Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1343-1347, April 1977

Biochemistry

Chromatin subunits from baker's yeast: Isolation and partial characterization (Saccharomyces cerevisiae/nucleosomes/histones/staphylococcal nuclease)

DANIEL A. NELSON, WILLIAM R. BELTZ, AND RANDOLPH L. RILL Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee, Florida 32306

Communicated by Irving M. Klotz, November 15, 1976

The organization of proteins along DNA in ABSTRACT chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease. Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 base pairs apart. Nucleoprotein fragments were resolved and isolated by centrifugation on linear, 5-20% sucrose gradients. The predominant UlS component appears to be identical to chromatin "subunits" or "nucleosomes" isolated from higher eukaryotes, containing a 150-160 base pair length of DNA and approximately equimolar amounts of four proteins that coelectrophorese with calf histones H2A, H2B, H3, and H4, plus small amounts of three proteins that electrophorese similarly to HI histones. Thus, the structural organization of the yeast genome is similar to that of more developed organisms, except for the smaller total repeat length. None of the yeast subunit proteins, including the possible HI proteins, contains cysteine.

Understanding of the complex genetic control mechanisms of higher organisms may be facilitated by examining primitive organisms that possess much lower degrees of gene complexity yet have developed advanced modes of gene transcription, replication, and organization. The primitive, unicellular Saccharomyces cerevisae (baker's yeast) is an attractive choice for such a model system. Similarities between the yeast transcriptional apparatus and that of differentiated cells include the presence of three distinct RNA polymerases (1), processing of ribosomal RNA from larger precursors (2), polyadenylylation of messenger RNA (3), and "capping" of the 5'-end of messenger RNA (4). Since yeast grows rapidly on simple nutrients, the patterns of gene regulation can be readily altered by changes in the medium (5). The reduced genetic complexity of yeast [about %/1oo that of typical mammalian cells (6)] facilitates isolation of specific genes, e.g., those coding for yeast ribosomal RNA (7). In addition, much of the yeast genome is active during exponential growth (8). Thus, yeast may be an excellent organism for studying gene control and for isolating and characterizing active and inactive portions of the genome. Before the transcriptional apparatus of yeast can be related with certainty to that of higher cells, similarities in genome organization must be established. Interphase chromatin fibrils of higher eukaryotes have a "beaded string" structure consisting of repeated arrays of globular particles (about 100 A diameter) connected by thin nucleoprotein filaments (9-12). Nucleases cleave these interconnecting regions, producing individual "subunits" and larger oligomeric nucleoprotein units that can be readily isolated (12, 13). Subunits from higher eukaryotes contain two copies each of histones H2A, H2B, H3, and H4 associated with about 140 base pairs of DNA (12, 14). Recent neutron diffraction studies (15) have confirmed earlier proAbbreviations: PMSF, phenylmethylsulfonylfluoride; NaDodSO4, sodium dodecyl sulfate. 1343

