Volume 6 Number 4 April 1979 Nucleic Acids Research Vlue6Nme4Api199NcecAisRsah
Neutron scattering on nuclei
Pierre Baudy and Stanley Bram* Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris, France
Received 17 November 1978
ABSTRACT Very small angle neutron scattering studies have been made on intact nuclei under a variety of solution conditions. Scattering maxima are observed at 30 to 40 nm and at 18 nm in most environments. Although the spacing, intensity and presence of the maximum near 40 nm varies considerably with environment the 18 nm is rather constant. The 30 to 40 nm maximum appears to be best interpreted by the presence of 35 to 50 nm diameter fibers in nuclei. An important result is that no scattering maximum was observed near 11 nm, suggesting that a tightly super coiled nucleofilament with such a pitch is not present.
INTRODUCTION Characteristic small angle scattering maxima at equivalent Bragg spacings near 11 and 5.5 nm (1 , 2) occupied an important part in studies of chromatin structure. Even though several rather different explanations for the origin of these maxima have been put forward (3, 4, 5, 6) their presence has helped to show that DNA was coiled in chromatin (2, 3) and to suggest a nucleosome subunit organization (7). Likewise, the existence of very small angle maxima near 40 and 20 nm (8, 9) are indicative of aspects of the super structure having dimensions similar to these spacings. These very small angle maxima were the first evidence that chromatin had a regular higher order structure (8, 9) and was not a random flexible chain. The very small angle reflections may also have several different explanations which we will briefly discuss. Our main purpose here is to describe small angle neutron scattering experiments on intact nuclei and on chromatin. In nuclei we also observe characteristic maxima near 40 and 20 nm spacings. The variation and disappearance of these maxima with the environmental conditions will imply which procedures are to be avoided if the native super structure is to be C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1721
Nucleic Acids Research
maintained. While studying the small angle region of the nuclei scattering, it is a also of interest to look for a 11 nm maximum as the solenoid ( 5), in of this peak observation upon the based tight 11 nm pitch super helix,is chromatin gels, but it has not been well documented in nuclei.
MATERIALS AND METHODS Nuclei were isolated from fresh calf thymus with the method of Panymin, Bilik, and Chalkley (10), except that Triton-X was omitted and the washing medium contained 2 mM instead of 10 mM MgCl2. After the 2.4 M sucrose pelleting the nuclei were suspended in .25 M sucrose. 10-4 M PMSF was added to most media but its presence did not alter the neutron scattering results. Nuclei were studied either suspended in various media at a concentration of a few percent chromatin or were examined after pelleting at 1,000 g for 15 minutes to a concentration of about 20 %. Phase examination and electron microscopy showed that more than 95 % of our nuclei were intact and largely free of cytoplasmic contamination (see figure 1). Chromatin was prepared by diluting nuclei to a chromatin concentration of .5 % in 1 mM NaCl with, or without a 12 hour mechanical stirring at 40 C. Rat liver nuclei were prepared by Dr. M. Leval with the method of Leval and Bouteille (11). The medium contained PMSF 10-4 M. Electron micrographs of these preparations can be found in ref. (11). Chromatin concentrations were obtained by diluting in 1 % sodium lauryl sulfate and .1 M NaCl, and determining the optical density at 260 nm. Electron microscopy was carried out on peleted nuclei after formaldehyde and osmium fixation, alcohol dehydration and embedding in methacrylate. 50 nm thick sections were then stained with uranyl acetate and lead citrate
(12). The neutron scattering on calf thymus nuclei was perfomed on the Dll diffractometer built by K. Ibel (13) at the I.L.L. in Grenoble. Sample distances of 5 and 10 meters and wave lengths of .6 to 1.0 nm were employed with a Ax/x of 9 %. Most experiments were at 20 to 250 C but some experiments were carried out over the range of 4 to 90° C. Typical runs lasted 15 minutes. In figures 2 and 3 the curves are presented after the background and solvent scattering were substracted as previously described (15, 8, 9 ). The 1722
Nucleic Acids Research
Figure 1 - Micrographs of calf thymus nuclei in 2 mM MgC12, .25 M sucrose: a) by phase-contrast of a suspension; b) by electron microscopy after pelleting, embedding and sectioning. The bars are 2 microns. very small angle scattering was approximately the same before and after subtraction since the high chromatin concentrations used gave a scattering several fold larger than that of a sample holder filled with water. Rat liver nuclei were studied at the C.E.N. Saclay with a ring detector diffractometer described by Cotton et al. (14). Experiments were performed at 40 C over a period of several hours. The scattering curves (fiq. 3) are smeared by a AX/A of 30%, consequently the actual maxima would be somewhat sharper and shifted to '10% larger spacings.
