Chromosoma (Berl.) 72, 1-14 (1979)
CHROMOSOMA 9 by Springer-Verlag 1979
Higher Order Structure of Chromosomes Tadashi A. Okada and David E. Comings Department of Medical Genetics, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010, U.S.A.
Abstract. Isolated Chinese hamster metaphase chromosomes were resus-
pended in 4 M ammonium acetate and spread on a surface of distilled water or 0.15 to 0.5 M ammonium acetate. The DNA was released in the form of a regular series of rosettes connected by interrossette DNA. The mean length of the rosette DNA was 14 ~tm, similar to the mean length of 10 gm for chromomere DNA of Drosophila polytene chromosomes. The mean interrosette DNA was 4.2 ~tm. SDS gel electrophoresis of the chromosomal nonhistone proteins showed them to be very similar to nuclear nonhistone proteins except for the presence of more actin and tubulin. Nuclear matrix proteins were present in the chromosomes and may play a role in forming the rosettes. Evidence that the rosette pattern is artifactual versus the possibility that it represents a real organizational substructure of the chromosomes is reviewed.
The most straight forward model of chromosome structure is a single DNA molecule folded upon itself eventually extending from one telomere to the other (DuPraw, 1965; Prescott, 1970; Comings, 1972; Kavenoff and Zimm, 1973). The discovery of nucleosomes has clarified the manner in which DNA is folded to interact with histones (Olins and Olins, 1974; Kornberg, 1974) and provided insights into the structure of the 100 to 250 A chromatin fiber (Griffith, 1976; Germond et al., 1975; Finch and Klug, 1976), The major remaining question is how is this fiber folded to form the chromatid, account for chromomeres and chromosome banding (Comings, 1978a), and for "scaffolding" proteins (Paulson and Laemmli, 1977; Adolph et al.,, 1977)? Electron microscopy studies of Drosophila oocyte and salivary gland chromatin (Sorsa, 1972, 1976; Comings and Okada, 1974) provide a starting point by suggesting that polytene chromomeres are formed by loops of chromatin separated by interchromomere or interloop DNA. However, a specific search for such a pattern in mammalian chromosomes was previously unrewarding (Comings and Okada, 1973). A recent report by McCready et al. (1977) that yeast chromatin was arranged in a supercoiled loop-interloop pattern when suspended in 4 M ammonium acetate and spread on water, stimulated us to try this method on isolated Chinese hamster chromosomes.
T.A. Okada and D. E. Comings
Materials and Methods Chinese hamster V 79 cells were grown in McCoy's medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin and streptomycin. Mitotic cells were collected by shaking to dislodge the cells in mitosis, after 3 h incubation in 0.06 gG/ml of colcemid. Chromosomes were isolated by the procedure of Wray and Subblefield (1970) with slight modification of the isolation buffer; 1.0 M hexylene gIycoI, 0.25 mM CaClz, 0.1 mM Pipes, pH 6.8. Isolated chr6mosomes were suspended in 4 M ammonium acetate, pH 7.0, centrifuged at ,10,000 x g for '10min and resuspended in l ml of the same solution. This suspension was diluted 0, 2, 5, and lO times with 4 M ammonium acetate and 100 ~tl mixed with 10 gl of cytochrome C (1 mg/ml). This was spread onto the surface of distilled water, 0.15 M, 0.25 M or 0.5 M ammonium acetate. Two to 4 min after spreading, copper grids coated with formvar and carbon were touched to .the surface of the hypophase. The grids were then placed- in 95% -ethyl alcohol, air dried and rotary shadowed with platinum-carbon. Chinese hamster V 79 DNA was isolated by the method of Marmur (1961). DNA was spread in the same manner as for isolated chromosomes. Electron micrographs were taken with a Hitachi HS 8-1 electron microscope at 50 KV. To reverse the image and increase the contrast, contact negatives of the negatives were made with DuPont graphic arts film 710 Cronar Ortho S Litho COS-4 7080 204. SDS slab gel electrophoresis was as described previously (Comings and Harris, 1975).
