Ultrastructure of chromatin. I. Negative staining of isolated fibers

J Cell Sci. 1991 May;99 ( Pt 1):99-106.

Abstract

The ultrastructure of chromatin fibers isolated from erythrocyte nuclei of Necturus maculosus and contrasted with a number of negative stains is described. Long (greater than 1000 nm) fibers are prepared under ionic conditions that promote fiber integrity, fixed with glutaraldehyde and negatively stained with aurothioglucose, ammonium molybdate, methylamine tungstate, sodium phosphotungstate, uranyl acetate and a uranyl acetate-sodium phosphotungstate sequence. All stains yield images of '30 nm' chromatin fibers, but aurothioglucose gives the most consistent diameter measurements (33 nm, S.D. 3.5 nm), and provides the clearest images of individual nucleosomes. Regions of fiber showing structural order are seen with all stains. The most commonly observed is a regular pattern of oblique cross-striations consistent with the visualization of the 'top' or 'bottom' of a helical structure. There is a significant relationship between fiber diameter and the cross-striation angle, consistent with an extensible chromatin fiber. Examination of power spectra prepared from selected ordered regions confirms the visual impressions, and indicates a striation spacing ranging from 11 nm to 18 nm, and dependent on the stain type. Fibers allowed to unfold slightly in a buffer containing 50 mM monovalent ions show evidence of a two-stranded helix-like organization. These results are discussed in terms of current models for the structure of the chromatin fiber.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Chromatin / ultrastructure*
  • Deoxyribonucleases
  • Erythrocytes / metabolism
  • Erythrocytes / ultrastructure*
  • Image Processing, Computer-Assisted
  • Microscopy, Electron
  • Necturus maculosus / metabolism*
  • Negative Staining
  • Osmolar Concentration

Substances

  • Chromatin
  • Deoxyribonucleases