Tightly bound DNA-protein complexes representing stable attachment sites of large DNA loops to components of the matrix

This study describes tightly bound DNA-protein complexes in DNA of matrices isolated from Friend erythroleukemia cells. When after radio-iodination of the associated proteins, such DNA is electrophoresed on agarose and the gel is subsequently subjected to autoradiography, the protein components of three or four complexes are visualized. Their two-dimensional electrophoretic analysis revealed that each possesses a simple but specific polypeptide composition, including a set of five non-histone proteins, characteristic for the matrix, and the core histones H3 and H4. Since the polypeptides dissociate from DNA by treatment with SDS, it is suggested that the linkage is not covalent. Reassociation and hybridization analysis of the DNA of the complexes indicated that it is enriched in highly repetitive, satellite sequences. The latter were found to be, to a great extent, similar to sequences localized at the base of large, dehistonized DNA loops obtained by high-salt extraction of isolated nuclei. Further experiments emphasized the complete conservation of this type of attachment throughout erythroid differentiation of Friend cells. It is proposed that the complexes represent attachment sites of basic, 30-100-kbp loop units of DNA.     

Matrix-associated DNA from maize is enriched in repetitive sequences

In order to elucidate some features of the topological organization of DNA within the plant nucleus, DNA fragments involved in the attachment of the DNA loops to the nuclear matrix in maize were studied. The matrix-associated DNA from dry embryo and meristematic cells after extensive digestion with DNase I and high salt treatment was about 2% of the total DNA, sized within the range of 50 and 250 bp. This DNA was found to be enriched in repetitive DNA sequences, both for nuclei from dry embryo and meristematic cells. The loop size of the DNA in cells of Zea mays appeared to be between 5 and 25 kbp.Abbreviations EDTA Diamino-ethanetetraacetic acid - EtBr Ethidium bromide - LIS Lithium diiodosalicylate - PMSF Phenylmethylsulfonyl fluoride - SDS Sodium dodecyl sulfate     

Histones of terminally differentiated cells undergo continuous turnover

In contrast to the widely accepted idea of the nearly absolute metabolic stability of histones, our experiments support the view that the histones of nonproliferating, terminally differentiated cells undergo continuous replacement. This conclusion is based on the incorporation of labeled amino acids into the histones of mouse kidney and liver cells after their intraperitoneal introduction. We have found that the intranuclear uptake of the histones made in the absence of replicative synthesis and their integration into chromatin proceed with striking delay. The metabolic rates of individual histones measured by calculating their half-lives suggest that each histone turns over at a specific rate. With regard to the basic chromatin structure, the nucleosome, such unequal turnover should mean that the histone core does not participate in this process as a single unit but rather as a protein mosaic in which each partner follows its own rate of removal. Additional experiments suggested that intact nucleosomes take part in the replacement, but the relative proportion of the nucleosomes involved should be limited. The nonnucleosomal H1A and H1 degree histones have been found to undergo faster replacement than the core histones. Moreover, in comparison to each other, these two histone subfractions are also replaced at a different rate. The results of autoradiography of isolated kidney and liver nuclei after continuous labeling with [3H]-thymidine suggest that the histone replacement is not associated with the repair of DNA.     

Distribution of tryptophan-containing proteins and of newly synthesized RNA in metaphase chromosomes. An autoradiographic study

Chinese hamster fibroblasts were labelled with ³H-tryptophan (for 15.5 h), with ³H-uridine (for 2 h) and with ³H-thymidine (for 15.5 h) in vitro. The distribution of the label was studied by autoradiography of isolated chromosomes. While ³H-thymidine-labelled chromosomes showed the well known uniform distribution of the label, in chromosomes labelled with ³H-tryptophan the label was unevenly distributed along the chromosomes. Quantitative measurements of the grain density over different segments of two easily identified chromosomes showed that each chromosome had a characteristic labelling pattern. ³H-uridine was incorporated in the same regions where ³H-tryptophan was localized. Control experiments showed that the observed labelling pattern was not due to non-specific adsorption of cytoplasmic ribonucleoproteins.     

