A Method for Determination of the in Situ Distribution of Chromosomal Proteins

Proc. Nat. Acad. Sci. USA Vol. 73, No. 2, pp. 423-427, February 1976 Biochemistry

A method for determination of the in situ distribution of chromosomal proteins (nonhistone chromosomal proteins/chromosome structure/Drosophila melanogaster/polytene chromosomes/immunofluorescence) LEE M. SILVER* AND SARAH C. R. ELGINt Committee on Higher Degrees in Biophysics and t Department of Biochemistry and Molecular Biology, The Biological Laboratories, Harvard University, Cambridge, Massac usetts 02138 Communicated by Matthew S. Meselson, December 1, 1975

ABSTRACT A technique has been developed for "stain- tracted from chromatin with 1.6 M NaCl-0.2 M HC1. The ing" cytological preparations by indirect immunofluorescent DNA and NHC proteins were solubilized in 0.05 M Tris, pH methods that permits determination of the in situ distribution of chromosomal proteins. The method is particularly ori- 8-1% sodium dodecyl sulfate (NaDodSO4), and the DNA ented to the use of polytene chromosome squashes from Dro- was removed by centrifugation (3). The supernatant con- sophila salivary glands. Control experiments indicate that tains the NHC proteins; this preparation will be referred to the fixation methods used allow little or no extraction or re- as NHCP-1. (ii) Chromatin was dissociated in 5 M urea-2 M arrangement of the chromosomal proteins. The results ob- NaCl-0.001 M sodium phosphate buffer, pH 7.0-1 mM so- tained demonstrate the specific in vivo chromosomal loca- tions of nonhistone proteins purified from isolated chroma- dium bisulfite. The histones, NHC proteins, and DNA were tin. The technique is apparently capable of resolution at the then eluted sequentially from a hydroxylapatite column level of the chromomere or band, the unit of genetic organi- with 0.001, 0.1, and 0.5 M phosphate (4). The fractions were zation in Drosophila. dialyzed extensively against 0.01 M acetic acid, Iyophilized, and stored at -20°. The nonhistone proteins, eluted at 0.1 M During the last several years studies on the organization of phosphate, will be referred to as NHCP-2. While the mass the eukaryotic genome have been greatly aided by the de- recovery of Drosophila NHC proteins isolated by this tech- velopment of the techniques of RNA-DNA and DNA-DNA nique was low (about 50%), the NHC protein population of in situ hybridization (1, 2). By producing a cytological visu- the fraction appeared to be essentially'the same as that of alization of the distribution of specific nucleic-acid se- the fraction isolated by technique i, as judged by analytical quences, these techniques have provided considerable new NaDodSO4 polyacrylamide gel electrophoresis. (method of information and have permitted the correlation of biochem- Laemmli, ref. 5). NHC proteins isolated by technique i are ical, genetic, and cytological data. We report here the devel- contaminated with histone (about 10%); NHC proteins iso- opment of a conceptually analogous technique which per- lated by technique ii appear to be free of histone contamina- mits the cytological visualization of the distribution of chro- tion. mosomal proteins using indirect