Electron microscope visualization of chromatin and other DNA-protein complexes.

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ELECTRON MICROSCOPE

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VISUALIZATION OF CHROMATIN AND OTHER DNA-PROTEIN COMPLEXES Jack D. Griffith Cancer Research Center, University of North Carolina Medical School, Chapel Hill, North Carolina 27514

Gunna Christiansen The Institute of Medical Microbiology, Bartholin Building, University of Aarhus, Aarhus 8000c, Denmark

INTRODUCTION Interest in techniques for visualizing DNA-protein complexes by electron micros copy (EM) has increased greatly with the impact of EM on studies of chromatin structure. Direct visualization of the natural repeating substructure of chromatin by EM has greatly contributed to our present knowledge of chromatin structure. The same techniques applied to in vitro complexes of RNA polymerase with DNA have brought new insights into the nature and location of specific binding sites. Direct examination of DNAs isolated from phage- or virus-infected cells, or from mitochondria, have revealed a class of proteins bound very tightly to the replication origins of these DNAs. The techniques used in these studies predate the more popular cyotchrome c surface spreading technique of Kleinschmidt (34) used to visualize pure DNA. In fact, some of the first direct visualizations of viruses and DNA were accomplished by Williams in 1945 (60, 63), who measured a dehydrated diameter of DNA of 15 A. The techniques reviewed here derive directly from his methods. The objective of this review is to provide a vehicle for those experienced with the cytochrome c methods to select the most suitable direct mounting technique for their needs. We have tried to provide an overview of the different procedures; to this end, typical micrographs have been kindly supplied 19

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by several of our colleagues. The references cited herein provide many more exam ples and detailed discussions of each procedure. Two areas not generally discussed in most publications are (a) common artifacts and how they are to be avoided, and (b) the problem of extrapolating from the structures seen in the micrographs to the actual structures as they are in solution. Although this relationship is well understood for DNA prepared by cytochrome c spreading, it is more difficult and hazardous for samples prepared by the direct mounting methods. Therefore, we have tried to emphasize the importance of proper fixation as the most reliable way of minimizing artifactual changes so as to bridge between what is visualized on the grid and what exists in solution. We have adopted a tenninology in which the procedures employing cytochrome c films spread on water surfaces will be termed basic protein film spreading, or spreading techniques, as distinguished from the techniques discussed in this review where samples are directly adsorbed onto the supporting films (to be called direct mounting). The latter would not include the BAC (benzldimethylalkylammonium chloride) method of Koller (see below) or the techniques of Miller described else where (30, 41). The term "high resolution" is nondescriptive and will be avoided. FIXATION

Samples prepared for EM by direct mounting techniques are necessarily exposed to harsh chemical and physical treatment during the preparative steps required for their visualization. For example, the hydrophobic carbon-supporting surface treated (charged) to bind the sample presents a disruptive environment not unlike that of strong ionic detergents. The washing steps expose the samples to very low ionic strength solutions, or if the samples are dried from buffer, they may be exposed momentarily to very high salt concentrations. Furthermore, on transfer ring the grids from one wash solution to the next, or during the final drying,

Figure 1 Intermediates in the in vitro replication of �X 174 DNA. (A) Initiation of �X 174 DNA replication begins with the binding of the cistron A protein to the origin of DNA replication on the supertwisted �X 174 RF I molecule, and cleavage of the viral strand to yield complexes of cistron A protein bound to relaxed �X 174 circles as shown here ( 20). (B) Upon addition of the additional proteins required for RF to single-strand synthesis in vitro, the viral strand is displaced as a "rolling loop," complexed with the E. coli DNA binding protein that makes it appear thicker than the duplex template circle. These intermediates were fixed by the addition of formaldehyde to 1% for 15 min on ice, followed by 0.6 % glutaraldehyde treatment for 15 min more on ice in a buffer containing 10 mM sodium phosphate, pH 7. 5 (12). Following fixation, the complexes were sedimented in sucrose gradients containing 1 M NaCI, absorbed onto glow-charged carbon grids in the sucrose, 1 M NaCI solution, and then placed in a mixture of 2mM spermidine, 0.1 5 M NaCI, 0.01 M tris(hydroxymethyl)aminomethane, pH 7. 5, for 2 min. The grids were next washed for 5 min each in baths of 0, 20, 5 0, and 100% ethanol in water, air dried, and shadowed with tungsten (with rotation). Bars equal 0. 5 pm (A) and 0.1/lm (B).