Resulis A small, s u b - t e l o c e n t r i c Chinese h a m s t e r m e t a p h a s e c h r o m o s o m e s u s p e n d e d in 4 M a m m o n i u m acetate a n d s p r e a d on w a t e r is s h o w n in F i g u r e 1. D N A fibers are s p r e a d u n i f o r m l y a r o u n d a' central structure r e s e m b l i n g the p r e p a r a tions d e s c r i b e d b y P a u l s o n a n d L a e m m l i (1977). The density o f t h i s structure is greater at the c e n t r o m e r i c regions t h a n in the s h o r t c h r o m o s o m e arms. A t a higher m a g n i f i c a t i o n the edge o f this d i s p e r s e d D N A shows the presence o f n u m e r o u s rosettes c o m p o s e d o f l o o p s o f D N A c l u s t e r e d a r o u n d an elevated center (lower p o r t i o n o f Fig. 2). In the u p p e r p o r t i o n o f this picture there are m a n y b e a d s r e p r e s e n t i n g i n c o m p l e t e l y e x t r a c t e d n u c l e o s o m e s . There are also c o a r s e r structures w h i c h largely r e p r e s e n t c h r o m a t i n t h a t has n o t b e e n dispersed. The striking feature o f the rosettes was t h a t they t e n d e d to be o f similar size a n d when a d e q u a t e l y s p r e a d there was a c o n n e c t i n g length o f interrossette D N A (Fig. 3 b d - f ) . Even when the rosettes were clustered they were s e p a r a t e entities Occupying defined d o m a i n s (Fig. 3 a, c). A m o n t a g e o f 9,pairs o f rosettes is s h o w n in F i g u r e 4 to illustrate the n o n r a n d o m a r r a n g e m e n t o f the D N A . M e a s u r e m e n t o f m a n y pairs s h o w e d the a m o u n t o f rosette a n d i n t e r r o s e t t e ~ D N A was fairly c o n s t a n t (Fig. 5). T h e m e a n length o f i n t e r r o s e t t e D N A was 4~2gm+l.7gm. The m e a n length o f rosette D N A (all loops) was 13.7 ~ t m + 4 . 8 ~tm. The m e a n n u m b e r o f rosette l o o p s was 2 0 . 7 + 5.3. The l o o p s were d e s t r o y e d b y D N a s e b u t n o t b y R N a s e t r e a t m e n t . W h i l e the well-defined p a t t e r n o f rosette a n d interrosette D N A .and the n o n r a n d o m a m o u n t o f D N A in a n d between t h e rosettes suggested a true o r g a n i z a t i o n a l structure was being observed, the w e l l - k n o w n p l e t h o r a o f artifacts t h a t can be o b t a i n e d by s p r e a d i n g D N A o r c h r o m a t i n u n d e r different c o n d i t i o n s necessitated m a n y c o n t r o l experiments.
Higher Order Structure of Chromosomes
Fig. 1. Chinese hamster sub-telocentric chromosome suspended in 4 M ammonium acetate and spread on distilled water. Some of the DNA has spread out around chromosome. The dense central portion is composed primarily of undispersed chromatin which is more dense at the centromeric heterochromatin. 6,000 x
1. To determine whether such structures were unique to the a m m o n i u m acetate treatment, isolated metaphase c h r o m o s o m e s were resuspended in 2 M NaC1, 10 m M Tris, p H 7.5, 0.2 m M MgCi2 and spread as with a m m o n i u m acetate treated chromosomes. The results were the same as with a m m o n i u m acetate. 2. To determine if the rosettes were artifactually p r o d u c e d by this type of treatment o f D N A , purified Chinese hamster D N A was suspended in 4 M a m m o n i u m acetate and spread on distilled water (Fig. 6a) and on 0.5 M a m m o nium acetate (Fig. 6b). N o rosettes were observed but d o u g h n u t and w o r m forms were seen, typical of a c y t o c h r o m e C effect producing aggregation of D N A fibers (Olins and Olins, 1971 ; Davis et al., 1971 ; Comings, 1978).