RESULTS AND DISCUSSION Very small angle maxima at about 40 and 18 nm are observed in the scattering from nuclei in environments which resemble those in situ. The 1723
Nucleic Acids Research maxima of the typical curves in figures 2 and 3 would be even more pronoun-
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Figure 2. Scattering curves from calf thymus nuclei in .25 M sucrose: pelleted in (s) 1 mM NaCl; 0.1 mM MgC12; (*) 2 mM EDTA pH 7. Nuclei suspended in (a) 2 mM MgCl2; 10 mM Tris pH 8; (A)1 mM MgCl2, 10 mM Tris pH 8; (o) 1 mM NaCl. (h = 4 ar/A Sin 0/2 where 0 is the scattering angle and X the wavelength of the neutrons). Note the pronounced shoulders at about h = .035 (A-1) or X 1 8 nm. 1724
Nucleic Acids Research
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Figure 3. Neutron scattering of nuclei from calf thymus pelleted in 0.25 M sucrose (s) 1 mM NaCl, 0.1 mM MgC12; (*) 2 mM EDTA pH 7. Calf thymus nuclei suspended without sucrose in (o) 1 mM NaCl. (M) Rat liver nuclei pelleted in .25 M sucrose, 2 mM MgC12, 10 mM Tris ph 8; (A) 40% D20 1 mM NaCl. ced after correction for the 10 % wavelength and 10 % collimation smearing. That they are clearly due to the scattering from nuclei is shown by their absence under different ionic environments (see Table 1). In Table 1 our
1725
Nucleic Acids Research results concerning the presence or absence of the 40 and 18 nm maxima are given for the various envirnmental conditions studied. The spacing and sharpness of the first scattering maxima (the one at the small values of h, or at larger spacings) is quite sensitive to the environmental conditions. Calf thymus nuclei pelleted at 1,000 g for 15 min. display a strong maxima near 38 nm while nuclei in a suspension at lOX lower concentration yield a broad maxima near 45 nm. Concentration into a pellet probably reduces the nuclear volume. Nuclei suspended in the absence of sucrose appear swollen in the phase microscope and do not exhibit a maximum at spacings larger than 20 nm. The difference between the spacing of the first maxima from rat liver and calf thumus nuclei (about 20%) may be due to inherent structural differences, or to the slightly different modes of preparation. The first scattering maximum is similar at 0.1 and 1 mM MgCl2 but sometimes is absent at higher divalent ion concentrations. It is important to note that the maxima at 18 nm does not vary much over the range of conditions studied, but disappears in .65 M NaCl. Chromatin when isolated without mechanical stirring can also yield strong characteristic maxima at the same spacings of intact nuclei (8, 9). However, after mild nuclease digestion these maxima, characteristic of some aspects of the native super structure in the nucleus, were never observed (15, 16). Scattering angles corresponding to spacings of up to 120 nm were studied. No other maxima at larger Bragg spacings were detected. As shown in figure 3, the very low angle maxima are prominent in 40% D20 where the neutron scattering from proteins is contrast matched. It is clear then that a non-chromatin associated protein component in the nucleus is not responsible for the maxima; conversely DNA is makinq a major contribution. u
Interpretation for the origin of the very small angle maxima. Fibers having diameters between 35 and 50 nm are the most prevalent structural entity seen in freeze fracture replicas (17, 18) and in recent classical electron micrographs (19) of nuclei and chromatin in the presence of divalent ions. The scattering from a gel or solution of cylinders or bundles of fibers having an outer diameter of 35 to 50 nm will exhibit a maximum at spacings between 25 and 40 nm and a second one near 18 nm (see ref. 20 for example). Although this is the explanation most consistent with all our results, others are possible and will be briefly considered. 1726
Nucleic Acids Research Table 1
Conditions Calf thymus nuclei, 0.25 M sucrose Pellet 1 imM NaCl, 10 4 M MgCl2 1 MM EDTA 650 nM NaCl
Suspension 1 mM NaCl, 10 nM Tris pH 8 1 mM KCI, 10 mM Tris 1 mM MgC12, 10 mM Tris 2 mM MgCl2, 10 mM Tris alf thymus nuclei no sucrose 1 mM NaCl 10 mM Hepes pH 8 Rat liver nuclei 0.25 M sucrose 10 4 M MgCl2 io- M MgC12
Calf thymus chromatin undispersed in 1 mM NaCl stirred 12 hours in 1 rM NaCl heated above 800 C and cooled to 20° C alf thymus or rat liver chromatin with brief nuclease digestion (-implies the maximum is absent)
Bragg spacing (nm) First max. Second maximum
38 38
18 18
45 45 45
18 18 18 18
-
-
18 18
32 30
18 18
40-45
18-20 Ref. (8,
Ref. (15,
91 16)
