Nuclear constituents such as polytene chromosomes and nuclear envelopes from Chironomus salivary gland cells can be isolated manually and this provides a sufficient criterion of purity for material in comparative structural studies. This was regarded as an important condition in testing earlier demonstrations and hypotheses, which were based on improved fixation and staining of intracellular carbohydrate-containing structures in thin sections of intact material. The suggestion was that the closely associated structures, chromosomes and nuclear pore complexes (NPCs) of the NEs, contain common basic components and subunits (cyclomeres and cyclosomes), which are involved in the folding and regulation of the DNA in eukaryotic chromosomes (Engelhardt and Pusa, 1972a).
The difficulties in extracting, with nonionic detergent and 2 M salt solution, and digesting with RNase and DNase such a small amount of manually isolated material like individual Chironomus polytene chromosomes and nuclear envelopes without losing them, was overcome by securing them on freshly cleaved mica pieces (Engelhardt and Plagens, 1980a). In this way, it was possible for the first time to isolate the matrix-scaffold from polytene chromosomes and compare it with the chromosomal band regions. Polytene chromosomes are amplified interphase chromosomes and the band regions are considered to be lateral bundles of chromomeres. Underlying core elements, assembled as "band-scaffolds" of the chromomeres, were uncovered. When these core elements were compared to similarly treated nuclear envelopes, similar basic structures could be shown in EM stereo pair study, by the following criterion. In both cases structurally similar scaffold elements were recognized in carbon or Pt-shadowed preparations as globular elements (with a mean diameter of 25-30 nm) and their ring-like assemblies (cyclomeres) of slightly variable diameter (60-90 nm), after the extraction and digestion. In the ring-like assemblies, the globular elements were somewhat wedge-shaped. In the NE, the ring-like assemblies could be identified as the NPCs and the globular elements as the annular subunits. When hyaluronidase was included as well, a vast amount of subunits, and also their linear and ring-like assemblies, were set free from the "band scaffold" and similarly treated NEs. In carbon replicas the basic elements of the scaffold could be recognized as having a dual appearance, especially when RNase was omitted, either globular or ring-like (globosomes, cyclosomes). Furthermore, when dehistonized polytene chromosomes were incubated with hyaluronidase alone (omitting nucleases), many rosettes were observed which consisted of rather short (1.2-2.2 kb) DNA loops folded around ring-like cores. Single loops associated with single globular elements and linear assemblies of such looped monomers were included, indicating that the rosettes are composite structures. On the basis of the results, a revised loop-and-rosette model of the polytene chromosomes is presented, which accounts for the size range and constrictions of polytene chromosome bands in more detail than earlier (Engelhardt, Plagens, Zbarsky, and Filatova, 1982).
To test and verify the results above, other approaches and material were used: metaphase chromosomes of human, elk, Chinese hamster ovary cells and Drosophila polytene chromosomes. Acid-isolation methods were developed to obtain highly pure and morphologically well-preserved chromosomes and nuclei, which were free from cellular debris. Aside from being suitable for ultrastructural cytogenetical karyotyping, they were found to be most revealing in high resolution stereoscopic EM analyses of internal chromosome structures and especially of chromatid coiling. In critical point-dried whole mounts or spreadings of metaphase and polytene chromosomes, an internal network of pore and ring-like structures was recognized in EM stereo pairs. The internal "scaffolding" network was completely resistant to the detergent-salt extraction and nucleases and devoid of any detectable DNA. Moreover, this network was indistinguishable from the NE and nuclear matrix of similarly treated nuclei or from the NE of intact acid-isolated nuclei. These results confirmed, accordingly, the results obtained above from manually isolated polytene chromosomes and NE. They are in agreement with a corresponding loop-and-rosette model for metaphase chromosomes presented earlier (Engelhardt and Pusa, 1978; Engelhardt, 1979). From the folding principle of this model natural reasons for chromatid coiling can be inferred.
All in all, similar basic elements have been found using quite different materials and EM techniques: (1) thin sectioning, (2) extraction and digestion of manually isolated polytene chromosomes and NEs, (3) critical point-dried whole mounts and spreadings of acid-isolated metaphase chromosomes, nuclei and polytene chromosomes. This has been successful only because of introducing new improved techniques in all these methods. In most interpretations the use of EM stereoscopic methods has been decisive. The structure of the chromosomal scaffold is a network of pore or ring-like structures, onto which the 10-nm DNA nucleosome "unit fibre" is folded in a loop-and-rosette configuration. Due to the short length of the loops, a superbead (supranucleosome) configuration of the "unit fibre," which condenses to the next-level 30-nm chromatin fibre, is seen as the only possibility. In view of the similarity with the network of NPCs in the NE, the internal interphase nuclear matrix, present as the scaffold in metaphase chromosomes, has presumably evolved as a detachable derivative of the NE during the evolution of the eukaryotic chromosomes.
Eukaryotic chromosome structure [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]