A new group of chromatin-associated proteins having a high content of acidic and basic amino acids have been isolated from the 0.35 M NaC1-extractable proteins from calf thymus.This new group, designated "high-mobility group" proteins have been partially separated and some interesting new proteins identified. One such protein contains over 55O/, acidic and basic amino acids.The major group of chromatin proteins, the histones, have received a great deal of attention over the past ten years [I]. Methods have been developed for their isolation in a pure form, and the complete amino acid sequence of all but one of the fractions is known. Since they appear to be non-specific gene repressors [2], possibly acting by causing some conformational change in the DNA which restricts transcription in a very non-specific manner, attention has turned to the so-called "acidic proteins" of chromatin in an attempt to find more specific interactions with DNA, which may have some relevance to specific gene derepression [3]. Work in this field has been generally unsuccessful since these proteins, once removed from the chromatin complex, seem to aggregate easily and are difficult to keep in solution.We have shown previously [5] that most of the non-histone proteins can be removed from calf thymus chromatin by extracting with 0.35 M NaCl and we have suggested that some of these were contaminants picked up from the cytoplasm during the preparative procedures. A recent paper [5] has indicated that this contamination is very small and our recent results agree with this but also indicate that those cytoplasmic contaminants which are present are in the low mobility group of proteins as determined by polyacrylamide gel electrophoresis [6]. This being so, we decided to separate the high and low mobility groups of proteins of the 0.35 M NaCl extract, quite arbitrarily, by precipitation with trichloroacetic acid. We found that once separated from the low mobility group of proteins, the high mobility group was easy to work with and quite soluble in dilute acids. It constitutes an easily defined group of chromatin proteins which, as far as we are aware, has not been described previously by other laboratories. This paper gives a preparative method for these proteins and an initial partial separation and characterisation. EXPERIMENTAL PROCEDURE AND RESULTS Polyacrylamide-Gel ElectrophoresisThe method used was that developed for the histones by Johns [ 7 ] with the modifications described by Dick and Johns [S]. The non-histone proteins were, however, dissolved in and applied to the gel in 0.1 N HCl, 9 M urea instead of the sample solvent described. The method for applying two samples to one gel for more exact comparative electrophoresis has also been described previously [9]. Preparation of Calf -Thymus ChromatinCalf thymus was obtained immediately after slaughter of the animal and frozen in solid CO, until required. It was then thawed and freed from membranes and connective tissue and minced using a domestic mincer. All subsequent operations were ...

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The group of calf thymus chromatin non-histone proteins which have a high electrophoretic mobility have been fractionated by CM-cellulose ion-exchange chromatography. The fractions thus obtained have been characterised by polyacrylamide gel electrophoresis, amino acid analysis, N-terminal amino acid analysis, and by their molecular weights. Using the CMcellulose chromatographic method, two proteins which have a high content of basic and acid amino acids have been isolated in a pure form, I n a previous report [l] it was shown that the non-histone proteins extracted from calf thymus chromatin with 0.35M NaCl can be conveniently fractionated into two groups by trichloroacetic acid precipitation. The non-histone proteins with low electrophoretic mobilities (low-mobility proteins) can be precipitated with 2O/, (w/v) trichloroacetic acid ; the remaining proteins with high electrophoretic mobilities (high-mobility proteins) can then be precipitated with loo/, (w/v) trichloroacetic acid or with acetone. Partial fractionation of the highmobility proteins by gel filtration revealed that they contained two very interesting proteins, designated protein 1 and protein 2, which contain over 55O/, of basic and acidic amino acids. These two proteins were previously identified as impurities in histone fractions [2,3] and more recently their existence has been confirmed by other workers [4] who have shown that they combine with histone Fl and polylysine.The gel filtration fractionation described previously [l] does not separate these two proteins and therefore, in view of the interesting properties of these two proteins, it was decided to develop a method for their isolation and separation. I n this report we describe a chromatographic fractionation of the high-mobility proteins in which proteins 1 and 2 can both be obtained in a pure form in relatively large amounts. EXPERIMENTAL PROCEDURE AND RESULTS Polyacrylamide-Gel ElectrophoresisPolyacrylamide gel electrophoresis using 20 O/,, acrylamide gels a t pH 2.4 was carried out as described previously [ 11. Amino-Acid AnalysisTotal amino acids were measured by the method of Moore et al.[5] using a Technicon autoanalyser. Samples were hydrolysed for 24 h in 6 N HC1 a t 110 "C. No corrections were made for hydrolytic losses. N-terminal Amino-Acid Analysiswas used to determine N-terminal amino acids. The dansyl technique described by Hartley [6]Determination of Molecular Weight Molecular weights were determined by a modification of the dodecylsulphate-polyacrylamide gel electrophoresis system of Weber and Osborn [7] using pure histone fractions as standards. l o o / , acrylamide gels (7 x 0.5 cm) containing 0.1 O/, sodium dodecylsulphate, 0.1 M sodium phosphate pH 7 were preelectrophoresed for 6 h a t 40 V with 0.1 O/, sodium dodecylsulphate, 0.1 M sodium phosphate buffer pH 7 in the electrode vessels. The electrode vessel solutions were then renewed and protein samples dissolved in 4 M urea, 0.1 O/, sodium dodecylsulphate, 0.1 ' J/, 2-mercaptoethanol, 0.01 O/, bromophenol blue, 0.01 ...

1. A new method has been devised for the separation of the histone fractions of calf thymus by electrophoresis in polyacrylamide gel at pH2.4. 2. The fractions have been characterized by their relative mobilities with respect to a marker protein, bovine plasma albumin. 3. A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye-histone complexes formed in the gel.

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