Evidence of nucleosomes in situ and their organization in chromatin and chromosomes of Chinese hamster cells

Evidence of Nucleosomes In Situ and their Organization in Chromatin and Chromosomes of Chinese Hamster Cells Lee S. Chai and Avery A. Sandberg

ABSTRACT: Freeze-fracture studies of intact metaphase Chinese hamster cells indicate that the chromosomes are composed of compactly aligned, fiat and hexagonally shaped bipartite disks. The overall dimensions of a bipartite disk are about 140 ± 25 A in height, 100 ± 20 A in width, and 54 + 4 ,~ in thickness. The center-to-center distance of two units of a bipartite disk is about 34 ± 3 A. The core of a disk appears to contain four substructures that measure about 25 ± 5 A in diameter. Through the center of the disk core there is an axial structure measuring about 20 ± 5 A in diameter, which spans and interconnects similar bipartite disks to form a continuous linear array. A structure measuring 20 ± 4 A in diameter is wound around the edges of the disk core and interconnects with the next bipartite disk. The connecting strand is parallel to the axial structure.. In addition, there appear to be bridgings between the curled structure and disk core. The chromatin of intact interphase cell nuclei shows similar bipartite disks with dimensions and features similar to those of metaphase chromosomes. The bipartite disks are aligned in a linear array in both chromatin and chromosomes.

INTRODUCTION C h r o m a t i n and c h r o m o s o m e s in t h i n section or spread on an air-water i n t e r p h a s e h a v e e x h i b i t e d structures m e a s u r i n g about 100 ,~ and 2 5 0 - 3 0 0 ,~ in d i a m e t e r [ 1 - 9 ] . T h e 100 ,~ fiber c o n s t i t u t e s a r e p e a t i n g u n i t that has a s p h e r i c a l structure and is k n o w n as the n u - b o d y or n u c l e o s o m e s [ 1 0 - 1 3 ] . E v i d e n c e d e r i v e d from X-ray diffraction [ 1 4 - 1 8 ] , n e u t r o n scattering [ 1 9 - 2 3 ] , b i o c h e m i c a l and o t h e r studies [ 2 4 - 3 4 ] s u p p o r t s the e x i s t e n c e of t h e s e r e p e a t i n g units. A n u m b e r of investigators h a v e prop o s e d v a r i o u s m o d e l s of c h r o m a t i n s t r u c t u r e based on the a b o v e studies [11, 3 5 - 4 6 ] . H o w e v e r , a l m o s t all the studies h a v e dealt w i t h d i s r u p t e d n u c l e i or i s o l a t e d c h r o m a t i n or c h r o m o s o m e s , the p r o c e d u r e s p o s s i b l y d e s t r o y i n g s o m e of the fine order of the n a t i v e structure. Thus, there is u n c e r t a i n t y as to the real s t r u c t u r e of the r e p e a t i n g u n i t s in the o r g a n i z a t i o n of c h r o m a t i n and c h r o m o s o m e s . In o r d e r to d e t e r m i n e w h e t h e r s u c h r e p e a t i n g u n i t s exist in situ, freeze-fracture studies of intact C h i n e s e h a m s t e r i n t e r p h a s e and m e t a p h a s e cells w e r e u n d e r t a k e n . As far as w e k n o w , this is the first s u c h report of in situ studies. T h e p r e s e n t results s h o w that c h r o m a t i n and c h r o m o s o m e s are, in fact, m a d e of r e p e a t i n g units. H o w From the Departments of Genetics and Endocrinology, Roswell Park Memorial Institute, Buffalo, New York. Address requests for reprints to: Dr. Avery A. Sandberg, Departments of Genetics and Endocrinology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N Y 14263. Received August 12, 1980; Accepted September 4, 1980.