immunofluorescence. Preparation of Antisera. Antiserum against NHCP-1, prepared by Stumph et al. (6), was used in the following MATERIALS AND METHODS designated experiments. Antiserum against NHCP-2 was Preparation of Chromosomal Proteins. Chromatin was prepared as follows. Two milligrams of NHCP-2 dissolved in prepared from 6- to 18-hr-old Drosophila melanogaster phosphate-buffered saline (0.01 M sodium phosphate, pH (Oregon R) embryos essentially as described (3). Dechorinat- 7.0-0.15 M NaCI)-0.1% NaDodSO4 were used in a 1:1 emul- ed Drosophila eggs were broken by nitrogen cavitation (re- sion with Freund's complete adjuvant (Difco Laboratories, lease from 300 lb. pressure) in buffer I (0.05 M Tris-HC1, pH Detroit, Mich.); this was injected subcutaneously into the 7.6-0.025 M KCI-0.005 M MgOAc-0.35 M sucrose). The neck region of an adult female rabbit. Four weeks later the suspension was filtered through two layers of Miracloth injection was repeated with 6 mg of NHCP-2. Rabbits were (Chicopee Mills, Inc.), and the nuclei were collected by low- bled from the major ear arteries between 6 and 14 days after speed centrifugation. After washing with saline-EDTA the booster injection. Antiserum was collected and stored at (0.075 M NaCl-0.024 M Na2EDTA, pH 8) the nuclei were -200 with 0.02% NaNs. It has been established that antisera lysed in 0.01 M Tris, pH 8, and the crude chromatin was col- raised against proteins injected in a solution of 0.1% NaDod- lected by centrifugation at 12,000 X g for 10 min. The chro- S04 will specifically interact with the native proteins (6). matin was washed twice with 0.005 M Tris, pH 8, and puri- Both of these sera interact with a wide range of Drosophila fied by centrifugation through 1.7 M sucrose. The purified NHC proteins, as shown by indirect immunofluorescent chromatin was washed twice with 0.01 M Tris, pH 8, staining of NaDodSO4 gels on which total NHC protein had sheared on a Virtis homogenizer at 20,000 rpm for 90 sec, been electrophoresed (ref. 6 and unpublished observations). and centrifuged at 12,000 X g for 30 min. The supernatant, IgG was prepared from the NHCP-2 sera by precipitation purified soluble chromatin, was used immediately for the with 1.75 M ammonium sulfate, dialysis to 0.0175 M phos- preparation of chromosomal proteins. About 30 mg of DNA phate buffer, and purification on DEAE-cellulose (7). as chromatin is obtained from 30 g of embryos. Preparation of Nuclei and Metaphase Chromosomes Total nonhistone chromosomal (NHC) proteins were pre- for Staining from D. melanogaster Tissue Culture Cells. pared by two techniques as follows. (i) Histones were ex- D. melanogaster tissue culture cells (Schneider's line 2) were grown as described by McKenzie et al. (8). Cells were arrest- Abbreviations: NHC proteins, nonhistone chromosomal proteins; ed in metaphase by two additions, 12 hr apart, of colchicine NaDodSO4 sodium dodecyl sulfate; phosphate-buffered saline, 0.01 (Sigma) at a final concentration of 2 ,ug/ml. Twelve hours M sodium phosphate (pH 7.0)-0.15 M NaCl. after the second inoculation cells were harvested by low- 423 Downloaded by guest on October 3, 2021 424 Biochemistry: Silver and Elgin Proc. Nat. Acad. Sci. USA 73 (1976)

