Histone-DNA contacts in the 167 bp 2-turn core particle.

The histone-DNA contacts in the 167 bp 2-turn core particle have been compared with those in the 146 bp 1.75-turn core particle by the methodology developed by Mirzabekov and his colleagues. The contacts in the 167 bp 2-turn core particle retain the essential features of those in the 146 bp 1.75-turn core particle but contacts for histones H3 and H2A were found in the 10 bp extension that discriminates the two particles. In addition the contact for histone H2A near the dyad axis was far more pronounced in the case of the 146 bp core particle.     

Biochemical and physiochemical characterization of chromatin fractions with different degrees of solubility isolated from chicken erythrocyte nuclei

Chicken erythrocyte chromatin was prepared according to two different methods [Fulmer, A. W., & Bloomfield, V. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5968-5972; Ausio, J., Borochov, N., Seger, D., & Eisenberg, H. (1984) J. Mol. Biol. 177, 373-398] to give three main common fractions, according to its solubility (S) or insolubility (I) in 0.15 M NaCl buffers or to its further solubility in 0.25 mM ethylenediaminetetraacetic acid (E). From the biochemical point of view, all of them have been found to be undistinguishable. Analytical ultracentrifugation shows that all of these fractions can reversibly undergo the transition from the low to the higher order structure, through a nearly identical way of folding. Thermal denaturation profiles yielded three transitions having the same Tm's for the three fractions. The percentage of DNA melting in the first transition decreased in the order S greater than I greater than E, and the amount in the second transition increased in the same order. Together with the different solubility of these fractions in the presence of divalent ions, these results indicate that in the three fractions of chromatin studied, the amount of linker DNA bound to the nucleosome varied.     

Estimation of domain length of chicken erythrocyte chromatin

Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease. The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy. These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T. and H.G. Zachau (1977) Cold Spring Harbour Symp. Quant. Biol. 42, 109–118). A domain length of 45 kbp was obtained.     

Alteration of domain architecture of chicken erythrocyte chromatin

The higher-order organisation of chromatin in chicken erythrocyte nuclei as a function of the ionic strength of the nuclear suspension buffer and also of the time of incubation in this buffer prior to nuclease digestion has been investigated. This organisation is described in terms of a physical parameter called the domain length. The 45-kbp-long domains of control nuclei were unravelled to give rise to domains of length 150 kbp on overnight equilibration at 0 degree C of the nuclei in standard isolation buffer containing 0.135 M NaCl prior to nuclease digestion. However, transition to the equilibrium state was preceded by a metastable and irregular domain architecture when the nuclei were incubated for only 1 h. In contrast, the domain length remained unchanged when nuclei were incubated in the isolation buffer alone for identical periods of time. The proteins dissociated at the higher ionic strength were characterised and their role in stabilising the domain structure is discussed.     

Asymmetry and polarity of nucleosomes in chicken erythrocyte chromatin.

Nucleosome dimers containing, on average, a single molecule of histone H5 have been isolated from chicken erythrocyte nuclei and the associated DNA fragments cloned and sequenced. The average sequence organization of at least one of the two nucleosomes in the dimers is highly asymmetric and suggests that the torsional, as well as the axial, flexibility of DNA is a determinant of nucleosome positioning. On average the nucleosome dimer is a polar structure containing linker DNA of variable lengths. The sequences associated with H5 containing nucleosomes and core particles are sufficiently different to indicate that removal of histone H5 (or H1) from chromatin may result in the migration of the histone octamer and a consequent exposure of sites for regulatory proteins.     

Effects of chromatin protein fractions on transcriptional activity of chicken thrombocyte and erythrocyte chromatin

1. Chromatin proteins of chicken thrombocytes and erythrocytes were separated into three fractions by successive extraction with 5 M urea containing various salt concentrations and pH values. Molecular composition of protein fractions was determined by SDS-polyacrylamide gel electrophoresis. 2. The efficiences of the chromatin residues after sequential protein extractions as well as those of reconstituted DNA-protein fraction complexes, in serving as a template for the in vitro RNA synthesis were measured in order to identify the effect of each fraction. 3. The different involvement of chromatin protein fractions of template properties of thrombocyte and erythrocyte chromatin was stated.     

Isolation and physical characterization of a stable core particle.

Core particles were prepared from mature chicken erythrocytes chromatin, according to the method of Lutter (J. Mol. Biol. 124, 391, 1978) with one major modification: after the second digestion, zonal centrifugation was used to isolate the core particle, instead of chromatography on Sepharose 6B. By using circular dichroism and electron microscopy, we were able to follow each step of the preparation and to offer an explanation of the discrepancies found in previous preparations and in our own preparations.     

