Nuclear and cytoplasmic fractions were isolated from various tissues of the rat by a nonaqueous technique . The high-mobility-group (HMG) proteins were extracted from these fractions with acid and separated by one-and two-dimensional PAGE . The concentrations of high-mobility-group proteins HMG1, HMG2, and HMG17 in the nucleus and cytoplasm were then estimated from the staining intensities of the electrophoretic bands . The cytoplasmic concentrations of these proteins were very low-usually less than '/3o of those present in the corresponding nuclear fractions . For the tissues studied (liver, kidney, heart, and lung), the concentrations of HMG proteins in the nucleus did not differ significantly from one tissue to another. Averaged over the four tissues investigated, there were 0.28 molecule of HMG1, 0 .18 molecule of HMG2, and 0.46 molecule of HMG17 per nucleosome . These values are considerably higher than those that have been reported previously .The high-mobility-group (HMG)' proteins are a family of well-characterized proteins which are present in relatively large amounts in the chromatin of all higher organisms (reviewed in reference 1) . The cells of higher eucaryotes contain four major HMG proteins : HMG1, HMG2, HMG14, and HMG17. These four proteins constitute two groups : one containing HMG1 and HMG2, and the other, HMG 14 and HMG17. The two proteins belonging to each group are of similar size, show considerable sequence homology, and may bind to similar or identical sites on the chromatin. Two major HMG proteins in trout testis, HMG-T and H6, have been extensively investigated. HMG-T is homologous to HMG 1 and HMG2, and H6, to HMG14 and HMG17.Considerable interest in the HMG proteins has been generated by reports that they are preferentially associated with transcriptionally competent portions of the genome (2-5), and a great deal of effort is currently being expended to define the role which these proteins play in transcription. Not all investigators have found such a relationship . Recently, Seale et al . (6) reported that there is little correlation between HMG protein content and transcriptional activity in HeLa cell chromatin, and Gabrielli et al . (7) have reported that, in mouse P815 cell chromatin, transcriptional activity is correlated with the presence of HMG 14, but not of HMG1 and HMG2 or of HMG 17 .' Ahbreviation used in this paper: HMG, high-mobility-group (protein) . 648Hypotheses concerning the function and metabolism of the HMG proteins frequently involve assumptions concerning their intracellular concentrations and subcellular distributions. Definitive information on these points, however, is unavailable. It has been suggested that there are -10' molecules of each of the major HMG proteins in a typical mammalian cell (8, 9), corresponding to about one molecule each of HMG1, HMG2, and HMG17 for every 30 nucleosomes. This value is apparently based on the yields of the HMG proteins which have been obtained from calf thymus using standard isolation techniques . Because yields were...

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Ubiquitin was radioiodinated and introduced into HeLa cells by the erythrocyte-mediated fusion procedure. Fractionation of injected HeLa cells and subsequent NaDodSO4/ polyacrylamide gel electrophoresis showed that HeLa nuclei contained two major labeled proteins: ubiquitin and the histone H2A-ubiquitin conjugate, protein A24. HeLa cytosol contained ubiquitin and a series of ubiquitin-protein conjugates of diverse molecular weights. When injected HeLa cells were treated with phenylhydrazine to denature the cotransferred hemoglobin, a series of prominent ubiquitin-globin conjugates appeared. The identity ofthese conjugates was established by microinjection experiments in which both proteins were labeled. At low doses of phenylhydrazine, the intracellular concentration of globin-ubiquitin conjugates was proportional to the rate of hemoglobin degradation. This result, together with the observation that ubiquitin conjugation to globin is markedly enhanced by phenylhydrazine-induced denaturation of hemoglobin, provides support for the hypothesis that the covalent attachment of ubiquitin to proteins signals proteolysis.Cells can selectively degrade soluble intracellular proteins (1-3). A fundamental question regarding the mechanism of selective proteolysis is whether the initial event is a proteolytic cleavage or a biochemical marking of the protein to be degraded. Evidence for the latter possibility was obtained by Hershko and his colleagues, who found that a small protein, subsequently identified as ubiquitin (4), was a necessary cofactor for the ATPdependent degradation ofproteins in rabbit reticulocyte lysates (5). Addition of radioiodinated ubiquitin to reticulocyte lysates and subsequent NaDodSO4/polyacrylamide gel analysis revealed that ubiquitin was conjugated to a number ofendogenous or exogenous proteins (6, 7).Although these results suggest that ubiquitin conjugation plays a role in protein degradation, they are not conclusive. Ubiquitin is conjugated to histones H2A and H2B (8-10), and yet these proteins are stable within cells (11). Even for proteins that are degraded, correlation between the extent of ubiquitin conjugation and degradation rate has not been demonstrated. Also, one could postulate that few proteins are modified by ubiquitin conjugation in intact reticulocytes but that upon lysis the conjugation system becomes uncontrolled, resulting in attachment of ubiquitin to many proteins. One could further hypothesize that proteins are denatured by conjugation and then degraded by proteases that normally act independently of ubiquitin.Evidence that ubiquitin is a signal eliciting proteolysis would be strengthened ifit were shown that ubiquitin conjugation followed protein denaturation rather than the converse. Hendil recently demonstrated that treatment of baby hamster kidney (BHK) cells with phenyihydrazine after microinjection of labeled globin produced a dramatic and selective increase in the degradation of the injected hemoglobin (12). If ubiquitin conjugation serves as a signal for prote...

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A method for isolating nuclei from tobacco leaves is described. Preliminary results indicate that the method may be generally applicable for isolating nuclei from plant tissues.