Metabolic behaviors of the core histones in proliferating Friend cells

This study examines the turnover of the core histones in proliferating Friend cells. It was calculated that these proteins turn over with half-lives of 21.6 days for H2A, 13.8 days for H2B, 43.3 days for H3, and 138.6 days for H4. The significant differences in the half-lives of the four core histones indicate that the protein moiety of the nucleosome is not replaced as one entire unit but as a "mosaic" in which each component follows its own rate of replacement. In some experiments the turnover rates of the variants of H2A, H2B, and H3 were compared. The results did not indicate any differences among these histone variants, suggesting that they are not excluded from the mechanisms controlling histone turnover. Metabolic heterogeneity was discovered, however, when the turnover rates of the acetylated and nonacetylated molecules of histone H4 were followed: it appeared that the acetylated molecules are replaced 2.5 times faster. The comparison of the rate of replacement of the histones in proliferating and differentiated cells from one site and their level of acetylation from another suggests that this postsynthetic modification might be involved in the control of histone metabolism. Such a conclusion is supported also by a number of model experiments.     

The induction of DNA strand breaks at specific sites by N-nitroso-N-ethylurea depends on the phases of the cell cycle

Synchronized root meristems of Pisum sativum were treated at each phase of the cell cycle with 6.25 mM N-nitroso-N-ethylurea. DNA extracted from treated cells and run in agarose gel electrophoresis exhibits a series of discrete fragments with length below 2500 bp and a significant number of unspecific single-stranded breaks (or alkali-labile sites). Experiments with micrococcal nuclease indicated that the nucleosomal organization of the chromatin is not responsible for the generation of the discrete fragments: it seems that their appearance is associated with a preferable attack of the mutagen at specific sites, characteristics for the plant genome. Moreover, a cell cycle dependent release of the discrete fragments was found with maximum at G1-S and minimum at mitosis. The model experiments designed to clarify this observation suggest that it might be determined from the cell cycle dependent fluctuation in the accessibility of the chromatin DNA and/or the process of excision-repair.     

The pool of histones in the nucleosol and cytosol of proliferating Friend cells is small, uneven and chasable.

This study examines the histones pools in the nucleosol and cytosol of proliferating Friend cells. By using the conventional approach, detectable amounts of these molecules were found in both compartments; however, only H3 and H2B were identified in nucleosol, and H3, H2B and H4 in cytosol. The authenticity of each of these histones was verified by two independent methods, migration in SDS/polyacrylamide gels and peptide mapping. When the sensitivity of the approach was increased by radiolabelling with 125I, two additional proteins, migrating as H2A and H4, were observed in nucleosol. Even by this approach, however, H1 was not detected. Direct quantitative measurements of the histones in both compartments indicated that these pools are uneven and small. This was found also in experiments involving inhibition of protein synthesis by cycloheximide. Considered together, our data do not support the idea of the existence of preformed histone heterocomplexes or octamers. Instead the assembly of nucleosomes during replication occurs by a successive deposition of individual core histones.     

Intranuclear distribution of the non-histone proteins: evidence for their compartmentalization

1. Qualitative and quantitative distribution of the non-histone proteins in nuclear matrix, chromatin, a new type of RNP-network and nucleosol of Friend cells have been investigated. 2. The specific territorial distribution and metabolism of these proteins found support for the idea of their exact compartmentalization. 3. Since the majority of the non-histone proteins belong to the protein moiety of nuclear RNP-structures their specific territorial distribution probably express a primary compartmentalization of the nuclear ribonucleoproteins.     

Mode of deposition of the histone subtypes during replication

By using various approaches, we have observed in cycling mouse erythroleukemia cells a characteristic sequential deposition of newly synthesized histones. Control experiments confirmed that this deposition is replicative and is not associated with a process of histone replacement. Interestingly, each of the newly synthesized variants of histones H3, H2B, and H2A was found to deposit simultaneously. This observation indicates that the mechanisms governing the nucleosome assembly do not discriminate among the variants and thus supports the idea that they have equal biological significance. In contrast, a different mode of deposition was found for the regular subtypes H1A and H1B, suggesting a structural and functional difference between these two histones.