speed centrifugation and washed twice with 10 ml of buff- buffered saline containing 3.7% formaldehyde. Slides were ered sucrose (0.1 M sucrose-0.5 mM CaC12-0.05 mM Pipes, held in the post-fixative for up to 3 hr, but never less than 20 pH 7) [Pipes is piperazine-N,N'-bis (2-ethanesulfonic acid) min. monosodium monohydrate]. The cells were resuspended in Indirect Immunofluorescence. The slides were removed the same buffer containing 1% Triton X-100, held at 20 for from the post-fixative solution and washed three times for 5 30 min, and homogenized with five strokes of a Teflon ho- min each in phosphate-buffered saline at ambient tempera- mogenizer. The suspension was centrifuged at 1500 X g for ture. Slides were not dried between any of the washes or in- 10 min in tubes with coverslips supported on the bottom; nu- cubations. The chromosome spreads were covered with an clei, chromosomes, and other cellular components pelleted appropriate dilution of specific antisera in phosphate-buff- onto the coverslips. Coverslips were immersed for 20 min in ered saline containing 1 mg/ml of nonspecific bovine phosphate-buffered saline containing 3.7% formaldehyde gamma globulin, and incubated in a humid atmosphere for and treated with antisera as described below. 30 min. The slides were washed again three times in phos- Fixation of Salivary Gland Nuclei. D. melanogaster lar- phate-buffered saline, and the chromosome spreads incubat- vae were grown at 180-200. Salivary glands were dissected ed with a 1:100 dilution of the fluorescein-conjugated IgG out of the larvae and were fixed by one of three methods. fraction of goat (anti-rabbit gamma globulin) serum (Miles All buffers and fixatives were made fresh from stock solu- Laboratories, Inc.) in phosphate-buffered saline for 30 min. tions on the day of the experiment. All stock solutions were This and subsequent operations were carried out under red stored with 0.01% NaNs. light. The slides were washed three times in phosphate-buff- Method I: Traditional Technique: Salivary glands were ered saline; a drop of a 9:1 solution of glycerin:1 M Tris-HCI dissected out of larvae in gland medium (25 mM disodium (pH 8.1) was placed directly on the wet slide, and a dust- glycerophosphate-10 mM KH2PO4-30 mM KCl-10 mM free coverslip was mounted. Slides were viewed with inci- MgCl2-3 mM CaCl2-162 mM sucrose) (9). Glands were in- dent ultraviolet illumination and with phase contrast optics cubated for 7 min in two changes of squashing solution (45% on a Leitz Orthoplan Microscope. Photographs were taken acetic acid-10 mM MgCl2-3.3% formaldehyde) (10) before using Tri-X film (Kodak). squashing as described below. Control: Fixation of Chromatin. Drosophila chromatin Methods II and III: Glutaraldehyde and Formaldehyde was treated with the above fixation reagents to establish the Techniques: Salivary glands were dissected out of larvae in effectiveness of the fixation procedure. Equal volumes of gland medium containing 1.0% or 0.5% Nonidet P40 (Shell soluble chromatin in gland medium and 2X concentrated Chemicals) for subsequent fixation in glutaraldehyde or fixative were mixed. After the appropriate fixation period formaldehyde, respectively, and were incubated in this solu- the chromatin was collected by centrifugation (17,000 X g, tion for 10-15 min. Care was taken not to damage the 10 min) and sequentially extracted with the squashing solu- glands during this initial incubation. Glands were trans- tion and with 0.05 M Tris, pH 8-1% NaDodSO4. Proteins ex- ferred on top of a small (3 mm square) piece of Nitex mate- tracted by the fixative were collected by