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On the structure of nucleohistone
J. Mol. Biol. (1971) .%,325-336
On the Structure of Nucleohistone S. BRAM~AND H. RIS Biophysics Laboratory and Department of Zoology University of Wisconsin, Madison, Wis. 53706, U.X.A. (Received 22 July 1969, and in revised form 22 June 1970) Nucleohistone from calf thymus was studied by both electron microscopy and small-angle X-ray scattering in solution. The over-all shape of nucleohistone was found to be approximated by a very long cylinder whose diameter varies from 80 to about 120 A, with a minor mass fraction of axial projections of similar thickness and 120 to 250 A long. In preparations where nucleohistone fibers were stretched before fixation, it was observed in the electron microscope that the diameter of some of the fibers decreases from 100 A to a minimum of 25 A. From our small-angle X-ray scattering in 0.80 mM-PO4 (pH 63) we obtained a cross-section radius of gyration and a minimum value for the mass per unit length. They were 30 A and 1100 daltons/A, respectively. The results were compared to the scattering curves calculated by computer from precise models. A structure consisting of one DNA double helix, irregularly supercoiled or folded with an average pitch of 45 A to make a thicker fiber was found to agree best.
1. Introduction In the chromosomes of higher organisms, DNA is associated with characteristic basic proteins, the histones. It has been suggested that histones have a role in the control of DNA transcription since the template activity of DNA is decreased when it is combined with histones as in native chromatin or in reconstituted nucleohistone. At present very little is known about the conformation of DNA or histones in either active or inactive chromatin. Physico-chemical studies indicate that a single DNA double helix is compactly folded in combination with an equal weight of
histones to form a fibrous structure of indeterminate length. No regularity in the distribution of the major histone fractions along the DNA has been discovered. X-ray diffraction studies of nucleohistone fibers and studies of small-angle scattering by nucleohistone in solution have yielded ambiguous and contradictory results. Electron micrographs suggest that chromatin and isolated nucleohistones consist of fibrils which average about 100 A in thickness. Digestion with pronase indicates that these fibrils contain a single DNA double helix which is irregularly folded when combined with histones (Ris, 1967). We have examined calf thymus nucleohistone by two independent techniques, small-angle X-ray scattering and electron microscopy. Smallangle X-ray scattering of nucleohistone fibers in solution supplies the cross-section radius of gyration and some suggestions on the packing within the fiber. Usually the mass per unit length of a long fiber can also be determined but, as we shall discuss, t Present address: Department de Biochimie Macromol&ulsire, 32.5
C.N.R.S., 34 Montpellier,
France.
326
S. BRARZ
AND
H.
RIS
such measurements are only qualitative for the nucleohistone preparations that have been obtained to date. It will be shown that the electron microscope and X-ray data on the same preparations are compatible.
2. Materials and Methods (a) Preparation
of nucleohistone
Calf thymus obt,ained from a slaughter house was placed in crushed distilled water-ice within 15 min of the death of the animal. No differences of physical properties were detected in nucleohistones propared wit,hin a 2hr period after slaughter. The isolation procedure was similar to that of Zubay & Doty (1959). Our modified method yields nucleohistone that retains the morphology of chromatin fibrils seen in sections of nuclei or in nuclei spread on distilled water (Ris, 1961). All procedures were carried out at 4°C. 25 g of tissue were minced with scissors and added to 200 ml. of a saline-EDTA solution consisting of 0.075 M-NaCl, 0.024 M-EDTA adjusted to pH 6.5 with 10 N-N&OH. The tissue was homogenized 3 consecutive times for 15 set (in a one quart single-speed Waring Blendor). The speed of the blendor was adjusted with a variable transformer set at 50 v. Between each homogenization the blendor was allowod to cool for several minutes. Tho homogenate was then filtered through a double thickness of cheese cloth. At this stage, it contained mostly free nuclei and cytoplasmic debris. The mixture was centrifuged at 755 g for 10 min and washed 3 times in saline-EDTA by brief blending and centrifuging at 1000 g for 15 min. The hnal pellet was rinsed with deionized distilled water, transferred to a blendor containing 300 ml. of water and stirred for 5 min at a speed just sufficient to create a vortex. The volume was then rapidly brought to 1 1. with cold water and the suspension was stirred gently for 10 min. The suspension was transferred to a beaker and stirred vigorously with a magnetic stirrer for 2 hr. It was then centrifuged at 60,000 g for 30 min. The upper two-thirds of the supernatant fraction was collected and used in our experiments. This fraction is water-clear and contains about 85% of the nucleohistone in the uncentrifuged suspension. The pellet,ed material will be referred to as “gel”. The supernatant was stored at 4°C. Within 7 days no change in the material could be observed either by electron microscopy or X-ray scattering. With longer storage, however, the fibers tend to clump and lose their sharp outlino. Therefore, no preparation older than 6 days was used in our experiments. We have also used thymus frozen in liquid nitrogen immediatoly after slaughter as a source of nucleohistone. Such preparations contain more than 400/, gel, and the fibrils are clumped and diffuse in cross-section. The same effect has been observed by Leith (1964). If isolated nucleohistone is frozen in liquid nitrogen and thawed up to 6 months later the appearance of the fibrils and the X-ray scattering are identical to those of fresh preparations. However, because freezing and thawing did incroaso the gel content, freshly propared nucleohistone was used in most of our measurements. In the original method of Zubay & Doty (1959) tho isolation medium is buffered at pH 8. We found that considerably more gel is formod at this pH than at pH 6.5, and the gel content increases much more rapidly with age of the preparation. The fibrils aro also more irregular in shape and diameter and often have a diffuse profile. Tho chemical composition and small-angle X-ray scattering, however, were identical in the two preparations.
(h) X-ray
scattering
Nucleohistone used for X-ray scattering was concentrated by ultrafiltration or by pelleting and redispersing in a smaller volume. It was then generally dialyzod against 0.8 mna-potassium phosphate, pH 6.8. Immediately before use, the larger aggregates (gel) were removed by centrifuging at 60,000 g for 30 min. The concentration of the sample was determined by chemical analysis of DNA and protein. The X-ray source, slit system and notation are doscribed in the proceding paper (Bram & Beeman, 1971).
STRUCTURE
327
OF NUCLEOHISTONE
(c) Chemical analysis Protein and DNA assays were the primary means of determining the nucleohistone concentrations in our preparations. DNA w&s determined by the diphenylamine reaction of Dische (1955). Deoxyadenosine-5’-phosphate (P-L Biochemicals) served as standard. Protein was determined with the Lowry procedure (Lowry, Rosebrough, Farr & Randall, 1951). Calf thymus histone extracted from calf thymus nuclei by the method of Johns, Philips, Simpson & Butler (1960) was used as standard. The concentration of the standard histone was known from amino-acid analysis (Spackman, Stein & Moore, 1958). We thank Dr T. Senshu of the Depurtment of Zoology for providing this standard. Phosphorus content wss determined by the method of Fiske & Subbarow (1925). Elementsal analysis for P, Ca, Mg, Al, Ba, Fe, Sr, B, Cu, Zn, Mn and Cr was performed on a pellet of nucleohistone rinsed in distilled water, dry ashed and dissolved in nitric acid, using a 1.5 m Jarrell-Ash 66,000 compact atom counter emission spectrometer (Christenson, Coon 8: Derse, 1967). (d) Xedimentation The sedimentation behavior of the nucleohistone was determined with the band sedimentation velocity method (Vinograd, Bruner, Kent & Wcigle, 1963). A 12-mm, type II, Kel-F cell (Beckman) was used. The sedimentation solvent was 0.80 mM-PO, in 90% douterium oxide, pH 6.8. Runs were made on two 0.015-ml. samples containing 0.41 and and 0.30 mg nucleohistone/ml. The mass averaged sedimentation coefficient for these samples was 58 and 60 s, respectively. From the width of the sedimentation band, it was apparent that the nucleohistone samples were very polydisperse. Large forward spreading of the band indicated that the sedimentation was concentration dependent even at these low concentrations. (e) Optical
density
The optical density was determined with a Hitachi Perkin-Elmer spectrophotometer. Solutions with optical densities less than 10 were measured in a l-mm cell to avoid errors due to incomplete dispersal of nucleohistone upon dilution. The molar extinction coefficient at 259 nm was 6.6 f 0.2 x lo6 cm2/mole P for dilute solutions of nucleohistone isolated at pH 6.5. This result agrees with tho extinction coefficient reported by Fredericq & Houssier (1967) and other workers. The absorbance at 259 nm for duplicate determinations on solutions that were initially more concentrated than about 1.5 mg nucleohistone/ml. differed by as much as 2076. Therefore, optical density was used only as an approximate measure of concentration. (f) Electron microscopy A drop of the nucleohistone suspension carbon-coated Formvar film. The grid was adjusted to pH 6.9 with NaOH. It was then ethanol and transferred to amyl acetate. The grid was then dried by Anderson’s specimen was observed with a Siemens condenser illumination. A 200 p condenser used. The magnification was determined diffraction grating.