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the samples may be subjected to very strong surface forces as the air-liquid interface wipes across the sample. Evidence that these forces do exist and can change chroma tin structure is documented below. Presently, the only practical solution is to stabilize the samples by ehemical fixation. Even so, fixation may not protect DNA from nicking or fragmenting during its preparation. Assuming that an optimal fixation can be achieved, it provides numerous advan tages beyond that of stabilizing the DNA-protein complexes for the final EM preparation. To cite one example, in a study of an in vitro DNA replication system (20), the enzyme reactions were carried out in the presence of high concen trations of bovine serum albumin and other proteins (which would have obscured visualization of the DNA). At various times, the replicating DNA complexes were fixed by a two-step procedure described below, then separated from the high concentration of free proteins by banding in CsCI density gradients (see Figure lA, B). Here, the fixation step made it possible to apply a powerful purification procedure to otherwise labile samples. DNA-protein samples are normally fixed with either formaldehyde or glutaral dehyde; these fixations most often provide adequate protection. In a recent study in this laboratory, very stringent criteria were applied to a well-characterized chro matin to define the most optimal conditions for fixation. The distinct sedimentation properties of the simian virus 40 (SV4O) minichromosome (12) combined with information about its structure deduced from previous EM studies (4, 12, 25, 28), in vitro reconstitution (24), and nuclease digestion studies (4) provided an assay for optimizing the conditions for chemical fixation of chromatin. Following an optimal fixation of the SV40 minichromosomes, they should (a) retain their native sedimentation rates, (b) band in CsCI eqUilibrium density gradients at the density of the 1:1 DNA:protein ratio established for chromatin, and (c) remain unchanged in appearance in the EM following harsh treatments with agents such Figure 2 The effect of different preparative conditions on the appearance of chromatin. When SV40 minichromosomes are treated with X rays to nick the DNA and mixed with 2 M NaCI just prior to mounting onto carbon supports, histone-free, relaxed DNA circles with the single tightly bound nonhistone protein complex (29) are seen (A). Open-fiber loops are also found if SV40 minichromosomes are mixed with urea (0.5-6 M), mounted onto the carbon supports, and taken through the same preparative steps (B). Here, no X-ray treatment is needed to observe the circles in the open form and the fibers appear thicker than the DNA, as :in (A). The same change is seen if rat liver or other chromatins are prepared in urea solutions. If the same SV40 minichromosomes had been fixed (see Figure 1) in solutions of 0.01 M NaCI (and no urea, which would have blocked fixation), chains of 21 chromatin beads are seen (C), consistent with the structure deduced from other physical studies. Rat liver (D) or SV40 chromatin fixed with only 0.5% formaldehyde for 10 min on ice and taken through the same steps is intermediate in structure between fully beaded and fully open fibers; longer fixation yielded fully beaded molecules. Samples were adsorbed onto glow-charged carbon supports for 3 min on ice, washed with 0, 20, 50, and 100% ethanol in water, air dried, and shadowed with tungsten (with rotation). Bar equals 0.5 /lm.



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as sodium dodecyl sulfate or high salt after fixation. Finally, structures observed by EM after such fixation should be in harmony with the structural information deduced from other physical studies. These criteria were best satisfied if the histone was first cross-linked to the DNA (in 0.01 M NaCI, 0.01 M sodium phosphate, pH 7.5) with 1% formaldehyde (freshly diluted from 37% stock solution following heating of the stock to 90°C for 5 min) for 15 min on ice. Formaldehyde treatment has been shown to link histone to DNA (10, 11). The resulting structures were then stabilized further with 0.6% glutaraldehyde (freshly diluted from an unheated 50% stock solution) for IS min more on ice [glutaraldehyde will form histone histone bonds but few DNA protein bonds (11)]. Following such a two-step fixation, the SV40 minichromosomes were found to satisfy all three criteria listed above (12). Samples fixed by this procedure are shown in Figures IA,B and 2G. What should be emphasized is that whatever fixation regime is used, stringent tests such as those above should be made a routine part of the sample preparation. We have found some apparently fresh bottles of the fixitives to be impotent or to become so with time; it is known that formaldehyde should not be exposed to light or to the cold. PREPARATION OF SAMPLES FOR DIRECT MOUNTING

