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Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the “tru” nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers.
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