361 Copyright © Elsevier North Holland, Inc., 1980 52 Vanderbilt Ave., New York, NY 1 0 0 1 7

Cancer Genetics and Gytogenetics 2, 381 - 380 (1980) 0165-4608/80/06036120502.25

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L . S . Chai and A. A. Sandberg ever, the repeating unit is not a sphere or a p l a t y s o m e but rather appears to be a hexagonal bipartite disk. The bipartite disks are stacked face to face and probably aligned in a linear array of 100 x 140 A in diameter. The detailed structural organization of the repeating units is discussed. A brief account of these results was presented at the 19th annual meeting of American Society of Cell Biology in Toronto [47].

MATERIALS AND METHODS A Chinese hamster cell line (Don) was used in this study. Culture conditions for growth in monolayers have been described [48,49]. Log-phase cultures were grown in RPMI m e d i u m 1640 [50] s u p p l e m e n t e d with 10% fetal calf serum, treated with 0.09 /~g/ml of Colcemid for 3 hr, and the metaphase cells shaken loose and pelleted at 600 x g for 5 min. The pelleted cells were either fixed with 0.5% glutaraldehyde alone or first fixed with ethanol-acetic acid (3 : 1) for 30 min to 2 hr, then w a s h e d once with buffer for 15 min and postfixed with 0.5% glutaraldehyde in Millonig phosphate buffer (pH 7.3) for 1 - 2 hr [51]. The pelleted cells were overlayered with 30% glycerine and 5% ethylene glycol in the buffer overnight; cells were not disturbed in order to m i n i m i z e distortion. The cell pellets were m o u n t e d on c o p p e r holders and frozen in liquid freon at -180°C. Freeze-fracture was carried out in a Polaron FreezeFracture apparatus at --115°C. Carbon-platinum was s h a d o w e d at a 45 ° angle and then i m m e d i a t e l y carbonized at a 90 ° angle. The replica was floated on water containing a few drops of 0.01% Bacitracin to m i n i m i z e s u d d e n dispersion. S p e c i m e n debris was removed in graded Clorox, followed by concentrated Clorox overnight [52]. The replica was picked up on uncoated copper grids and examined in a JEM-7 electron microscope at an accelerating voltage of 80 or 100 kV. Micrographs were taken at the instrumental magnification of 4 0 - 1 0 0 , 0 0 0 x. RESULTS Metaphase Cells Freeze-fracture replicas of a Chinese hamster metaphase cell showed several chromosomes. A fracture profile along the longitudinal length of a chromatid revealed clear delineation of the c h r o m o s o m e from the c y t o p l a s m with a narrow region that appeared to be the centromere (Fig. 1). A chromatid fracture in cross section showed a nearly oval profile (Figs. 1 and 2). Cross sections accounted for more than 80% of the c h r o m o s o m e fractures, with the r e m a i n d e r occurring p e r p e n d i c u l a r to the arms of the chromosomes. At low magnification, the chromatids a p p e a r e d to consist of globular structures. Fractures on different planes showed distinctly different facets of the structural alignment. Fracture occasionally resulted in the appearance of chromosomal detail at different plateaus (Fig. 3). In other areas, the orientation of the globular structures suggested a lattice arrangement (Figs. 4 and 5c), with some of them possessing characteristics of bipartite disks (Fig. 5, see also Fig. 7). The disks a p p e a r e d to be stacked face to face and possibly interconnected by one or more strands (Fig. 5). The disks in face view showed hexagonal features (Fig. 5). Details of the disks and their substructure became evident w h e n the figures were enlarged further using a Nikon Profile Projector. In Figure 6 are shown several aligned disks appearing to have hexagonal shapes. A d i s k shown in Figure 6a has partially emerged from the plane and clearly shows a w o u n d strand along the perimeter. In this disk, the core and strand were separated but were possibly connected by bridgings (Fig. 6a,b). In Figure 6a a unit is shown that has four electron-dense struc-

F i g u r e 1 Freeze-fracture replica of an intact Chinese hamster m e t a p h a s e cell showing several chromosomes. A fracture that occurred along the longitudinal length of a chromatid shows clear delineation of the c h r o m o s o m e from the cytoplasm; the narrow region appears to be the centromere (c). Two chromatids in cross section are s h o w n at the bottom of Figure 1. Bar indicates

1.0/~m. F i g u r e 2 A chromatid in cross-fracture shows two straight sides and remaining edges with semicircle. There are globular structures (in low magnification) throughout. Bar indicates 0.1 ~m.