A side by side packing would give rise to maxima at spacings equal to the separation (d) and d/a/i. This is not observed. An important result is that the 40 and 18 nm peaks do not shift to larger spacings together uponswelling and that the 18 nm maxima may be quite strong while the -. 40 nm reflection is absent. This finding suggests that they are of different origin. (We were not aware of this behavior in 1974 when we suggested an interpretation based upon a 40 nm pitch coil (8, 9)). The scattering from 20 to 30 nm spherical groupings of nucleosomes, which have been called "super beads" (21), should show a very sharp scattering maximum near 15 nm. The spacings and the breadth of the shoulders in the experimental data do not seem to fit such a model. 1727
Nucleic Acids Research We have carried out a limited number of neutron scattering experiments on intact calf thymus tissue. Spacings on the order of 40 nm are often observed. It is probably that these maxima are the same as those from isolated nuclei. An 11 nm maximum is not observed in the neutron scattering from nuclei under the environment conditions so far studied by us (see figure 3 and table 1). If, as suggested by several authors (4, 5, 6) the 11 nm maximum arises from a tight super helix or solenoid with a corresponding pitch of X, 11 nm, the results strongly suggest that such structures are rare or absent in nuclei. Our results do not conflict with most studies reporting the 11 nm maximum, as these were carried out on isolated chromatin, but they are not in accord with one X-ray study of nuclei (22). In this study (22) it may have been difficult to measure well a maximum on a film which was superimposed on the central parasitic scatter; in addition scattering curves were not presented. Consequently, direct comparison is difficult; still it is possible that an 11 nm maximum would be present at conditions different from those we employed. In any case, with gels of isolated chromatin the first scattering maximum is observed near 11 nm at concentrations of about 40%, but it is at X~ 13 nm at concentrations of 10-20% (21, 16) and is shifted toward 20 nm upon further dilution (23). *
To whom correspondence should be addressed. Currently on leave at: Biochemistry Department, University of California, Berkeley, Ca. 94720, U.S.A. A preliminary report of these findings was presented at the Harwell Symposium on Neutrons in Biology in July, 1976.
ACKNOWLEDGEMENTS We are grateful to Dr. K. Ibel and the staff of the Institut Laue Langevin and to Dr. J. P. Cotton and the staff at the laboratory Leon Bruillion for their assistance. We thank Drs. M. Leval and M. Bouteille for proividing rat liver nuclei. Financial assistance was provided by an A.T.P. chromatine. We acknowledge that we became aware of similar X-ray scattering experiments by Dr. John Langmore after we completed our study.
1728
Nucleic Acids Research REFERENCES 1) Luzzati, V., and Nicolaieff (1963) J. Molec. Biol. 7, 142. 2) Wilkins, M.H.F., Zubay, G., and Wilson, H. R. (1959) J. Molec. Biol. 1, 179. 3) Pardon, J. F., Richards, B. M., and Wilkins, M.H.F. (1967) Nature 215, 508. 4) Carlson, R. D., and Olins, D. E. (1976) Nucleic Acids Research 3, 89. 5) Finch, J. T., and Klug, A. (1976) Proc. Natl. Acad. Sci. (U.S.A.) 73, 1897. 6) Carpenter, B. G., Baldwin, J. P., Bradbury, E. M., and Ibel, K. (1976) Nucleic Acdis Research 3, 1739. 7) Kornberg, R. D. (1974) Science 184, 865. 8) Bram, S., Baudy, P., Butler-Browne, G., and Ibel, K. (1974) Biochimie 56, 1449. 9) Bram, S., Butler-Browne, G., Baudy, P., and Ibel, K. (1975) Proc. Natl. Acad. Sci. (U.S.A.) 72, 1043. 10) Panymin, S., Bilik, N., and Chalkley, R. (1971) J. Biol. Chem. 246, 4206. 11) Leval, M., and Bouteille, M. (1973) Exper. Cell Res. 76, 337. 12) Renoylds, E. S. (1963) J. Cell Biol. 17, 111. 13) Ibel, K. (1974) J. Appl. Cryst. 9, 296. 14) Cotton, J. P., Decker, D., Benoit, H., Farnoux, B., Higgens, J. Jannink, G., Ober, R., Picot, C., and des Cloisaux, J. (1974) Macromolecules 7, 863. 15) Bram, S., Baudy, P., Lepault, J., and Hermann, D. (1977) Nucleic Acids Research 7, 2275. 16) Baudy, P., and Bram, S. (1978) Nucleic Acids Research, 5, 3697. 17) Lepault, J. (1978) Thesis, University of Paris VI. 18) Lepault, J., Bram, S., Escaig, J. and Wray, W., In Preparation. 19) Sedat, J., and Manyelidis, L. (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 331. 20) Kratky, 0. (1963) Prog. in Biophysics and Molecular Biology 13, 105. 21) Hoizier, J., Nehls, P. and Renz, M. (1977) Chromosoma 62, 301. 22) Olins, D. E., and Olins, A. L. (1972) J. Cell Biol. 53, 715. 23) Bram, S., Butler-Browne, G., Bradbury, E. M., Baldwin, J. P., Reiss, C., and Ibel, K. (1974) Biochimie 54, 987.