Preparation of Supporting Films

Carbon supporting films are almost universally used in direct mounting techniques. Formvar or collodion, ideal for DNA spreading techniques, are too electron opaque and unstable for the higher magnification viewing needed for visualizing protein free DNA. An illustration of the preparation of carbon films can be found in an earlier review (27). Sogo et al (52) have examined films of aluminum and beryllium: these may have advantages for very fine structure studies, for routine examination of chromatin; however, carbon appears to be most easily prepared and generally suitable. It has been said to be important to use very thin carbon films to attain the highest contrast between DNA and·its support. Although true for examinations of positively stained or unstained samples, the thickness of the carbon film is relatively unimportant in studies of shadowed DNA or chromatin. To monitor the thickness of carbon films, we place plastic microscope coverslips (2 X 2 cm) over one portion of the mica sheets during the deposition of the carbon layer. The absorbance can then be used to judge the thickness of the film, by placing the plastic coverslip in the normal cuvette position of a spectrophotometer and reading the optical density at 350 p.m. We find that films of A350 of 0.175 or greater are thicker than is required for most direct mounting purposes. Films of AlSO between 0.075 and 0.150 (0.100 is ideal) provide good supporting strength when placed over 4OO-mesh copper screens, without noticeably reducing the con trast as compared to thinner films, when examining shadowed chromatin or DNA. Films of A350 of 0.015 to 0.030 are useful for positive staining studies when sup ported over plastic meshes.


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Treatment 0/ the Supporting Films to Facilitate DNA Binding

DNA fibers adhere poorly to the surface of hydrophobic, untreated carbon films. To facilitate binding, the films must be treated or "activated." Sogo et al (52) suggest these treatments operate by applying a charge opposite that of the DNA to the surface of the grid. A common physical approach is to subject the grids to a 1O,OOO-V discharge in an air atmosphere of 200 11m of Hg pressure (27) (glow charging). Dubochet et al (IS) have introduced the use of amylamine atmos phere during glow charging, which has been adopted by several groups (5-S, 14, 15, 39, 44, 51). In either variation, it is important to optimize the charging time for each configuration of electrodes and grid placement. A chemical approach is to apply cationic compounds to the grids that facilitate DNA binding. Williams (61) has obtained good results by coating carbon films with a layer of polylysine (by touching the grids to a drop of a polylysine solution and air drying). Koller et al (37) introduced the use of ethidium bromide for the same purpose,a technique that has become popular (1,32,65); more recently they have used divalent cations for the same purpose (47). In our studies we have obtained good results by glow charging the carbon films in air, followed by adsorbing the DNA to the grids in a solution of 2 mM spermidine, 0.15 M NaCl, 0.01 M tris(hydroxymethyl)amino methane, pH 7.5 ( 1, 20). Contrast Enhancement by Heavy Metal Shadowing or Staining

Following adsorption to treated carbon films and washing and drying,the samples are usually visualized by heavy metal shadowing or staining. Shadowing,a physical process,displays any material protruding from the surface of the support. Because the metal layer is uniform over the grid, it provides a good picture of the bulk nature of the sample and makes it possible to follow DNA over long distances. The shadowing process,however,imposes a limit on the ultimate resolution. Dimers of Escherichia coli DNA polymerase I bound to DNA (each monomer is 109,000 daltons and 65 A in diameter) can be distinguished from monomers (26). In chains of E coli DNA binding protein tetramers bound to single-stranded DNA, the individual SO,ooo-dalton tetramers can be resolved (see Figure lA,B). Proteins smaller than this become difficult to discern when bound to DNA. Furthermore, subunit structure of proteins is usually not resolved after shadowing because of the granular structure of the metal film and the fact that the shadowed metal does not penetrate into the interior of the proteins as do liquid stains. Most labora tories have used either platinum or platinum-palladium mixtures (14, 15, 24, 32, 44, 51, 54-56, 66). Recently several authors (14, 44, 65) have prestained their samples with uranyl acetate prior to shadowing. Tungsten has been exclusively used in our work. The details of tungsten shadowing and a resistance monitor needed for optimization of the shadowing have been described (27). Positive or negative staining of viruses or large proteins has allowed the visualiza tion of subunits as small as, perhaps, 30,000 daltons. Staining, therefore, offers a significant advantage over shadowing in resolution. As the dimensions of the object


