The histone variants and high-mobility-group (HMG) proteins of a transcribing fraction of chromatin, described in the preceeding paper of this journal, have been analysed qualitatively and quantitatively by a combination of one-dimensional and two-dimensional gel electrophoresis. The stoichiometry of the four core histones (all variants included) in this fraction is equimolar and is not detectably different from that in the nontranscribing fraction or in total chromatin. The molar ratio of histone H I to the core histones is markedly lower, by approximately 72 %, than that in the nontranscribiiig fraction. A minor histone variant identified as M1(an H2A variant) is detected only in the transcribing fraction, while variant H3.1 is found only in the nontranscribing fraction. Proteins A24, H M G l and H MG2 are essentially absent from the transcribing fraction ; HMG14 is found uniquely in this fraction, while HMG17 occurs at a relatively lower level.In preceding papers [l -31 we have described the separation and properties of a fraction of chromatin which, by a number of criteria, is greatly enriched in transcribing chromatin. Although it is clear [4] that transcribing chromatin possesses a nucleosome-like organisation, the possibility that nucleosomal structure is different in regions which are undergoing transcription, due to inclusion of high-mobilitygroup (HMG) proteins [5-71 or of specific histone variants [8,9], or to differences in tlic relative amounts of histone HI [lo-131, has frequently been discussed (reviewed in [4]). T o examine these possible differences, we have analysed quantitatively the histones and H M G proteins in the transcribing chromatin fraction from mouse cells. MATERIALS AND METHODS MaterialsChemicals and materials were as follows: Triton N-101, 2-mercaptoethanol and sodium dodecyl sulfate (Sigma); acrylamide, bisacrylamide and PhMeS02F (Fluka) ; pronase (Serva) ; [4,5-3H]lysine, [G-3H]tryptophan, I4C-labelled amino acid mixture and aquasol (NEN); PPO (Merck); X-Omat R X-ray film (Kodak). The sources of other chemicals arc given in the preceding paper [ 3 ] . Preparation unlf Fractionation qf ChromatinThe proteins of growing mouse P815 cells were labelled during at least three generations of growth using [3H]lysine (2-10 FCi/ml), [3H]tryptophan (6.6 pCi/ml) or a 14C-labelled amino acid mixture(0.25pCi/ml).To monitor chromatin fractionation, DNA was labelled at a low level using [14C]thyAbbreviutions. HMG, high-mobility-group; PhMeSOZF, phenylmethylsulfonyl fluoride. midine (4 pCi/l) when 'H-labelled amino acids were used. Growth conditions, chromatin preparation and fractionation were as described [2]. The appropriate chromatin fractions were pooled, precipitated with MgClz [3] and stored at -70 C. Extraction of Clwomatin ProteinsTotal Chromatin proteins were extracted with phenol [14], precipitated overnight at -20°C by addition of 6 vol. of acetone at -20 "C, washed three times with acetone, and resuspended in 0.8 M urea, 10 % 2-mercaptoethanol and stored at -70 "C until loading onto g...

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RNA extracted from total and membrane-bound polysomes of mouse liver was capable of directing protein synthesis in a homologous cell-free system in the presence of a 0.5 M KCl ribosomal wash fraction. Analysis of the products by polyacrylamide gel electrophoresis and immunoprecipitation showed that newly formed albumin could account for up to 8% of the total protein synthesized.

A method is described for the preparation of active "run-off" 80S ribosomes and 40S and 60S subunits of mouse liver. A polysome preparation was incubated at 37 degrees C for 10 min under the condition for protein synthesis (4 mM Mg2+, 100 mM KCL). Puromycin (10 mM)and 2 M KCL were added to a final concentration of 0.1 mM and 500 mM, respectively, and the reaction mixture was further incubated at 37 degrees C for 10 min. This latter treatment destabilized small polysomes and "stuck" 80S particles, which were remaining after the first incubation, leading to complete release of 40S and 60S particles. Thus, the present method minimized variations in yield of subunits due to polysome preparations and preincubation conditions. The subunits were separated by sucrose density-gradient centrifugation or recovered by precipitation following reassociation into 80S particles (run-off 80S). The reformation of 80S particles from the subunits occurred spontaneously at 5 mM Mg2+ and 100mM KCL. The isolated 40S and 60S subunits, separately, showed low phenylalanine-incorporating activity in the presence of poly(U), but when recombined, polymerized up to 10 phenylalanine residues per couple.