precipitation with rial (nylon monofilament bolting cloth) to fixative solution 5% perchloric acid; the pellet was washed with 95% ethanol (100 mM NaCl-2 mM KCI-10 mM MgCl2-10 mM sodium and lyophilized. The squashing solution extracts were di- phosphate, pH 7.0-2% Nonidet P40) containing either 0.6% alyzed and lyophilized. The Tris-NaDodSO4 extracts were glutaraldehyde or 2% formaldehyde. The glands were incu- dialyzed to NaDodSO4 gel sample buffer. All extracts were bated for 15 min in the glutaraldehyde fixative or for 30 analyzed by NaDodSO4 gel electrophoresis (5). min in the formaldehyde fixative. The glands were then incubated for 10-15 min in squashing solution (45% acetic acid-10 mM MgCl2), and transferred (without the Nitex) to RESULTS AND DISCUSSION 10 pil of squashing solution on a siliconized coverslip. The The results obtained with this staining technique are pre- glands should be dissected free of any extraneous tissue dur- sented in Figs. 1 and 2. The general principles of such a ing the incubation in squashing solution. technique are partially analogous to those of in situ nucleic Preparation of Polytene Chromosome Spreads. A clean acid hybridization (1, 2). The fixation of the chromosomes or microscope slide was touched to the drop on the coverslip nucleus must preserve the arrangement of macromolecules and the slide was placed, coverslip up, on top of a piece of as it exists in vivo. The analytical treatment must be such black filter paper to make the glands visible. A rubber- that it does not adversely affect the cytological integrity of tipped probe was used to move the coverslip rapidly back the specimen. The sensitivity of the probe should be as high and forth horizontally on the slide, applying minimal verti- as possible; ultimately one may wish to detect molecules cal pressure. This action broke open the glands and dis- present only once per haploid genome. The detection should persed the nuclei; in addition, it was usually sufficient to be such as to permit resolution at the level of the band or break the nuclei and spread apart the chromosomes of the chromomere. traditionally fixed glands. Extensive tapping of the coverslip These starting criteria for establishing the technique ap- with the eraser end of a pencil was used to break open the pear to have been met. A high degree of spatial localization glutaraldehyde- and formaldehyde-fixed nuclei and spread of the staining is evident. Specific staining of nuclei and po- the chromosome arms. Continuous thumb pressure was used lytene chromosomes with little or no staining of other cellu- as the final step in the preparation of glutaraldehyde- and lar components is observed. Similar results, not shown, were traditionally fixed chromosome spreads. Flat spreads are es- obtained for metaphase chromosomes and nuclei of Droso- sential for good phase contrast morphology. phila tissue culture cells. At high magnification of the poly- When the chromosome preparations were spread suffi- tene chromosomes it appears possible to distinguish staining ciently (requiring a maximum of 10 min), the slide was of individual bands or chromomeres. This indicates signifi- plunged into liquid nitrogen for 15 sec. The coverslip was cant resolution, since the chromomere is the apparent unit of immediately pried off with a razor blade, and the slide was genetic (11-13) and transcriptional activity (14, 15, 8). Each placed horizontally into post-fixative solution, phosphate- chromomere of the polytene chromsomes is stained (or not Downloaded by guest on October 3, 2021 Biochemistry: Silver and Elgin Proc. Nat. Acad. Sci. USA 73 (1976) 425

