The solubilization of nucleosomes and histone H1 with increasing concentrations of NaC1 has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCI. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaC1 concentrations in content of nonhistone chromosomal proteins. 40-60% of the nucleosomes were released by 0.3 M NaC1 with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaC1. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaC1. These fractions contained the DNA least available for micrococcal nuclease attack. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by involved in a special function.KEY WORDS nucleosome fractionation nucleosome packing 9 histone H1 interaction with nucleosomes 9 histone H1 stoichiometry 9 chromatin condensation mechanism Chromatin in the interphase nucleus in eukaryotic organisms is organized morphologically in more condensed and less condensed structures. RNA synthesis apparently occurs in the less condensed regions (30), and satellite DNA is thought to be present in condensed chromatin. Recent evidence 0.2 M NaC1 suggest that this population is that more than 85% of the nuclear DNA, including the actively transcribing genes as well as satellite DNA sequences, is complexed with histones in particulate structures called nucleosomes (3,6,23,24,39,42,46,52) suggests that the more-and less-condensed morphological features of the nucleus may reflect differences in the macromolecular composition and/or the packing of the nucleosomes. The differential condensation is preserved in nuclei isolated in the presence of divalent cations (51), and dilution or chelation of divalent cations causes the native nuclear organi-J. CELL BIOLOGY ~) The Rockefeller University Press
We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.
Total cell polypeptides synthesized, in cultured Drosophila cells under control (25°C) and heat-shock (37°C) conditions have been compared in two different two-dimensional polyacrylamide gel electrophoresis systems which, together, resolve polypeptides having a wide range of isoelectric points, including the most basic polypeptides of the cell . The electrophoresis of basic proteins showed that the most prominent basic polypeptide synthesized in heat shock comigrated with histone H2b. This heat-shock polypeptide was identified as histone H2b by two criteria: (a) it comigrated with authentic histone H2b in Triton-urea-acetic acid acrylamide gel electrophoresis after solubilization from nuclei with acid ; and (b) partial proteolysis peptide maps of the basic heat-shock protein and histone H2b were identical . The synthesis of histone H2b was induced threefold in heat shock, whereas synthesis of the other histones was reduced from two-to tenfold . The noncoordinate synthesis of histones in Drosophila in heat shock provides an interesting system in which to investigate transcriptional and translational controls of histone synthesis as well as assembly of histones into chromatin.The heat-shock response in Drosophila was first observed as a puffing response at nine specific loci on the polytene salivary chromosomes in larvae that were transferred from 25°C to 37°C (1). Subsequently, it has been found that the response involves the induction of synthesis of a number of RNAs, several of which code for a series of polypeptides synthesized during heat shock, the heat-shock proteins (2-6) . The mechanism of induction and the physiological meaning of the response are not understood, and up to this time none of the heat-shock polypeptides has been identified .I have characterized the heat-shock polypeptides in Drosophila by two-dimensional polyacrylamide gel electrophoresis (PAGE) of basic as well as neutral and acidic polypeptides from solubilized whole cells. These experiments have led to the identification of the smallest and most basic heat-shock protein as histone H2b . MATERIALS AND METHODS Cell Culture and Labeling ConditionsThe A4E6 line of Drosophila Kc cells (7) was obtained from Peter and Lucy Cherbas, of Harvard University, and was maintained at 25°C in tissue culture flasks in D22 medium (8) supplemented with 10% heat-inactivated fetal calf serum (FCS) at densities between l and 10 x 10' cells/ml. For heat-shock experiments the cells were centrifuged, washed once with D22 minus methionine, and resuspended at a density of l x 10' cells/ml in D22 minus methionine containing 10% dialyzed FCS. The culture was divided in half and 100 iuCi/ml ['Slmethionine (Amersham Corp., Arlington Heights, Ill .) was added to each culture 20 min after the beginning of the incubation period at 25°C (control) or 37°C (heat shock). At the end of the incubation the cells were centrifuged and the medium was removed .THE JOURNAL Of CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 579-583 © The Rockefeller University Press " 0021-9525/81/1...
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.