posals (10, 11) that these histones form a "core" about which the unit length of DNA is wrapped. Lysine-rich histones HI (and H5 in erythrocytes) appear to be associated primarily with the "spacer" DNA, 30-60 base pairs long, between globular subunits (14, 16, 17). Yeast chromatin potentially may have a subunit structure since yeast nuclei contain several major basic proteins that electrophorese similarly to histones (18, 19). In fact, staphylococcal nuclease digests yeast chromatin at discrete intervals, indicating that proteins are arranged along the DNA in-a periodic manner (18, 20, 21). However, some caution is warranted in interpreting these results because of reports that yeast lacks histones Hi and H3 (22) and that yeast ribosomal proteins may be coisolated with or otherwise mistaken for histones (23). As described below, we have confirmed the periodic structure of yeast chromatin and have isolated and partially characterized monomeric subunits and oligomer units from yeast that appear to be nearly identical to subunits from higher eukaryotes. MATERIALS AND METHODS Isolation and Digestion of Yeast Nuclei. Saccharomyces cerevisiae strain A8209B was obtained from Dr. Gerald Fink (Cornell University). Yeast cells and protoplasts were prepared as described by deKloet et al. (24). Prior to isolation of nuclei, protoplasts (OD6Wj nm = 1-1.5) were incubated for 45 min at 300 in 20 mM KH2PO4 (pH 6.2)/2 mM MgCl2/2% glucose/ 0.3% casamino acids/20% sorbitol to permit return to a metabolically active state (24). The protoplasts were then rapidly cooled to 40 and washed with cold 20% sorbitol. Nuclei were isolated (at 0-40) by a method modified slightly from that of Blobel and Potter (25). Protoplasts were washed once in TKMC buffer (50 mM Tris-HCI, pH 7.5/25 mM KCI/5 mM MgCI2/1 mM CaCI2) containing 0.25 M sucrose, then twice in the same solution containing 0.5% Triton X-100. The crude nuclear pellet was purified by centrifugation through dense sucrose in TKMC buffer plus 0.1 mM phenylmethylsulfonylfluoride (PMSF) as described by Blobel and Potter (25). The nuclear pellet was washed twice in digestion buffer (250 mM sucrose/60 mM KCI/15 mM NaCl/10 mM MgCl2/1 mM CaCl2/15 mM Triscacodylate at pH 6.5) and resuspended in digestion buffer at a concentration of 100 Mg of DNA per ml [determined by the modified diphenylamine method of Burton (26)]. The integrity of the nuclei during isolation was assayed with a light microscope after staining with pyronin B-methyl green. Nuclei were incubated at 370 with staphylococcal nuclease (29,000 units/mg, Worthington) at a concentration of 50 units of nuclease per 50 ,g of DNA. One-tenth volume of 0.15 M EDTA (pH 7.5) was used to terminate the digestion, and the samples were thereafter kept at 0-40 (the termination buffer was also made 1 mM in PMSF unless samples were to be treated with protease).

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Fractionation of Nucleoprotein Fragments of Digested Nuclei. Digested nuclei were lysed by dialysis against 10 mM Tris/0.1 mM EDTA/0.1 mM PMSF at pH 7.5. Then 0.2-ml aliquots were layered onto 12-ml, 5-20%, linear sucrose gradients containing the above buffer. Gradients were centrifuged in an International SB-283 rotor at 40,000 rpm for 10 hr at 40 Analysis of Yeast Nuclear Proteins. Protein electrophoresis was done on 18% polyacrylamide gels containing sodium dodecyl sulfate (NaDodSO4) as described by Laemmli (27) and modified by Bonner and Pollard (28). Gels were stained with 0.25% Coomassie blue in 50% methanol/10% acetic acid, destained, and equilibrated prior to scanning in the above solution containing a small amount of dye. Whole nuclear proteins were electrophoresed directly from nuclei washed with cold ethanol, dried, and resuspended in NaDodSO4 sample buffer (1% NaDodSO4/20% glycerol/ 0.005% bromphenol blue/10 mM Tris/5 mM EDTA/0.5% 2-mercaptoethanol/1 mM PMSF at pH 7.5). Samples were incubated at 370 for 30 min, heated to 1000 for 1.5 min, then applied to the gel. Acid-extractable nuclear proteins were isolated from nuclei treated with RNase A (50 units/ml, Worthington) and staphylococcal nuclease (50 units/ml) and dialyzed into 10 mM Tris-HCI, pH 7.5/0.1 mM EDTA/0.1 mM PMSF. Chilled samples were made 0.2 M in H2SO4 and centrifuged. Protein in the supernate was precipitated with four volumes of cold acetone. Crude nucleohistone was prepared from nuclei digested with staphylococcal nuclease for 30 min at 370 and dialyzed into 10 mM Tris-HCI, pH 7.5/0.1 mM EDTA/0.1 mM PMSF. Nuclear debris was removed by centrifugation and the supernate was made 3 mM in MgCl2. The aggregated material was pelleted, washed with cold ethanol, resuspended in NaDodSO4 sample buffer, and applied directly to gels. Nucleoproteins in gradient fractions were precipitated by addition of MgCI2 to a final concentration of 3 mM, followed by addition of two volumes of ethanol. After standing overnight at -200, the aggregated histone-DNA complexes were pelleted, washed with cold ethanol, dried under reduced pressure, resuspended in NaDodSO4 sample buffer, and applied directly to gels. Protein Sulfhydryl Group Modifications. Oxidation-reduction of calf thymus and chicken erythrocyte histones and proteins in crude yeast nucleohistone was done as described by Marzluff et al. (29). Samples (5 mg of protein per ml) were oxidized for 16 hr at 370 in 6 M guanidine-HCI/0.3 M Tris-HCI at pH 8.3, and dialyzed overnight against 10 mM Tris-HCl, pH 7.5/0.1 mM EDTA/0.1 mM PMSF. Twenty volumes of NaDodSO4 gel sample buffer was then added and samples were electrophoresed on NaDodSO4 gels. Carboxymethylation using ICH214COOH (15.25 mCi/mmol; New England Nuclear, Boston, MA) was done in NaDodSO4 gel sample buffer (pH 8) without 2-mercaptoethanol. Samples (1 ,ug of protein per 8 ,ul) were made 0.1 mM in dithiothreitol and incubated for 30 min at 370, then incubated another 30 min after the addition of 'Ao volume of 4 mM ICH214COOH in 10 mM Tris-HCI, pH 8.5/5 mM EDTA. The reaction was stopped with an excess of dithiothreitol and samples were run on NaDodSO4 histone gels. Stained gels were scanned to quantitate the amount of histone in the gel. Bands were cut from the gels, treated with 0.5 ml hydrogen peroxide for 10-12 hr at 60°, then neutralized with 50 l of 1 M Tris (pH 8.5). Radioactivity of samples was determined after addition of 4 ml of water plus 10 ml of Triton-toluene scintillation fluid (2/1, vol/vol Triton-

