Comparative studies on the structure of soluble and insoluble chromatin from chicken erythrocytes

Comparative studies on the structure of soluble and insoluble chromatin from chicken erythrocytes Holger Notbohm Institut .fife Medizinische Molekularbiologie der Medizinischen Universitiit zu Liibeck, Ratzeburger Allee 160, D 2400 Liibeck, FRG

(Received 1 July 1985; revised 8 October 1985) Two ,[i'actions oJ" chicken erythrocyte chromatin differing in solubility, I and S chromatin, have been analysed using physicochemical and biochemical methods. Structural transition from an extended to a compact structure with increasing salt concentrations up to physiological conditions was studied using synchrotron radiation small angle scattering. Structural properties couhl be deduced fi'om circular dichroism spectra which reflect DN A packing in nucleosomes. Differences in elipticity of chromatin under varying salt conditions demonstrate that I and S ehromatin have different structures at equal salt concentrations. Biochemical analysis of the protein composition revealed the absence of HMG 1/2 proteins in I chromatin and a lower content ofhistone H5 in S chromatin. Both differences in chromatin composition might be responsible Jbr or at least contribute to the solubility ol S chromatin at physiological ionic" strength. Keywords: DNA; chromatin structure: synchrotron X-ray small angle scattering; circular dichroism

Introduction In the electron microscope as well as in the light microscope, chromatin in the nucleus shows structures with different densities. Apparently, these areas also differ with respect to their genetic activity. Chromatin containing active genes or those that can be activated in a distinct cell type should have a structure different from that of inactive parts. The structural requirements of these functionally different types of chromatin are unknown. Investigations are difficult because there is as yet no reliable way to isolate defined chromatin fractions, although many attempts to obtain functionally different kinds of chromatin have been described in the literaturet - 6 With a number of different techniques especially active chromatin has been characterized (1) as being hypersensitive to DNAse I digestion 7. (2) Nucleosomes of active genes are believed to be enriched in H M G 14/17 or H M G 1/2 non-histone proteins* and they may lack histone H1 (or H5) s'9. However, no difference of active to bulk chromatin in H M G 14/17 content was reported by Seal et al. t° (3) Acetylation and ubiquination are enhanced ~ (4) Transcribing sequences may be associated with the nuclear matrix and nuclear membrane 12. (5) Finally, in higher eukaryotes the D N A of expressed chromatin in general has a lower content of 5methylcytosine ~3 * ,4hhreciations" HMG, High Mobility Group non-histone nuclear proteins: PMSF, PhMeSO2 F, phenylmethylsulphonyl fluoride: SDS, sodium dodecyl sulphate. Enzymes: M icrococcal nuclease (EC 3.1.31. I1, 0141 8130/86/020114- 07503.00 1986 Butterworth & Co. lPublishers) Ltd

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Chromatin is usually isolated in low ionic strength buffers and separated from the nuclear debris by centrifugation. The pelleted material still contains a considerable part of the chromatin which is cosedimented with nuclear matrix and membrane material partially bound to it. Most structural investigations have been performed with the whole soluble chromatin obtained in the supernatant. From model experiments done with isolated chromatin it is known that higher order structures are dependent on the concentration of mono- or divalent cations, on the presence of a full set of core histones, and probably also on some non-histone proteins. In EDTA containing buffers the D N A packing ratio in the chromatin is 6 and the structure is rather extended. F r o m electron micrographs a beaded string picture is deduced, while on the basis of small angle scattering data an extended helical arrangement with an internucleosomal distance of 2 0 n m 1"~ is postulated. A more recent approach to fractionate chromatin is to make use of differences in chromatin solubility; thus two kinds of chromatin can be obtained. One is soluble only in monovalent salt concentrations below 60 mM (I chromatin) while the other fraction is soluble even in 150mM (S chromatin) (nomenclature according to Fulmer and Bloomfield 15). In this report X-ray scattering data and c.d. spectra are presented indicating specific structural differences between the two kinds of chromatin. A satisfying explanation for the phenomenon of differing solubility is, as yet, not available. As Fulmer and Bloomfield 15 already pointed out the core histone composition is identical in both fractions. However, there are differences with respect to the H M G 1/2 content and the ratio of core histones to

Structure of S and I chromatin: H. Notbohm histone H5, which may contribute to or even be essential for the higher solubility of S chromatin as a result of nonneutralized charges.