T.A. Okada/md D. E. Comings
Fig. 2. An edge of the chromosome shown in Figure 1 illustrating the several rosette structures in the lower portion of the figure. The upper portion shows undisrupted nucleosomes and areas of undisrupted chromatin. 26,000x
3. Because of the t e n d e n c y for D N A to f o r m rosettes w h e n spread o n a h y p o p h a s e of less t h a n 0.2 M a m m o n i u m acetate (Davis et al., 1971), c h r o m o s o mes were spread o n a h y p o p h a s e of distilled water a n d 0.15, 0.25 a n d 0.5 M a m m o n i u m acetate. All four p r o d u c e d the same rosette-interrossette D N A patterns.
Higher Order Structure of C h r o m o s o m e s
Fig. 3a-e. Representative samples of the rosette-interrosette pattern of D N A from Chinese hamster chromosomes, a A cluster of rosettes close to the undispersed chromosome and two free rosettes. 24,000 x . b Two rosettes attached to a third by interrosette D N A . 32,000x. c A cluster of incompletely dispersed rosettes 35,000 x . d and e Two typical examples of a rosetteinterrosette D N A pattern. Both 41,000 x .
T.A. Okada and D. E. Comings
4. If the chromosomes were not first placed in high ionic strength media to dissociate the histories no D N A fibers were released. The chromatin of such preparations is too condensed to provide any information about the higher order structure of the D N A fibers.
Higher Order Structure of Chromosomes
g Fig. 4. A montage of line drawings of typical examples of the rosette-interrosette DNA pattern
5. On the assumption that nonhistone proteins may be involved in the maintenance of the rosettes in these preparations (see below), pure calf thymus DNA was complexed with calf thymus histones by progressive dialysis form 2 M NaC1 and urea. This preparation was then suspended in 2 M NaC1 and water spread. Although rosettes were formed by this procedure they had a different appearance with a flat central region and formed a much less complex structure (see Fig. 7d, Comings and Okada, 1976). 6. Numerous experiments published previously on whole mount electron microscopy of DNA-nuclear matrix complexes show similar matrix-DNA rosettes (Comings and Okada, 1976). To examine the nonhistone proteins involved, isolated chromosomes and isolated chromosomes pelleted from the 4 M ammonium acetate suspension were compared to V 79 nuclei, also isolated by the hexylene glycol technique, and such nuclei pelleted from 4 M ammonium acetate. To identify the nuclear matrix proteins this complex was isolated from Chinese hamster liver by the procedure used previously (Comings and Okada, 1976; Berezney and Coffey, 1974). SDS gel electrophoresis in 12 and 9% gels (Fig. 7) showed the following: 1. The nonhistone proteins of interphase and metaphase chromosomes are al-
T.A. Okada and D. E. Comings I n t e r Rosette DNA Length = 4.2 ~ m S.D. = + 1.7
Jm Total Rosette DNA
~, = 13.7 S.D. = +- 4.8 40O/o 9 ."~, ~i~'
.~.... :: ......