Proc. Natl. Acad. Sci. USA 74 (1977)

FIG. 1. Electrophoretic patterns of (A) histones from chicken erythrocyte nuclei, (B) total yeast nuclear proteins, (C) proteins acid-extracted from yeast nuclei, and (D) proteins from "crude nucleohistone" isolated from yeast nuclei as described under Materials and Methods. Samples were electrophoresed simultaneously on 18% polyacrylamide gels containing 0.1% NaDodSO4.

toluene, 0.4% Omnifluor; New England Nuclear, Boston, MA). Electrophoretic Analysis of DNA Fragments. DNA isolated as described previously (13) was electrophoresed on 1.4% agarose gels or 6% acrylamide gels by the method of Loening (30). DNA from the gradient fractions was run on 6% polyacrylamide gels with the gels and tray buffer made 0.1% in NaDodSO4. DNA gels containing NaDodSO4 were scanned at 260 nm. Gels without NaDodSO4 were stained with Stains-all (Eastman), destained, and scanned at 610 nm. Sizes of DNA fragments were determined by comparisons with coelectrophoresed fragments of PM2 DNA cleaved with endonuclease Hae III (from Haemophilus aegyptius). Sizes of these PM2 fragments have recently been recalibrated by comparisons with electrophoretic mobilities of Hind and Hae III fragments of simian virus 40 DNA and Hind fragments of bacteriophage lambda DNA (R. T. Kovacic and K. E. Van Holde, private communication). The most recent values are 5-15% higher than values cited previously by Van Holde and coworkers (11, 14, 20) and Rill et al. (31). RESULTS Yeast Nuclear Proteins. Electrophoretic patterns of total yeast nuclear proteins and acid-extracted nuclear proteins are compared in Fig. 1. Four major proteins band in nearly identical positions to chicken erythrocyte histones H3, H2B, H2A, and H4, in agreement with recent observations (18, 19). In a separate experiment, purified calf thymus histones H3 and H4 were added to yeast acid-extracted proteins and coelectrophoresed with the putative H3 and H4 histones of yeast (data not shown). Fig. 1 also illustrates that acid extraction is not selective for obtaining histone analogs from yeast. Since elec-

Biochemistry:

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Proc. Natl. Acad. Sci. USA 74 (1977)

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z 0 U)