1729
Neutron scattering on nuclei
Pierre Baudy and Stanley Bram* Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris, France
Received 17 November 1978
ABSTRACT Very small angle neutron scattering studies have been made on intact nuclei under a variety of solution conditions. Scattering maxima are observed at 30 to 40 nm and at 18 nm in most environments. Although the spacing, intensity and presence of the maximum near 40 nm varies considerably with environment the 18 nm is rather constant. The 30 to 40 nm maximum appears to be best interpreted by the presence of 35 to 50 nm diameter fibers in nuclei. An important result is that no scattering maximum was observed near 11 nm, suggesting that a tightly super coiled nucleofilament with such a pitch is not present.
INTRODUCTION Characteristic small angle scattering maxima at equivalent Bragg spacings near 11 and 5.5 nm (1 , 2) occupied an important part in studies of chromatin structure. Even though several rather different explanations for the origin of these maxima have been put forward (3, 4, 5, 6) their presence has helped to show that DNA was coiled in chromatin (2, 3) and to suggest a nucleosome subunit organization (7). Likewise, the existence of very small angle maxima near 40 and 20 nm (8, 9) are indicative of aspects of the super structure having dimensions similar to these spacings. These very small angle maxima were the first evidence that chromatin had a regular higher order structure (8, 9) and was not a random flexible chain. The very small angle reflections may also have several different explanations which we will briefly discuss. Our main purpose here is to describe small angle neutron scattering experiments on intact nuclei and on chromatin. In nuclei we also observe characteristic maxima near 40 and 20 nm spacings. The variation and disappearance of these maxima with the environmental conditions will imply which procedures are to be avoided if the native super structure is to be C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1721
Nucleic Acids Research
maintained. While studying the small angle region of the nuclei scattering, it is a also of interest to look for a 11 nm maximum as the solenoid ( 5), in of this peak observation upon the based tight 11 nm pitch super helix,is chromatin gels, but it has not been well documented in nuclei.