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become smaller, the ease of resolving structure becomes much more difficult. DNA itself is not generally visualized by negative staining and must be seen by positive staining techniques. Because the stains are selective, components abundant in the sample and easily detected by shadowing might not be visualized at all by staining because of their small size or poor staining properties. Also, because the staining film varies in thickness over the surface of the grid, it is often difficult to follow DNA molecules for more: than short distances. Finally, although shadowed samples (though limited in resolution) are not further degraded by the electron beam, visualization of the finest details by staining requires great attention to minimizing the exposure of the sample to the electron beam through minimal beam exposure techniques, such as those of Williams & Fisher (62). With these reservations, once a sample is well characterized, careful staining techniques appear to provide significantly finer structural resolution than shadow ing. Two excellent examples of staining are shown in Figure 3A,B. Uranyl acetate and uranyl formate have been most widely used (3, 5, 6, 23, 35, 42); tantalum chloride (36) has also been described as a positive stain. Adequate fixation is particularly important if the samples are to be stain,ed, as the uranyl compounds are effective only at very low pH (2-3.5). For example, efforts to visualize the solution structure of DNA-polyamine complexes by staining with uranyl acetate or uranyl formate may be futile because they cannot be fixed with aldehydes. and at such low pHs the DNA polyamine complexes undergo a marked collapse in structure (J. D. Griffith, unpublished data). OTHER TECHNIQUES

BAe Spreading Direct mounting procedures do not allow the visualization of single-stranded nu cleic acids unless extended by proteins such as the E. coli DNA binding protein or the bacteriophage T4 gene 32 protein (17) (see Figures 1A,B and 4B). A method similar in operation to the basic protein spreading techniques, but using a small molecule, benz1dimethylalkylammonium chloride (BAC) instead of cytochrome c, has been introduced by Vollenweider, Sogo & Koller (58). In this method, single-stranded DNA or RNA are extended in the presence of formamide and can be visualized by shadowing. The thickness of the shadowed DNA is such that moderate-sized proteins attached to it can be seen. Double-stranded DNA or RNA is also visualized and is somewhat greater in width (an example is shown in Figure 4B). Several subsequent reports have presented valuable applications of this technique (33, 57). In general, the method appears to demand very careful )

Figure 3

Examples of DNA-protein complexes visualized with uranyl stains. (AJ Lambda

repressor was visualized bound to wild-type A DNA by staining the DNA-protein complexes with 0.5% uranyl formate (freshly prepared at pH 4) for 30 to 45 sec following adsorption

of the samples to glow-charged carbon grids (8). Courtesy of Christine Brack. (BJ Chromatin from freshly disrupted chicken erythrocytes was stained with 5 mM uranyl acetate in water in this micrograph of the nu body structure of chromatin. Courtesy of Ada and Don Dlins . Bars equal 0.1 /lID.


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Figure 4 Examples of the visualization of single-stranded nucleic acids. (A) In this example from Brack et al (6), PM2 DNA was denatured with T4 gene 32 protein by mixing nicked PM2 DNA and gene 32 protein together in 2 mM ethylenediarninetetraacetic acid solutions at 37°C. The complexes we,re fixed with glutaraldehyde, purified by gel filtration. mounted onto carbon supports, and shadowed. The single-stranded regions are well extended and thickened relative to the duplex DNA, by gene 32 protein. Courtesy of Christine Brack.

(B) Complexes of Q� replicase bound to Q� RNA were prepared and fixed

as

described in

Vollenweider et al (57) and then spread with the BAC technique (58). Courtesy of Vollenweider and Th. Koller. BarS equal 0.5 /JIll.

H. J.


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attention, in particular to the purity of the water used. For visualization of duplex DNA the direct mounting techniques are probably easier. If, however, single stranded nucleic acid-protein complexes are to be studied directly, this is the only method presently available.


