One ultimate problem in chromosome research is the folding of DNA. An interphase nucleus (diameter 5-10 mm) of a diploid mammalian cell contains about 2 m (2 x 106 µm) of DNA. At metaphase, this 2 m of DNA is packed into the 46 chromosomes of man, for instance, with a total length of about 200 µm. This necessitates a reduction in length of approximately 104 : 1.
In eukaryotic chromosomes, the way the DNA is folded is unclear, especially at the higher levels of foldings, i.e. beyond the packing of DNA (2.5 nm in diameter) into the 10-nm unit thread by the histones of nucleosomes (packing ratio 6 : 1),1and next into the 30-nm chromatin (solenoid? superbeads?) fibre of the interphase (packing ratio 40 : 1).2
1160 - 200 bp/nucleosome (10 bp = 3.4 nm) = 16.0 x 3.4 - 20.0 x 3.4 nm/11 nm (size of a nucleosome) = 5 : 1 - 7 : 1, average value 6 : 1
2Assumed to compose of about 6 - 7 nucleosomes. Then every µm of DNA is still compacted by 6 - 7 x 6 : 1, average value 40 : 1
Earlier, indications were obtained of the fine structure of residual cores in various, whole-mounted chromosomes after salt extraction (Stubblefield and Wray, l971; Solari, 1972; Lezzi and Robert, 1972) and enzyme digestion (Abuelo and Moore, 1969; Comings and Okada, 1971; Solari, 1972), but these were difficult to analyse.
Later, successful extraction and digestion have been done on nuclei which could be isolated and treated in bulk, then analysed by electron microscopy. It has been shown that in the nuclei there is an internal structural framework that is largely composed of nonhistones: the nuclear protein matrix (Berezney and Coffey, 1974, 1976, 1977). The analogous structure of metaphase chromosomes is the scaffold (Adolph, Cheng, Paulson and Laemmli, 1977).
The existing ultrastructural data are in any case incomplete and controversial. Fibrous and granular structures have been reported (Comings and Okada, 1976; Berezney, 1980), but it is unclear how they constitute the matrix. And if real, how is the matrix-scaffold related to interphase chromosomes, particularly to polytene chromosomes and especially the banding pattern? It is probably connected with the modes of DNA folding (cf. Paulson and Laemmli, 1977) and the apparently proteinaceous fixing points of DNA loops. The ultrastructure of these fixpoints, the modes of DNA folding on them (compare, e.g. Paulson and Laemmli, 1977; Mullinger and Johnson, 1980) and the size of the loops and their state during the cell cycle are still unknown.
New evidence has recently demonstrated topoisomerase II (an enzyme which regulates the supercoiling of DNA, see Gellert, 1981; Liu, 1983 for review), in intact mitotic chromosomes, chromosomal scaffolds and interphase nuclei (Earnshaw and Heck, 1985; Earnshaw, Halligan, Cook, Heck, and Liu, 1985), in the nuclear matrix and polytene chromosomes (Berrios, Osheroff, and Fisher, 1985). These data support the existence in situ of the matrix-scaffold and characterizes some of its subunits.
Based on evolutionary considerations and comparative electron microscopic investigations Engelhardt and Pusa (l972a, b) presented the hypothesis that the nuclear envelope (NE) and chromosomes have common subunits (for review see Maul 1977). When NEs are treated with salt-detergent and nucleases, the nuclear pore complex-lamina fraction stays intact (Aaronson and Blobel, 1975; Dwyer and Blobel, 1976; Scheer, Kartenbeck, Trendelenburg, Stadler and Franke, 1976). However it is not clear whether the bands of the residual polytene chromosomes and metaphase chromosome scaffold, visible after salt-detergent dehistonization, exhibit similar structures after additional nuclease (RNase and DNase) treatments.
Bulk isolation does not necessarily give clean preparations. Important details or loose elements are easily lost in washings and during different steps. Only aggregated parts that hang together are carried further. Manually isolated NEs have been used for protein analyses (Krohne, Franke and Scheer, 1978). Manual isolation could also be a purity criterion for chromosomes in comparing the structure of the chromosome scaffold and the NE.
Polytene chromosomes and similar material which cannot be obtained in bulk or in acceptable purity must be isolated manually and treated individually. A new method has been invented in which the material is secured on freshly cleaved mica, which keeps them accurately in place during subsequent treatments (Engelhardt and Plagens, 1980a, b). In this way unwanted loss and collapse of material can be avoided.
The methods used developed as follows. Under the light microscope, the bands of manually isolated polytene chromosomes and NEs of Chironomus tentans salivary gland cells were remarkably resistant to salt dehistonization and detergent (Triton X-100). This observation was made possible by sticking the material to glass fibre filters and inspecting them after ethidium bromide staining in incident fluorescent light. With a sensitive protein analysis method (using radioactive iodine labelling), both the NE and the polytene chromosomes displayed the same residual protein patterns (electrophoretic mobility) on SDS gels after treatment (Plagens, 1978).
In pilot experiments with dehistonized polytene chromosomes whole-mounted on grids (with formvar-carbon made hydrophilic with cytochrome c), a large amount of unfolded DNA hid most of the band structures (Fig. 1). Attempts to digest the material further with nucleases and to identify residual band structures were not feasible because of complete loss of the chromosomes.
However, the new mica technique helped to keep the manually isolated polytene chromosomes and NEs firmly in place for subsequent salt extraction and enzyme digestions which in turn could be monitored by light-microscopy (LM). The residual core material or scaffold of manually isolated polytene chromosomes and NEs was uncovered. These were also successfully subjected to electron microscopic (EM) study.
The problem of studying and comparing these bulky whole-mounts was solved by using stereo inspection, a method which proved essential in their interpretation.
These improvements in the methods led to the conclusion that the chromosome scaffold and the pore complexes of the NE are made of morphologically similar particles. Some tentative ultrastructural data on these elements fit with those that can be found on gyrase (topoisomerase II of prokaryotes).
These results are not restricted to polytene chromosomes and nuclear matrix (Engelhardt and Plagens, 1980a, b; Engelhardt, Plagens, Zbarsky and Filatova, 1982), since similar basic elements can be shown also in the scaffold of meiotic (Engelhardt, Plagens and Pusa, 1980), and metaphase chromosomes (Plagens, Engelhardt and Müller, 1982).
In developing new methods for ultrastructural cytogenetical analyses further unexpected confirmation of these results have been obtained from stereo inspection of intact and treated (extracted and digested) acid-isolated critical point-dried whole-mounts and spreadings of metaphase chromosomes, interphase nuclei of many different species and Drosophila polytene chromosomes.
Furthermore, in chromosomes the DNA is folded around these elements in a loop-and-rosette configuration. The results presented here lead to the proposal of a loop-and-rosette model for the polytene and metaphase chromosome structure.
Eukaryotic chromosome structure [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]