MATERIALS AND METHODS Nuclei were isolated from fresh calf thymus with the method of Panymin, Bilik, and Chalkley (10), except that Triton-X was omitted and the washing medium contained 2 mM instead of 10 mM MgCl2. After the 2.4 M sucrose pelleting the nuclei were suspended in .25 M sucrose. 10-4 M PMSF was added to most media but its presence did not alter the neutron scattering results. Nuclei were studied either suspended in various media at a concentration of a few percent chromatin or were examined after pelleting at 1,000 g for 15 minutes to a concentration of about 20 %. Phase examination and electron microscopy showed that more than 95 % of our nuclei were intact and largely free of cytoplasmic contamination (see figure 1). Chromatin was prepared by diluting nuclei to a chromatin concentration of .5 % in 1 mM NaCl with, or without a 12 hour mechanical stirring at 40 C. Rat liver nuclei were prepared by Dr. M. Leval with the method of Leval and Bouteille (11). The medium contained PMSF 10-4 M. Electron micrographs of these preparations can be found in ref. (11). Chromatin concentrations were obtained by diluting in 1 % sodium lauryl sulfate and .1 M NaCl, and determining the optical density at 260 nm. Electron microscopy was carried out on peleted nuclei after formaldehyde and osmium fixation, alcohol dehydration and embedding in methacrylate. 50 nm thick sections were then stained with uranyl acetate and lead citrate
(12). The neutron scattering on calf thymus nuclei was perfomed on the Dll diffractometer built by K. Ibel (13) at the I.L.L. in Grenoble. Sample distances of 5 and 10 meters and wave lengths of .6 to 1.0 nm were employed with a Ax/x of 9 %. Most experiments were at 20 to 250 C but some experiments were carried out over the range of 4 to 90° C. Typical runs lasted 15 minutes. In figures 2 and 3 the curves are presented after the background and solvent scattering were substracted as previously described (15, 8, 9 ). The 1722
Nucleic Acids Research
Figure 1 - Micrographs of calf thymus nuclei in 2 mM MgC12, .25 M sucrose: a) by phase-contrast of a suspension; b) by electron microscopy after pelleting, embedding and sectioning. The bars are 2 microns. very small angle scattering was approximately the same before and after subtraction since the high chromatin concentrations used gave a scattering several fold larger than that of a sample holder filled with water. Rat liver nuclei were studied at the C.E.N. Saclay with a ring detector diffractometer described by Cotton et al. (14). Experiments were performed at 40 C over a period of several hours. The scattering curves (fiq. 3) are smeared by a AX/A of 30%, consequently the actual maxima would be somewhat sharper and shifted to '10% larger spacings.
RESULTS AND DISCUSSION Very small angle maxima at about 40 and 18 nm are observed in the scattering from nuclei in environments which resemble those in situ. The 1723
Nucleic Acids Research maxima of the typical curves in figures 2 and 3 would be even more pronoun-
Loge J
61
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.
-
*
.. *
*l
3*
t
3*-
*
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3
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0~ 0
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1
2
3
h.102(A-1)
Figure 2. Scattering curves from calf thymus nuclei in .25 M sucrose: pelleted in (s) 1 mM NaCl; 0.1 mM MgC12; (*) 2 mM EDTA pH 7. Nuclei suspended in (a) 2 mM MgCl2; 10 mM Tris pH 8; (A)1 mM MgCl2, 10 mM Tris pH 8; (o) 1 mM NaCl. (h = 4 ar/A Sin 0/2 where 0 is the scattering angle and X the wavelength of the neutrons). Note the pronounced shoulders at about h = .035 (A-1) or X 1 8 nm. 1724
Nucleic Acids Research
0*
Logel 0
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Figure 3. Neutron scattering of nuclei from calf thymus pelleted in 0.25 M sucrose (s) 1 mM NaCl, 0.1 mM MgC12; (*) 2 mM EDTA pH 7. Calf thymus nuclei suspended without sucrose in (o) 1 mM NaCl. (M) Rat liver nuclei pelleted in .25 M sucrose, 2 mM MgC12, 10 mM Tris ph 8; (A) 40% D20 1 mM NaCl. ced after correction for the 10 % wavelength and 10 % collimation smearing. That they are clearly due to the scattering from nuclei is shown by their absence under different ionic environments (see Table 1). In Table 1 our
1725
Nucleic Acids Research results concerning the presence or absence of the 40 and 18 nm maxima are given for the various envirnmental conditions studied. The spacing and sharpness of the first scattering maxima (the one at the small values of h, or at larger spacings) is quite sensitive to the environmental conditions. Calf thymus nuclei pelleted at 1,000 g for 15 min. display a strong maxima near 38 nm while nuclei in a suspension at lOX lower concentration yield a broad maxima near 45 nm. Concentration into a pellet probably reduces the nuclear volume. Nuclei suspended in the absence of sucrose appear swollen in the phase microscope and do not exhibit a maximum at spacings larger than 20 nm. The difference between the spacing of the first maxima from rat liver and calf thumus nuclei (about 20%) may be due to inherent structural differences, or to the slightly different modes of preparation. The first scattering maximum is