Gene 32 Protein Mapping

A novel method that takes advantage of the affinity of the bacteriophage T4 gene 32 protein for single-stranded nucleic acids over duplex molecules (17) to map single-stranded regions on DNA-DNA or DNA-RNA hybrids was described inde pendently in 1975 by Brack et al (6) and Wu & Davidson (65). In this procedure, gene 32 protein is added to hybred molecules or to covalently closed, supertwisted DNAs and the protein is fixed in place with glutaraldehyde and then prepared by the direct mounting procedures with either ethidium bromide (37, 65) or amyla mine charging (6) [an example by Brack et al (6) is shown in Figure 4A]. Other direct mounting procedures would seem equally suitable. This technique has several advantages over the classic surface spreading techniques by using cytochrome c and formamide (16, 27): it affords considerably finer structural resolution in locating the juncture of single-stranded and duplex segments; the contrast between RNA DNA duplexes and single-stranded DNA is excellent as compared to the poor definition between the two in the older procedures; and it would appear possible to stabilize and locate much smaller regions of discontinuity (single stranded in otherwise duplex molecules or vice versa) than could have been detected previously. A variation of this method would use the E coli DNA binding protein (49), which might be more easily obtained by using the purification of Weiner et al (59). E coli DNA binding protein. however. foreshortens the single-stranded mole cules by a factor of 2.5:1 over duplex DNA, which might prove disadvantageous for some applications [the gene 32 protein does not foreshorten single-stranded molecules (17)]. (Examples of the E coli binding protein assembled onto single stranded DNA loops attached to duplex loops during DNA replication are in Figure lA,B.) Applications of the gene 32 mapping technique can be found in Wu & Davidson (65) where it was used to map rRNA and tRNA genes on 80 phage genomes, in Wu et al (66) where it was used to map the position of histone genes on two recombinant plasmids, in Pellegrini, Manning & Davidson (46) where it was used to map ribosomal genes of Drosophila, and in Wu, Roberts & Davidson (67) where the terminal sequences of adenovirus DNA were examined. RECENT APPLICATIONS

Visualization of Polymerases Bound to their Templates

Two properties of the E. coli RNA polymerase molecule, its large size (about 500,000 daltons) and its tight binding to promoter sites, has facilitated visualization of polymerase-promoter complexes. Bordier & Dubochet (5) have studied the bind ing of E coli RNA polymerase to T7 DNA with amylamine charging of carbon films and positive staining. Similar studies by Koller, Sogo & Bujard (37) and


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Portmann et al (48) used the divalent cation and ethidium bromide treatment of carbon films (an example from the study of Portmann et al is in Figure SA). Williams (61) applied the polylysine treatment of carbon films and tungsten shadow ing to the same system. Similar polymerase-DNA complexes were visualized on polyoma DNA (39). These studies have provided information about the multiplicity and location of promoter sites that would have been difficult to obtain through usual genetic techniques. Furthermore, with this method, promoter sites can be mapped on DNAs for whom little genetics exists. Although somewhat smaller (109,000 daltons), DNA polymerase 1 of E. coli binds to free 3' OH tennini at nicks or gaps in duplex DNAs. The binding is tight, particularly in low salt and in the absence of divalent metals. Visualization of this polymerase bound to nicked DNA by direct mounting and tungsten shadow ing (26) provides a tool for localization of nicks in DNA. An excellent example of the use of the BAC technique was the visualization of Q(3 replicase bound to a Q(3 template (57) (Figure 4B). In this case, the gene 32 technique would have extended the single-stranded RNA, but would have hidden the replicase molecules. Intermediates in the in vitro replication of C/JX 174 phage DNA have been examined in a collaboration by Eisenberg, Griffith & Kornberg (20), by using spermidine-treated carbon films and tungsten shadowing. In an early stage of repli

cation, C/JX 174 cistron A protein binds to the duplex form 1 C/JX 174 DNA molecule, which relaxes the DNA through a niCking of the viral (+) strand. The cistron A protein then remains very tightly bound at the nick. Visualization of the cistron A-relaxed DNA complex following cleavage by two restriction endonucleases al lowed the localization of the binding site to within ±20 base pairs on the C/JX 174 genome. Visualization of later stages in replication demonstrated that if>X 174 DNA replicates in a fully closed rolling circle mode, a fact that would have been difficult to demonstrate by other physical techniques (Figure lA,B). Visualization of Replication Origin Complexes

An important application of the direct mounting techniques has been to visualize proteins bound to DNA following mild extraction of the DNA from cells or from virus particles. Extraction of SV40 DNA from nuclei of lytically infected

Figure 5 Examples of shadowed DNA-protein complexes. (A) E. coli RNA polymerase is visualized bound to T7 DNA: following ihcubation of the enzyme and DNA, the complexes were directly adsorbed onto mica sheets in the incubation buffer by using the divalent cation mounting technique described by Portmann & Koller (47). Following fixation of the polymer ase-DNA complexes on the mica sheets, the samples were washed and dehydrated, and a carbon-platinum replica was prepared. Courtesy

of R.