F i g u r e 3 A fracture s h o w i n g different p l a t e a u s of a c h r o m o s o m e . Bar i n d i c a t e s 50 n m . F i g u r e 4 O r i e n t a t i o n of globular s t r u c t u r e s of a c h r o m o s o m e in a lattice pattern. Bar indicates 50 n m .

F i g u r e 5 Enlargement of a portion of Figure 3 showing the alignment of disks in continuous succession. They appear to be stacked face to face and interconnected by a strand or strands (arrows). Disks, w h i c h appear to be in a face view, show hexagonal features (thick arrows) (a and b). In c are s h o w n various facets of the disk; some of t h e m appear compact and reveal a lattice packing (arrows). Bar indicates 100 A.

F i g u r e 6 Details of the disks and their substructures become evident when the figures are enlarged further. Several aligned disks are shown in the center of a. Another disk, which has partially emerged from the plane, is also shown (right bottom). Polygonal characteristics of the disks are evident (arrow heads). In this disk the core and strand that make up the polygonal perimeter are separated but interconnected by bridging arms (white arrows). Some disks appear to (upper left) consist of four electron-dense structures (white bars). A rod-like structure, which extends between these electron-dense structures (opposing white arrows), is also seen. In addition, a curled structure encircles the above units. The insert shows a double coil that appears to be left-handed. In b, of the several disks shown, one possesses a slightly elongated hexagonal shape. There are extending arms from the core that associate with the strand making up the hexagon (white arrow heads). The disks in c clearly show hexagonal features. The two opposing disks, which are in face view, show an association point (small arrow). There is a short strand (long arrow) that interconnects one disk (profile) to the next (face view). Bar indicates 100 A.

F i g u r e 7 T h e bipartite characteristics (double arrow h e a d s in a, b, a n d d) a n d h e x a g o n a l i t y (large arrow h e a d s in d) of d i s k s are s h o w n . T h e r e are two c o n n e c t i n g s t r a n d s b e t w e e n bipartite d i s k s (arrows in a, b, c, d, a n d e). O n e of t h e c o n n e c t i n g s t r a n d s is located at t h e axial position, w h e r e a s t h e o t h e r is located at the p e r i p h e r y of t h e disk. T h e bipartite disks, w h i c h a p p e a r to be split or set apart, s h o w a n e l e c t r o n - d e n s e s t r u c t u r e in t h e center, s u g g e s t i n g a c o l l a p s e d axis (open arrow in h), w h e r e a s s o m e s h o w a h o l e in t h e center (open arrows in a a n d f). W h e n d i s k s are s e e n from a n o t h e r angle (possibly from t h e top), a c o n n e c t i n g " S " s h a p e d s t r a n d (arrows in f a n d g) c a n be seen. Bar i n d i c a t e s 100 ,~.

