Isolation and properties of structured chromatin from Guerin ascites tumour and rat liver (original) (raw)
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Molecular Biology Reports, 1982
Hamster liver nuclei were fractionated by digestion with micrococcal nuclease into nuclease released (SP) and nuclease resistant (PP) fractions varying in chemical composition and transcriptional activity. Electrophoretic analysis of histones from SP and PP showed no qualitative and quantitative differences. Apparently chromatin-bound protease activity can be found in both fractions. Nonhistone chromatin proteins isolated from SP and PP under mild conditions were fractionated by hydroxyapatite chromatography into NHCP1, NHCP2, NHCP3 and molecular heterogeneity and specificity were tested by SDS-polyacrylamide gel electrophoresis.
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
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.
Native chromatin and damage induced by nuclease
Biochemical and Biophysical Research Communications, 1988
Differential scanning calorimetry, gel electrophoresis and polarized light scattering of chromatin prepared by different methods have been carried out at low and high ionic strength, before and after shearing. These noninvasive studies, when compared to the ones similarly conducted in the corresponding native nuclei, conclusively point to the artefactual nature of chromatin prepared by limited nuclease digestion, which has no resemblence with the in situ chromatin-DNA structure being instead preserved by lysis of native nuclei and by subsequent sedimentation and suspension of the viscous chromatin mass. Native nucleofilaments appear longer than 200 nucleosomes and yield, from thermodynamic and optical standpoints, a tight quaternary structure mantained even at 0.01 M.
Nucleic Acids Research, 1980
It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.
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.
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.
Evidence for a subunit structure of chromatin in mouse myeloma cells
Chromosoma, 1975
If micrococcal nuclease is allowed to digest ehromatin as it exists inside intact nuclei isolated from mouse myeloma tissue culture cells, more than 60% of the DNA can be isolated as a homogeneous fragment on a sucrose gradient. Analytical ultracentrifugation indicates that the protected DNA is native, unnicked, and about 140 • 10 base pairs long. After less extensive nuclcase digestion, the protected DNA migrates in gels in lengths which are integral multiples of this 140 base pair "monomer" band. A submonomer band, 105 i 10 base pairs long, can also be detected. Similar digestion patterns were obtained by two different nuclear isolation procedures and even when intact ceils were gently lysed directly in the digestion medium. These results confirm and extend the chromatin digestion studies of previous investigators and provide support for a subunit model for eukaryotic chromatin. The single strand specific Si nuclease did not digest intranuclear chromatin under the conditions used.