was placed on a grid that was covered with a floated for 20 set on a solution of 10% formalin rinsed in distilled water, dehydrated in absolute All of these operations were performed at 4°C. critical point method (Anderson, 1951). The Elmiscope I electron microscope with double aperture and a 50 p objective aperture were with a carbon replica of a 54,800 lines/in.
3. Results (a) Chemical composition The content
RNA
content
determined
by phosphorus
assay.
of our
nucleohistone
by the diphenylamine We obtained
an RNA
was
obtained
reaction content
from
by
subtracting
the DNA
of 0 f
2%.
content
the
DNA
calculated
328
S. BRAM
AND
H. RIS
In calculating the protein and DNA content we assumed that nucleohistone consisted solely of nucleic acid and protein. The protein content (as histone) for six independent determinations on four different preparations of nucleohistone isolated at pH 6.5 was 55 f 2%. Although in our hands the protein content of nucleohistone isolated at pH 8.0 was generally lower than that obtained at pH 6.5. the difference was only slightly larger than the experimental error. The atomic emission results showed less than one calcium, or magnesium, ion is present per 100 atoms of phosphorus. These results imply that divalent cations do not have an important structural role in the 100 A nucleohistone.
(b) Electron microscopy Electron microscope observations show that the nucleohistone preparation does not contain individually dispersed nucleohistone molecules. Most of the material observed forms a network of fibers (Plate I) though short pieces of individual fibers are occasionally seen. The small fragments are more common in preparations that have been extensively sheared. At low magnification, our nucleohistone fibers have a knobby appearance due to numerous short side branches which are randomly spaced along the fiber and project 80 to 200 A from the fiber axis (Plate I, arrows). These protuberances are about 100 A thick and appear to comprise about one-fourth to one-third of the fiber mass. When the fibers are viewed in stereo electron micrographs, the protuberances can be seen to project randomly in all directions from the fibril. The cross-section of the non-projecting portion of the fibers is distinct in outline and is generally between 80 and 120 A in diameter. We should note that this diameter agrees with that obtained from our X-ray scattering cross-section radius of gyration. The electron microscope had another important role in our studies, since it could show that the gross morphology of nucleohistone was unchanged by our handling techniques and X-ray procedures. Plate II(a) shows an electron micrograph of material which had been used in an X-ray run. The structure appears identical to that of freshly prepared nucleohistone. From this and many other pictures taken at various points in our procedures, we conclude that the X-ray exposure does not alter the basic structure of nucleohistone as seen in the electron microscope. It appears that the nucleohistone complex is relatively stable. If the nucleohistone fibers are stretched-and this can be accomplished by streaking a formvar covered grid over the surface of a nucleohistone solution-one observes regions where the 100 A fibers get progressively thinner until a minimum of about 25 to 30 A in diameter is reached (Plate II(b)). The fibers appear to have semi-elastic properties. That a minimum thickness of 25 A is observed implies that a non-extendable structure of this diameter is coiled or folded into the fiber. There is evidence that similar thin regions may exist in the non-stretched state. Short thin regions were also observed in sections of nuclei and nucleohistone pellets (Bram, 1968). (c) X-ray scattering X-ray scattering curves were measured using the equipment and procedures described in the preceding paper (Bram & Beeman, 1971). Data were taken with four-slit symmetrical diffractometers having successive slits either 50 or 10 cm apart,
I'LATR
dissolved 4z.xoo.
I. 1UOx fibrils from calf thymus c*hromosomes isolated in saline-EDTA at pH 6.5 and in water. C’riticitl l)oint dried. Note the knobby protuberances marked by axrows.
(b)
STRUCTURE
OF NUCLEOHISTONE
329
and slit widths either 0.03 or 0.09 cm. CuKa (154 A) was the wavelength used in all measurements. Data extended from a scattering angle of about 3 to almost 200 mrad. The equivalent Bragg spacings are from 513 to 7.7 8. Figure 4 shows the entire scattering curve. It is a composite of several runs using different concentrations of nucleohistone and different instrumental parameters. Scattering data at the smaller angles, the radius of gyration region, were taken at as low a nucleohistone concentration as possible and with 0.03 cm slit width settings. In Figure 2 the scattered intensity, I(+), in the radius of gyration region has been plotted for a nucleohistone sample of concentration 1.75 mg/ml. The same data are plotted first as log [$*I (#)I vers’suB$” and then as log [I(+)] versus 4”. The first plot is that which would exhibit the cross-sectional radius of gyration of a long cylinder while the second is that which would exhibit the total radius of gyration of a globular particle. In both plots there is an initial short straight line of large slope followed by a more extended line of much smaller slope. Only in the first plot is this second line reasonably straight. The double straight line is superficially similar to that observed in the preceding study of sonicated DNA (Bram & Beeman, 1971) but we shall argue that the core and shell interpretation of the DNA data cannot be applied to the nucleohistone curve. We believe the initial straight line represents scattering from globular rather than fibrous regions of the preparation. It might be related to the knobby protuberances seen on the fibers in electron micrographs or it might be the remnant of a more intense scattering at very small angles from clusters of nucleohistone fibers. The second straight line we shall interpret as cross-section scattering. From the slope of the first straight line we calculate a total radius of gyration of approximately 12.5A. The exact result depends on how one subtracts an extrapolated background due to the second straight line. This is in reasonable agreement with the size of the knobby projections seen in the electron microscope. The cross-section radius of gyration derived from the slope of the second straight line is 36 f 2 A. If the first straight line is treated as the scattering from a long rod (which we think it is not) we deduce a cross-section radius of gyration of 52 A. The entire scattering curve is reproducible from one preparation to another and is independent of small changes in the temperature and conditions of solution providing the concentration of nucleohistone is kept below about 5 mg/ml. At higher concentrations the first straight line gradually disappears although the scattering at large angles remains unchanged. Samples in 20 m&r-PO, and 06 mM-PO, and at 4 and 22°C give similar scattering curves. Samples dialyzed against distilled water show a 10 to 20% increase in the slopes and intercepts of both the first and second straight line. In one experiment a sample was dialyzed against 10% formalin at pH 6.9 and then redialyzed against 06 mM-PO, buffer. The usual scattering curve was observed. Thus the formalin fixative used in our electron microscopy introduces no irreversible changes in conformation. This has also been noted by Nicolaieff (1967). At angles larger than about 20 mrad even quite high nucleohistone concentrations seem not to alter the scattering. A gel containing 260 mg nuoleohistone/mI. gave a curve almost identical with that of Figure 1. The distinguishing features of the scattering at larger angles are an inflection at 35 to 40 mrad (41 A Bragg spacing), a shoulder at 65 to 70 mrad (23 A), a distinct peak at 125 to 130 mrad (12.0 A). and and a shoulder at 172 mrad (8.9 A).
330
S. BRA&I
AND
II.