Materials and methods Chromatin is prepared from mature chicken erythrocyte nuclei following the procedure of Shaw et al. 16. Nuclei are digested for 5 min with micrococcal nuclease (Worthington, 80 units/ml, 37°C) and then dialysed against 13 mM KC1, 7 mM NaC1, 5 mM Tris-HC1, 0.4 mM MgCI2, 0.1 mM EGTA, pH 7.5 overnight. After 30 min centrifugation at 5000x9 the chromatin in the supernatant is dialysed for 30 min against a buffer containing 150 mM NaC1, 0.1 mM EDTA, 0.2 mM PMSF, 5mM Tris, pH 7.5. During this procedure insoluble I chromatin aggregates. It can subsequently be collected from the dialysis bag membrane. The soluble S chromatin is carefully removed with a pipette and then centrifuged again for a short time to get rid of contaminating traces of I chromatin. Then both chromatin fractions are dialysed against buffers of varying NaCI concentrations containing in addition 0.1 mM EDTA, 0.2 mM PMSF and 5 mM Tris-HCl, pH 7.5. The amount of S chromatin varies with the time the nuclei have been dialysed after nuclease digestion, but it does not increase linearly. With higher yield (maximum is about 50~ of total nuclear chromatin with constant nuclease digestion conditions) the amount of S chromatin remains unchanged at a final portion of about 20~ (Fioure 1). This is 10~o of whole nuclear chromatin allowing no S chromatin in the nuclear pellet. So, S chromatin can be extracted from the nuclei more easily, while the amount of I chromatin is much more dependent on the time the nuclei have been dialysed. Sedimentation coefficients are determined with the analytical ultracentrifuge (MSE) and a computer based evaluation of sedimentation coefficient distributions. This is a useful tool for investigating heterogeneous samples of unfractionated chromatin preparations. S-values for S chromatin are about 35 while isolated I chromatin has most abundant sedimentation coefficients of 45-55S (both kinds of S-values for chromatin in EDTA). Therefore, both chromatin fractions have the appropriate

size to form quaternary structures because the number of nucleosomes is above 6 17. The c.d. and synchrotron X-ray scattering data presented here are taken from the same preparation with a 35~o content of S chromatin, and sedimentation coefficients were 37 and 50 for S and 1 chromatin.

Preparation of acid-soluble chromatin proteins The method applied follows the procedu re described by Rabbani et al. 18 Gel electrophoresis Acid-soluble chromatin proteins are analysed by polyacrylamide gel electrophoresis on acidic urea gels ~9 in a vertical slab gel apparatus (LKB-Instruments) (5-6 h, 300V). Subsequently, the proteins are stained with Coomassie blue. Histone content of the samples is analysed in 18~o polyacrylamide SDS gels 2°. Small angle scattering Synchroton radiation small angle scattering was performed at the EMBL-Outstation in Hamburg. Camera conditions are the same as described in Reference 14. The detector sample distance is 2 m, exposure time 5 rain. An area sensitive detector is used and data reduction is performed with the OTOCO program of EMBL. The difference counting rate in the experiments is so high that error bars cannot be visualized on the graph. The normalized scattering angle is s=2"sin(20)/2, 20 scattering angle, 2 wavelength, s = 1/d(d= Bragg spacing). Circular dichroism C.d. spectra are taken from a Jasco spectral polarimeter in the range of 255-320 nm in a 1 cm cuvette adjusting the sample concentration to an optical density of A26o = 1.0. Measurements are repeated four times. The observed elipticity can be reproduced with a variation of about +/150deg cm2/dmol with samples of different preparations. Light scatterin9 The data from quasielastic and total light scattering experiments as well as the experimental conditions have been published by Meuel and Notbohm 21.

