V istlalixution of Chromatin u-B odies ADA L. OLINS The University of Tennessee-Oak Ridge Graduate School of Biomediml Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
I. Introduction In 1973 Woodcock ( 1 ) and Olins and Olins ( 2 ) reported that chromatin particles, ubodies, could be visualized in electron micrographs. The new innovations which permitted this observation were: (1) Samples were applied to an electron microscope grid by the method developed by Miller and Beatty ( 3 , 4 ) . This method used a detergent (Kodak Photo-Flo) to minimize surface tension during the drying procedure, and prevent tangling of the chromatin fibers (2). A negative stain was used to resolve individual particles which are very close to each other (3). Micrographs were made at higher magnifications so that the viewers’ attention could be focused on 80-d; structures. As it turned out none of these are essential steps in visualizing v-bodies. Monomers produced by nuclease digestion of nuclei can be visualized without fixation, without use of Photo-Flo, by using a positive stain or shadowing and at a fairly low magnification. However, the visualization of particles in untangled chromatin fibers streaming out of an identifiable nucleus was necessary for positive identification of v-bodies and their correlation with the nuclease digest product. Why didn’t previous methods show the existence of the particle? Thinsection preparations suffered from (1) dehydration by organic solvents, (2) a difficulty in following fibers and knowing the direction of section with respect to individual fibers, and (3) the absence of a technique such as negative staining. Chromatin fiber spreads, however, came close to visualizing the real chromatin structure, but drying procedures involving organic solvents probably destroyed the structure of v-bodies. Furthermore, the use of concentrated stains with poorly extended fibers (i.e., where connecting strands are still tightly associated with the ubody) make the visualization of the particles impossible. 61
ADA L. OLINS
A. v-Bodies in Isolated Nuclei (5,6) Nuclei are suspended in 0.2 M KCl to give a concentration of lo8
nuclei/ml. A hemocytometer is used to determine the concentration. At this stage the nuclei should not be clumped or aggregated. The nuclei are then diluted 200-fold by adding 0.001 M ethylenediaminetetraacetic acid (EDTA) (PH 7.0), and the tube is gently inverted several times to allow mixing. Rough treatment at this stage could cause aggregation and should be avoided. The nuclei are allowed to swell for 10 minutes on ice, followed by the addition of fresh 10% formaldehyde, pH 7.0 (NaOH), to a final concentration of 0.9% formaldehyde. The nuclei can be kept in this state for several hours. The microcentrifugation chamber (7)(inner diameter 4 mm and height 6 mm) is filled with 10% formaldehyde (PH 7.0). A freshly glowed carboncoated grid is carefully inserted into the bottom of the centrifugation chamber so that no air bubbles are trapped under the grid. Two or three drops of the formaldehyde are removed from the top of the centrifugation chamber with a Pasteur pipette and replaced with one, two, or three drops of the swollen, fixed nuclei. There should be a convex meniscus standing slightly above the top level of the centrifugation chamber. This excess sample is squeezed out with a coverslip and blotted. This procedure tends to form a seal between the coverslip and the centrifugation chamber. The nuclei are then centrifuged at 800 g for 4 minutes in a swinging-bucket centrifuge. At the end of the centrifugation the coverslip is removed, 10% formaldehyde is added drop by drop to give a convex meniscus, the centrifugation chamber is turned upside down, and the grid is removed whenit settles on the meniscus. Immediately thereafter the grid is washed in approximately 50 ml of water containing three drops of Kodak Photo-Flo and one drop of pH 10 borate buffer (this solution is made fresh daily) and drained by touching the edge of the grid to the edge of bibulous paper. When the grid is dry it is stained.
B. v-Bodies in Chromatin, Small Chromosomes (Viral), and Nuclease Digestion Products The sample is diluted to an A,,, = 0.5-3.0 with 0.001 M EDTA (pH 7.0).
Aim for a concentration which covers the carbon film well but gives few overlapping molecules. The concentration needed varies in an unpredictable way. Apply a drop of the sample to a freshly glowed carbon film for 30 seconds, wash in dilute Photo-Flo (as with nuclei), drain with bibulous
VISUALIZATION OF CHROMATIN u-BODIES
paper, and dry. Stain. Fixation is not necessary for smaller samples and may cause cross-links which make visualization of chromatin particles more difficult.
C. Stains Many stains can be used successfully to visualize v-bodies. Ethanol and methanol solvents will not affect the general morphology of the v-bodies at this stage. Positive stains give excellent contrast but do not resolve ubodies which are in contact with each other. High-concentration negative stains (1% or more) have too much contrast to visualize the small amounts of stain between close particles. Low-concentration negative stains produce less contrast but tend to be more delicate and show more detail. Our standard and most reliable staining procedure at present is to put a small drop of 0.01 M uranyl acetate in water, on the sample side of the grid for 30 seconds and touch the edge of the grid to the edge of bibulous paper to drain as thoroughly as possible. Low-angle rotary shadowing has been used successfully in several laboratories. It is a high-contrast method and makes the electron microscopy much more rapid. However, the details of the internal v-body structure are lost, and subtle differences in morphology are unattainable.
D. Supporting Films When low-contrast, dilute negative stains are used, it is important to use the thinnest carbon film support possible. Both 45% glycerin drained from a warm glass slide and mica have been used successfully as a smooth surface on which the carbon is evaporated. A shield 1 x 2 cm placed about 3 cm below the evaporating carbon produces a more uniform film and makes it somewhat easier to control the thickness of the film. I have kept carbon film for several months without any trouble. In order to render the carbon film hydrophilic, it is glowed at a pressure of 50 pm of mercury for 1 minute a short time before it is used.