RI9
4. Discussion (a) Wide-angle
X-ray
scattering
The scattering at larger angles can be compared to the results of Wilkins, Zubay $ Wilson (1959) and Pardon, Wilkins & Richards (1967) on X-ray scattering from gels and drawn fibers of nucleohistone. Our inflection at 41 A may correspond to the spacings of 35 to 38 A which the above authors find to be fairly intense. They find larger spacings that are perhaps between fibers in the more concentrated preparations they used?. The shoulder at 23 A and the peak at 12.0 and shoulder at 9-O A appear in the results of Wilkins et aE. (1959). The latter two maxima are almost certainly due to DNA. The shape and position of these two maxima of the scattering curve agree well with those of the calculated scattering curve for DNA with the B form co-ordinates12.6 and 9.3 A-but very poorly with the scattering curve from the A form (see Bram & Beeman, 1971 for A and B form scattering curves). This implies that the major portion of the DNA in nucleohistone has a structure similar to B form. The observation of a spacing at 3.4 A from nucleohistone fibers supports this conclusion, but their finding does not indicate the relative amount of B form present. (b) Small-angle
X-ray
scattering
The spherically averaged X-ray scattering from an isotropic sample does not, in general, provide detailed information on the structure of the sample. One can be certain only of the density function (the distribution of the magnitudes of Patterson vectors) which is the Fourier inversion of the scattering curve. Additional information is available from the X-ray scattering to the extent that independent evidence justifies treating the sample in terms of at least a partially defined model. For instance, frequently one knows that the sample is a well-dispersed collection of identical particles. In the preceding work on DNA a wealth of chemical evidence plus electron micrographs of the actual samples used in the X-ray studies support the model of a well-dispersed collection of large rods of identical and uniform cross section. The import of our electron microscopy of nucleohistone is quite different. We see most of the material as fibers of fairly uniform diameter but with a great many knobby protuberances distributed apparently at random. There is much clustering of the nucleohistone into relatively dense regions a few 1000 d across. Insofar as these structures are also present in the dilute solution they must make their contributions to the X-ray scattering and an interpretation in terms of rigid rods alone is unjustified. Many features of the X-ray scattering support the electron microscope results. This is a welcome confirmation that the structures observed are not artifacts due to specimen preparation for the microscope but the structure is too complex to permit independent, quantitative interpretation of the X-ray results. The second straight line of Figure 2 is, we believe, clearly the cross-section scattering from the fibrous regions of the sample. The plot of log (+*1(#)] is much more linear than the plot of log I(+). The cross-section radius of gyration of 30 J%corresponds to a solid cylinder diameter of 85 d and is in reasonable agreement with the electron microscope observations. t A very recent finding is that our material clearly showed low-angle X-ray reflections at 105, 55, 38 and 22 11 when dried and then examined at high relative humidity in the laboratory of Dr J. F. Pardon. Thus, our proprtration proordur~s MC not rcsponnihlc for the absence of the 105 and 55 L% spacings from our aolutions.
STRUCTURE
OF NUCLEOHISTONE
331
The first linear region of Figure 2 is short and equally straight in the two kinds of plot but for the following reasons we believe it is not cross-section scattering. First a cross-section radius of gyration of 52 A gives a solid cylinder diameter of about 150 A which is larger than we observe in the electron microscope. A globular radius of gyration of 125 A is about that to be expected from the larger protuberances. Second, the dependence of the observed radius of gyration on the nucleohistone concentration is quite different for the first and second straight lines (both treated as cross-section scattering). In a range of low nucleohistone concentrations from O-5 to 5 mg/ml. the first radius of gyration decreases about 4% per mg/ml. increase in concentration. The decrease of the second radius of gyration is scarcely detectable, less than 0.676 per mg/ml. It should be noted that in the DNA measurements neither radius of gyration shows a concentration dependence greater than O*25°/0per mg/ml. With nucleohistone concentrations above 5 mg/ml., the slope of the first straight line decreases rapidly. One expects a smaller concentration dependence of a cross-section radius of gyration since only those rods whose axes are perpendicular to the scattering vector make an appreciable contribution to the observed scattering. Thus, although there are good arguments that the curve at the smallest angles does not represent cross-section scattering, its assignment to the knobby protuberances is less certain. The clusters a few 1000 A across, acting as scattering units, should contribute strongly at angles much smaller t’han we are able to reach but perhaps the t,ail of this scattering is present even at 3 to 5 mrad. Quite possibly cluster scattering was observed in the light-scattering measurements of Zubay & Doty (1959) on similar nucleohistone preparations. They reported a radius of gyration of about 1700 A. (c) illms per unit length Unfortunately, the model we propose makes it very diiiicult to extract useful information from our absolute intensity measurements. One might attempt a resolution of the scattering curve into components and the assignment of an absolute scattering power to t,hat part which seems to come from the long fibers. (The ratio of t,he intercepts of the two straight lines indicates that the fiber contribution is between 65 and 75% of the total.) This still does not permit a calculation of mass per unit length unless one knows what fraction of the total mass concentration of nucleohistone is contributing to the cross-section scattering. Our results can be stated as follows. Let us assume that the second straight line of Figure 2 represents cross-section scattering from the fibrous part of the preparation with only minor contributions from other structures. Extrapolation of this line to zero nngle provides the I,, of equation (4) of the preceding paper. We calculate for nucleohistone an I = 0.53 electrons per dalton assuming a 45% DNA content. For the partial specific volume we use 0.68 cm3/g from the work of Zubay & Doty (1959). Equation (4) now provides the mass per unit length of the fibers if the concentration of fibers in the solution is known. If we assume that all the dissloved nucleohistone contributes to the fiber scattering we obtain M/L = 1100 daltons/a. If only half contributes M/L = 2200 daltons/A. We find the intensity of scattering in the second straight line region to be proportional to nucleohistone concentration. This implies that the fraction of the sample contributing to the cross-section scattering is independent of concentration but it does not say what that fraction might be. However, M/L changes only slightly witSI concentration, source and method of preparation and with high speed centri-
332
S. BRAN
Ah’D
H.
RI8
fugation of the solution, although electron microscope observations show that these factors do alter the packing of the nucleohistone. This may indicate that most of the nucleohistine in fact, contributes to the cross-section scattering. We remind the reader that these computations use the two-component, not the more correct three-component formalism. These effects are probably small with nucleohistone which has a smaller charge and smaller surface to volume ratio than DNA. (d) Comparison with previous studies Our results are in fair agreement with the work of Luzzati & Nicolaieff (1963). They report a cross-section radius of gyration of 26 A for dilute solutions of calf thymus and chicken erythrocyte nucleohistone, and a mass per unit length of about 1500 daltons/A. Their scattering data are processed and presented without slit correction and are shown as plots of log scattered intensity versus log scattering angle as in our Figure 1. They do not report an initial steep slope as in the scattering curve seen in Figure 2, nor do they present electron micrographs of their preparations. It seems reasonable to assume that their preparations had structural complexities similar to ours and that t.heir mass per unit length results are subject to similar very large uncertainties. The 1500 daltons/ A reported should represent a minimum value. (e) Consideration of ,models All of these results are quite low compared to what one calculates for a solid cylinder of diameter 100 A and average density 14 g/cm3. Such a cylinder has a mass per unit length of 6600 daltons/A. If an AU/L of 1100 to 1500 daltons/,& is correct, it shows that nucleohistone has an extensively solvated, loosely packed structure. This M/L also requires that the mass per unit length due to DNA be three to four times that of the B form. Luzzati & Nicolaieff (1963) f ound that the small-angle X-ray scattering was consistent with a model containing four strands of DNA per cross-section. However, such a model is inconsistent with the results of our electron microscopy and other pertinent physical studies on nucleohistone. A structure containing four DNA’s per cross-section would have a minimum diameter of at least 50 A. In the electron microscope we observe that the nucleohistone consists of 25 A strands in regions which appear stretched or unravelled or have been partially digested with pronase (Ris, 1967). Multi-loop nodes, which would be expected from a multi-stranded nucleoprotein complex after pronase digestion, are not observed. The substructure observed at high resolution also does not agree with a multi-stranded model. If we reject multi-stranded models, the M/L can only be achieved by a super coiling or folding of the DNA. Incontrovertible evidence for super coiling, according to Pardon et al. (1967) is provided by the disappearance of small-angle spacings from nucleohistone fiber patterns upon stretching. Hydrodynamic studies by Zubay & Doty (1959) and Giannoni & Peacocke (1963) indicate that nucleohistone consists of single DNA molecules. Flow birefringence and dichroism experiments by Ohba (1966) show a characteristic shortening of the DNA in nucleohistone which implies a super helical configuration. With this evidence in mind we will show that a super-coiled model would also agree with all of our X-ray scattering data. Based on our electron microscopy, we will use 30 A as the thickness of the fiber. The cross-section radius of gyration-30 A-then determines that the distance from the center of the coiled fiber to the axis of the super
STRUCTURE
/
,
j
OF
,
,
NUCLEOHISTOSE
Tl?r
--,-
.-.