368

L . S . Chai and A. A. Sandberg tures measuring 25 ± 5 A in diameter. In addition, there was a strand-like structure that curled a r o u n d the above units. In other areas, the w o u n d structure a p p e a r e d in the form of a double coil (Fig. 6a inset). The overall d i m e n s i o n s of a hexagonal disk were about 140 ± 25 ,~ in height, 100 ± 20 ,~ in width, and 54 ± 4 A in thickness; the disks possessed two long sides measuring about 70 -+ 15 A each and four short sides measuring about 37 ± 7 A each. At higher magnification the bipartite characteristics of a disk became more evident (Fig. 7 a - d ) . A unit (one disk of the double set) of a bipartite disk measured about 20 A in thickness (small arrows in Fig. 7 a - c) and the center to center distance was about 34 ,~. There were two connecting strands between the bipartite disks (Fig 7a, c, e), one located p e r i p h e r a l l y and the other more centrally (Fig 7a, c, e). The one in the central position and the peripheral strands measured about 20-+ 4 A; however, the size of the strand in the central position was more variable. The spacing between adjoining bipartite disks was also variable, in some areas measuring up to 80 A, while in others the disks were more compact and showed hardly any space at all. The peripheral strand was often S-shaped (Fig. 7f,g). An electron-dense structure was present in the center of some of the unit disks (Fig. 7h, open arrow), whereas others often showed only a hole in the center (Fig. 7a,f, open arrows). Inspite of the presence of such holes, the disks retained their usual structure. There were association points between the core and the w o u n d strand that surr o u n d e d it; d e p e n d i n g on the angle of fracture, up to eight bridgings were noted (Fig. 8).

Interphase Cells Freeze-fracture replicas of Chinese hamster interphase cells showed irregularly distributed chromatin (Fig. 9a). Enlargement revealed various aspects of the bipartite disks (Figs. 9b, c, d, and e). Thus, the bipartite disks were stacked face to face, interconnected by two strands (Figs. 9b and c) and oriented in rows in the form of a lattice (Fig. 9b, white arrowheads). There were bridgings between the disk core and w o u n d strand that made up the hexagonal perimeter (Figs. 9d,e), as well as periodic crosslinkings between two adjoining rows of stacked disks (Fig. 9f). A single row (a linear array) measured about 100 x 140 A in diameter; two rows in parallel cross-link measured about 200 or 280 A in diameter.

DISCUSSION Bipartite Disks There is concensus that nucleosomes are spherical structures that are aligned like "beads on a string" [10-13]. Recent reports consider nucleosomes to be platysomes aligned edge to edge [45,46]. Our results provide evidence that in interphase chromatin and metaphase chromosomes a nucleosomes in situ may assume the form of a flat, slightly elongated hexagonal bipartite disk. The overall d i m e n s i o n s of a bipartite disk in face view and profile are given in Figure 10. The d i m e n s i o n s are similar to those obtained with X-ray and neutron scattering studies [ 1 4 - 23]. The height to w i d t h ratio of a hexagonal disk was about 2 : 1. Others have reported a similar ratio [53,54]. The disk may resemble a platysome, i.e., both are flat, but the n u c l e o s o m e is differentiated by being a hexagonal bipartite disk. Similar features (hexagonality, bridgings between the core and the hexagonal perimeter, and stacking of the disks) may be seen in p u b l i s h e d figures of freeze-fracture studies of isolated chromatin (see Figures 1 and 2 of [35] for stacking of the nucleosomes and Figure 3 for hexagonal forms and

Nucleosomes in DON Chromatin and Chromosomes

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Figure 8 An association between the core and strand, which makes up the hexagon, is further shown. A number of bridging arms are to be noted (arrows and white arrow heads), the number depending on the angle of fractures. Bar indicates 100 A. bridgings). The authors, however, failed to point out these characteristics [36]. Nevertheless, the results are mutually confirmatory. Our results are also in agreement with some of the features observed in purified nucleosome cores, e.g., the stacking of bipartite disks and hexagonal lattice alignment [55,56]. However, most of the studies to-date have not reported the hexagonal bipartite characteristics. Conformation of the DNA There is much evidence to support the concept that DNA is w o u n d around a histone core [20-22,26]. In our studies, a strand, which measured about 20 A in diameter, w i n d i n g along the edge of the disk core and making-up the hexagonal perimeter was evident. In agreement with earlier evidence, we interpret this structure to be DNA. Crick and Klug have suggested that DNA could be kinked at some points [57], and hence, we postulate that the DNA may be kinked at each corner of the hexagon at a 60 ° angle. The kinking of the DNA double helix at a 60 ° angle was tested by using Corey-Pauling-Koltun (CPK) models. Preliminary results indicate that it is feasible, indeed, on the basis of these models for the DNA to be w o u n d with kinks at certain intervals around the disk as shown in Figure 6a. A more detailed analysis of this area will be published separately.

