The distribution of nuclear proteins and transcriptionally-active sequences in rat liver chromatin fractions (original) (raw)
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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.
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
Biochimica et biophysica acta, 1979
Mouse liver non-histone proteins, isolated by hydroxyapatite chromatography, were fractionated by hydrophobic chromatography using omega-amino-decyl-agarose omega-amino butyl-agarose, decyl-agarose, butyl-agarose, phenyl-Sepharose, and CPAD-Sepharose. Two column loading techniques were used. In the 0.35 M NaCl technique, the proteins were dialized into 0.35 M NaCl, applied to the column and initially eluted with 0.35 M NaCl. In the 40% (NH4)2SO4 technique, the non-histone proteins were mixed with the hydrophobic agarose, dialized against 40% (NH4)2SO4, and initially eluted with 40% (NH4)2SO4. In both cases the columns were subsequently eluted with 10 mM Tris-HCl, pH 7.5, 0.35, 1.0 and 5.0 M LiBr, and finally with 1% sodium dodecyl sulfate. The 0.35 M NaCl technique, using decyl-agarose and phenyl-Sepharose, resulted in a single step marked enrichment of the major hnRNA proteins (1 M LiBr fraction). The 40% (NH4)2SO4 technique resulted in a single step isolation of a pair of 15-20 00...
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.
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.
Cell biology international reports, 1981
When rat liver nuclei are subjected to endogenous nuclease digestion in the presence of 5 mM Mg++, they release a chromatin fraction containing DNA of well defined fragment lengths and DNA-dependent RNA polymerases. The DNA is enriched 4.4-fold for sequences complementary to cDNA prepared to cytoplasmic poly-A-containing RNA when compared with whole cell DNA. This method of obtaining a transcriptionally enriched chromatin fraction is compared with other reported nuclease methods.
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.
The Journal of Cell Biology, 1976
High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(...