,
333
i.~T-’
.,---.
.
-r-
-t _
0 ccl 0 “0
-2 -
e a R e 0
-3 -
-4 -
L Scottermg
angle
(mrod)
Fro. 1. The slit corrected intensity plotted against the scattering angle for four runs of nucleohistone. (0) Data from 3 to 10 mred taken with 300 p slits, 1.7 mg nucleohistone/ml. at 4°C; from 10 to 45 mrad, 900 p slits were used, on sample containing 5.9 mg nuoleohistone/ml. at 4°C; (0) 8.2 mg nucleohistonejml. in 20 mM-PO,; 22”C, 900 p slits; ( A) 16 mg nueIeohistone/ml. in 0.80 mxPO,, 22’C. 900 p slits.
331
FIG. 2. The scattering curve of a typical run of nucleohistone after background subtraction slit correction (1.75 mg/ml., 4°C). (0) Intensity times scatt,cring angle; (0) intensity.
and
coil will be about 28 A. We must adjust the pitch of a helix to give the observed M/L using : N/L of DNA in nucleohistone Contour length ----~~~ _ JL(kgy + q pitch M/L of DNA in B form The minimum M/L of DNA in nucleohistone = 0.45 g DNA/g nucleohistone * 1100 daltons/A = 495 daltons/A. For a regular helix one then calculates t,hat the pitch would have to be 50 to 70 A to agree with the observed M/L of 1500 to 1100 daltons/A. Since these values for the M/L are minimum values, 50 to 70 if represent maximum values for the pitch. To obtain further information on t)he structure of nucleohistone in solution WC compared our wide-angle curve to computer-calculated scattering curves for several models using equations of the prevTious paper. (The models were chosen to have the observed cross-section radius of gyration.) Poor agreement was observed with cylindrical models. Figure 3 shows the curve (b) for a model simulating a regular helix, 600 A long with a pitch of 50 A. Eight scattering points spaced inside a square 20 A on a side centered at a radial distance of 30 A was t,he basic unit for constructing the computer model. The co-ordinate of the unit center was raised by A 2 (5 A here) and rotated by A Z/pitch. A more detailed discussion of the model calculations is given by Bram (1968). The calculated scattering curve shows two sharp maxima at spacings of 48 and 23 8. As can be seen in Figure 3 the nucleohistone curve (a) shows a distinct inflection at 35 to 40 mrad (44 to 38 A) and a distinct shoulder at 68 mrad (22 8). The in%ections of the experimental curve correspond in both position and relative intensity to those from a model of a regular helix, similar to Figure 3 (curve b), but having a pitch of 46 A and radius of 30 A. This pitch agrees with the small-angle X-ray scattering
STRUCTURE
OF
K UCLEOHISTONE
335
.
30
50 70 Scattering angle ( mrad)
FIG. 3. Comparison of the calculated scattering curve from a model simulrtting a super coil of pitch 50 A, radius 30 A and fiber thickness 22 A to the nxperimental c~~rvc. The filled circles represent a composite of 3 runs in 0.80 mx-PO,.
mass per unit length and the observation of a semi-meridional spacing at 38 A from nucleohistone fibers (Wilkins et al., 1959). The 30 A radial dimensions would agree well with the presence of the intense 30 A equatorial reflections in the nucleohistone fiber diagrams. Since the electron micrographs showed few regions which would be interpret’ed as regular, orderly coils, we considered the scattering from non-uniform helices. A nonuniform helix was simulated by allowing the pitch and radial distance to vary along the axis. The maxima from the computer-calculated scattering curves for irregular coils were considerably broader t’han t’hose observed from regular helices and fit better to the experimental curves.
5. Conclusions Carefully prepared calf thymus nucleohistone appears in electron micrographs to be a network of fibrils about 100 A in diameter. There are numerous knobby protuberances where the fibril is locally thickened to 200 or 300 A. The network seems to be continuous but interconnected (or overlapping) in a complex, apparently random, manner. The compactness of the network varies greatly. In some areas individual fibrils can be followed for several 1000 A. In others clustering is so great that individual fibrils are resolved with difficulty. Occasionally we see a fibril thinned down to a diameter of about 25 A. A model for nucleohistone containing one double helix of DNA and associated histone non-uniformly coiled with an average pitch of 45 A is most consistent with our results. X-ray and light scattering suggest that the same nucleohistone preparations in dilute solutions are a network of fibrils similar to what is seen in the electron microscope. A part of the X-ray curve behaves as the cross-section scattering from long rods and the measured radius of gyration is in good agreement with the electron microscope diameter. The X-ray scattering at the smallest angles, we believe, is produced by the protuberances or by the large clusters.
336
S. BRAN
ASI)
II.
RTS
We have made an absolute intensity calibration of our scattering curve but can report only a minimum mass per unit length of 1100 daltons/A. It could be several times larger than this if only a small fraction of the dissolved sample contributes to the cross-section scattering. We thank Professor W. W. Beeman This research has been supported Institutes of Health. One of us (H.R.) acknowledges the Award (KG-GM-21, 948) and research Health.
for numerous helpful conversations. by research and training grants of the National awards of a Public Health Service Career Program grant, (GM-04738) from U.S. National Institutes of
REFERENCES Anderson, T. (1951). N.Y. AC&. Sci., 13, 130. Bram, S. (1968). Thesis, University of Wisconsin. Bran-r, S. & Beeman, W. W. (1971). J. MOE. Biol. 55, 311. Christensen, R. E., Coon, F. B. & Derse, P. H. (1967). P&burgh Conference 012AnaZyfiicoZ Chemiat?y. (Available from Jarrell-Ash Company as reprint no. 74). Dische, Z. (1955). In The NucZeic Acids, cd. by E. Chargaff 8: J. N. Davidson. vol. 1, p. 285. New York: Academic Press. Fiske, C. H. & Subbarow, Y. (1925). J. BioZ. Chem. 66, 375. Frederiq E. & Houssier, C. (1967). Europ. J. Biochem. 1, 51. Giannoni, G. & Peacocke, A. R. (1963). Biochem. biophys. Acta, 68, 157. Johns, E. W., Philips, D. M. P., Simpson, P. & Butler, J. A. V. (1960). Biochem. J. 77, 631. Leith, D. (1964). Ph.D. Thesis, University of Wisconsin. Lowry, 0. H., Rosebrough, M. .J., Farr, ,4. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265. Luzzati, V. & Nicolaieff, A. (1963). J. Mol. BioZ. 7, 142. Nicolaieff, A. (1967). Sm.uZZ-Angle X-Ray Scattering, cd. by H. Brumberger. New York: Gordon & Breach. Ohba, Y. (1966). Biochem. biophys. Acta, 23, 76. Pardon, J. F., Wilkins, M. H. F. & Richards, B. M. (1967). Nature, 215, 508. Ris, H. (1961). Canad. J. Genet. 3, 95. Ris, H. (1967). Regulation of Nucleic Acid and Protein Biosynthesis, ed. by V. Konigsberger & L. Bosch. Amsterdam: Elsevier. Spa&man, D. H., Stem, W. H. & Moore, S. (1958). Andyt. Chem. 30, 1190. Vinograd, J., Bruner, R., Kent, R. & Weigle, J. (1963). Proc. Nat. Acad. Sci., Wwh. 49, 902. Wilkins, M. H. F., Zubay, G. & Wilson, H. R. (1959). J. Mol. BioZ. 1, 179. Zubay, G. t Doty, P. (1959). J. Mol. BioZ. 1, 1.