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F i g u r e 9 Freeze-fracture replica of part of an intact Chinese hamster interphase cell nucleus in which the chromatin is irregularly distributed. (b) An enlargement of area from a showing bipartite disks (large arrows). Some of the disks are connected by strands (small arrows). The disks are stacked face to face (open arrow on upper left) and aligned in rows (a group of white arrows). (c and d) Higher magnifications of area from a showing bipartite disks (small arrowheads), which are stacked face to face and interconnected by two strands (arrows in c). There are bridging arms between the disk core and the strand (white arrowheads in d and e). Two rows of stacked disks are periodically associated with each other (arrowheads in f). Bars indicate 0.1 ~m and 100 A, respectively.

A h e x a g o n a l bipartite d i s k p o s s e s s e s four long and four short facets of D N A p l u s a c r o s s - o v e r s e g m e n t b e t w e e n the t w o u n i t s (of a bipartite disk) and an inter-bipartite strand. T h e l e n g t h of a long facet is e q u i v a l e n t to about 20 base pairs (bp) of DNA; that of a short facet a b o u t 10 bp. Each s e g m e n t of the D N A was p l a c e d in a corres p o n d i n g p o s i t i o n a n d a s c h e m a t i c p r e s e n t a t i o n of the bipartite d i s k is g i v e n in Figure 11a. It s h o w s 10 kinks at i n t e r v a l s of 10 and 20 bp w i t h the total a m o u n t of D N A on the p e r i m e t e r of a bipartite d i s k being e q u i v a l e n t to a b o u t 140 bp (1 and 6/8 turns). T h i s is in a g r e e m e n t w i t h b i o c h e m i c a l data [29,31 - 33,58,59]. T h e f r e q u e n c y of kinks

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L . S . Chai and A. A. Sandberg

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I 201:4~ Figure 10 Schematic illustration of a nucleosorne in a flat face view and in profile, according to the description in the text. At right, the hexagonal perimeter represents the DNA. The broken lines and extended segment at the bottom indicate the DNA cross-ever within the bipartite disk and inter-bipartite regions. The four units within the hexagonal frame represent four histone complexes (in a bipartite disk, it would be octamer histone complexes). The open circle at the center is the axis and represents histone H1. At left, a bipartite disk possesses a narrow space of 10 A or less. The center to center distance of two units of a bipartite disk is about 34 A. The axial arrow indicates one molecule of histone H1. The arrowhead (15 -+ 5 A) dipicts the amino terminal region and the tail of the arrow the carboxyl terminal region (50 -+ 15 ~.). (The knob of the hydrophobic region of the histone H1 is not shown.)

occurred at multiples of 10 bp, w h i c h is also in agreement with the 10 bp DNA fragments resulting from e n z y m e treatment [26,59,60]. The inter-bipartite strand consists of about 40 bp of DNA. However, the latter was not visualized where the disks were compactly stacked. The variation in the inter-bipartite region may have been due to the angle of fracture. Therefore, it was not clear w h e t h e r the 40 bp strand was present in every inter-bipartite region. It has been reported that some of the linker DNA is usually short, i.e., 25 bp in actively dividing cells and 60 bp in resting cells [61].

Interaction between DNA and Histone Complex Our results indicate that there are four substructures w i t h i n a single unit of a bipartite disk w i t h points of association w i t h the w o u n d strand forming the hexagon. In some of the material observed by us, up to eight association points could be seen, but this was not consistent, possibly due to the angle of fracture and/or orientation of the disks (a schematic model of the interaction between the DNA and histone is proposed in Fig. 11) The present results agree with the formation of a tetramer [11, 62, 63]. These reports indicate that a tetramer is c o m p o s e d of one molecule each of histone H2a, H2b, H3, and H4. Each histone molecule may assume a globular structure possessing a n u m b e r of facets. Histones with hydrophobic facets may face each other and thus form a tetramer and then an octamer. Another facet, possibly hydrophilic, may face out and be exposed to the inter-bipartite region. The remaining facet from a central hydrophobic globular region may extend two arms, one bearing an amino and the other a carboxyl terminal [64-66] and may interact with the DNA [67-72]. The postulated 16 points of association of an octamer per unit bipartite disk is further il-