Release of a globin gene enriched chromatin fraction from chicken erythrocyte nuclei following DNase II digestion

Mild digestion of chicken erythrocyte nuclei with deoxyribonuclease II results in the release of a chromatin fraction which is 4- to 13-fold enriched for the globin coding sequences when compared to total chicken DNA. The remaining nuclear pellet is depleted in these sequences. A maximum of 25% of the globin genes have been recovered in the released fraction. The addition of 5 mM sodium butyrate to the digestion buffer is required to obtain reproducible globin gene enrichment. The released fraction contains equimolar amounts of the four core histones and a subset of the nonhistone chromosomal proteins. The globin genes are released as large chromatin fragments which exceed the 1.6 kilobase size of the transcribed portion of the gene.     

Isolation and characterisation of a chicken gelatinase (type IV collagenase).

The proform of chick gelatinase (type IV collagenase) was isolated and purified to a high specific activity of 12,071 U/mg from cultured embryonic skin fibroblasts stimulated with cytochalasin-B. The enzyme was activated in the presence of 4-aminophenylmercuric acetate with a fall in molecular weight from 66,000-58,000 on non-reducing polyacrylamide gel electrophoresis and was active over the pH range of 6.0-8.9 against a number of substrates. Further biochemical characterisation showed that the organomercurial activated form of the enzyme behaved like a typical mammalian gelatinase, actively degrading gelatin, soluble type I collagen, collagenase generated type I fragments, type IV collagen (producing 3/4 and 1/4 fragments) and type V collagen, whilst having little effect on laminin. The enzyme was inhibited by metal chelators such as EDTA and 1,10-phenanthroline, but not by inhibitors is suggested that this may be TIMP-2. An antiserum was raised to the proenzyme and was found to localise intra- and extra-cellularly in both tissue sections and cell cultures.     

cis-diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle

The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins.     

Isolation and characterisation of a biotin-binding protein from the pregnant-rat serum and comparison with that from the chicken egg-yolk

A biotin-binding protein exhibiting partial immunological cross-reactivity with the purified chicken egg-yolk biotin-binding protein has been detected, for the first time, in the sera of pregnant/estrogenised female rats but not of the normal males. This protein, purified by affinity chromatography on a biotin-AH-Sepharose was homogeneous by electrophoretic and immunological criteria. It was a glycoprotein of Mr 66,000 without any detectable subunites, had a pI 4.1, and specifically bound [14C]biotin. Several structural and functional features of the biotin-binding protein of rat and chicken were found to be similar. These included immunological cross-reactivity, acidic and glycoprotein nature, ability to tightly bind [14C]biotin, estrogen stimulation for their appearance in circulation, and the pattern of distribution of radioiodinated peptides upon proteolysis with trypsin.     

A novel magnesium-dependent structural transition of chicken erythrocyte chromatin in situ

Lowering magnesium concentration below the value of 1 mM leads to a structural transition of chicken erythrocyte chromatin in situ, which results in a change in its fragmentation by pancreatic DNAase (DNAase I) from double-nucleosome to 100-basepairs mode. At 0.75 mM MgCl2, the pattern of chromatin fragmentation by DNAase I is similar to that generated by DNAase II, and it is further changed at lower concentrations of magnesium. This transition is, at least partly, reversible, and is, presumably, related to packing of the 25-30 nm chromatin fiber into higher-order structures.     

The structure of the chromatin core particle in solution.

The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A.     

Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin

An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour.     

Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.     

Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin.

Formaldehyde (HCHO) fixation of chicken erythrocyte chromatin produces a marked decrease in its positive circular dichroism (CD), above 260 nm, and the appearance of s small negative ellipticity around 295 nm. The ultraviolet spectrum of chromatin is unaffected, nor does HCHO produce any changes in the uv or CD spectra of chicken erythrocyte DNA. The extent of the circular dichroism transition from the native chromatin to the suppressed spectrum is dependent on the concentration of HCHO and salt concentration. The kinetics of the reactions are complex, implicating at least two reactive species. Studies of the reaction of HCHO with chromatin in ethylene glycol and CD measurements of aqueous chromatin solution with added glutaraldehyde preclude simple dehydration and general cross-linking effects as causes of the CD changes observed. The results are interpreted as indicating a conformational change of the DNA in chromatin caused by histone-DNA or histone-histone cross-linking.