Portmann and Th. Koller. (B) The

nucleosome structure of chromatin was visualized by adsorbing a dilute chromatin solution onto glow-activated carbon films, washing the samples briefly with water, and air drying. The samples were shadowc:d with 80% platinum and 20% palladium (4:1 mixture) at an angle of 7° with rotation. Courtesy of Alex Varshavsky.



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cells or from virus particles with solutions of high salt (but not ionic detergents) ' followed by mounting onto glow-charged carbon films and tungsten shadowing led to the identification of a single large protein complex attached to each circular SV40 DNA molecule at its origin of replication (29). Kasamatsu & Wu have obtained evidence from further EM studies that this protein complex may have a nicking function (32, 33). A similar tightly bound protein complex that holds the two ends of the linear adenovirus DNA molecule together was detected by surface spreading techniques (50). T. Broker (personal communication) has applied a ferritin-avidin-biotin labeling technique (2,40) to confirm this result. The origin of DNA replication in adenovirus is close to one end. A complex that apparently contains both membrane and protein components was visualized tightly bound to the replication origin of HeLa cell mitochondrial DNA by using both spermidine and ethidium bromide treatments of carbon films and platinum or tungsten shadow ing (1). As described above, the cistron A protein can also be found tightly bound to the replication origin of C/>X 174 DNA. Visualization and mapping of these proteins has been important in advancing our knowledge of the biology of the respective DNAs. Their detection by techniques other than EM would likely have been difficult because, in several cases, no functional activities have yet been established. Visualization of Chromatin

Direct visualization techniques have contributed greatly to our current knowledge of chromatin structure. It is now agreed that the four core histones (H2a, H2b, H3, and H4) complex DNA to generate a repeating chain of beads or nucleosomes each about 110 A in diameter and spaced by about 50 base pairs of protein-free DNA from the next. This repeating chain of beads has been visualized by numerous investigators studying viral (4, 13, 14, 21, 25, 28) and cellular chromatins (23 25, 43, 44, 53 55, 64) and chromatin reconstituted in vitro (24) and by using both negative staining and shadowing techniques (see Figure 2G, 3A, and 5B). The chain of beads structure is observed in the absence of histone HI and when the chromatin is fixed in salt solutions of either low (0.01 M NaCl) or moderate (0.6Q, M NaCl) salt. At intermediate salt concentrations (0.15 M NaCl) or in the presence of divalent cations, the beaded chain is found to collapse into a more compact structure, consistent with sedimentation changes observed over the same salt ranges (12). Little internal detail has been detected in the nuc1eosomes by shadowing. Negative staining (42) shows a hole in the center of the beads or "nu bodies" (see Figure 3A). Monomers or oligomers of two, three, and four nucleosomes have been visualized by staining with uranyl acetate by Finch et al (23). Examination of the particles by STEM methods (38) has provided evidence that the particles may be disk shaped, although other physical methods suggest a more spherical particle (45). Visualizations of chromatin containing histone HI have been more varied, possi bly because of the fact that HI may be lost upon exposure to low salt in the presence of acceptor molecules of HI (31). Evidence from my laboratory by using both sedimentation analysis and EM shows that with proper fixation, chromatin ,

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containing the native complement of HI always appears condensed within the salt range of 0.02 M NaCI to about 0.25 M NaCI (12, 25). Although it is certain that the fundamental structure of HI-depleted chromatin (in low salt) is that of a chain of beads, it is less certain what the structure(s) of chromatin containing histone HI is, in particular as it exists in the interphase nucleus. Bram & Ris (9) have shown that under some conditions, interphase chro matin spread on water surfaces can be seen as networks of fibers of about 12o-A diameter. Thicker fibers, about 250-300 A in diameter, are routinely found upon examination of water spread or critically dried metaphase chromosomes (19). Finch & KIug (22) have proposed a model of one higher order arrangement of the nucleosomes based on a solenoid of 250-A diameter and a pitch of 110 A. At this (early) stage of investigation, it is important not to assume that certain struc tures that are rare but easily visualized (as possibly the 120-A fiber) represent the bulk arrangement of interphase chromatin. PROBLEMS IN DIRECT MOUNTING CHROMATIN