Results

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FigureI The amount ofS chromatin obtained in a preparation is plotted against the total mass of isolated chromatin. The data points are from different preparations, which usually vary in the yield of isolated chromatin

Small anyle scatterin9 Synchroton small angle scattering studies were done for analysing the salt dependence of I and S chromatin structures. With synchrotron radiation one is able to investigate within one day about 30 samples derived from a single preparation using different salt concentrations; so degradative effects due to nucleases and proteases during the measurements can be neglected. Also samples with rather low concentrations of chromatin can still be measured. Due to the very short exposure time radiation damage is negligible. Fiyure 2a shows scattering curves of S chromatin in buffers of different ionic strength. In Figure 2b the same is shown for I chromatin. The main difference in the scattering curves of chromatin can be observed at the maximum s=0.05 n m - ~ or 20 nm equivalent Bragg spacing. This maximum is decreasing while raising the ionic strength of the buffer. According to the interpretation of Perez-Grau et al. ~4 this is an indication

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Figure 2 (a) Synchroton radiation scattering curves of soluble chicken erythrocyte chromatin at different monovalent salt concentrations. The scattering angle position s=0.05 nm-1 is marked as a characteristic maximum for chromatin in buffers containing no or very low salt. In model calculations an extended helical arrangement of nucleosomes having a pitch of about 20 nm is deduced from these data 14 (see also Figure 7). (b) Corresponding small angle synchrotron scattering of I chromatin

for an internucleosomal spacing of a b o u t 20 nm which fades as a result of close nucleosome stacking with a pitch of 11 nm (solenoid). Model calculations of Bordas et al. found the data being consistent with a helical arrangement of nucleosomes with a pitch of 30 nm and 2.5 nucleosomes per turn. During this transition the mass per unit length increases as indicated by the higher scattering intensity at lowest angles. The structural transition from an extended helical structure to a more c o m p a c t one clearly takes place between 5 and 20 mM for I chromatin (new data show that I chromatin is already condensed at 10 raM) while soluble chromatin is still in an extended form at 20 mM m o n o v a l e n t salt concentration.

Circular dichroism There are differences in the absolute values of elipticity at wavelengths 275 and 285 nm (see Figures 3u and b). In 0.2 mM E D T A the elipticity is somewhat higher for I chromatin, but in 40 mr, I chromatin is lower c o m p a r e d with S chromatin in a buffer containing 150mM m o n o v a l e n t cations. The major difference in c.d. spectra of ! and S chromatin is the ratio of the maxima at 275 and 285 nm (Figure 4). The elipticities of l chromatin at 275 and 285 nm are already different at very low ionic strength

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Structure of S and I chromatin: H. Notbohm and are quickly diverging when the salt concentration increases. Elipticities of S chromatin are equal at low ionic strength and start to diverge only above 20mM monovalent salt concentration. At about 60-70raM monovalent salt concentration I chromatin begins to precipitate. This process was monitored by measuring dynamic light scattering 21. The results obtained are comparable with those already published by Ausio et a/. 22"23, although in their study precipitation started at 75 mM monovalent cations rather then at 60 mM. Total light scattering increases while the diffusion coefficient decreases. Both observations indicate an increase of molecular mass which macroscopically ends up with precipitation. This different structural behaviour of the two chromatin fractions must depend on some essential differences in chromatin components. The histone composition is nearly identical in I and S chromatin except that the histone H5 content is higher in I chromatin (Figure 5). A more complicated problem is an analysis of non-histone proteins in I and S chromatin, especially since many of these proteins are not tightly bound to nucleosomes. Furthermore, they show a considerable variety and their amounts are in general very low. Exceptions are the High Mobility Group proteins (HMG) which can be detected quantitively by electrophoresis if one decreases histone concentration on the gel (Fioure 6). In addition to the residual histone bands, which do not represent histones in their original stoichiometry, those for H M G 1/2 can be recognized. As a marker a preparation of total calf thymus proteins using the acidic extraction method of Rabani et al.1 a was used (lane Thy). The position of the bands for H M G 1/2 in relation to residual H1 agrees well with data in the literature ~8. After sedimentation of S chromatin in the ultracentrifuge (55 000 rev/min, 4 h, in a Beckman SW65 rotor) the same protein pattern was observed for the pellet, showing that H M G 1/2 non-histones are bound to S chromatin. Furthermore the difference in H M G protein content was also observed by Komeiko and Felsenfeld 24. As can be seen in Figure 6, the concentration of the remaining histones is higher for I chromatin, although the chromatin samples from which the proteins had been H2b