9S II S 17 S 22 S SEDIMENTATION -b-

FIG. 3. Sucrose density gradient centrifugation patterns of nucleoprotein fragments from (A) chicken erythrocyte nuclei digested for 30 min with staphylococcal nuclease (5 units/50 g of DNA) and from yeast nuclei digested for (B) 5, (C) 15, and (D) 30 min with the nuclease (1 unit/jg of DNA). Fraction 1 ("monomer") and fraction 2 ("oligomer") were pooled for compositional analysis from all the gradients shown. Approximate S20,u values are given along the abscissa. MIGRATION

FIG. 2. (A) Comparison of the sizes of DNA from (top) yeast nuclei digested for 5 min with 1 unit of staphylococcal nuclease per ,ug of DNA and (bottom) chicken erythrocyte nuclei digested for 30 min with 5 units of nuclease per 50 ,ug of DNA. Samples were electrophoresed simultaneously on 16-cm gels of 1.4% agarose, stained with Stains-all, and scanned at 610 nm. (B) Electrophoretic analysis, on 6% polyacrylamide gels, of the time course of digestion of DNA in yeast nuclei by staphylococcal nuclease. Samples were digested for 1, 5, 30, and 60 min (bottom to top). The large amount of rapidly moving material was present in undigested control samples and was resistant to RNase A. Possibly this material is poly(A). No nucleic acid of this mobility was found in isolated subunits. Migration positions of some of the Hae III fragments of PM2 DNA electrophoresed with the samples are indicated along the abscissas of the figures. The sizes of these fragments, in base pairs, are: 854 (D), 794 (E), 642 (F), 592 (G), 498 (H), 322 (I), 288 (J), 263 (K), 160 (L), 145 (M), 117 (N), and 94 (0).

trophoretic mobilities are not sufficient criteria for distinguishing ribosomal proteins from histones (23), a better criterion for identifying these proteins as histones is a demonstration that they are organized along yeast DNA in subunits such as those found in higher eukaryotes. That these proteins are histones is suggested by the finding that a "nucleohistone" fraction significantly enriched in putative yeast histones is obtained by digesting nuclei with nuclease, dialyzing into low ionic strength buffer, centrifuging to remove nuclear membrane debris, and precipitating the remaining nucleoprotein fragments with Mg++ and ethanol (see Fig. 1D). The DNA content of the precipitated material determined from the absorbance at 260 nm and from analyses using diphenylamine were identical within experimental error, indicating that mostly DNA-protein complexes were precipitated. This result is rather remarkable considering that yeast nuclei contain roughly four times as much RNA as DNA (32). The bulk of the yeast nuclear RNA must be rapidly digested by staphylococcal nuclease and the oligoribonucleotides removed by the Mg++ precipitation of the nucleohistone. RNAassociated proteins, other proteins, and, possibly, resistant RNA were apparently removed by the first centrifugation step since

the bulk of the nonhistone protein was found in the nuclear debris fraction. Nuclease Digestion of Yeast Nuclei and Subunit Isolation. To determine if yeast chromatin contains repeated subunits, we digested yeast nuclei with staphylococcal nuclease and separated and characterized the resulting DNA and nucleoprotein fragments. As shown in Fig. 2, electrophoresis resolved DNA from digested nuclei into a series of discrete bands that are integer multiples of a basic repeat length. In agreement with the most recent estimates of Thomas and Farber (18) and Lohr and Van Holde (20,21), the basic repeat length is about 160 base pairs, considerably smaller than the approximate 190-200 base pair repeat found in chicken erythrocyte and rat liver nuclei (refs. 12, 14, 33, and 34; see also Fig. 2A). As the digestion proceeds, the initially sharp band at about 160 base pairs splits into a doublet, with another band appearing at -about the 140 base pair position (Fig. 2B). Density gradient centrifugation of nucleoprotein fragments further demonstrates the yeast chromatin subunit structure (Fig. 3). The sedimentation position (about 11S) of the monomer subunits of yeast and chicken erythrocyte chromatin are virtually identical, but the sedimentation coefficients of dimer and higher oligomer particles of yeast are noticeably smaller than those of erythrocyte oligomers, presumably due to the shorter repeat length. The lengths of the major DNA species from the "monomer" and "oligomer" (predominantly dimer) gradient fractions were determined to be 160 and 320 base pairs, respectively, in agreement with the sizes of "monomer" and "dimer" DNA isolated from whole digests. Treatment of these nucleoproteins with RNase A did not alter the DNA electrophoresis patterns, indicating that the particles were not ribonucleoproteins. This finding and the large oligonucleotide peak at the top of the gradients again suggest that most RNA and ribonucleoproteins are rapidly degraded. Proteins isolated from monomer and dimer subunits are resolved into four major components that electrophorese, on