The preparative steps used in direct mounting exert harsh forces on DNA-protein complexes that may change their native structure. Some samples are able to with stand these changes better than others. For example, the binding of E. coli DNA polymerase 1 to DNA is enhanced by very low salt conditions, such that even if the protein is denatured by the drying steps, it remains bound to the DNA without using any fixation (26). In other instances, proteins whose association with DNA was labile at low salt might be lost during the washes. Changes in chromatin structure can occur when unfixed or poorly fixed chroma tin is prepared by direct mounting. The association of the four core histones with DNA that forms the nucleosome is strong-withstanding heating to 65°C and considerable shear forces (12). The association of histone HI with DNA, however, is much weaker and the HI-dependent organization of the beaded chain, therefore, is likely to be much more labile to disruptive forces. It is reasonable that prepara tions of chromatin containing Hi, which had not been properly fixed, might show a loss of HI-dependent structure, but would retain the basic nuc1eosomal structure. This apparently does occur because several reports of HI containing chromatin have shown beaded chains identical in appearance to H I-stripped chromatin. Without fixation, the nucleosome may dissociate during the preparative steps, thereby yielding an extended DNA-like fiber slightly thicker in diameter than protein-free DNA. An example of an SV40 minichromosome that has undergone such a change is in Figure 2B. This change in unfixed chromatin is very likely the origin of the 30-A nucleohistone fiber reported in earlier studies of chromatin structure (68). In work in this laboratory it was found that exposure to very low salt, removal of histone HI, removal of divalent cations, and addition of urea all stimulated the change in unfixed chromatin to the 30-A fiber form. Mount ing chromatin onto carbon films in the presence of divalent cations and histone HI and avoiding distilled water washes, all reduced the tendency of unfixed chroma tin to change to 30-A extended fibers. By combining poor fixation with preparation

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of chromatin in certain salt mixtures, a broad spectrum of different structures could be created during the preparative steps (Figure 2D), whose relation to the original structures was ambiguous. An excellent example of such difficulties is the problem of visualizing the structure of chromatin in urea solutions. Because urea and aldehydes chemically react with themselves to eliminate the fixitive (if the urea is in excess), we have found no way to fix the structure of chromatin in urea solutions such that criteria for proper fixation as those described above could be met.

CONCLUSION In this review we have tried to cover the direct mounting techniques currently use and, through micrographs kindly supplied by our colleagues, to illustrate uses of the different methods. For the novice, it is important to select one technique, taking time to become acquainted with its use in visualizing simple test samples before applying it to examine more complex structures. We have tried to emphasize the importance of controlling the fixation for two reasons. First, confidence in the fixation step allows one to correlate structures observed by EM with structures present in solution. Second, fixation of otherwise labile samples allows one to apply powerful purification techniques.

in

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Visualization of ribosome--single-stranded DNA complexes in the electron microscope.

Electron microscope visualization of the products of Bacillus subtilis transformation.

Visualization of fingerprints in the scanning electron microscope.

Visualization of polymercurimethane-labeled for bacteriophage in the scanning transmission electron microscope.

Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA.

Electron microscopic visualization of DNA and DNA-protein complexes as adjunct to biochemical studies.

Electron microscope specimen tilt stage.

Quantitation and resolution in electron microscope radioautography.

Scanning electron microscope studies of turkey semen.

Economy of effort in electron microscope morphometry.

Scanning electron microscope observation of canal cleanliness.

Chromatin structure visualization by immunoelectron microscopy.

Preparation of extrapulmonary tissues and body fluids for quantitative transmission electron microscope analysis of asbestos and other mineral particle concentrations.

A comparative study in the transmission electron microscope and scanning electron microscope of intracellular structures in sheep heart muscle cells.

Dental wear: a scanning electron microscope study.

A simplified technique for electron microscope autoradiography.

Topographical contrast in the transmission electron microscope.

SNF chromatin remodeling complexes and cancer.

Transmission and scanning electron microscope studies of calcified cartilage resorption.

Scanning and transmission electron microscope study of pellicle morphogenesis.

Visualization of genes by electron microscopy.

Electron microscopy of defined lengths of chromatin.

Cryo-electron microscopy of chromatin biology.

Tools and procedures for visualization of proteins and other biomolecules.