCI-20 mM (NH4)2SO4-2 mM dithiothreitol-2.6% glycerol-100 p.g of bovine serum albumin per ml at 4°C for 30 min. After centrifugation, the supernatant was removed and used for the enzyme assay.…”

“…Chromosome IV was prepared from isolated nuclei and used in the indirect immunofluorescence staining assay essentially as previously described (11,25,26). The chromosomes were fixed for 0 to 20 s. After staining, the chromosomes were mounted in the presence of p-phenylenediamine to retard fading during microscopy (20).…”

Establishing an EU framework for diabetes

“…An early approach to measuring co‐transcriptional intron removal was though micro‐dissection of chromatin puffs, regions of insect polytene chromosomes undergoing active transcription known as ‘Balbiani Rings’ (BR) 34. Analysis of nascent RNA isolated from contiguous segments of the BR1 gene found that intron 3 is completely excised prior to transcriptional termination, while the fourth and terminal intron, which is positioned within 1 kb of the polyadenylation site, is only excised in 5–10% of the same transcripts 35.…”