No abstract

Two-dimensional gel profiles of the 0.2 M H2SO4-soluble proteins of monomer nucleosomal fractions were found to contain protein A24. Protein A24 is of interest because it is composed of histone 2A and "ubiqnitin," apparently joined by an isopeptide linkage [Goldknopf, I. L. & Busch, H. (1977) Proc. NatL Acad. Sci. USA 74,[864][865][866][867][868]; Hunt, L. T. & Dayhoff, M. 0. (1977) Biochem. Biophys. Res. Commun. 74,[650][651][652][653][654][655]. Monomer nucleosomal fractions were obtained by sucrose densi: gradient centrifugation of micrococcal nuclease digests of rat liver nuclei. As shown by their DNA size, the monomer fractions were highly purified. Proteins A24 and Bu, another protein of unknown characteristics, were found along with histones 1, 2A, 2B, 3, and 4 in the monomer fractions in relative amounts similar to those found in extracts from whole nuclei and chromatin. Other-acid-soluble proteins found in the nuclear and chromatin extracts were essentially absent from the monomer fraction. Inasmuch-as protein A24 and Bu were found in lesser amounts than the histones, it is suggested that they are associated with specialized subsets of nucleosomes. Protein A24 (1), a conjugated chromatin protein (2), has a -branched structure in which one arm and the stem are composed of histone 2A (3) and the other arm contains "ubiquitin" (4-7). The carboxyl terminus of ubiquitin is attached to a Gly-Gly peptide which is in an isopeptide linkage to the E-NH2 group of lysine residue 119 of histone 2A (8). Lysine 119 is a part of the very basic carboxyl-terminal amino acid sequence of histone 2A (9, 10). The overall structure of protein A24 is (8): Ubiquitin-( X)-Gly-Gly 7Histone 2A:R-Lys-Lys-Thr-Glu-Ser-His-His-Lys-Ala-Lys-Gly-Lys. 119 129The presence of histone 2A in protein A24 as well as its histone-like chromatin binding properties (1) suggested that protein A24 may be a component of nucleosomes (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).In the present study, the 0.2 M H2SO4-soluble proteins of purified rat liver monomer nucleosomes were analyzed by two-dimensional polyacryla'mide gel electrophoresis. On the basis of their electrophoretic mobility (24)(25)(26), protein A24, protein Bu, and the histones were present in the nucleosome extracts. Proteins A24 and Bu were present in smaller amounts than the histones and may be associated with specialized subsets of nucleosomes. MATERIALS AND METHODSRat liver nuclei were isolated by centrifugation in 2.2 M sucrose/3 mM calcium acetate as described previously (27). The monomer nucleosome fraction was obtained by a modification of the procedure of Lacey and Axel (28) and Sahasrabuddhe and Van Holde (17). Nuclei were suspended in 25 ,uM CaCl2/5 mM sodium phosphate/i mM phenylmethylsulfonyl fluoride, pH 6.7, at an OD20 of 5.0. Digestion was at 370 for 10 min with micrococcal nuclease (Sigma grade VI) at 5 Mg/ml. One-fourth volume of 25 mM EDTA/1 mM phenylmethylsulfonyl fluoride, pH 7.0, was added to stop the reaction and the digest was concentrated by...

The operator of the gal operon is located about 60 base pairs preceding the startpoints of the transcription of the two gal promoters. This location contrasts with the location of the operator in other phage or bacterial operons where the repressor binds more closely to the respective transcription initiation sites. Models explaining how the repressor-operator interactions may control the two gal promoters are presented.