Tightly Bound Non-histone Proteins in Nucleosomes from Pig-Liver Chromatin (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
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...
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.
Proteins of chromatin in genetic restriction
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1972
Nonhistone proteins from the condensed (nucleolar) and diffuse (extranucleolar) fractions of rat liver and kidney chromatin preparations were compared by electrophoresis in polyacrylamide gels. Although considerable differences were seen between the rat liver and kidney nonhistone chomatin proteins, the nucleolar and extranueleolar nonhistone chromatin proteins from the same source were found to be qualitatively similar. The tissue-specific electrophoretie distribution of nonhistone proteins was not affected by extraction of the liver and kidney chromatin preparations with 0.3 M NaC1. This was interpreted to indicate that the electrophoretic tissue specificity of nonhistone proteins was not caused by cytoplasmic contamination. The electrophoretic patterns of histones were practically identical in nucleolar and extranucleolar chromatin samples. The close similarity of these two types of chromatin was further substantiated by analysis of their derivative thelmal denaturation profiles. The modified thelmal stability of nucleolar chromatin was found to be caused by its content of RNA which partially destabilized the DNA-histone interactions. Nucleolar chromatin is substantially heterochromatic, as compared to the euchromatic extranucleolar chromatin, and the striking structural differences between these two chromatin types do not appear to be reflected in their protein contents at the present level of resolution.
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.
Nucleosome mono-, di-, tri-, and tetramers from chicken embryo chromatin
Nucleic Acids Research, 1977
The fractionation of gram quantities of nuclease digested chromatin from chicken embryos into nucleosome mono-, di-, tri-, and tetramers is described in detail. Each of these nucleosomal species contains a fraction soluble in 0.1 M KCl that decreases with increasing repeat number. Less histone Hi is associated with the nucleosome fractions soluble as compared to the respective fractions precipitated in 0.1 M KC1. Thermal denaturation profiles of the four nucleosomal species are monophasic. The same T of 78 C has been determined for the KCl-soluble nucleosomes m and for the KCl-insoluble monomer. The T of the KCl-insoluble o m oligomers is 79.8 C. Multiphasic melting curves were recorded for nucleosomal material that was concentrated by lyophilisation or stored at 4°C in 0.25 mM EDTA. Total nucleosome mono-, di-, tri-, and tetramers (consisting of both the fraction soluble and insoluble in 0.1 M KCI) have been analyzed concerning their sedimentation, diffusion, partial specific volume, and molecular weight and compared with the' sedimentation and molecular weight data of KCl-soluble nucleosome mono-and tetramers.
Experimental Cell Research, 1986
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.
A set of non-histone proteins isolated from the nuclei of various rat tissues
European Journal of Biochemistry, 1983
A set of non-histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat. Following moderate digestion of thoroughly washed nuclei with DNase 1 or micrococcal nuclease, EDTA was added to 5 m M to the reaction mixture and the preparation centrifuged. We found that the supernatant contained a limited amount of non-histone proteins (fraction SI). Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A -D) each possessing closely spaced doublets or a triplet. Their molecular weights were A, 76100-80000; B, 48200-49500; C, 44500-45200 and D, 39500-41 500. The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus. Two-dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges. Some were labelled with [32P]orthophosphate in vivo, or with [y-32P]ATP and purified nuclear protein kinase NII in vitro. The released proteins B -D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations a t similar sites in the nucleus. The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B -D constitute a group of proteins that have several common characteristics.
Isolation and properties of structured chromatin from Guerin ascites tumour and rat liver
European journal of biochemistry / FEBS, 1976
The method proposed by Hancock for isolation of structured chromatin from tissue culture cells is modified and used for isolation of chromatin from Guerin ascites tumour and rat liver. Micrococcal nuclease digestion patterns and thermal denaturation of these chromatins are studied and compared wiith those of chromatins prepared by precipitation and extraction with salts (salt chromatins). In contrast to the multiphasic melting profiles and salt chromatins, the structured chromatins exhibit relatively homogeneous denaturation patterns under a variety of conditions, suggesting thhat their DNA is uniformly stabilized by histones and there are no free independently melting DNA stretches. Digestion of structure of the deoxyribonucleoprotein is intact. No discrete fragments are formed upon digestion of salt chromatin.