Cytological analysis of the effect of treatments visualizing the chromosome core on non-histone nuclear proteins (original) (raw)
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The non-histone proteins of chromatin, their isolation and composition in a number of tissues
Biochimica et biophysica acta, 1972
I. A method is described for the fractlonation of salt urea-dissociated chromatin using hydroxylapatite With the exception of experiments using chromatms prepared from "citric acid" nuclei, high yields of acidic non-hlstone proteins, relatively free of RNA, can be obtained by this procedure 2. The non-hlstone proteins of a number of chromatms were compared by electrophoresis m sodium dodecyl sulphate-urea polyacrylamlde gels employing a discontinuous buffer system. Proteins trom mouse chromatins prepared from "citric acid" nuclei were found to be extremely heterogeneous, but in the case of calf thymus the proteins were mainly low molecular weight. On the other hand, the non-hlstone plotems of chromatin from "sucrose" nuclei appeared to contain fewer high molecular weight species in the tissues studied, with the exception of brain Preparation of nuclei by the double-detergent procedure of Penman (J Mol B,ol, 17 (1966) 117) gave chromatm with a low protein to DNA ratio These proteins also appeared to be predominantly low molecular weight. Duck erythrocyte nuclei prepared by lysls also contained low molecular weight chromatm non-hlstone proteins. 3 Using salt fractlonation techmques attempts were also made to remove "cytoplasmic" and "residual" acidic proteins from chromatin. The proteins which remained with the DNA and histones were found to be mainly low molecular weight in kidney and liver, but in the case of brain a wide spectrum of proteins was seen, 4. Little tissue or species specificity of non-hlstone proteins were found on comparison of "sucrobe" nuclei chromatms prepared from a number of mouse and bovine tissues 5 It is concluded that the non-hlstone proteins which remain tightly bound to DNA in chromatin are of the same approximate size as the basic histones Because of the procedural variation in the heterogeneity of these chromatin proteins, detection of ~mgle species of regulatory proteins appears to require other technique~ INTRODUCTION Recent investigations on chromatin have linked the non-histone fraction with the organ-specific restriction of the DNA templatO 4. This fraction consists largely of Present address Serum and Vaccine institute, \Varaaw, Poland Btochzm Btophys ~4cta, 277 (I97~') 384-4 °2
Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry
Chromosoma, 1984
A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%-30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.
Tightly Bound Non-histone Proteins in Nucleosomes from Pig-Liver Chromatin
European Journal of Biochemistry, 1981
Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine. By this method non-histone proteins have been found to be associated with the core particles. Proteins tightly bound to the core particle DNA (i.e. dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71 000 to 20000. They show, furthermore, a peculiar amino acid composition. Other tightly bound proteins have been shown to be present only in the spacer regions. The existence of two different classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are compatible or incompatible, respectively, with the simultaneous binding of the histone octamer.
Experimental Cell Research, 1973
SDS Gel electrophoresis of mouse nuclei washed with various salt solutions and of Chinese hamster metaphase and interphase chromatin led to the following conclusions. 1. The pattern of non-histone proteins was the same after nuclei were washed with solutions varying markedly in ionic strength and cation content. 2. The pattern of the nuclear sap and the non-histone proteins showed many similar bands suggesting they are not totally distinct entities. The nuclear sap appears to be composed of several classes of proteins varying in their affinity for DNA. 3. Some of even the most tightly binding non-histone proteins could be removed from the DNA by low ionic strength salts simply by repeated washing of the chromatin. By contrast the histones were not removed by repeated washing. 4. When nuclei were washed with a salt solution that closely mimicked normal intranuclear conditions the lysine-rich histone was removed from the chromatin suggesting this histone is loosely bound to DNA in vivo. 5. It is pointed out that even though some nuclear sap proteins appear to be readily washed off the chromatin this does not mean they have no gene regulatory function. 6. The acid-soluble non-histone proteins reported to be unique to metaphase chromosomes are shown to be the result of the adsorption of cytoplasmic proteins onto the chromosomes during the isolation procedure. 7. Electrophoresis of well washed metaphase and interphase chromatin indicated their nonhistone proteins were quite similar.
Chromosome Structure as Revealed by a Combined Chemical and Immunochemical Procedure
Proceedings of the National Academy of Sciences, 1973
Human metaphase chromosomes were photooxidized in the presence of methylene blue, a process that destroys guanine residues in DNA. Indirect immunofluorescence showed that such chromosomes reacted with a cytosine-specific antibody revealing a consistent fluorescent banding pattern by which each chromosome could be identified. The observed fluorescent patterns were the reverse of those produced in formamide-denatured chromosomes treated with an antibody specific for adenine and of the patterns obtained with quinacrine and with Giemsa staining by the G-banding techniques. The patterns were identical to Giemsa R-banding patterns. The chromosome banding patterns, therefore, appeared to reflect DNA base composition, indicating the feasibility of a combined chemical-immunochemical investigation of the chemical organization of chromosomes.
Immunofluorescent study of chromatin proteins in cultured fibroblasts
Experimental Cell Research, 1974
Antibodies against chromatin from 3T6 mouse fibroblasts and WI-38 human diploid fibroblasts were prepared by immunization of rabbits. Immunofluorescent studies showed species-specificity of these antichromatin antibodies. Furthermore, using anti-3T6 chromatin antibodies against 3T6 cells and anti-WI-38 chromatin antibodies against WI-38 cells, we observed, by immunofluorescent techniques, granular fluorescence i&the diCfusely stained nucleus and diffuse fluorescence in the cytoplasm. These results raise the possibility of the presence of a cytoplasmic pool of chromatin proteins.