Electron tomographies of metaphase chromosomes depleted of histones and DNA (by acid extraction and DNase I digestion) show distinct scaffolding macro coils, 30-nm thick fibers and 30-nm particles (Engelhardt P. et al,1994a, 1994b). No fluorescence can be detected in the chromosomes after DNase I digestion whereas controls show intensive DNA-fluorescence with e.g. DAPI. Immunoblottings of electrophoresis do not show positive staining for histones H1 or H2b, as extracted proteins, used as a positive control, demonstrate. Substructural details of the chromosome scaffold have not been shown earlier.

We have developed new 3D-preservation methods for whole-mounts of chromosomes. These were modified from tannic-acid methods that have been used to minimize cell shrinkage after glutaraldehyde (Robards A.W. & Wilson A.J. 1993). This was because, we had observed significant reduction of the mean diameter in 3D-reconstructions of some viruses (Rota, HIV, Uukuniemi), used as test objects, although substitution from solid (frozen) t-butanol (analogous to critical-point drying) results in good preservation, shrinkage is not avoided (Engelhardt P. et al, 1994b). Also negative staining, used to show the finest details of whole-mounted viruses, show in 3D-reconstruction that the viruses are very flattened (50%). Our new preservation methods combine the advantage of both the tannic and negative staining methods and reveal new details in 3D-reconstructions of chromosomes.

With the new methods the chromosomes show thicker (30-60-nm) chromatin fibres, with very distinct decorations, and regularly appearing and clearly observable 30-60-nm domains or particles with similar decorations. Also the scaffold fibres appear thicker (30-60-nm) and show regular patterns though less distinct than in the chromatin fibres where DNA is present.

In high resolution electron tomographies with MEM we can detect that the 30-60-nm scaffold fibers are composed of coiled chains of small toroid-like structures of about 8nm in outer diameter with a central hole of about 4nm. The toroidal structures could be manifestations of scaffolding proteins e.g. topoisomerase II. In some instances we can also see a very thin network-like layer appearing to cover the 30-60-nm scaffolding fibres. What all these structures truly represent is under investigation.

References

Engelhardt P. et al, (1994a) 3D Imaging Sciences in Microscopy, Munich
Engelhardt P. et al, (1994b) Electron tomography of chromosomes and viruses. CSC News, 6(4) 14-20
Robards A.W. & Wilson A.J. (1993) Procedures in Electron Microscopy, II, p. 11: 4.9