um The Number of Loops/Rosette = 20.74 S.D. = +_ 5.35
| u.JIW.p...I,. i
Fig. 5. Interrosette DNA length, rosette DNA length and number of DNA loops/ rosette 20
most identical except for larger amounts of actin and tubulin associated with the chromosomes. 2. The nuclear matrix proteins are also present in the chromosomes. 3. H1 histone is removed by the a m m o n i u m acetate procedure but the H2, H2A, H2B, and H4 histones are not completely removed. This is expected since nucleosomes can be seen in the more central portions of the chromosomes where the D N A has not been completely released. 4. More nonhistone proteins are present than in the studies of Paulson and Laemmli (1977) on chromosomes treated with dextran sulfate and heparin. Discussion
These results show that when isolated Chinese hamster chromosomes are resuspended in 4 M a m m o n i u m acetate and then spread on distilled water or 0.15
Higher Order Structure of Chromosomes
Fig. 6a and b. Purified Chinese hamster DNA suspended in 4 mM ammonium acetate and spread a on distilled water, and b on 0.5 M ammonium acetate. No rosette-interrosette patterns were seen but the aggregation of DNA typical of cytochrome C effect is seen. See text. 20,000x
to 0.5 M ammonium acetate, the D N A that is released forms a rosette-interrosette pattern. The major question is whether these patterns are artifacts of the procedure or whether they represent a real organizational substructure of chromosomal DNA,
The Case for Artifact 1. The apparent nonrandom amount of interrosette D N A may be a sampling artifact. Singificantly longer interrosette stretches may not be seen because the longer D N A fibers break and much shorter stretches are not recorded because the interosette D N A could bot be seen be scored. The apparent nonrandom amount of D N A in the rosettes may also be a sampling artifact. Smaller rosettes may be unstable and lost during spreading, larger amounts of D N A may naturally form two rosettes instead of one. 2. Similar rosettes have been observed in the spreading of viral, bacterial, mitochondrial and kinetoplast D N A (Olins and Olins, 1971; Wolstenholme et al., 1974; Renger and Wolstenholme, 1972; Van Bruggen et al., 1968; Laurent and Steinert, 1970 ; Caro, 1965). Certainly these cannot be considered chromomeres.
T.A. Okada and D. E: Comings
Fig. 7. SDS acrylamide gel electrophoresis of the nonhistone proteins of isolated Chinese hamster chromosomes, chromosomes after suspension in 4M ammonium acetate (A chromosomes), isolated V79 nuclei, isolated nuclei after suspension in 4M ammonium acetate (A nuclei), and isolated Chinese hamster liver nuclear matrix proteins 3. Each nucleosome results in one turn of the D N A helix ( G e r m o n d et al., 1975). W h e n c h r o m a t i n is exposed to high salt conditions that simultaneously release m a n y nucleosomes, the resulting supercoiled D N A becomes twisted into artifactual rosettes which have no structural meaning. 4. Exposure of D N A to c y t o c h r o m e C and varying amounts of a m m o n i u m acetate plus dehydration in ethanol can result in collapse and aggregation of
Higher Order Structure of Chromosomes
DNA to form artifactual tubes and loops (Olins and Olins, 1971; Lang, 1973; Comings, 1976). These aggregations may be resistant to DNase and some of the scaffolding like structure seen in these preparations may be this type of artifact. 5. Under Other conditions of removal of histones, such as with dextran sulfate and heparin (Paulson and Laemmli, 1977) such rosettes are not seen.
The Case for the Rosette-Interrosette Pattern being Real 1. There seems to be a nonrandom distribution of loop and interloop DNA. The values of 4.2 g m + 1.7 ~tm for ihterrosette DNA and 13.7 btm_+4.8 ~tm for rosette DNA are tighter than would be expected from purely artifactual formation even given some sampling bias. 2. Similar rosettes were not produced when pure DNA was spread under the same conditions. 3. Spreading conditions of low or high pH, or specific concentrations of ammonium acetate, known to favor the formation of rosettes with DNA or DNA plus histones (Olins and Olins, 1971 ; Lang, 1973) were either not present or were shown to have no effect on the rosette formation. 4. Domains of super coiled DNA have been reported in numerous eukaryotes (Cooke and Brazell, 1975; Inde et al., 1975; Benyajati and Worcel, 1976; Pifion and Salts, 1977). The rosettes may represent such domains. 5. Similar loops of DNA have been observed with DNA-nuclear matrix complexes in interphase nuclei (Comings and Okada, 1976). SDS gel electrophoresis of ammonium acetate treated chromosomes shows that the nuclear matrix and numerous other non-histone proteins are present in metaphase chromosomes. 