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isolated for electrophoresis originally had had the same optical densities. This reproducible observation might indicate a decreased binding strength of the histones in I chromatin. H M G 14 and H M G 17 bands may be observable below the H4 band; however, degradation products can also be located in that range.

Discussion The superstructure of DNA in chromatin is largely dependent on the content of cations in the surrounding solute. This dependence is well known for pure DNA. Stiffness of these macromolecules is higher at low ionic strength. The negative charges of the phosphodiester backbone repel each other, thus a stiff rod is formed 25. In the nuclear chromatin of eukaryotic cells, DNA interacts with basic histones thus neutralizing part of the charges in the backbone. In this way, bending of DNA into a circle of l l nm diameter is facilitated. As a constituent of nucleosomes DNA structure is largely independent of the surrounding salt contents. So, for the formation of higher order structures of chromatin other components must be responsible. An important one, histone H l, is located in the region where both DNA strands leave the core particle to enter the adjacent nucleosome (in chicken erythrocytes historic H5 has a similar function). Furthermore, the protein-free linker DNA is involved. In contrast to the DNA of the core particle the flexibility of linker DNA should be sensitive to the surrounding salt concentration; in salt-free EDTA containing buffers the linker DNA forms a rather stiff rod. Apparently, H M G proteins which in general are supposed to have more regulatory functions - mainly act on the surface of the core particle. They are probably not part of every nucleosome, and their content varies with cell type or species 26. These proteins can interact with H1 histones as well as with the neighbouring DNA. Histone H 1 is probably directing the DNA strand at its crossover point at the nucleosome in a distinct way and determining further folding. Increasing the concentration of mono- or divalent cations causes neutralization of linker DNA and of these parts of the nucleosomal DNA which contain non-saturated negative charges. As a consequence, transition of the linker DNA from a stiff rod to a more flexible chain takes place and repelling forces at the nucleosomes disappear, hydrophobic interactions become more effective and the structure collapses. With further raising of the ionic strength interparticle attraction increases, and finally chromatin gets precipitated. In our study we could demonstrate that the two fractions of isolated chromatin differ in their dynamics of structural transition when raising the ionic strength. This is visualized schematically in Figure 7. However, the gross overall structure itself is not changed as can be demonstrated by electron micrographs 15 and X-ray scattering data. From the model for structural transition (as briefly discussed above) one can conclude that in both chromatin fractions apparently the net negative charge is different. It should be less in I chromatin because the charge neutralization is completed when precipitation takes place at about 6070 mM monovalent salt concentration, while S chromatin does not precipitate at all in the range up to 150mM monovalent salt. Furthermore, the process of chromatin compaction is beginning earlier (Fiy,re 7). For the observed decrease of elipticity in tile c.d. spectrum of

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DNA in chromatin the most probable explanation” is the influence of a PSI-type band caused by the scattering resonance phenomenon. This results from closely and parallel packed DNA strands, as it is also known from the nucleosome structure. For chromatin it was stated that a smaller positive elipticity is invariably accompanied by a higher 0285/0275 ratio28 and a stronger negative band at 295nm. This can be visualized by simply subtracting a PSI band spectrum with maximum at about 275 nm from an appropriate DNA spectrum. However, the results as shown in Figurr 4 cannot be interpreted simply in this way, because even when the positive elipticity of S and I chromatin at 285 nm is the same or higher in I chromatin, is smaller in S chromatin. This ratio the ratio of O,,,/O,,, can even become higher than 1.0 for chromatin from bovine neurons (Notbohm, unpublished). So, the phenomenon can only be explained by a different type of

