Electron microscopy of defined lengths of chromatin.

Abstract

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.

Subunit Structure of Simian‐Virus‐40 Minichromosome

“…However, our observations are in conflict with the results of Griffith [17], who did not observe any DNA bridges connecting the nucleosomes when the SV40 minichromosomes were visualized at an ionic strength of 0.15 M. Our electron microscope measurements show that, for SV40 minichromosomes consisting of 20 nucleosomes, the total internucleosomal DNA length represents about 15% of the SV40 genome length, irrespective of the ionic strength. This result is in keeping with the report of Finch et al [9] who did not find any dependance upon ionic strength of the internucleosoinal distance in cellular chromatin fractions of defined length. From our electron microscope measurements, a nucleosome of SV40 minichromosomes purified from infected cells contains on the average 190-200 base pairs.…”

“…All of our results support the chromatin model, which proposes that the same length of DNA is present in the nucleosome and in the biochemical unit [6,9]. First, electron microscopic measurements indicate that a nucleosome contains 190-200 base pairs of DNA.…”

“…In this model, almost all of the DNA in native chromatin is contained in the nucleosome. The suggestion that the nucleosome and the biochemical unit are identical has been supported by electron These results were presented at the International Symposium on 0rgani;ution utzd E~yprrssion q / Etkuuyoric Gmornr, University de Teheran, May 2--6, 1976 and at the Dahlem Workshop on microscopy of complexes formed in vitro between adenovirus 171 or Simian virus 40 (SV40) DNAs [8] and histones and by counts of nucleosomes in chromatin fractions containing defined lengths of DNA [9]. On the other hand several groups [lo-141 have interpreted the electron microscopy and nuclease digestion results in terms of a 'bead and bridge' model.…”

The structure of the chromatin core particle in solution

“…We first considered spherical models because the appearance of the chromatin subunit (nucleosome) in the electron microscope both when isolated [25][26][27] and in chromatin26,2830 is circular in profile and thus resembles the bead structure proposed by Olins and Olins 28 and by Kornberg31. As an example of the scattering from such models we include in figure 1 both neutron and X-ray profiles for a sphere with overall diameter 106X having a histone core with diameter 791.…”

Chromatin structure at the 44D larval cuticle gene locus inDrosophila: the effect of a transposable element insertion

“…For example, in a case where such preferential cutting based on nucleosome positions can be clearly distinguished from enhanced cutting based on sequence preference, cleavage of chromatin by MPE and by micrococcal nuclease gave identical patterns (13). Micrococcal nuclease has been used extensively in chromatin structure analysis, and the nature of its digestion products indicates that it preferentially cleaves a given DNA sequence when it falls between nucleo-,somes (21). Furthermore, the average interval between the MPEFe(II) cleavages observed at the larval cuticle gene cluster is 200 base pairs, an interval associated with a classical nucleosome repeat.…”