Later in 1975, P.M. Oudet and others used the term nucleosome for these particles. Electron microscopy was also done on chromatin subjected to nuclease digestion, and the nucleosome structure was confirmed. The structure of DNA suggests, that DNA alone can not form repeats at intervals of 10 nm, which meant that the association of histones must have conferred the repeating organization to chromatin. Association of histones is also suggested firstly, from the knowledge that histones are positively charged and will associate with negatively charged groups in DNA, and secondly from the knowledge that histones perform their function in chromatin by binding to DNA. Therefore, it suggested that repeating units in chromatin result due to association of DNA with histones and that these repeats occur at intervals of 10 nm (1 Å = 10^-7 mm = 10^-10m).

Chromatin reconstitution experiments
X-ray diffraction patterns of native chromatin could be compared with those of chromatin, reconstituted from DNA and histones to find out the degree of successful reconstitution. It was shown by T. Kornberg (T. KornfTerg is different from A. Kornberg of DNA replication fame), that 90% reconstitution was achieved, when DNA was mixed with unfractionated histone preparation. However, when purified histones were used, reconstitution failed suggesting that some denaturation of histones takes place during purification, which destroys the compactly folded native conformation of chromatin.

Nuclease digestion and regular distribution of histones
Nucleases are a class of enzymes which will cause fragmentation of DNA at those sites which are not protected by proteins like histones. Therefore, it was expected that through a study of DNA fragments, location of these sites of naked DNA will be possible and information will thus be available about the nature of association of histones with DNA. This technique of nuclease digestion was utilized in 1973 by Hewish and Burgoyne for fragmentation of chromatin of rat liver cells. The fragments were separated by gel electrophoresis. It was found that fragments of different sizes were produced which formed distinct bands representing sizes which were multiples (two, three, four times, etc.) of size of the smallest unit. This information came from further detailed study of bands (Figs. 27.1, 27.2). On this other hand, when DNA alone and not the chromatin, was fragmented with same nuclease enzyme, uniform smear father than bands were observed on gel. Appearance of uniform smear rather than bands meant that fragments obtained due to nuclease digestion of naked DNA, gave a gradient of size, rather than being multiples of smallest unit size as shown through distinct bands in nuclease digestion of chromatin. These studies of rat liver chromatin clearly demonstrated that rat nuclease cleaved chromatin at sites spaced at regular intervals along the DNA, while naked DNA was cleaved at random. Therefore, it was concluded that histones confer a regular pattern of protection against nuclease and, that the proteins should be distributed along the DNA in a regular periodicity.

Fig. 27.1. Multimeric series of DNA bands after nuclease digestion of chromatin followed by gel electrophoresis.

Fig. 27.2. (a) Results of sedimentation of nucleosome preparation on sucrose gradient showing separation of monomers, dimers, trimers and tetramers. (b) Gel electrophoresis of DNA purified from fractions in (a) above suggesting that each multimer had a multiple of unit DNA length, (redrawn from Lewin's 'Genes IV).

Since, histones were believed to be distributed along DNA in a regular periodicity, their organization in chromatin, was an important aspect for study. Some information about this organization came with the help of gel filtration technique. This technique is just opposite to gel electrophoresis, because in gel electrophoresis, heavier molecules move slowly, while in gel filtration, heavier molecules move faster. In gel filtration, small molecules can enter gel particles and their movement, therefore, is obstructed, but the bigger molecules pass around the particles and thus move more rapidly down the column. When histones were subjected to gel filtration, these were clearly separated into two groups, one consisting of H1, H3 and H4 and the other group consisting of H2A and H2B. This was considered to be surprising, because HI was known to be double the size of the other proteins, and therefore, should have moved faster and separated itself from others. Therefore, it was speculated that H3 and H4 could associate into a dimer to have a size equal to that of H1.

Relative distribution of different histones in repeat unit
There are certain chemicals which help in chemical cross linking and thus facilitate in the study of cross linking between molecules, if any. When this technique was applied to histones in solution, it was discovered that H3 and H4 were present as a tetramer (or a pair of dimers) i.e. (H₃)₂.(H4)₂ and not as a simple dimer (H3.H4). This tetramer nature was subsequently confirmed in chromatin also. The relationship of this tetramer with other histones and DNA was also studied. It was shown that chromosomes of most organisms had equal number of molecules of histones H2A, H2B, H3 and H4. It was also shown that 25 nucleotides of DNA were present, per histone molecule. Since H3 and H4 are present as tetramers, (H3)₂.(H4)₂, each repeating unit may have one such tetramer (described as kernel) and two molecules each of H2A and H2B (because in chromatin H2A = H2B = H3 = H4). Histones will thus form an octamer, (H3)₂.(H4)₂/(H2A.H2B)₂ associated with 200 bases in each repeating unit. One molecule of H1 is also associated with each octamer in a nucleosome unit. However, H1 was not linked with other histones, so that in core particle H1 was absent.