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PSI band in S chromatin which has already been found indeed to occur in other c.d. spectra of special types of chromatin”. So, we suggest that the difference in c.d. spectra of I and S chromatin is due to variations in histone-DNA interactions mainly due to the action of HMG l/2 proteins which are present only in S chromatin. HMG l/2 binding to the nucleosomes may change the ordering of DNA in the nucleosome, especially in the linker region. The deficit of histone H5 has a similar effect29. This implies that in addition to or instead of the effect of the histone H 1 and H5 electrostatic forces act on the DNA close to the nucleosome core. Our synchroton scattering data show that for I chromatin net charge neutralization of DNA is already completed at lower ionic strength, indicated by a disappearing 20nm maximum. Therefore, the basic HMG l/2 proteins present only in S chromatin and in the nuclear pellet do not interact in the

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same way that histones do with DNA. Actually, it is known that H M G 1/2 are proteins having acidic as well as basic domains and therefore have the ability to bind to DNA without neutralizing it 28. This could contribute to the observed increased solubility. Further attempts were made to determine whether phosphorylation might play a role in the solubility of chromatin. However, we did not succeed in determining phosphorus quantitively in isolated histones. Other modifications which could have changed the net charge of chromatin are acetylation of histones. During the isolation of chromatin acetyl groups are usually lost. Appropriate buffers containing Na-butyrate are necessary to suppress acetylase activity. However, acetylation seems not to be essential for the observed structural differences of I and S chromatin because the probable loss of acetyl groups apparently did not alter the properties of the two types of chromatin. Specific chromatin fractions have also been investigated by Rose and Garrard 3° using genetic hybridization techniques as probes to test genetic activity of chromatin extracted after nuclease digestion. These authors found that the DNA in short pieces ofchromatin which had moved through the nuclear membrane and of chromatin partially derived from matrix-bound material hybridized most efficiently with specific gene probes. These results correspond to the H M G 1/2 patterns in protein analysing gels from S chromatin and the nuclear pellet using our fractionation procedure. The functional role of H M G 1/2 is still not clear; one is tempted to associate it now with transcriptional activity. In the same way H1 or H5 deficiency can be considered as an indication of activated chromatin 9. Recently, Komaiko and Felsenfeld 24 used the same approach to obtain I and S chromatin, although their method of preparation differs considerably from the one used here. They found that the fl-globin gene is enriched in S chromatin. Their objection was - and this might also be true for our fractionation p r o c e d u r e - that histone H I and H5 migrate from smaller chromatin pieces to longer ones (also reported by Thomas et al.31). Solubility should then be more a result of in vitro histone transfer than reflecting structural in vivo conditions. On the other hand, the

constant amount of S chromatin released from the nucleus, while the total amount of isolated chromatin varies (Figure I), is an indication for S chromatin being a real in vivo constituent of the nucleus. From the results obtained one can conclude that in the presence of physiological monovalent salt, chromatin in the nucleus might exist in two states: as S chromatin in a loose superstructure (solenoid, superbead) which is or can easily be made accessible for modifying enzymes that initiate transcription; and as I chromatin which is very compact already at low ionic strength. So, under physiological conditions it may aggregate with neighbouring chromatin fibres comparable to precipitation in vitro. In this way, it might correspond to heterochromatic regions of the nucleus. The solubility of S chromatin is supported by H M G 1 and 2 which have the potential to bind to DNA without neutralizing it. Binding to DNA is accompanied by displacement of H l(H5) while the acidic domains preserve solubility in water. The mechanism of HI(H5) displacement is unknown. Of course, this is a simplified picture neglecting other factors as for example the role of divalent cations. Certainly, their influence on chromatin structure is much stronger as compared to that of monovalent cations. In a physiological surrounding they have the potential to change chromatin superstructure already by minimal variations of their ionic strength. On the other hand, it is hard to imagine that ionic strength changes should play such an influential role in switching genes on and off. So, changes in the ionic environment of chromatin may contribute to a raw control of gene activity, while the accurate tuning occurs via other mechanisms.

