Analysis of E.col chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli de-oxibonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17 000 and 9,000 (proteins I and 2, respectively).2) Proteins 4 and 2 occur in approximately equal molar amounts in the isolated E coli chromosome, and their relative content corresponds to one molecule of protein I plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deozyribonucleoprotein suggesting a fairly uniform distriution of the proteins I and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein UJ which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes itE staphylococcal nuclease proceeds throu-ghdiscrete deoxyribonucleoprotein intermediates (in particular, at~120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite concl-usns about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.

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Nucleosomes and subnucleosomes separated either by sucrose gradient ultracentrifugation or by polyacrylamide gel electrophoresis contain proteins incorporating [3H]tryptophan, i.e. non-histone proteins. The fractions of mononucleosomes MN3 and MN2 are enriched in these proteins as compared to the MNI fraction. Two-dimensional gel electrophoresis of chromatin digests reveals a number of non-histone proteins comigrating with deoxyribonucleoprotein particles in the first direction (in non-dissociating conditions). A significant fraction of these proteins corresponds to basic non-histone proteins, so-called HMG (high-mobility-group) proteins. Two HMG proteins are present in mononucleosomes MN3 exclusively and three others in mononucleosomes MN3 and MN2. One of them is recovered also in subnucleosomes SN2, and another in SN3 subnucleosome fraction, At least three HMG proteins are rapidly released from the oligonucleosome fractions as well as from the insoluble DNA . protein residue. Thus, they are located in chromatin readily available to DNAase action.Apart from HMG proteins, a number of other non-histone proteins are present in mononucleosomes but their relative content in the oligonucleosome fraction is much higher. The conclusion has been drawn that many non-histone proteins, in particular HMG proteins, interact with linker DNA in chromatin.Recent studies show that eukaryotic chromatin is organized in repeated units, nucleosomes, containing about 200 base pairs per repeat on the average. Each nucleosome contains a 'core particle' consisting of a histone octamer and a DNA strand 140-145 base pairs in length (for references see [l]). Core particles are connected by linkers whose length may vary among different species [2], tissues [3] and probably among different nucleosomes in the same cell [4]. Histone H1 in DNAase digests of chromatin was found to be combined with a linker DNA fragment 30 base pairs long [5,6]. However, the question about the location of non-histone proteins remained open, in spite of the appearance of a number of conflicting data [6-113.The evidence about the existence of non-histone proteins in mononucleosomes has been obtained in experiments conducted with nucleosomes poorly purified by sucrose gradient centrifugation or gel chromatography. The resolving power of these techniques is rather low. Therefore, two-dimensional gel electroAbbreviations. CeMesNBr, cetyltrimethyl ammonium bromide; DNAase, deoxyribonuclease; DNA . protein, deoxyribonucleoprokin; HMG proteins, high-mobility-group proteins; dodecylsulfate, sodium dodecylsulfate.Enzymes. Staphylococcal nuclease (EC 3.1.4.7) ; pancreatic deoxyribonuclease or DNAase I (EC 3.1.4.5); spleen deoxyribonuclease or DNAase I1 (EC 3.1.4.6).phoresis was employed in this work for analysis. In the first direction, deoxyribonucleoprotein particles were separated in the low-ionic-strength non-dissociating conditions. This procedure gives a high resolution, separating mononucleosomes into three, or even more, subcomponents. In the second direction, the ...

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165--180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170--190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.

When chromosomal proteins in chromatin or in mononucleosomes were extensively cross-linked with an imido ester, the H1-containing nonameric histone complex was revealed. In this complex, histone H1 is connected with the octamer of core histones. The cross-linking of H1 of the octamer is realized preferentially through H2a and H3 histones. Some HMG (high mortality group) proteins located presumably in the linker regions of a nucleosome fiber also take part in the formation of dimers, possibly with the histones of a nucleosomal core. The results suggest mutant interactions between some linker-associated proteins and intranucleosomal histones. Experiments involving extensive cross-linking of proteins in the purified mononucleosome subfractions demonstrated differences in the organization of core histones between 'complete' nucleosomes and nucleosomes lacking H1.