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(B) Figure ll (A) Conformation of DNA, location of octamer histones and their association with each other. (The position of histones is postulated according to [41] and [95].1 DNA kinks at each hexagonal corner at a 60 ° angle in respect to the dyed axis of the double helix. The formation of kinks is related with the occurrence of the major groove; the minor groove is exposed to the outside. There are 10 kinks within a bipartite disk at 20, 10, 10, 20, 20 (intra-bipartite segment) 20, 10, 10, and 20 bp intervals {total of 140 bp with 1 and 6/8 turns). There is an interbipartite strand of about 40 bp. Kinks and segments of the DNA are numbered according to the path of DNA and positions of the respective histone. Dimers (upper and lower dimers, respectively) seem to have a greater affinity for each other than tetramers. A split octamer shown in the figures is characterized by displacement of the axis, histone H1, from the tetramer on the right. The displacement of the axis has no disruptive effect on the rest of the histone complexes. (Histone H4 possibly stabilizes the incoming DNA and histone H3 stabilizes the outgoing DNA [95].) H2a and H2b alternate within a bipartite disk [41]. (B) There are two association points for each histone with the DNA double helix: one at the amino terminus and the other at the carboxyl terminal. The amino terminus associates with only one given strand (the solid line/ and the carboxyl terminus with the opposing strand (broken line). There are no interchanges or random associations. Furthermore, every 10 bp intervals of the DNA double helix or every 20 base intervals of the DNA single strand are stabilized by each arm.

l u s t r a t e d i n F i g u r e 11. T h e a m i n o t e r m i n u s a s s o c i a t e s w i t h o n l y o n e g i v e n s t r a n d (for e x a m p l e t h e s o l i d line) a n d t h e c a r b o x y l t e r m i n u s w i t h t h e o p p o s i n g s t r a n d ( b r o k e n line). T h e r e are n o i n t e r c h a n g e s or r a n d o m a s s o c i a t i o n s . F u r t h e r m o r e , t h e D N A is a s s o c i a t e d w i t h e a c h a r m at 10 b p i n t e r v a l s w h e n i n a d o u b l e h e l i x or at 20 b p intervals when in a single strand. A f u r t h e r p o s s i b i l i t y is t h a t t h e h y d r o p h i l i c facets o n b o t h s i d e s of a n o c t a m e r ( b o t h s i d e s of a b i p a r t i t e disk) m a y h a v e t h e c a p a c i t y to i n t e r a c t w i t h n o n - h i s t o n e p r o t e i n s . T h e r e is i n c r e a s i n g e v i d e n c e t h a t u b i q u i t i n t r a n s i t i o n a l l y i n t e r a c t s w i t h hist o n e H 2 A [73]. T h u s , t h e site of u b i q u i t i n i n t e r a c t i o n w i t h t h e H 2 A m a y w e l l b e at t h e e x p o s e d h y d r o p h i l i c f a c e t of t h e i n t e r - b i p a r t i t e region. A c e t y l a t i o n , m e t h y l a t i o n , or p h o s p h o r y l a t i o n m a y o c c u r at t h i s r e g i o n .