6. Pachytene chromosomes (Lucciani et al., 1976) and banding of prometaphase chromosomes (Yunis, 1976) show that chromosome bands are composed of multiple sub-bands.~ The rosettes observed here may represent the ultimate sub-division of the chromomere and be comparable to the chromomeres of polytene chromosomes. Given the presence of 3 x 10 12 grams of DNA per haploid mammalian genome (Vendrely, 1955), 3,000 visible bands in mammalian prometaphase chromosomes (Yunis, 1976), and approximately 2/3 of the DNA being in the chromomeres, each prometaphase chromomere would contain 6.5x 10-15 gms of DNA, or 6.4x l0 s base pairs (640 kb), or 213 btm of DNA. Since an average rosette contains about 14 btm or 4.2x 104 base pairs it is about 15 times smaller than the average prometaphase band and there would be 50,000 per genome. It is intriguing that this is very similar to the estimated average of 3,0 x 104 base pairs, or 10 gm of DNA per Drosophila polytene chromomere (Edstr6m, 1964). If mammalian chromosomes could be polytenized these rosettes and interrosettes might from the same bands and interbands seen in the Diptera. Such loop and interloop DNA can provide a model of mammalian chromosome structure that can explain the presence of chromosome bands (Comings, 1977, 1978a). 7. If the loops are formed by sealing the base of the loop together with nuclear matrix or other nonhistone proteins, the absence of such rosettes in
T.A. Okada and D. E. Comings
Fig. 8. Possibilities of rosette formation (A) from anchores, (B) from non-anchored loops. See text
the dextran-heparin preparations may be due to the extraction of greater amounts of nonhistone protein including those sealing the base of the loops together. The SDS gel electrophoresis of these ammonium acetate treated chromosomes show many more nonhistone proteins than the dextran-heparin treated chromosomes (Paulson and Laemmli, 1977). 8. Sonnenbichler (1969) reported similar structures in calf thymus chromatin. 9. While osmotic lysis of chick mitochondrial DNA usually produces free supercoiled circles, lysis of yeast mito6hondria consistently produces rosettes (Van Bruggen et al., 1968). This suggests the rosettes may be due to a specific type of DNA-membrane or DNA-nuclear matrix association rather than the spreading conditions. 10. The occurrence of rosettes very similar to these, when a crude or purified preparation of circular kinetoplast DNA was water spread (Renger and Wolstenholme, 1972; Wolstenholme et al., 1974) could be taken as evidence that these are artifacts. However, these rosettes were shown to be the result of topologically interlocking 0.8 ~tm segments of circular DNA. This suggests that such rosettes may form only under special conditions such as the presence of circular or effectively circular DNA. DNA loops held together at the base by protein (or RNA) are effectively circular DNA. Figure 8A shows one mechanism by which periodic loops of chromatin formed by attachment to nuclear matrix or other nonhistone proteins, then supercoiled by the release of nucleosomes, could result in the rosette- interrosette pattern seen here. The sealing of chromatin into loops is not considered essential to form these rosettes. One aspect of chromomeres that is beyond dispute is that they are more condensed than interchromomere DNA. Figure 8B illustrates how such a region of chromatin condensation without a sealed loop could also form rosettes after osmotic lysis.
Higher Order Structure of Chromosomes
We feel that neither of the above arguments overwhelms the other. The following experiments would help to resolve the question of whether rosettes represent a true structural element of chromosome. 1. Since the G-bands tend to be composed of relatively At-rich DNA and the R-bands composed of relatively GC-rich DNA (Comings, 1978a), the demonstration of differences in mean base composition of rosette versus interrosette DNA would suggest these are real structures. 2. Examination of meiotic chromosome may show a highly non-random arrangement of rosettes aligned along the axial filament or lateral element of the synaptonemal complex with interrosette DNA forming the lampbrush-like loops, or with rosettes homologously paired. 3. In polytene chromosomes the rosettes may align themselves to form the bands with interrosette DNA forming the interbands. Such arrangements would provide strong evidence that the rosette-interrosette patterns are real structures. Studies of these systems are in progress. Acknowledgement. This work was supported by NIH Grant # G M 15886 and the Arnold Buckley Research Fund.
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Received September 12, 1978 / Accepted September 30, 1978 by J.G. Gall Ready for press December 12, 1978