On the Structure of Nucleohistone S. BRAM~AND H. RIS Biophysics Laboratory and Department of Zoology University of Wisconsin, Madison, Wis. 53706, U.X.A. (Received 22 July 1969, and in revised form 22 June 1970) Nucleohistone from calf thymus was studied by both electron microscopy and small-angle X-ray scattering in solution. The over-all shape of nucleohistone was found to be approximated by a very long cylinder whose diameter varies from 80 to about 120 A, with a minor mass fraction of axial projections of similar thickness and 120 to 250 A long. In preparations where nucleohistone fibers were stretched before fixation, it was observed in the electron microscope that the diameter of some of the fibers decreases from 100 A to a minimum of 25 A. From our small-angle X-ray scattering in 0.80 mM-PO4 (pH 63) we obtained a cross-section radius of gyration and a minimum value for the mass per unit length. They were 30 A and 1100 daltons/A, respectively. The results were compared to the scattering curves calculated by computer from precise models. A structure consisting of one DNA double helix, irregularly supercoiled or folded with an average pitch of 45 A to make a thicker fiber was found to agree best.
1. Introduction In the chromosomes of higher organisms, DNA is associated with characteristic basic proteins, the histones. It has been suggested that histones have a role in the control of DNA transcription since the template activity of DNA is decreased when it is combined with histones as in native chromatin or in reconstituted nucleohistone. At present very little is known about the conformation of DNA or histones in either active or inactive chromatin. Physico-chemical studies indicate that a single DNA double helix is compactly folded in combination with an equal weight of
histones to form a fibrous structure of indeterminate length. No regularity in the distribution of the major histone fractions along the DNA has been discovered. X-ray diffraction studies of nucleohistone fibers and studies of small-angle scattering by nucleohistone in solution have yielded ambiguous and contradictory results. Electron micrographs suggest that chromatin and isolated nucleohistones consist of fibrils which average about 100 A in thickness. Digestion with pronase indicates that these fibrils contain a single DNA double helix which is irregularly folded when combined with histones (Ris, 1967). We have examined calf thymus nucleohistone by two independent techniques, small-angle X-ray scattering and electron microscopy. Smallangle X-ray scattering of nucleohistone fibers in solution supplies the cross-section radius of gyration and some suggestions on the packing within the fiber. Usually the mass per unit length of a long fiber can also be determined but, as we shall discuss, t Present address: Department de Biochimie Macromol&ulsire, 32.5
C.N.R.S., 34 Montpellier,
France.
326
S. BRARZ
AND
H.
RIS
such measurements are only qualitative for the nucleohistone preparations that have been obtained to date. It will be shown that the electron microscope and X-ray data on the same preparations are compatible.
2. Materials and Methods (a) Preparation
of nucleohistone
Calf thymus obt,ained from a slaughter house was placed in crushed distilled water-ice within 15 min of the death of the animal. No differences of physical properties were detected in nucleohistones propared wit,hin a 2hr period after slaughter. The isolation procedure was similar to that of Zubay & Doty (1959). Our modified method yields nucleohistone that retains the morphology of chromatin fibrils seen in sections of nuclei or in nuclei spread on distilled water (Ris, 1961). All procedures were carried out at 4°C. 25 g of tissue were minced with scissors and added to 200 ml. of a saline-EDTA solution consisting of 0.075 M-NaCl, 0.024 M-EDTA adjusted to pH 6.5 with 10 N-N&OH. The tissue was homogenized 3 consecutive times for 15 set (in a one quart single-speed Waring Blendor). The speed of the blendor was adjusted with a variable transformer set at 50 v. Between each homogenization the blendor was allowod to cool for several minutes. Tho homogenate was then filtered through a double thickness of cheese cloth. At this stage, it contained mostly free nuclei and cytoplasmic debris. The mixture was centrifuged at 755 g for 10 min and washed 3 times in saline-EDTA by brief blending and centrifuging at 1000 g for 15 min. The hnal pellet was rinsed with deionized distilled water, transferred to a blendor containing 300 ml. of water and stirred for 5 min at a speed just sufficient to create a vortex. The volume was then rapidly brought to 1 1. with cold water and the suspension was stirred gently for 10 min. The suspension was transferred to a beaker and stirred vigorously with a magnetic stirrer for 2 hr. It was then centrifuged at 60,000 g for 30 min. The upper two-thirds of the supernatant fraction was collected and used in our experiments. This fraction is water-clear and contains about 85% of the nucleohistone in the uncentrifuged suspension. The pellet,ed material will be referred to as “gel”. The supernatant was stored at 4°C. Within 7 days no change in the material could be observed either by electron microscopy or X-ray scattering. With longer storage, however, the fibers tend to clump and lose their sharp outlino. Therefore, no preparation older than 6 days was used in our experiments. We have also used thymus frozen in liquid nitrogen immediatoly after slaughter as a source of nucleohistone. Such preparations contain more than 400/, gel, and the fibrils are clumped and diffuse in cross-section. The same effect has been observed by Leith (1964). If isolated nucleohistone is frozen in liquid nitrogen and thawed up to 6 months later the appearance of the fibrils and the X-ray scattering are identical to those of fresh preparations. However, because freezing and thawing did incroaso the gel content, freshly propared nucleohistone was used in most of our measurements. In the original method of Zubay & Doty (1959) tho isolation medium is buffered at pH 8. We found that considerably more gel is formod at this pH than at pH 6.5, and the gel content increases much more rapidly with age of the preparation. The fibrils aro also more irregular in shape and diameter and often have a diffuse profile. Tho chemical composition and small-angle X-ray scattering, however, were identical in the two preparations.
(h) X-ray
scattering
Nucleohistone used for X-ray scattering was concentrated by ultrafiltration or by pelleting and redispersing in a smaller volume. It was then generally dialyzod against 0.8 mna-potassium phosphate, pH 6.8. Immediately before use, the larger aggregates (gel) were removed by centrifuging at 60,000 g for 30 min. The concentration of the sample was determined by chemical analysis of DNA and protein. The X-ray source, slit system and notation are doscribed in the proceding paper (Bram & Beeman, 1971).
STRUCTURE
327
OF NUCLEOHISTONE
(c) Chemical analysis Protein and DNA assays were the primary means of determining the nucleohistone concentrations in our preparations. DNA w&s determined by the diphenylamine reaction of Dische (1955). Deoxyadenosine-5’-phosphate (P-L Biochemicals) served as standard. Protein was determined with the Lowry procedure (Lowry, Rosebrough, Farr & Randall, 1951). Calf thymus histone extracted from calf thymus nuclei by the method of Johns, Philips, Simpson & Butler (1960) was used as standard. The concentration of the standard histone was known from amino-acid analysis (Spackman, Stein & Moore, 1958). We thank Dr T. Senshu of the Depurtment of Zoology for providing this standard. Phosphorus content wss determined by the method of Fiske & Subbarow (1925). Elementsal analysis for P, Ca, Mg, Al, Ba, Fe, Sr, B, Cu, Zn, Mn and Cr was performed on a pellet of nucleohistone rinsed in distilled water, dry ashed and dissolved in nitric acid, using a 1.5 m Jarrell-Ash 66,000 compact atom counter emission spectrometer (Christenson, Coon 8: Derse, 1967). (d) Xedimentation The sedimentation behavior of the nucleohistone was determined with the band sedimentation velocity method (Vinograd, Bruner, Kent & Wcigle, 1963). A 12-mm, type II, Kel-F cell (Beckman) was used. The sedimentation solvent was 0.80 mM-PO, in 90% douterium oxide, pH 6.8. Runs were made on two 0.015-ml. samples containing 0.41 and and 0.30 mg nucleohistone/ml. The mass averaged sedimentation coefficient for these samples was 58 and 60 s, respectively. From the width of the sedimentation band, it was apparent that the nucleohistone samples were very polydisperse. Large forward spreading of the band indicated that the sedimentation was concentration dependent even at these low concentrations. (e) Optical
density
The optical density was determined with a Hitachi Perkin-Elmer spectrophotometer. Solutions with optical densities less than 10 were measured in a l-mm cell to avoid errors due to incomplete dispersal of nucleohistone upon dilution. The molar extinction coefficient at 259 nm was 6.6 f 0.2 x lo6 cm2/mole P for dilute solutions of nucleohistone isolated at pH 6.5. This result agrees with tho extinction coefficient reported by Fredericq & Houssier (1967) and other workers. The absorbance at 259 nm for duplicate determinations on solutions that were initially more concentrated than about 1.5 mg nucleohistone/ml. differed by as much as 2076. Therefore, optical density was used only as an approximate measure of concentration. (f) Electron microscopy A drop of the nucleohistone suspension carbon-coated Formvar film. The grid was adjusted to pH 6.9 with NaOH. It was then ethanol and transferred to amyl acetate. The grid was then dried by Anderson’s specimen was observed with a Siemens condenser illumination. A 200 p condenser used. The magnification was determined diffraction grating.
was placed on a grid that was covered with a floated for 20 set on a solution of 10% formalin rinsed in distilled water, dehydrated in absolute All of these operations were performed at 4°C. critical point method (Anderson, 1951). The Elmiscope I electron microscope with double aperture and a 50 p objective aperture were with a carbon replica of a 54,800 lines/in.
