Involvement of histone H1 in the organization of the chromosome fiber.

Abstract

ABSTRACTnaked DNA, and that it is the cooperative binding of histone Hi that is responsible for the folding of the thin chromosome fiber. The results of four independent experimental approaches provide evidence that supports this view: (i) competition between long and short nucleosome chains for histone Hi analyzed by filter binding; (ii) the distribution of histone Hi in mixtures of long and short chromosome fibers separated by sucrose gradient velocity sedimentation; (iii) the sedimentation behavior of long chromosome fiber fragments as a function of NaCl concentration in the range of the transition; (iv) electron microscopy of chromosome fibers above and below the transition. MATERIALS AND METHODSCell nuclei were isolated from bovine lymphocytes (13) and stored in 2 mM MgCl2, 5 mM Tris.HCl (pH 7.5), and 66% (vol/vol) glycerol at -600 until needed, but not longer than 10 days. Radioactively labeled nuclei were obtained from bovine lymphocytes that had been stimulated by phytohemagglutinin P (Difco) in medium containing [3H]thymidine after 60 hr of incubation.Fragmentation of chromatin in nuclei by micrococcal nuclease (Boehringer or Worthington) was carried out at 00 in 0.2 M sucrose, 1 mM CaCl2, 5 mM Tris-HCI (pH 7.5), and either 80 or 60 mM NaCl at a concentration of 2.4-108 nuclei per ml. The digestion reaction was terminated and nuclei were lysed by gently adding the same volume of a solution containing 5 mM EDTA, 5 mM Tris-HCl (pH 7.5), and 80 or 60 mM NaCl. The nuclear debris was pelleted at 5000 X g for 8 min. The supernatant contained 50-80% of the nuclear DNA in the form of fragmented chromosome fibers. Histone-HI-depleted fiber fragments were made by the aid of tRNA (14). Histone HI was prepared by the trichloroacetic acid extraction procedure (15).The filter binding assay was carried out as described earlier (12). Stock solutions of histone H1 were diluted into 0.5 ml samples of 5 mM Tris-HCl (pH 7.5) and 40 mM NaCl containing equal weights of labeled and unlabeled histone-Hldepleted nucleosomes. After incubation for 30 min at 00, the reaction mixture was filtered through nitrocellulose membrane filters at a flow rate of 0. 1 ml-sec-'. The filters were washed three times with 0.7 ml of buffer, dried, and monitored for radioactivity. The values given are the means of three experiments; the standard deviations were less than 10% of the mean values. Filters retained 20 (+5)% of the histone-Hi-depleted nucleosome trimer (background).Sucrose gradient analyses were made by layering fragmented chromosome fiber samples (0.5 ml) on preformed linear gradients from 10 to 30% sucrose containing 1 mM sodium phosphate (pH 6.8), 0.2 mM EDTA, and NaCl at the same concentration as in the samples. Nitrocellulose tubes with 0.5 ml cushions of 86% (vol/vol) glycerol were spun in a Beckman SW 40 rotor at 3°. The gradients were analyzed with the use of a turbulence-free flow cell (ISCO).DNA sizes were analyzed electrophoretically on 1.4% agarose gels as previously described (16

Higher order structure in a short repeat length chromatin.

“…Furthermore, it was not possible to investigate the effects of chromatin concentration and sedimentation coefficients have not, therefore, been corrected for this parameter. Analysis was carried out in a buffer containing 80 mM NaCI, which provided the conditions necessary to induce higher order folding of the chromatin (3,9,30,36) . A summary of the results of this study are shown in Fig .…”

Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

“…A possible approach to the detection of critical sites of DNA modification in the mammalian genome may be to study the mode of distribution of specific carcinogen-DNA adducts in relation to defined chromatin fractions, e.g., in the DNA of actively transcribed versus inactive chromatin and/or chromatin of various folding levels. While the precise structure of transcriptionally active chromatin is still unclear, chromatin is now generally accepted to be organized in the form of condensed, 20-30 nm thick fibers (Finch and Klug, 1976;Renz et al, 1977), representing the next higher folding level of the 11 nm thick nucleosomal fiber (Kornberg, 1977). We have previously shown that in rat brain chromatin briefly exposed to the ethylating N-nitroso carcinogen, N-ethyl-N-nitrosourea (Et-NU) in vitro, the alkylation product 06-ethyl-2'-deoxyguanosine (O6-EtdGuo) is formed at a lower rate in the DNA IRL Press Limited, Oxford, England.…”

The role of charge neutralization and cooperative binding of linker histone in. the higher‐order structure of chromatin

“…Accordingly, the solenoid model suggests the cooperative mode. Some authors have suggested the non-cooperative mode [4,5] to be the correct description, whereas the cooperative form [6,7] was proposed by others. Chromatin samples of less than 30 nucleosomes in length, often shorter than dodecamers, were used in these experiments.…”

“…Examination of the mode of interaction between chromatin and linker histone would reduce the possibility of higher order structures of chromatin. However, the reported results would still require clarification; some workers have suggested a noncooperative mode [4,5] while others have proposed a cooperative type [6,7].…”