Fractionation of unfixed chromatin by buoyant-density centrifugation (original) (raw)
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Partial Purification of the Template-Active Fraction of Chromatin: A Preliminary Report
Proceedings of the National Academy of Sciences, 1974
A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl 2 . The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA·RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA.
On the heterogeneity of chromatin fractions after gel filtration
Journal of Molecular Biology, 1974
Partially salt-extracted nucleoproteins in the eluted fractions after gel filtration in 0.6 to 1-O M-NaCl are heterogeneous in terms of the protein to DNA ratio. This heterogeneity is due to a redistribution within the gel column of the proteins associated with the DNA. If the fractions of the nucleoprotein peak are pooled before the NaCl is removed, then the resulting material is homogeneous.
Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide
1978
Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absenoe ox presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of SNA was found In the light complex (at the density of 1.14-1.16 g/om 3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy oomplex (1.19-1.21 g/om 3). The eleotrophoretio profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhlstone proteins were present only In the heavy component.
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
Fractionation of chromatin based on strength of binding with the matrix
1980
Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14+17IDNA ratios but were not enriched in active gene sequences (albumin and c-Ha-rasl genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgClz (monomer nucleosomes and polynucleosomes) contained in addition to the histones. HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14+17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction. 0 19R6 Academic Press. Inc.
Experimental changes in the width of the chromatin fibers from chicken erythrocytes
Experimental Cell Research, 1971
Widths of chromatin fibers prepared by spreading erythrocyte chromatin on water have been measured in different experimental conditions. Chromatin fibers from hemoglobin-free. EDTApretreated isolated nuclei, prepared by direct negative staining with man;1 acetate show an average width of 37 A with a standard deviation of 13 A. The same chromatin fibers. when previously treated with ethanol show a change in the average width from 37 to 138 A. The same chromatin, when floated on a hemoglobin solution and then treated with ethanol shows a further enlargement of its average width, from 37 to 313 A. These changes were compared with the measurements of chromatin fibers from whole erythrocytes spread on water. The average width of these fibers after ethanol treatment is 244 A. These results show that the 240-250 A chromatin fibers are the result of conformational changes of a thinner elementary fibril 30-40 8, wide, which are mainly dependent on the action of ethanol on this fibril and on the presence of additional proteins like hemoglobin.
Electrophoresis of Chromatin on Nondenaturing Agarose Gels Containing Mg[IMAGE]
Journal of Biological Chemistry, 1995
We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg 2؉ , chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg 2؉ . In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg 2؉ and form cylindrical structures with a diameter of ϳ33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.
Ultrastructure of transcriptionally competent chromatin
Nucleic Acids Research, 1990
We have examined a salt-soluble, transcriptionally competent gene-enriched fraction of chicken erythrocyte chromatin and compared it to bulk chromatin using the unique microanalytical capabilities of Electron Spectroscopic Imaging (ESI). The saltsoluble fraction is enriched 48 fold in 03-globin gene sequences and is also enriched in histones that are post-synthetically modified, including acetylation and ubiquitination. Differences between the two fractions are also apparent in the distribution of the two major forms of nucleoprotein structures, including (1) particles which present a circular profile and possess protein and DNA content nearly identical to that of the canonical nucleosome and account for 89% of particles in the bulk fraction but account for only 66% of the particles in the competent fraction, and (2) u-shaped particles which possess about 20% less protein mass than particles of circular profile and are about 1Ox more prevalent in the transcriptionally competent fraction than in the bulk. Additionally, elongated particles with protein and DNA content similar to the u-shaped objects are also seen in the competent fraction.
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.
Molekuliarnaia biologiia
Chromatin fractions from rat liver nuclei digested by nucleases were separated by differential solubility into several fractions. Material solubilized during digestion (predominantly monomer nucleosomes and polynucleosomes) had the highest HMG14+17IDNA ratios but were not enriched in active gene sequences (albumin and c-Ha-rasl genes). Material soluble in a low ionic strength buffer containing 0.2 mM MgClz (monomer nucleosomes and polynucleosomes) contained in addition to the histones. HMG14 and 17 plus a 41K non-histone protein. This fraction was depleted in active gene sequences and enriched in inactive sequences. The insoluble material was highly enriched in active sequences and had the lowest HMG14+17/DNA ratio. This fraction could be further fractionated into a histone-containing 2 M NaCl-soluble fraction and a 2 M NaCl-insoluble matrix-bound fraction, both of which were enriched in active sequences. The results show that the HMG proteins do not partition with active sequences during fractionation of chromatin. The 41K protein may be associated with inactive chromatin fraction. 0 19R6 Academic Press. Inc.