3. Results (a) Chemical composition The content
RNA
content
determined
by phosphorus
assay.
of our
nucleohistone
by the diphenylamine We obtained
an RNA
was
obtained
reaction content
from
by
subtracting
the DNA
of 0 f
2%.
content
the
DNA
calculated
328
S. BRAM
AND
H. RIS
In calculating the protein and DNA content we assumed that nucleohistone consisted solely of nucleic acid and protein. The protein content (as histone) for six independent determinations on four different preparations of nucleohistone isolated at pH 6.5 was 55 f 2%. Although in our hands the protein content of nucleohistone isolated at pH 8.0 was generally lower than that obtained at pH 6.5. the difference was only slightly larger than the experimental error. The atomic emission results showed less than one calcium, or magnesium, ion is present per 100 atoms of phosphorus. These results imply that divalent cations do not have an important structural role in the 100 A nucleohistone.
(b) Electron microscopy Electron microscope observations show that the nucleohistone preparation does not contain individually dispersed nucleohistone molecules. Most of the material observed forms a network of fibers (Plate I) though short pieces of individual fibers are occasionally seen. The small fragments are more common in preparations that have been extensively sheared. At low magnification, our nucleohistone fibers have a knobby appearance due to numerous short side branches which are randomly spaced along the fiber and project 80 to 200 A from the fiber axis (Plate I, arrows). These protuberances are about 100 A thick and appear to comprise about one-fourth to one-third of the fiber mass. When the fibers are viewed in stereo electron micrographs, the protuberances can be seen to project randomly in all directions from the fibril. The cross-section of the non-projecting portion of the fibers is distinct in outline and is generally between 80 and 120 A in diameter. We should note that this diameter agrees with that obtained from our X-ray scattering cross-section radius of gyration. The electron microscope had another important role in our studies, since it could show that the gross morphology of nucleohistone was unchanged by our handling techniques and X-ray procedures. Plate II(a) shows an electron micrograph of material which had been used in an X-ray run. The structure appears identical to that of freshly prepared nucleohistone. From this and many other pictures taken at various points in our procedures, we conclude that the X-ray exposure does not alter the basic structure of nucleohistone as seen in the electron microscope. It appears that the nucleohistone complex is relatively stable. If the nucleohistone fibers are stretched-and this can be accomplished by streaking a formvar covered grid over the surface of a nucleohistone solution-one observes regions where the 100 A fibers get progressively thinner until a minimum of about 25 to 30 A in diameter is reached (Plate II(b)). The fibers appear to have semi-elastic properties. That a minimum thickness of 25 A is observed implies that a non-extendable structure of this diameter is coiled or folded into the fiber. There is evidence that similar thin regions may exist in the non-stretched state. Short thin regions were also observed in sections of nuclei and nucleohistone pellets (Bram, 1968). (c) X-ray scattering X-ray scattering curves were measured using the equipment and procedures described in the preceding paper (Bram & Beeman, 1971). Data were taken with four-slit symmetrical diffractometers having successive slits either 50 or 10 cm apart,
I'LATR
dissolved 4z.xoo.
I. 1UOx fibrils from calf thymus c*hromosomes isolated in saline-EDTA at pH 6.5 and in water. C’riticitl l)oint dried. Note the knobby protuberances marked by axrows.
(b)
STRUCTURE
OF NUCLEOHISTONE
329
and slit widths either 0.03 or 0.09 cm. CuKa (154 A) was the wavelength used in all measurements. Data extended from a scattering angle of about 3 to almost 200 mrad. The equivalent Bragg spacings are from 513 to 7.7 8. Figure 4 shows the entire scattering curve. It is a composite of several runs using different concentrations of nucleohistone and different instrumental parameters. Scattering data at the smaller angles, the radius of gyration region, were taken at as low a nucleohistone concentration as possible and with 0.03 cm slit width settings. In Figure 2 the scattered intensity, I(+), in the radius of gyration region has been plotted for a nucleohistone sample of concentration 1.75 mg/ml. The same data are plotted first as log [$*I (#)I vers’suB$” and then as log [I(+)] versus 4”. The first plot is that which would exhibit the cross-sectional radius of gyration of a long cylinder while the second is that which would exhibit the total radius of gyration of a globular particle. In both plots there is an initial short straight line of large slope followed by a more extended line of much smaller slope. Only in the first plot is this second line reasonably straight. The double straight line is superficially similar to that observed in the preceding study of sonicated DNA (Bram & Beeman, 1971) but we shall argue that the core and shell interpretation of the DNA data cannot be applied to the nucleohistone curve. We believe the initial straight line represents scattering from globular rather than fibrous regions of the preparation. It might be related to the knobby protuberances seen on the fibers in electron micrographs or it might be the remnant of a more intense scattering at very small angles from clusters of nucleohistone fibers. The second straight line we shall interpret as cross-section scattering. From the slope of the first straight line we calculate a total radius of gyration of approximately 12.5A. The exact result depends on how one subtracts an extrapolated background due to the second straight line. This is in reasonable agreement with the size of the knobby projections seen in the electron microscope. The cross-section radius of gyration derived from the slope of the second straight line is 36 f 2 A. If the first straight line is treated as the scattering from a long rod (which we think it is not) we deduce a cross-section radius of gyration of 52 A. The entire scattering curve is reproducible from one preparation to another and is independent of small changes in the temperature and conditions of solution providing the concentration of nucleohistone is kept below about 5 mg/ml. At higher concentrations the first straight line gradually disappears although the scattering at large angles remains unchanged. Samples in 20 m&r-PO, and 06 mM-PO, and at 4 and 22°C give similar scattering curves. Samples dialyzed against distilled water show a 10 to 20% increase in the slopes and intercepts of both the first and second straight line. In one experiment a sample was dialyzed against 10% formalin at pH 6.9 and then redialyzed against 06 mM-PO, buffer. The usual scattering curve was observed. Thus the formalin fixative used in our electron microscopy introduces no irreversible changes in conformation. This has also been noted by Nicolaieff (1967). At angles larger than about 20 mrad even quite high nucleohistone concentrations seem not to alter the scattering. A gel containing 260 mg nuoleohistone/mI. gave a curve almost identical with that of Figure 1. The distinguishing features of the scattering at larger angles are an inflection at 35 to 40 mrad (41 A Bragg spacing), a shoulder at 65 to 70 mrad (23 A), a distinct peak at 125 to 130 mrad (12.0 A). and and a shoulder at 172 mrad (8.9 A).
330
S. BRA&I
AND
II.
