Irregular orientation of nucleosomes in the well-defined chromatin plates of metaphase chromosomes.

In previous studies with partially denatured metaphase chromosomes, we detected platelike structures instead of the chromatin fibers currently considered in different structural models for chromosomes. Here we have observed that dilution of compact metaphase chromosomes with hyposmotic solutions can transform whole chromatids into extended plates formed by many layers. Since this treatment is soft and it does not change the ionic conditions, these observations indicate that native chromosomes are formed by stacked plates. This strengthens our hypothesis about the multilayer structure of chromosomes, which was originally based on results obtained using stronger denaturing conditions. We have investigated the structure of plates emanated from chromosomes using electron tomography. Our three-dimensional reconstructions demonstrate conclusively that the surface of the plates is very smooth and do not show repetitive structures supporting any regular organization of nucleosomes; even the nucleosomes in plate edges show irregular orientations. Furthermore, we have used polarizing microscopy for the study of whole chromosomes in metaphase cells in aqueous solution. Our results show that condensed chromosomes are not birefringent under structuring ionic conditions similar to those used with plates. This observation is incompatible with the existence of parallel columns of nucleosomes within chromosomes. In summary, we have not detected any regular orientation of nucleosomes, but at the same time, our results indicate that the bulk of chromatin in native chromosomes is organized forming very well-defined plates, in which the nucleosomes of the successive layers are interdigitated. Presumably, this dense structure is required for safe transfer of DNA to daughter cells.