E. Electron Microscopy High-resolution microscopy demands the utmost from the microscope and the microscopist. It is therefore advisable to maintain the microscope at its best. A good vacuum, a cold finger, correct microscope alignment, astigmatism correction, and short exposure to the electron beam each add a small increment toward improving the quality of the data. The condensor setting should give the smallest beam spot possible which does not give
ADA L. OLINS
FIG. 1. (A-D) Isolated rat liver nuclei; (E-H) isolated chicken erythrocyte nuclei (8).(A) and (E), Isolated nuclei in CKM buffer; (B) and (F), nuclei from (A) and (E) swollen in water; (C) and (G), nuclei from (A) and (E) suspended in 0.2 M KCl; (D) and (H), nuclei from (C) and ( G ) swollen in water.
VISUALIZATION OF CHROMATIN U-BODIES
FIG. 2. Electron micrograph of a swollen and fixed chicken erythrocyte nucleus centrifuged onto a glowed carbon-coated grid, dried in Photo-Flo, and dried in 0.5% ammonium rnolybdate (pH 7.4). The chromatin fibers spilling out of the nucleus are well separated at the edge of the nucleus. From Olins ez ul. (6), with permission.
rapid contamination of the sample. Micrographs should be taken at x 40,000 magnification or higher so that astigmatism and proper focus (at or slightly under exact focus) can be checked before each micrograph is made. A pointed filament is recommended.
111. Demonstration Isolated nuclei are extremely sensitive to variations in salt concentration. Figure 1 is a phase-light micrograph which demonstratesthis point. Although the nuclei did not swell very much in 0.2 M KCl, they allow more complete swelling when placed in water than do nuclei which go directly from magnesium into water. In order to assure complete swelling we have recently used 1 mM EDTA (pH 7.0) instead of water for the final swelling; thus nuclei in CKM buffer [O.OOS M MgC1,-0.025 M KC1-0.05 M cacodylate (pH 7.5)]
FIG. 3. Electron micrograph of the edge of a rat thymus nucleus centrifuged onto a carbon-coated grid. A few chromatin fibers are stretched out from a region of higher chromatin concentration. Negative stain with 0.5% ammonium molybdate (pH 7.4).
are spun, resuspended in 0.2 M KCl, and diluted 1 :200 with 0.001 MEDTA (pH 7.0). At low magnification it is very difficult to be certain that v-bodies are present on chromatin fibers; however, this is a useful magnification for scanning the grid and identifying the source of the chromatin fibers (Fig. 2). Classically, a negative stain piles up around the specimen, so contrast is
FIG. 4. High-resolution electron micrograph of a spread chicken erythrocyte nucleus. Note the u-bodies with clear internal structure. Many chromatin fibers exhibit a zigzag configuration with the u-bodies lying on alternate sides of the connecting strand. The specimen preparation is similar to Fig. 2, except that the sample was stained with aqueous 0.2% uranyl acetate. From Olins et ul. (6),with pernksion.
FIG.5. Electron micrographs of monomer u-bodies obtained after micrococcal nuclease digestion and fractionation by sucrose gradient ultracentrifugation. From Olins et al. (lo), with permission.
ADA L. OLMS
achieved by an outline effect (Fig. 3). A similar effect is seen by using 1-2% uranyl acetate or 1% PTA (pH 7) and drying face down on bibulous paper. If too much stain is left on the carbon film less and less structural information remains visible. The staining method we prefer is highly reproducible and seems to give more detail than other methods (see Section IIC). Although the stain is not washed off the grid, so little stain remains on the grid that the outline formed and the direct staining of the connecting strand might not immediately identify this as a negative staining technique. It is known (9) that uranyl can bind strongly to the phosphates in DNA, and this is an alternative explanation for the staining observed in Figs. 4 and 5. We can identify more detail in the shape of v-bodies using this method than any other method we have tried. Isolated monomer v-bodies, the product of micrococcal nuclease digestion, are shown in Fig. 5. Note that the internal structure of v-bodies in chromatin fibers is still clearly visible. ACKNOWLEDGMENT I wish to thank 0. J. Miller, Jr. for teaching me how to use the electron microscope, Howard G. Davies for helping me become a more critical microscopist, Donald E. Olins for many important discussions, and Mayphoon Hsie for excellent technical assistance.
REFERENCES I . Woodcock, C. L. F., J. Cell Biol. 59, 368a (1973). 2. Olins, A. L., and O h , D. E., J. Cell Biol. 59, 252a (1973). 3. Miller, 0. L., Jr., and Beatty, B. R., J. Cell. Physiol. 74, (Suppl. I), 225 (1969). 4. Miller, 0. J., Jr., and Beatty, B. R., Science 164, 955 (1969). 5. Olins, A. L., and Olins, D. E., Science 183, 330 (1974). 6. Olins, A. L., Breillatt, J. P., Carlson, R. D., Senior, M. B., Wright, E. B., and Olins, D. E., in “The Molecular Biology of the Mammalian Genetic Apparatus,” Part A (Paul 0. P. Tso’,ed.). ElseviedNorth Holland Biomed. Press, Amsterdam, 1976. 7. Miller, 0. J., Jr., and Bakken, A. H., Acta Endocrinol. Suppl. 168, 155 (1972). 8. Olins, D. E., and Olins, A. L., J. Cell Biol. 53, 715 (1972). 9. Zobel, C. R., and Beer, M., J. Biophys. Biochem. Cytol. 10, 335 (1961). 10. Olins, A. L., Senior, M. B., and Olins, D. E., J. Cell Biol. 68, 787 (1976).