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Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.  相似文献   

3.
Previous studies (Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN1) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN2) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN3) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180–200 bp). Most of the MN3 particles are rapidly converted by nuclease into mononucleosomes MN1 and MN2 There exists, however, a relatively nuclease-resistant subpopulation of the MN3 mononucleosomes. These 200 bp MN1 particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN3 particles and the smaller mononucleosomes MN1 and MN2.There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN1 is a set of naked double-stranded DNA fragments ~20 bp long. The SN2 is a complex of a specific basic nonhistone protein (molecular weight ~16,000 daltons) and a DNA fragment ~27 bp long. The SN3 contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment ~27 bp long. The SN4 contains histones H2a, H2b, H4 and a DNA fragment ~45 bp long. The SN5 contains histones H2a, H2b, H3 and a DNA fragment ~55 bp long. The SN6 is a complex of histone H1 and a DNA fragment ~35 bp long. Subnucleosomes SN7 and SN8 each contain all the histones except H1, and DNA fragments ~100 and ~120 bp long, respectively.Nuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.  相似文献   

4.
The estrogen receptor of MCF-7 cells labeled with high specific activity estradiol was used to mark the chromatin binding sites for this regulatory molecule. Many of these sites are especially sensitive to nuclease, and produce on digestion a series of uniquely sedimenting products. Several of these have been examined in some detail in this paper. These include a form of receptor that sediments in trace digests at 9S but in more extensive digests at 7S, fast mononucleosomes of about 12.5S, and a species at 15S. Two components of digests, fast mononucleosomes and dinucleosomes were isolated and subjected to further digestion. Much of the hormone on these isolated particles was found to be sensitive to additional hydrolysis, although some was nuclease resistant. It appears that a major fraction of the hormone receptor complexes bound to MCF-7 cell chromatin occurs at nucleosome-free regions which can be detected as transient hydrolysis intermediates.  相似文献   

5.
Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp). Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion. This was confirmed by stripping the protein from chromatin with Dowex resin. Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion. Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes. Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes. Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome. Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx. 10 kilodaltons. Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease. Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.  相似文献   

6.
We have characterized the biochemical nature of the Ku protein, the antigen recognized by autoantibodies from certain patients with scleroderma-polymyositis overlap syndrome. From extracts of HeLa cells labeled with [32P]orthophosphate, anti-Ku antibodies precipitated a high molecular weight nucleic acid identified as DNA because of sensitivity to DNase I and resistance to RNase. From extracts of cells labeled with [35S] methionine, these antibodies precipitated two polypeptides of 70,000 and 80,000 Da. These proteins were purified using immunoaffinity column chromatography. In immunoblots most sera containing anti-Ku antibodies recognized both Ku proteins but one serum bound only to the 70,000-Da subunit. When nucleosomal segments of chromatin were used as antigen, anti-Ku antibodies precipitated dinucleosomes and larger forms of chromatin but not mononucleosomes. Thus, the Ku antigen is a novel DNA-binding protein that is at least partially exposed on nucleosomal segments of chromatin.  相似文献   

7.
In murine L-cell nuclei micrococcal nuclease causes chromatin fragmentation with predominant liberation of dinucleosomes. Analysis of dynamics of rat liver nuclear chromatin cleavage by micrococcal nuclease revealed that the "dinucleosomal" mode of fragmentation is due to the pretreatment of nuclei with the non-ionic detergent Triton X-100 in the course of the isolation procedure. The set of particles detected in nuclease hydrolysates of nuclear chromatin pretreated with Triton X-100 and those isolated by the standard procedure was shown to be significantly different. In Triton X-100 treated nuclei the dichromatosome is the main hydrolysate component under various experimental conditions of nuclease hydrolysis and the sole component under "mild" conditions, whereas sucrose-treated nuclei contain three types of dinucleosomes. In Triton-treated nuclei prolongation of hydrolysis results in the liberation of the chromatosome which is absent in chromatin hydrolysates of sucrose-treated nuclei. Hydrolysis of Triton-treated nuclear chromatin by micrococcal nuclease is unaccompanied by the liberation (up to the stage of "deep" hydrolysis) of the core particle, the major component of the "sucrose" nuclear hydrolysate under the conditions used. The sharp differences in the accessibility of various types of dinucleosomes observed during pretreatment of nuclei with Triton X-100 are interpreted in terms of the localization of histone H1. The non-random type of the histone H1 molecule orientation along the nucleosome fibril is postulated.  相似文献   

8.
Overall pathway of mononucleosome production.   总被引:13,自引:0,他引:13  
Five electrophoretically distinguishable classes of mononucleosomes (MI, MII, ...MV) are produced upon treatment of mammalian nuclear chromatin with micrococcal nuclease. These five forms differ in their initial DNA lengths, relative mass proportions, stability, contents of histone H1, and presence of certain nonhistone proteins. A new "chromatin fingerprinting" technique has been developed in order to trace nuclease-mediated interconversions between these mononucleosomes and their polynucleosomal precursors. Application of this technique, together with earlier findings from this laboratory, has made possible the elucidation of the overall pathway of nuclease cleavage of chromatin which leads to the production and interconversion of these mononucleosomes, and has permitted reconstruction of the organization of these mononucleosomes in undigested chromatin...  相似文献   

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Electrophoresis methods used to study the fragments of chromatin revealed under the effect of Ca,Mg-dependent endonuclease on it have permitted establishing that stability of chromatin to the nucleosome level increases with aging. Changes in the compactness of the chromatin structure with aging make the accessibility of the linker DNA to nuclease lower the size of DNA fragments corresponding to mononucleosomes increasing from 192 +/- 5 pairs of bases to 209 +/- 5 pairs. Stabilization of the chromatin structure begins from certain nucleosomes which become more compact with the age. When analyzing basic proteins of chromatin by electrophoresis in different systems no qualitative changes were found in the subfraction composition of histones with aging, that permits supposing nonhistone proteins of chromatin and histone H1 to participate in the change of the chromatin structure compactness with the age.  相似文献   

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