NaDodSO4 gels, nearly identically with chicken erythrocyte histones H3, H2B, H2A, and H4 (Fig. 4). Although we have not measured the weight ratio of protein to DNA in the subunits directly, this ratio appears to be nearly identical for yeast and

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Froc. Natl. Acad. Sci. USA 74 (1977) treatment with labeled iodoacetic acid, about 1.8 times as many counts per minute were incorporated into calf H3 as chicken H3, in agreement with results of Marzluff et al. (29).

MIGRATION )FIG. 4. Electrophoretic patterns of proteins from "monomer" gradient fraction 1 (top curve) and "oligomer" gradient fraction 2 (bottom curve) isolated as shown in Fig. 3. Migration positions of chicken erythrocyte histones electrophoresed with these samples are indicated along the upper margin.

chicken erythrocyte subunits, as judged by a comparison, using subunit preparations from yeast and erythrocytes, of the ratio of the total area under the protein bands to the total amount of DNA placed on the gels (determined with diphenylamine). In a similar manner we have found that the amounts of total histone (H3, H2B, H2A, and H4) relative to DNA are nearly the same in yeast, calf thymus, and chicken liver nuclei. Thus, as in higher cells, the histone to DNA ratio in yeast must be about 1 to 1 (wt/wt). This correlates well with the report of Lohr et al. (21) that at least 80-90% of the yeast genome is in a repeating structure.

The monomer and oligomer subunits of yeast appear to be somewhat depleted of histones H2A and H2B relative to the subunit of chicken erythrocytes. This appearance may be due to differences in dye binding of the yeast histones since the H2A and H2B bands are less intense than the H3 and H4 bands in gel patterns of whole yeast nuclear proteins, acid-extractible yeast proteins, and crude yeast nucleohistone. In addition, the gradient patterns indicate that a substantial amount of 9S "submonomer" nucleoprotein was coisolated with the "monomer" fraction used for histone and DNA analysis. We have recently found that this submonomer fragment from chicken erythrocyte and mouse myeloma chromatin is depleted of one copy each of H2A and H2B; thus, histone gels of the yeast monomer region might be expected to show a depletion of these histones. It is also of interest to note that the yeast monomer and oligomer nucleoprotein fractions contain small amounts of three proteins that electrophorese very similarly'to HI and H5 histones of chicken erythrocytes. Analyses for Cysteine Residues. To further assess the similarities between yeast subunit proteins and histones, we examined the "crude nucleohistone" fraction for cysteine residues by oxidation and reduction and by carboxymethylation with ICH214COOH. None of the putative yeast histones, including the Hl-like proteins, aggregated under mild oxidizing conditions and none was carboxymethylated. Chicken erythrocyte and calf thymus histones isolated and treated in the same manner gave the expected results-chicken erythrocyte H3 was nearly quantitatively oxidized to the dimer and most of the calf H3 (which contains two cysteines) remained in the slightly shifted monomer band, presumably forming an internal disulfide bond. None of the other histones was altered. After