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A Possible Role of Histone H1 in the Alignment of the Nucleosomes The present results indicate an axial structure which is located at the center of the disk and which may be responsible for the face-to-face stacking of the bipartite disks and their linear array. Furthermore, we postulate that histone H1 is a major compon e n t of the axial structure (schematics are shown in Fig. 12; the lengths depicted are not in scale). This interpretation is derived from the following considerations. The tertiary structure of histone H1 consists of three regions: a relatively short hydrophilic region located near the amino terminal, followed by a relatively small globular hydrophobic region and ending in a relatively long hydrophilic region composed of about one-half of the total polypeptides [74- 80]. Thus, histone H1 can be viewed as a hydrophilic rod with a small hydrophobic knob (amino terminal residues of 4 0 - 1 1 5 , which is about one-half of the other core histones) near one end. It is suggested that the basic residues of the globular region of H1 are on the surface; such specific spatial configurations of positive charges may act as a n i o n receptors [78]. It is, therefore, possible that the globular region of histone H1 and the core histones may have ionic interaction. The presence of a hole in the center of the disk indicates that the axis may be easily removed without disrupting the core histone complex, which agrees with the easy displacement of histone H1 u n d e r certain ionic conditions. Reports on the formation of chemical cross-links between histone H1 with all four nucleosomal histone cores [81 -83] and with histone H2a [84] have appeared. Additionally, others have reported the formation of dimers and polymers of histone H1 in chemical crosslinking and high salt concentration [85 - 87,96]. Therefore, an axial structure through the disk core consisting of histone H1 is consistent with present knowledge. Since there is one histone H1 to one octamer complex [88], the formation of a continuous axial structure requires a H1-H1 interaction. The short a m i n o terminus of one molecule could interact with the relatively long carboxyl terminus of the next, probably by ionic bonding. The inter-bipartite region is, therefore, bisected by the short head of the amino terminus (which belongs to the preceding bipartite disk) and the long tail of the carboxyl terminus (which belongs to the following bipartite disk). Moreover, histone H1 stabilizes inter-bipartite DNA strands. It is, therefore, possible that an inter-bipartite DNA strand may associate with the axial histone H1 w h e n the DNA strand recoils and is then located between bipartite disks.

Figure 12 Profile of bipartite disks and their inter-bipartite regions are illustrated. Where the inter-bipartite DNA strand is short, the DNA may be aligned parallel to the axis (right). Where the inter-bipartite DNA strand is long and/or the axial structure has changed its conformation, the inter-bipartite DNA may recoil between two bipartite disks (left). It is possible that DNA may associate with the axial structure when the DNA is recoiled. Length and conformation of the inter-bipartite DNA strand may be correlated with variations of histone H1. Thus, length of H1 may also be variable. There is directionality in the orientation of histone H1. The short arrowhead indicates the amino-terminal region and the long tail end indicates the carboxylterminal region. The dots between the head and tail indicate some kind of interaction.

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Nucleosomes in DON Chromatin and Chromosomes

Higher Order Packing The bipartite disks are stacked face to face (100 × 140 A in diameter) and the axial structure going through the center of the disks probably maintains the disks in a linear array. The bipartite disks in one array are periodically cross-linked with the edges of bipartite disks of the adjoining array. The profile diameter is about 280 fi~ (see Figs. 5a and 9f}. There are two axes of orientation in these parallel arrays: a straight linear one (profile) and another one wavy or zigzagging (top view), the axes being p e r p e n d i c u l a r to each other. These features are schematically illustrated in Figure 13. Others have reported a similar structure measuring 2 5 0 - 300 A in diameter (not in freeze-fracture material) [7]. It is interesting to note that the same structure was converted to two t h i n n e r linear structures of 100 A in diameter in the presence of a chelator (EDTA), indicating that divalent cations, presumably Ca 2÷ and Mg 2÷, are involved in the formation of a thicker 2 5 0 - 300 A fiber [7]. The thicker fiber may represent side by side association of two t h i n n e r 100 A fibers [7, 89, 90]. Freezefracture studies of nuclei and metaphase chromosomes have also shown that the 250 A fiber appears to consist of two 130 A units [91]. On the other hand, the 100 A fibers can be converted into 200 A fibers by the addition of Mg 2* ions [92]. The parallel alignment of two linear arrays in the present results agrees with those of other observations. Furthermore, it is possible that the parallel alignment of two linear arrays may be formed by their doubling back. The periodic interaction between two parallel linear arrays appeared to occur at intra- and inter-bipartite regions of DNA crossover. Possible, divalent cations may be b o u n d to phosphate of the parallel strand, thus causing their interaction (Fig. 13). There was variation in the length of the linear arrays. At a given angle, the alignment of a linear or a parallel array was irregular. In some areas, the disks were aligned in such a way as to give the appearance of a lattice, as illustrated in Figure 14. It is in