RI9
4. Discussion (a) Wide-angle
X-ray
scattering
The scattering at larger angles can be compared to the results of Wilkins, Zubay $ Wilson (1959) and Pardon, Wilkins & Richards (1967) on X-ray scattering from gels and drawn fibers of nucleohistone. Our inflection at 41 A may correspond to the spacings of 35 to 38 A which the above authors find to be fairly intense. They find larger spacings that are perhaps between fibers in the more concentrated preparations they used?. The shoulder at 23 A and the peak at 12.0 and shoulder at 9-O A appear in the results of Wilkins et aE. (1959). The latter two maxima are almost certainly due to DNA. The shape and position of these two maxima of the scattering curve agree well with those of the calculated scattering curve for DNA with the B form co-ordinates12.6 and 9.3 A-but very poorly with the scattering curve from the A form (see Bram & Beeman, 1971 for A and B form scattering curves). This implies that the major portion of the DNA in nucleohistone has a structure similar to B form. The observation of a spacing at 3.4 A from nucleohistone fibers supports this conclusion, but their finding does not indicate the relative amount of B form present. (b) Small-angle
X-ray
scattering
The spherically averaged X-ray scattering from an isotropic sample does not, in general, provide detailed information on the structure of the sample. One can be certain only of the density function (the distribution of the magnitudes of Patterson vectors) which is the Fourier inversion of the scattering curve. Additional information is available from the X-ray scattering to the extent that independent evidence justifies treating the sample in terms of at least a partially defined model. For instance, frequently one knows that the sample is a well-dispersed collection of identical particles. In the preceding work on DNA a wealth of chemical evidence plus electron micrographs of the actual samples used in the X-ray studies support the model of a well-dispersed collection of large rods of identical and uniform cross section. The import of our electron microscopy of nucleohistone is quite different. We see most of the material as fibers of fairly uniform diameter but with a great many knobby protuberances distributed apparently at random. There is much clustering of the nucleohistone into relatively dense regions a few 1000 d across. Insofar as these structures are also present in the dilute solution they must make their contributions to the X-ray scattering and an interpretation in terms of rigid rods alone is unjustified. Many features of the X-ray scattering support the electron microscope results. This is a welcome confirmation that the structures observed are not artifacts due to specimen preparation for the microscope but the structure is too complex to permit independent, quantitative interpretation of the X-ray results. The second straight line of Figure 2 is, we believe, clearly the cross-section scattering from the fibrous regions of the sample. The plot of log (+*1(#)] is much more linear than the plot of log I(+). The cross-section radius of gyration of 30 J%corresponds to a solid cylinder diameter of 85 d and is in reasonable agreement with the electron microscope observations. t A very recent finding is that our material clearly showed low-angle X-ray reflections at 105, 55, 38 and 22 11 when dried and then examined at high relative humidity in the laboratory of Dr J. F. Pardon. Thus, our proprtration proordur~s MC not rcsponnihlc for the absence of the 105 and 55 L% spacings from our aolutions.
STRUCTURE
OF NUCLEOHISTONE
331
The first linear region of Figure 2 is short and equally straight in the two kinds of plot but for the following reasons we believe it is not cross-section scattering. First a cross-section radius of gyration of 52 A gives a solid cylinder diameter of about 150 A which is larger than we observe in the electron microscope. A globular radius of gyration of 125 A is about that to be expected from the larger protuberances. Second, the dependence of the observed radius of gyration on the nucleohistone concentration is quite different for the first and second straight lines (both treated as cross-section scattering). In a range of low nucleohistone concentrations from O-5 to 5 mg/ml. the first radius of gyration decreases about 4% per mg/ml. increase in concentration. The decrease of the second radius of gyration is scarcely detectable, less than 0.676 per mg/ml. It should be noted that in the DNA measurements neither radius of gyration shows a concentration dependence greater than O*25°/0per mg/ml. With nucleohistone concentrations above 5 mg/ml., the slope of the first straight line decreases rapidly. One expects a smaller concentration dependence of a cross-section radius of gyration since only those rods whose axes are perpendicular to the scattering vector make an appreciable contribution to the observed scattering. Thus, although there are good arguments that the curve at the smallest angles does not represent cross-section scattering, its assignment to the knobby protuberances is less certain. The clusters a few 1000 A across, acting as scattering units, should contribute strongly at angles much smaller t’han we are able to reach but perhaps the t,ail of this scattering is present even at 3 to 5 mrad. Quite possibly cluster scattering was observed in the light-scattering measurements of Zubay & Doty (1959) on similar nucleohistone preparations. They reported a radius of gyration of about 1700 A. (c) illms per unit length Unfortunately, the model we propose makes it very diiiicult to extract useful information from our absolute intensity measurements. One might attempt a resolution of the scattering curve into components and the assignment of an absolute scattering power to t,hat part which seems to come from the long fibers. (The ratio of t,he intercepts of the two straight lines indicates that the fiber contribution is between 65 and 75% of the total.) This still does not permit a calculation of mass per unit length unless one knows what fraction of the total mass concentration of nucleohistone is contributing to the cross-section scattering. Our results can be stated as follows. Let us assume that the second straight line of Figure 2 represents cross-section scattering from the fibrous part of the preparation with only minor contributions from other structures. Extrapolation of this line to zero nngle provides the I,, of equation (4) of the preceding paper. We calculate for nucleohistone an I = 0.53 electrons per dalton assuming a 45% DNA content. For the partial specific volume we use 0.68 cm3/g from the work of Zubay & Doty (1959). Equation (4) now provides the mass per unit length of the fibers if the concentration of fibers in the solution is known. If we assume that all the dissloved nucleohistone contributes to the fiber scattering we obtain M/L = 1100 daltons/a. If only half contributes M/L = 2200 daltons/A. We find the intensity of scattering in the second straight line region to be proportional to nucleohistone concentration. This implies that the fraction of the sample contributing to the cross-section scattering is independent of concentration but it does not say what that fraction might be. However, M/L changes only slightly witSI concentration, source and method of preparation and with high speed centri-
332
S. BRAN
Ah’D
H.
RI8
fugation of the solution, although electron microscope observations show that these factors do alter the packing of the nucleohistone. This may indicate that most of the nucleohistine in fact, contributes to the cross-section scattering. We remind the reader that these computations use the two-component, not the more correct three-component formalism. These effects are probably small with nucleohistone which has a smaller charge and smaller surface to volume ratio than DNA. (d) Comparison with previous studies Our results are in fair agreement with the work of Luzzati & Nicolaieff (1963). They report a cross-section radius of gyration of 26 A for dilute solutions of calf thymus and chicken erythrocyte nucleohistone, and a mass per unit length of about 1500 daltons/A. Their scattering data are processed and presented without slit correction and are shown as plots of log scattered intensity versus log scattering angle as in our Figure 1. They do not report an initial steep slope as in the scattering curve seen in Figure 2, nor do they present electron micrographs of their preparations. It seems reasonable to assume that their preparations had structural complexities similar to ours and that t.heir mass per unit length results are subject to similar very large uncertainties. The 1500 daltons/ A reported should represent a minimum value. (e) Consideration of ,models All of these results are quite low compared to what one calculates for a solid cylinder of diameter 100 A and average density 14 g/cm3. Such a cylinder has a mass per unit length of 6600 daltons/A. If an AU/L of 1100 to 1500 daltons/,& is correct, it shows that nucleohistone has an extensively solvated, loosely packed structure. This M/L also requires that the mass per unit length due to DNA be three to four times that of the B form. Luzzati & Nicolaieff (1963) f ound that the small-angle X-ray scattering was consistent with a model containing four strands of DNA per cross-section. However, such a model is inconsistent with the results of our electron microscopy and other pertinent physical studies on nucleohistone. A structure containing four DNA’s per cross-section would have a minimum diameter of at least 50 A. In the electron microscope we observe that the nucleohistone consists of 25 A strands in regions which appear stretched or unravelled or have been partially digested with pronase (Ris, 1967). Multi-loop nodes, which would be expected from a multi-stranded nucleoprotein complex after pronase digestion, are not observed. The substructure observed at high resolution also does not agree with a multi-stranded model. If we reject multi-stranded models, the M/L can only be achieved by a super coiling or folding of the DNA. Incontrovertible evidence for super coiling, according to Pardon et al. (1967) is provided by the disappearance of small-angle spacings from nucleohistone fiber patterns upon stretching. Hydrodynamic studies by Zubay & Doty (1959) and Giannoni & Peacocke (1963) indicate that nucleohistone consists of single DNA molecules. Flow birefringence and dichroism experiments by Ohba (1966) show a characteristic shortening of the DNA in nucleohistone which implies a super helical configuration. With this evidence in mind we will show that a super-coiled model would also agree with all of our X-ray scattering data. Based on our electron microscopy, we will use 30 A as the thickness of the fiber. The cross-section radius of gyration-30 A-then determines that the distance from the center of the coiled fiber to the axis of the super
STRUCTURE
/
,
j
OF
,
,
NUCLEOHISTOSE
Tl?r
--,-
.-.