DISCUSSION Histones in chromatin of higher organisms are organized into specific complexes along DNA, forming structural repeat units consisting of a globular "core particle" (14) containing 140 base pairs of DNA wrapped about a core complex of two copies each of histones H2A, H2B, H3, and H4 plus a more extensible approximately 40-60 base pair "spacer" region associated with HI histones. Recent reports have shown that three fungi-yeast (18, 19), Neurospora (35), Aspergillus (36), and the macronucleus of the ciliate protozoan Tetrahymena (36) contain four to five major classes of basic proteins that appear analogous to histones. Staphylococcal nuclease digests DNA in nuclei of these organisms at periodic intervals, suggesting a repeating organization of these proteins in simple eukaryotes (18, 20, 21, 37-39). The results presented here demonstrate directly that the repeat units of yeast chromatin are, in fact, similar to those found in higher eukaryotes in terms of sedimentation coefficient (about 1 IS), histone composition, and histone stoichiometry, and strongly support the concept of a universal subunit organization of chromatin based on an octameric complex of core histones. DNA lengths in repeat units of all three fungi are 30-50 base pairs smaller than repeat lengths reported for higher eukaryotes (generally 185-205 base pairs). This feature may be characteristic of fungi, as suggested by Thomas and Farber (18); however, the repeat length in chromatin of cultured Chinese hamster ovary cells is only 173 (±4) base pairs (40), indicating that small repeats are not restricted to lower eukaryotes. Variations in the repeat unit DNA size both in higher eukaryotes (40) and in lower eukaryotes (18, 38, 39) appear to be restricted to the spacer regions since in all cases core size (about 140 base pairs) DNA eventually becomes the predominant product of staphylococcal nuclease digestion of chromatin, regardless of the source. Chemical and physical criteria for identifying histones in higher organisms frequently have proven unsatisfactory when applied to lower eukaryotes because of high background levels of other basic proteins and possible changes in certain histone properties (e.g., solubility). As the universal nature of the subunit structure of eukaryotic chromatin becomes more evident, histones H2A, H2B, H3, and H4 may be more meaningfully identified in terms of their structural role, i.e., by their occurrence in a specific complex associated with DNA in a classic subunit structure. By this criterion yeast clearly has a normal complement of these histones. Establishing that a protein belongs to the Hi class is more difficult because of its location in the rapidly digested spacer regions. Three proteins that lack cysteine residues and electrophorese similarly to Hi histones occur in modest amounts in yeast monomer and oligomer subunit preparations. Although this result is suggestive, further characterization is required to determine if these are HI histones. The finding that yeast subunit preparations are virtually free from other contaminating basic proteins may be of practical importance for isolating pure yeast histones. Sequence analyses have shown that the four core histones, particularly H3 and H4, have been extremely conserved. These studies demonstrate one significant evolutionary difference in yeast histone H3-the absence of cysteine. Knowledge of the total sequences of yeast histones will be of considerable interest for assessing the function of particular local sequences in the core particle structure. Even without sequence information it

Biochemistry:

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is interesting to note that yeast histones band in virtually.; the same locations as higher eukaryote histones on NaDodSO4 gels, suggesting a conservation of molecular weight, and on acid-urea gels (32), suggesting a general conservation of charge. Furthermore, the conservation of core particle DNA size, sedimentation coefficient, composition, and internal digestion sites suggests a preservation of specific histone-histone and histone-DNA interactions and of a particular folded conformation of the DNA. Thus, an extremely efficient structure evolved at an early stage, facilitating the storage of information by reducing the genome length while preserving potential for full genome activity and regulation. In this respect folding of the DNA on the outside of the histone core in an easily recognizable conformation is obviously advantageous. The common patterns of chromosomal organization and similarities in transcriptional systems indicate that lower eukaryotes should be -eminently suitable models for studying certain gene control mechanisms operative in higher organisms. We thank Drs. S. R. deKloet and W. F. Marzluff for their advice. This work was supported by a grant from the National Institutes of Health and a grant from the Energy Research and Development Administration to the Institute of Molecular Biophysics. R.L.R. is the recipient of a National Institutes of Health Career Development Award. 1. Schultz, L. D. & Hall, B. D. (1976) Proc. Natl. Acad. Sci. USA 73, 1029-1033. 2. Retel, J. & Planta, R. J. (1970) Biochim. Biophys. Acta 224, 458-469. 3. McLaughlin, C. S., Warner, J. R., Edmonds, J., Nakazato, H. &

Vaughan, M. H. (1973) J. Biol. Chem. 248, 1466-1476. 4. deKloet, S. R. & Andrean, B. A. C. (1976) Biochim. Biophys. Acta 425, 401-408. 5. Geer, H. & Fink, G. R. (1976) in Methods in Cell Biology, ed. Prescott, D. M. (Academic Press, New York), Vol. 9, pp. 247279. 6. Lewin, B. (1974) Gene Expression-2, Eukaryotic Chromosomes (John Wiley & Sons, London), p. 7. 7. Lusby, E. W. & deKloet, S. R. (1976) Arch. Biochem. Biophys. 174, 187-191. 8. deKloet, S. R. (1970) Arch. Biochem. Biophys. 136,402-412. 9. Olins, A. L. & Olins, D. E. (1974) Science 183,330-332. 10. Kornberg, R. (1974) Science 184, 868-871. 11. Van Holde, K. E., Sahasrabuddhe, C. S. & Shaw, B. R. (1974) Nucleic Acids Res. 1, 1597-1607. 12. Noll, M. (1974) Nature 251, 249-251.