(A)

Figure 13 Packing of bipartite disks is illustrated in a linear profile (A) and in a top view (B): A 280 A unit is formed by doubling back of the 100 × 140 A unit and by the disks facing each other at the inter-bipartite DNA regions, thus exposing angulated hexagonal facets. The direction of the bipartite disk packing is indicated by the axial arrowhead. The short bars between adjoining DNA strand indicate possible ionic interactions.

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Figure 14

A face view alignment of bipartite disks in a paracrystalline lattice. The interbipartite DNA strand regions face each other and are enclosed within but expose hexagonal facets (see Figure 13a), which form a unit of about 280 A in diameter. Incoming and outgoing DNA strands are indicated by segments of double line. A possible ionic interaction is indicated between them. Exposed hexagonal facets may also be neutralized by ionic interactions (dots). Separation of the linear arrays of the bipartite disks depend on environmental conditions, e.g., it can result in a single linear array of 140 A units or an enclosed 280 A or multiples of it. It is possible that the lattice packing may not encompass the entire chromosome. It is estimated that 6 - 8 cm long DNA may be packed within a 5/~m long chromatid if the DNA were packed according to our schematics.

a g r e e m e n t w i t h the crystal lattice o b s e r v e d in t h i n sections of p u r i f i e d h i s t o n e cores [55, 56]. W h e t h e r t h e lattice p a c k i n g e n c o m p a s e s the entire c h r o m o s o m e or not, espec i a l l y in r e l a t i o n to n o n - h i s t o n e proteins, is not clear. W h e n the p a c k i n g of the bipartite disks stacked in a l i n e a r array is seen from three different angles, a linear (profile), a w a v y or zigzagging (top view), and a lattice (face v i e w ) o r i e n t a t i o n are seen.

The Interphase Chromatin and the Metaphase Chromosomes A l i g n m e n t of the bipartite disks in the c h r o m a t i n of i n t e r p h a s e n u c l e i was s i m i l a r to that in m e t a p h a s e c h r o m o s o m e s . Others h a v e r e p o r t e d similarities of certain aspects of n u c l e o s o m e a l i g n m e n t in i n t e r p h a s e c h r o m a t i n v e r s u s m e t a p h a s e c h r o m o s o m e s [93, 94]. Differences o b s e r v e d in the p r e s e n t w o r k p r o b a b l y r e s i d e in the e x t e n t of c o m p a c t i o n . T h e arm of a c h r o m a t i d in cross-fracture s h o w s d e f i n e d d i m e n s i o n s of about 1.0 × 1.5 ~ m in d i a m e t e r . E v e n t h o u g h there was s o m e p a c k i n g of the n u c l e o s o m e s in i n t e r p h a s e c h r o m a t i n , similar to that s e e n in the m e t a p h a s e c h r o m o s o m e s , the fractured i n t e r p h a s e c h r o m a t i n was i r r e g u l a r l y d i s p e r s e d . Yet, t h e r e was lattice p a c k i n g of bipartite disks in s o m e areas in a m a n n e r similar to that in m e t a p h a s e c h r o m o s o m e s . Thus, c h r o m a t i n of i n t e r p h a s e n u c l e i was s h o w n to be c h a r a c t e r i z e d by partial lattice packing, the extent and pattern p o s s i b l y d e p e n d i n g On the p h a s e of the n u c l e u s in the cell cycle.

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The authors are grateful to Drs. K.S. McCarty, H. Weinfeld, and S. Matsui for critical reading of the manuscript.

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