,
333
i.~T-’
.,---.
.
-r-
-t _
0 ccl 0 “0
-2 -
e a R e 0
-3 -
-4 -
L Scottermg
angle
(mrod)
Fro. 1. The slit corrected intensity plotted against the scattering angle for four runs of nucleohistone. (0) Data from 3 to 10 mred taken with 300 p slits, 1.7 mg nucleohistone/ml. at 4°C; from 10 to 45 mrad, 900 p slits were used, on sample containing 5.9 mg nuoleohistone/ml. at 4°C; (0) 8.2 mg nucleohistonejml. in 20 mM-PO,; 22”C, 900 p slits; ( A) 16 mg nueIeohistone/ml. in 0.80 mxPO,, 22’C. 900 p slits.
331
FIG. 2. The scattering curve of a typical run of nucleohistone after background subtraction slit correction (1.75 mg/ml., 4°C). (0) Intensity times scatt,cring angle; (0) intensity.
and
coil will be about 28 A. We must adjust the pitch of a helix to give the observed M/L using : N/L of DNA in nucleohistone Contour length ----~~~ _ JL(kgy + q pitch M/L of DNA in B form The minimum M/L of DNA in nucleohistone = 0.45 g DNA/g nucleohistone * 1100 daltons/A = 495 daltons/A. For a regular helix one then calculates t,hat the pitch would have to be 50 to 70 A to agree with the observed M/L of 1500 to 1100 daltons/A. Since these values for the M/L are minimum values, 50 to 70 if represent maximum values for the pitch. To obtain further information on t)he structure of nucleohistone in solution WC compared our wide-angle curve to computer-calculated scattering curves for several models using equations of the prevTious paper. (The models were chosen to have the observed cross-section radius of gyration.) Poor agreement was observed with cylindrical models. Figure 3 shows the curve (b) for a model simulating a regular helix, 600 A long with a pitch of 50 A. Eight scattering points spaced inside a square 20 A on a side centered at a radial distance of 30 A was t,he basic unit for constructing the computer model. The co-ordinate of the unit center was raised by A 2 (5 A here) and rotated by A Z/pitch. A more detailed discussion of the model calculations is given by Bram (1968). The calculated scattering curve shows two sharp maxima at spacings of 48 and 23 8. As can be seen in Figure 3 the nucleohistone curve (a) shows a distinct inflection at 35 to 40 mrad (44 to 38 A) and a distinct shoulder at 68 mrad (22 8). The in%ections of the experimental curve correspond in both position and relative intensity to those from a model of a regular helix, similar to Figure 3 (curve b), but having a pitch of 46 A and radius of 30 A. This pitch agrees with the small-angle X-ray scattering
STRUCTURE
OF
K UCLEOHISTONE
335
.
30
50 70 Scattering angle ( mrad)
FIG. 3. Comparison of the calculated scattering curve from a model simulrtting a super coil of pitch 50 A, radius 30 A and fiber thickness 22 A to the nxperimental c~~rvc. The filled circles represent a composite of 3 runs in 0.80 mx-PO,.
mass per unit length and the observation of a semi-meridional spacing at 38 A from nucleohistone fibers (Wilkins et al., 1959). The 30 A radial dimensions would agree well with the presence of the intense 30 A equatorial reflections in the nucleohistone fiber diagrams. Since the electron micrographs showed few regions which would be interpret’ed as regular, orderly coils, we considered the scattering from non-uniform helices. A nonuniform helix was simulated by allowing the pitch and radial distance to vary along the axis. The maxima from the computer-calculated scattering curves for irregular coils were considerably broader t’han t’hose observed from regular helices and fit better to the experimental curves.
5. Conclusions Carefully prepared calf thymus nucleohistone appears in electron micrographs to be a network of fibrils about 100 A in diameter. There are numerous knobby protuberances where the fibril is locally thickened to 200 or 300 A. The network seems to be continuous but interconnected (or overlapping) in a complex, apparently random, manner. The compactness of the network varies greatly. In some areas individual fibrils can be followed for several 1000 A. In others clustering is so great that individual fibrils are resolved with difficulty. Occasionally we see a fibril thinned down to a diameter of about 25 A. A model for nucleohistone containing one double helix of DNA and associated histone non-uniformly coiled with an average pitch of 45 A is most consistent with our results. X-ray and light scattering suggest that the same nucleohistone preparations in dilute solutions are a network of fibrils similar to what is seen in the electron microscope. A part of the X-ray curve behaves as the cross-section scattering from long rods and the measured radius of gyration is in good agreement with the electron microscope diameter. The X-ray scattering at the smallest angles, we believe, is produced by the protuberances or by the large clusters.
336
S. BRAN
ASI)
II.
RTS
We have made an absolute intensity calibration of our scattering curve but can report only a minimum mass per unit length of 1100 daltons/A. It could be several times larger than this if only a small fraction of the dissolved sample contributes to the cross-section scattering. We thank Professor W. W. Beeman This research has been supported Institutes of Health. One of us (H.R.) acknowledges the Award (KG-GM-21, 948) and research Health.
for numerous helpful conversations. by research and training grants of the National awards of a Public Health Service Career Program grant, (GM-04738) from U.S. National Institutes of
REFERENCES Anderson, T. (1951). N.Y. AC&. Sci., 13, 130. Bram, S. (1968). Thesis, University of Wisconsin. Bran-r, S. & Beeman, W. W. (1971). J. MOE. Biol. 55, 311. Christensen, R. E., Coon, F. B. & Derse, P. H. (1967). P&burgh Conference 012AnaZyfiicoZ Chemiat?y. (Available from Jarrell-Ash Company as reprint no. 74). Dische, Z. (1955). In The NucZeic Acids, cd. by E. Chargaff 8: J. N. Davidson. vol. 1, p. 285. New York: Academic Press. Fiske, C. H. & Subbarow, Y. (1925). J. BioZ. Chem. 66, 375. Frederiq E. & Houssier, C. (1967). Europ. J. Biochem. 1, 51. Giannoni, G. & Peacocke, A. R. (1963). Biochem. biophys. Acta, 68, 157. Johns, E. W., Philips, D. M. P., Simpson, P. & Butler, J. A. V. (1960). Biochem. J. 77, 631. Leith, D. (1964). Ph.D. Thesis, University of Wisconsin. Lowry, 0. H., Rosebrough, M. .J., Farr, ,4. L. & Randall, R. J. (1951). J. BioZ. Chem. 193, 265. Luzzati, V. & Nicolaieff, A. (1963). J. Mol. BioZ. 7, 142. Nicolaieff, A. (1967). Sm.uZZ-Angle X-Ray Scattering, cd. by H. Brumberger. New York: Gordon & Breach. Ohba, Y. (1966). Biochem. biophys. Acta, 23, 76. Pardon, J. F., Wilkins, M. H. F. & Richards, B. M. (1967). Nature, 215, 508. Ris, H. (1961). Canad. J. Genet. 3, 95. Ris, H. (1967). Regulation of Nucleic Acid and Protein Biosynthesis, ed. by V. Konigsberger & L. Bosch. Amsterdam: Elsevier. Spa&man, D. H., Stem, W. H. & Moore, S. (1958). Andyt. Chem. 30, 1190. Vinograd, J., Bruner, R., Kent, R. & Weigle, J. (1963). Proc. Nat. Acad. Sci., Wwh. 49, 902. Wilkins, M. H. F., Zubay, G. & Wilson, H. R. (1959). J. Mol. BioZ. 1, 179. Zubay, G. t Doty, P. (1959). J. Mol. BioZ. 1, 1.