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13. Q Oosterhof, D. K., Hozier, J. C. & Rill, R. L. (1975) Proc. Natl. Acad. Sci. USA 72,633-637. 14. Shaw, B. R., Herman, T. M., Kovacic, R. T., Beaudreau, G. S. & Van Holde, K. E. (1976) Proc. Natl. Acad. Sci. USA 73, 505509. 15. Pardon, J. F., Worchester, D. L., Wooley, J. C., Tatchell, K., Van Holde, K. E. & Richards, B. M. (1975) Nucleic Acids Res. 2, 2163-2176. 16. Varshavsky, A. J., Bakayev, V. V. & Georgiev, G. P. (1976) Nucleic Acids Res. 3, 477-491. 17. Whitlock, J. P. & Simpson, R. T. (1976) Biochemistry 15, 3307-3314. 18. Thomas, J. 0. & Farber, V. (1976) FEBS Lett. 66,274-279. 19. Moll, R. & Wintersberger, E. (1976) Proc. Natl. Acad. Sci. USA 73, 1863-1867. 20. Lohr, D. & Van Holde, K. E. (1975) Science 188, 165-166. 21. Lohr, D., Kovacic, R. T. & Van Holde, K. E. (1977) Biochemistry, 16,463-471. 22. Franco, L., Johns, E. W. & Navlet, J. M. (1974) Eur. J. Biochem. 45,83-89. 23. Leighton, T., Leighton, F., Dill, B. & Stock, H (1976) Biochim. Biophys. Acta 432,381-394. 24. deKloet, S. R., Van Wermeskerken, R. K. A. & Konigsberger, U. S. (1961) Biochim. Biophys. Acta 47, 138-143. 25. Blobel, G. & Potter, V. R. (1966) Science 154, 1662-1665. 26. Burton, K. (1968) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. 12, Part B, pp. 163-166. 27. Laemmli, U K. (1970) Nature 227,1-6. 28. Bonner, W. M. & Pollard, H. B. (1975) Biochem. Biophys. Res. Commun. 64, 282-288. 29. Marzluff, W. F., Sanders, L. A., Miller, D. M. & McCarty, K. S. (1972) J. Biol. Chem. 247, 2026-2033. 30. Leoning, U. E. (1967) Biochem. J. 102, 251-257. 31. Rill, R. L., Oosterhof, D. K., Hozier, J. C. & Nelson, D. A. (1975) Nucleic Acids Res. 2, 1525-1538. 32. Wintersberger, U., Smith, P., & Letnansky, K. (1973) Eur. J. Biochem. 33, 123-130. 33. Axel, R. (1975) Biochemistry 14,2921-2925. 34. Sollner-Webb, B. & Felsenfeld, G. (1975) Biochemistry 14, 2915-2920. 35. Goff, C. G. (1976) J. Biol. Chem. 251, 4131-4138. 36. Felden, R. A., Sanders, M. M. & Morris, N. R. (1976) J. Cell Biol. 68,430-439. 37. Gorovsky, M. A. & Keevert, J. B. (1975) Proc. Natl. Acad. Sci. USA 72, 3536-3540. 38. Noll, M. (1976) Cell 8,349-355. 39. Morris, N. R. (1976) Cell 8, 357-365. 40. Rill, R. L., Nelson, D. A., Oosterhof, D. K. & Hozier, J. C. (1977) Nucleic Acids Res., in press.

Chromatin subunits from baker's yeast: isolation and partial characterization.

Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1343-1347, April 1977 Biochemistry Chromatin subunits from baker's yeast: Isolation and partial chara...
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