Acknowledgements The author thanks Dr M. H. C. Koch who has carried out the experiment at the EMBL Outstation at DESY in Hamburg and Professor Dr E. Harbers for critical discussion. The technical assistance of Mrs C. Sesseimann and Mrs G. Wegener is gratefully acknowledged.

References 1 2 3

4 5 6 7 8 9 10 II 12 13 14 15 16

Frenster, J. H., Allgrey, V. G. and Mirsky, A. E. Proc. Natl. Acad. Sci. USA 1963, 50, 1026 Brust, R. and Harbers, E. Biochem. Biophys. Res, Comm. 1981, 101,359 Harbers, E. and Vogt, M. in "The Cell Nucleus - Metabolism and Radio-Sensitivity', (Ed. H. Bush), Academic Press. London, 1966, p. 165 Gottesfeld, J. M., Garrard, W. T., Bagi, G.. Wilson, R. F. and Bonner, J. Proc. Natl. Acad. Sci. USA 1974. 71.2193 Str~itling, W. H., Van, N. T. and O'Malley, B. W. Et,'. J. Bioclwm. 1976, 66, 423 Davie, J. R. and Saunders, C. A. J. Biol. Chem. 1981. 256, 12574 Elgin, S . C . R . C e l l 1 9 8 1 , 2 7 . 4 1 3 Weisbrod, S. and Weintraub, H. Cell 198 I. 23, 391 Jackson, J. B. and Rill, R. L. Bio('hemistr)" 1981, 20. 1042 Seale, R. L., Annunziato, A. T. and Smith, R. D. Bioehemistrr 1983, 22, 5008 Levinger. L. and Varshavsky, A. Cell 1982, 28, 375 Robinson, S. I., Nelkin, B. D. and Vogelstein. B. Cell 1982, 28. 99 Naveh-Many. T. and Cedar, H. Proe. Natl, Aead. Sci. USA 198 I. 78, 4246 Perez-Grau. L., Bordas. J. and Koch. M. H. J. Nm'leie Acids Res. 1984. 12. 2987 Fulmer, A. W. and Bloomfield, V. A. Proc. Natl. Acad. Sci. USA 1981, 78. 5968 Shaw, B. R.. Herman. T. M.. Kovacic. R. T.. Beaudreau. G. and van Holde, K. Proc. Natl. Acad. Sci. USA 1976, 73, 505

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S t r u c t u r e o[ S and I chromatin: H. N o t b o h m 17 18

24 25

Thomas, J. O. and Butler, J. G. J. Mol. Biol. 1980, 144, 889 Rabbani, A., Goodwin, G. H. and Johns, E. W. Biochem. J. 1978. 173, 497 Panyim, S. and Chalkley, R. Biochemistry 1969, 8, 3972 Loening, V. E. Biochem. J. 1967, 102, 251 Meuel, B. and Notbohm, H. Z. NatmJbrsch. 1982, 38, 126 Borochov, N., Ausio, J. and Eisenberg, H. Nucleic Acids Res. 1984, 12, 3089 Ausio, J., Borochov, N., Seger, D. and Eisenberg, H. J. Mol. Biol. 1984, 177, 373 Komaiko, W. and Felsenfeld, G. Biochemistry 1985, 24. 1186 Manning, G. S. Biopolymers 198h 20, 1751

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19 20 21 22 23

26 27 28 29 30 31

Gordon, J. S., Rosenfeld, B. 1., Kaufman, R. and Williams. D. L. Biochemistry 1980, 19, 43 Cowman, M. K. and Fasman. G. D. Proc. Natl. Acad. Sci. USA 1978, 75, 4759 Prevelige, P. E., Jr. and Fasman, G. D. Biochim. Biophys. Acre 1983, 739, 85 Carballo, M., Puigdomenech, P. and Palau, J. EMBO J. 1983. 2(10), 1759 Rose, S. M. and Garrad, W. T. J. Biol. Chem. 1984, 259, 8534 Thomas, J. O.,Rees, C. and Pearson, E. C. Eur. J. Biochem. 1985, 147, 143