Super-helical Model for Nucleohistone

Pardon, J. F.; Wilkins, M. H. F.; Richards, B. M.

doi:10.1038/215508a0

Cited by 187 publications

(53 citation statements)

References 8 publications

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“…1, model II) and contain 200 base pairs of DNA, giving a packing ratio of about 7:1. The length of the unit in this model comes from x-ray diffraction, which shows a 100 A repeat in the fiber direction (5,6), and the width comes from electron micrographs showing fibers about 100 A thick (7,8). The amount of DNA per unit is derived from biochemical results (9,10) and has been substantiated by studies involving brief micrococcal nuclease digestion (11).…”

mentioning

confidence: 99%

Electron microscopy of defined lengths of chromatin.

Finch¹,

Noll²,

Kornberg³

1975

Proc. Natl. Acad. Sci. U.S.A.

139

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Defined lengths of chromatin were prepared by brief digestion with micrococcal nuclease and fractionation in a sucrose gradient. A length containing a given number of 200 base pair repeating units appeared as the same number of 100 A beads in the electron microscope. The distance between beads within a length was small, usually less than about 20 A. Several independent lines of work have led to the idea of beadlike units in chromatin, but there is disagreement over the spacing and DNA content of the beads. Two models have been put forward. One stems from a particulate appearance of chromatin fibers in the electron microscope, observed by Olins and Olins (1) The aim of the work described here was to determine the relationship between the bead observed in the electron microscope and the 200 base pair repeating unit inferred from biochemical evidence. Counts of beads in chromatin containing defined lengths of DNA, in particular SV40 chromatin (12) and complexes formed in vitro between adenovirus DNA and histones (13), have suggested that the bead and the biochemical unit are the same. Chromatin containing defined lengths of DNA can also be obtained by brief digestion with micrococcal nuclease and fractionation in a sucrose gradient (11). Here we report the counts of beads in such fractions. In the course of this work we have also made measurements of inter-bead distances in an effort to decide between the extended and compact models described above. MATERIALS AND METHODSChromatin (A26o = 46) was prepared as described (ref. 15, with the use of 110 units/ml of micrococcal nuclease for 2 min at 370 and lysis in half the stated volume of 0.2 mM NaEDTA) and 0.8 ml was layered on each of five 36.3 ml isokinetic sucrose gradients, containing 1 mM NaEDTA, pH 7, with ct = 5%, c, = 28.8% and Vm = 33 ml (16). The gradients were centrifuged for 22 hr at 27,000 rpm and 40 in a Beckman SW27 rotor, and analyzed with the use of a turbulence-free flow cell (Molecular Instruments Co, Evanston, Ill.). Corresponding fractions of the gradients (Fig. 2) were pooled, and dialyzed overnight against 1 mM NaEDTA, pH 7. The purity at this stage, determined as described in the text, was 95, 87, 78, and 67% for monomer, dimer, trimer and tetramer fractions. An aliquot (3 ml) of each fraction was further purified on a second sucrose gradient identical to the first.For electron microscopy, the purified fractions were dialyzed against 0.5 mM NaEDTA, pH 7, adjusted to the required ionic strength by the addition of ammonium acetate, and fixed if required in 5% formaldehyde, pH 7, for 5-10 min at 300. Drops of solutions were applied to thin carboncoated grids and the excess solution was withdrawn with an edge of filter paper.

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mentioning

confidence: 99%

Electron microscopy of defined lengths of chromatin.

Finch¹,

Noll²,

Kornberg³

1975

Proc. Natl. Acad. Sci. U.S.A.