similar at 0.1 and 1 mM MgCl2 but sometimes is absent at higher divalent ion concentrations. It is important to note that the maxima at 18 nm does not vary much over the range of conditions studied, but disappears in .65 M NaCl. Chromatin when isolated without mechanical stirring can also yield strong characteristic maxima at the same spacings of intact nuclei (8, 9). However, after mild nuclease digestion these maxima, characteristic of some aspects of the native super structure in the nucleus, were never observed (15, 16). Scattering angles corresponding to spacings of up to 120 nm were studied. No other maxima at larger Bragg spacings were detected. As shown in figure 3, the very low angle maxima are prominent in 40% D20 where the neutron scattering from proteins is contrast matched. It is clear then that a non-chromatin associated protein component in the nucleus is not responsible for the maxima; conversely DNA is makinq a major contribution. u
Interpretation for the origin of the very small angle maxima. Fibers having diameters between 35 and 50 nm are the most prevalent structural entity seen in freeze fracture replicas (17, 18) and in recent classical electron micrographs (19) of nuclei and chromatin in the presence of divalent ions. The scattering from a gel or solution of cylinders or bundles of fibers having an outer diameter of 35 to 50 nm will exhibit a maximum at spacings between 25 and 40 nm and a second one near 18 nm (see ref. 20 for example). Although this is the explanation most consistent with all our results, others are possible and will be briefly considered. 1726
Nucleic Acids Research Table 1
Conditions Calf thymus nuclei, 0.25 M sucrose Pellet 1 imM NaCl, 10 4 M MgCl2 1 MM EDTA 650 nM NaCl
Suspension 1 mM NaCl, 10 nM Tris pH 8 1 mM KCI, 10 mM Tris 1 mM MgC12, 10 mM Tris 2 mM MgCl2, 10 mM Tris alf thymus nuclei no sucrose 1 mM NaCl 10 mM Hepes pH 8 Rat liver nuclei 0.25 M sucrose 10 4 M MgCl2 io- M MgC12
Calf thymus chromatin undispersed in 1 mM NaCl stirred 12 hours in 1 rM NaCl heated above 800 C and cooled to 20° C alf thymus or rat liver chromatin with brief nuclease digestion (-implies the maximum is absent)
Bragg spacing (nm) First max. Second maximum
38 38
18 18
45 45 45
18 18 18 18
-
-
18 18
32 30
18 18
40-45
18-20 Ref. (8,
Ref. (15,
91 16)
A side by side packing would give rise to maxima at spacings equal to the separation (d) and d/a/i. This is not observed. An important result is that the 40 and 18 nm peaks do not shift to larger spacings together uponswelling and that the 18 nm maxima may be quite strong while the -. 40 nm reflection is absent. This finding suggests that they are of different origin. (We were not aware of this behavior in 1974 when we suggested an interpretation based upon a 40 nm pitch coil (8, 9)). The scattering from 20 to 30 nm spherical groupings of nucleosomes, which have been called "super beads" (21), should show a very sharp scattering maximum near 15 nm. The spacings and the breadth of the shoulders in the experimental data do not seem to fit such a model. 1727
Nucleic Acids Research We have carried out a limited number of neutron scattering experiments on intact calf thymus tissue. Spacings on the order of 40 nm are often observed. It is probably that these maxima are the same as those from isolated nuclei. An 11 nm maximum is not observed in the neutron scattering from nuclei under the environment conditions so far studied by us (see figure 3 and table 1). If, as suggested by several authors (4, 5, 6) the 11 nm maximum arises from a tight super helix or solenoid with a corresponding pitch of X, 11 nm, the results strongly suggest that such structures are rare or absent in nuclei. Our results do not conflict with most studies reporting the 11 nm maximum, as these were carried out on isolated chromatin, but they are not in accord with one X-ray study of nuclei (22). In this study (22) it may have been difficult to measure well a maximum on a film which was superimposed on the central parasitic scatter; in addition scattering curves were not presented. Consequently, direct comparison is difficult; still it is possible that an 11 nm maximum would be present at conditions different from those we employed. In any case, with gels of isolated chromatin the first scattering maximum is observed near 11 nm at concentrations of about 40%, but it is at X~ 13 nm at concentrations of 10-20% (21, 16) and is shifted toward 20 nm upon further dilution (23). *
To whom correspondence should be addressed. Currently on leave at: Biochemistry Department, University of California, Berkeley, Ca. 94720, U.S.A. A preliminary report of these findings was presented at the Harwell Symposium on Neutrons in Biology in July, 1976.
ACKNOWLEDGEMENTS We are grateful to Dr. K. Ibel and the staff of the Institut Laue Langevin and to Dr. J. P. Cotton and the staff at the laboratory Leon Bruillion for their assistance. We thank Drs. M. Leval and M. Bouteille for proividing rat liver nuclei. Financial assistance was provided by an A.T.P. chromatine. We acknowledge that we became aware of similar X-ray scattering experiments by Dr. John Langmore after we completed our study.
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