Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.     

Mapping and positioning DNA-binding proteins along genomic DNA. Structure of D. melanogaster ribosomal 'Alu-repeats' and 1.688 satellite chromatin.

Chromatin structure of so-called 'Alu-repeat' in D. melanogaster ribosomal non-transcribed spacer that contains sequences homologous to the promoter of ribosomal genes has been studied. Using the 'protein image' hybridization assay based on UV-light-induced DNA-protein crosslinking and 2-D gel retardation electrophoresis, two proteins of the molecular mass of 50 kD (rABP50) and 70 kD (rABP70), associated with 'Alu-repeat' DNA have been found. Exo III mapping of crosslinking sites and DNase I footprinting have provided a detailed map of H1, rABP50 and rABP70 contacts within the 'Alu-repeat' and H1 and a non-histone protein contacts on satellite DNA. These data indicate precise positioning of non-histone proteins, histone H1 and nucleosomes within genomic regions studied and account for the presence of unusual 240 bp long nucleosomal particles in 'Alu-repeats'. The same approach can be adapted for successive mapping and positioning proteins on genomic DNA.     

Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.

UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.

High speed supernatants of Xenopus laevis oocyte nuclei efficiently assemble DNA into nucleosomes in vitro under physiological salt conditions. The assembly activity cofractionates with two histone complexes composed of the acidic protein N1/N2 in complex with histones H3 and H4, and nucleoplasmin in complex with histones H2B and H2A. Both histone complexes have been purified and their nucleosome assembly activities have been analysed separately and in combination. While the histones from the N1/N2 complexes are efficiently transferred to DNA and induce supercoils into relaxed circular plasmid DNA, the nucleoplasmin complexes show no supercoil induction, but can also transfer their histones to DNA. In combination, the complexes act synergistically in supercoil induction thereby increasing the velocity and the number of supercoils induced. Electron microscopic analysis of the reaction products shows fully packaged nucleoprotein structures with the typical nucleosomal appearance resulting in a compaction ratio of 2.8 under low ionic strength conditions. The high mobility group protein HMG-1, which is also present in the soluble nuclear homogenate from X. laevis oocytes, is not required for nucleosome core assembly. Fractionation experiments show that the synergistic effect in the supercoiling reaction can be exerted by histones H3 and H4 bound to DNA and the nucleoplasmin complexes alone. This indicates that it is not the synchronous action of both complexes which is required for nucleosome assembly, but that their cooperative action can be resolved into two steps: deposition of H3 and H4 from the N1/N2 complexes onto the DNA and completion of nucleosome core formation by addition of H2B and H2A from the nucleoplasmin complexes.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7     

Amplified ribosomal spacer sequence: structure and evolutionary origin

A novel class of repeated sequences consisting of tandem arrays of ribosomal spacer sequence has been discovered in a mouse genome. Comparison to normal ribosomal DNA reveals that one repeat unit consists of two separate parts of spacer sequence. This amplified spacer sequence has a pseudogene-like structure but is distinct from the previously reported pseudogenes and orphons in regions lacking coding sequences. So far the amplified spacer sequence has been found only in the BALB/c mouse genome but not in ten other laboratory strains and several wild-type mouse stocks. Surprisingly, a part of the amplified spacer sequence unit had a higher homology to the corresponding part of the ribosomal DNA sequence of Mus musculus molossinus, a Japanese wild-type mouse, than to the corresponding part of the rDNA of the BALB/c mouse. These findings suggest that the amplified spacer sequence of the BALB/c mouse might have partly originated in M. m. molossinus or in a related subspecies.     

Isolation of yeast histone genes H2A and H2B

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.     

Histone modifications in simian virus 40 and in nucleoprotein complexes containing supercoiled viral DNA.

Simian virus (SV40) nucleoprotein complexes containing circular supercoiled viral DNA were extracted from infected cells and purified by differential centrifugation. The protein content of these complexes was compared by electrophoresis on 15% acrylamide gels with the protein content of purified SV40 virions and with histones from virus-infected cells. The electrophoretic patterns of histones from each of the sources revealed several major differences. SV40 virions contained histones H3, H2B, H2A, and H4 but not H1. Nucleoprotein complexes and host cells contained all five major histone groups. Relative to cellular histones, virion and nucleoprotein complex histones were enriched 15 to 40% in histones H3 and H4. In addition to the major classes of histones, several subfractions of histones H1, H3, and H4 were observed in acrylamide gels of proteins from SV40 virions and viral nucleoprotein complexes. Acetate labeling experiments indicated that each subfraction of histones H3 and H4 had a different level of acetylation. The histones from SV40 virions and nucleoprotein complexes were acetylated to significantly higher levels than those of infected host cells. No apparent differences in phosphorylation of the major histone groups were observed.