139

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“…It has been suggested that this conformation is a supercoil [2]. Marked differences are also found in the optical rotatory dispersion and circular dichroic spectra of DNA and nucleohistone [3-51.…”

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confidence: 99%

The Partial Dissociation of Nucleohistone by Salts

Henson

Walker

1970

European Journal of Biochemistry

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The circular dichroism of calf thymus deoxyribonucleohistone was compared with that of samples partially depleted of histone in solutions of sodium chloride. Differences in the near ultraviolet circular dichroism of DNA and deoxyribonucleohistone were associated with the presence of the f2b, f2a2 and f 3 histone fractions in the complex. The near ultraviolet circular dichroic spectra of both DNA and deoxyribonucleohistone were very sensitive to the solvent environment. Thus differences in the circular dichroic spectra of DNA and deoxyribonucleohistone cannot necessarily be associated with long-range effects, such as supercoiling, but may be due to short-range effects, such as might be caused by histone-DNA interactions.The absorbance and the circular dichroic melting profiles of deoxyribonucleohistone were biphasic. A new dichroic species was formed during the melting of the first of these steps and evidence is presented that this is a new histone-DNA complex produced by the migration of some or all of the fractions f2b, f2a2 and f 3 from denatured t o native regions of the DNA during the melting process. Thus the biphasic melting profile of deoxyribonucleohistone does not necessarily imply that these fractions are arranged heterogeneously on the DNA.Studies on the hydrodynamic properties of nucleohistone have shown that certain histone fractions maintain nucleohistone in a more compact conformation than DNA [l]. It has been suggested that this conformation is a supercoil [2]. Marked differences are also found in the optical rotatory dispersion and circular dichroic spectra of DNA and nucleohistone [3-51. I n order to see whether the histone fractions reponsible for the characteristic difference in hydrodynamic properties were also responsible for the difference in optical properties, we have investigated the circular dichroic spectra of nucleohistone partially depleted of specific histone fractions by salt.The histone fractions which are implicated in the supercoiling of nucleohistone also produce biphasic melting profiles in the complex [1,6]. Therefore we have studied the circular dichroism of native and partially depleted nucleohistone as a function of temperature in order to gain further information about the contribution of specific histone fractions to the conformation of the native nucleoprotein. Apreliminary account of this work has been published [7]. EXPERIMENTAL PROCEDUREThe preparation, characterisation and experimental methods, other than circular dichroism, have been described previously [11. The solvent used in all cases was 0.7 mM sodium phosphate, p H 6.8, except where otherwise statcd. Deoxyribonucleohistone samples that have been partially depleted of histone will be identified by the molarity of the sodium chloride solution used for the depletion. Thus a sample depleted in 0.7 M NaCl is designated 0.7 deoxyribonucleohistone. The extinction coefficients for the heat-treated samples were obtained by phosphorus analysis [S]. Circular dichroic spectra were measured in a Jouan Dicrographe Model C...

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“…The similarity between the layer line spacings found in the optical diffraction patterns of H4 fibers and the spacings found in x-ray diffraction patterns of chromatin (8)(9)(10)(11)(12)(13) raises the possibility that the structure of the assembled histone fibers is closely related to the substructure of chromatin, most probably to the continuous 100-A fibers. The fibers described here consist of H4 only, but similar fibers are formed by other single histones and by mixtures of histones either in pairs or all four together (2,3).…”

Section: Methodsmentioning

confidence: 58%

Arrangement of subunits in assembled histone H4 fibers.

Sperling¹,

Amos²

1977

Proc. Natl. Acad. Sci. U.S.A.

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The structure of assembled histone H4 fibers has been studied by analysis of electron micrographs, including optical diffraction. An individual fiber has the appearance of an 80WA wide ribbon, twisted at intervals of about 330 A. Thicker fibers which have been observed seem to be bundles of ribbons. In diffraction patterns from both kinds of fiber, layer lines at axial spacings of about 1/55 A-1, 1/37 A-1, and 1/27 A-' were most consistently observed. The

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Super-helical Model for Nucleohistone

Cited by 187 publications

References 8 publications

Electron microscopy of defined lengths of chromatin.

Electron microscopy of defined lengths of chromatin.

The Partial Dissociation of Nucleohistone by Salts

Arrangement of subunits in assembled histone H4 fibers.

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