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FIG. 1. Polytene chromosome spread prepared by formaldehyde fixation and stained with antisera against NHCP-2, viewed by phase contrast (a) and UV dark field optics (b).

stained) uniformly along the width of the chromosome, as rescent pattern of each polytene chromosome spread is re- anticipated; the polytene chromosomes are the consequence producible from one set of polytene chromosomes to the of replication without chromatid separation, so that each next in a given salivary gland squash. A comparison of the chromomere consists of a lateral amplification of the chro- fluorescent patterns obtained with chromosomes treated by matin by a factor of up to 1024 (16). The complete fluo- each of the fixation techniques indicates that the formalde- Downloaded by guest on October 3, 2021 426 Biochemistry: Silver and Elgin Proc. Nat. Acad. Sci. USA 73 (1976)

. 4 I . 4t

It. I

.f.0by t

4 i....-I *: i " I. 2a. 1t ' 4* 40prK. *

FIG. 2. Polytene chromosome spread prepared by the traditional technique and stained with antisera against NHCP-2, viewed by phase contrast (a) and UV dark field optics (b).

hyde and glutaraldehyde techniques yield the same qualita- suits suggest that all such analyses should be confirmed both tive fluorescent patterns, at the present level of resolution, with traditionally squashed and with formaldehyde-fixed with the two NHC protein antisera (Fig. 3). A few differ- polytene chromosomes. That the fixation is effective for the ences were noted with the traditional technique, perhaps polytene chromosomes is confirmed by the observation that due to extraction of some NHC proteins. such chromosomes are stained using anti-histone 1 sera, The traditional techniques for preparing polytene chro- while traditionally squashed chromosomes are not (unpub- mosome spreads of good morphology utilize a squashing so- lished observations). lution containing 45% acetic acid without other fixation. Un- That the staining pattern observed is a consequence of fortunately, about 50% of the total histone content of chro- specific antibody-antigen interactions is substantiated by matin is extracted by 45% acetic acid within 30 sec (17). several control experiments. No staining is observed if the However, as determined by disc gel electrophoresis, no de- chromosome preparation is initially incubated with phos- tectable protein was extracted by the acetic acid squashing phate-buffered saline, preimmunization rabbit sera, or rab- solution used here from Drosophila chromatin pretreated bit antisera against bovine serum albumin before incubation with the formaldehyde or glutaraldehyde fixatives. Most of with the fluorescein-conjugated goat antibody against rabbit the histones and about 10% of the NHC proteins were ex- IgG. Absorption of the anti-NHC protein sera with chroma- tracted from untreated Drosophila chromatin by the squash- tin prior to its use eliminates staining. However, preincuba- ing solution even if it contained 3.3% formaldehyde. About tion of the antisera with an excess of Drosophila DNA or 50% of the NHC proteins could be subsequently extracted yeast RNA has no observable effect. from the latter material with 1% NaDodSO4. Less than 10% The fluorescent patterns that we have observed are highly of the NHC proteins could be extracted from formaldehyde- complex, but several distinguishing features deserve men- fixed chromatin, and no detectable NHC proteins could be tion. The staining patterns are independent of the mass dis- extracted from glutaraldehyde-fixed chromatin, by 1% Na- tribution. The chromocenter is brightly stained. Most puffs DodSO4. Previous work of others has shown that the neutral are brightly stained, but the puff 2B5-6 is not stained bright- detergent Nonidet P40, used prior to fixation, affects cellu- ly. Occasionally, large puffs have been observed stained only lar membranes without extracting or greatly rearranging in a subsection. Many chromosomal regions are conspicuous- chromosomal proteins (18, 19). ly unstained, including the 21D region of chromosome 2 Although the glutaraldehyde technique provides the most (Fig. 3), which contains satellite DNA sequences as demon- complete fixation of chromatin, it is difficult to obtain flat, strated by in situ hybridization (20). Such understained re- well spread polytene chromosomes (and, therefore, good gions could be a consequence of limited accessibility of some phase contrast morphology) from glutaraldehyde-fixed sali- antigenic determinants after fixation, as well as a conse- vary glands. This problem is reduced with formaldehyde quence of differential distribution. The staining patterns of fixation. While the phase contrast morphology of the poly- different larvae are very similar, but quantitative differ- tene chromosomes prepared by the traditional technique is ences in the fluorescence of particular bands are apparent. excellent, some loss of contrast and resolution is revealed in Preliminary analysis suggests that the staining may be stage this case by fluorescent antibody staining. The present re- specific for some components of the NHC protein fraction. Downloaded by guest on October 3, 2021 Biochemistry: Silver and Elgin Proc. Nat. Acad. Sct. USA 73 (1976) 427

larly in the polytene chromosomes of Drosophila. Initial studies of this type will focus on the major, probably struc- tural nonhistone chromosomal proteins. Distributions of var- 3a ious enzyme activities could also be ascertained; such studies could serve as additional positive control experiments (i.e., RNA polymerase should be found to be at sites of RNA syn- thesis and may be found at additional sites). The technique should also allow confirmation of the validity of in vitro re- 3b constitution of defined portions of DNA with chromosomal proteins; the constituents of the complex at a minimum should show overlapping distributions in the native polytene chromosomes. It is hoped that this correlation of cytological, genetic, and biochemical results possible for the Drosophila chromosomes will lead to increased understanding of molec- 3c ular structure/function relationships of the eukaryotic genome.

We thank David Miller, Steve Henikoff, and J. Jose Bonner for technical suggestions. This work was supported by grants to S.C.R.E. from the Jane Coffin Childs Memorial Fund for Medical 3d Research (Project no. 305), the National Institutes of Health (GM20779), and the American Cancer Society (Grant NP-184) and by NIH Training Grant ST01 GM 00782-17 (L.M.S.).

1. Gall, J. G. & Pardue, M. L. (1969) Proc. Nat. Acad. Sct. USA 63,378-3. 3e 2. Pardue, M. L. & Gall, J. G. (1969) Proc. Nat. Acad. Sct. USA 64,600-604. 3. Elgin, S. C. R. & Hood, L. E. (1973) Biochemistry 12, 4984- palm 4991. 4. MacGillivray, A. J., Cameron, A., Krauze, R. J., Rickwood, D. F IG. 3. Comparison of the staining of chromosome arm 2L (re- & Paul, J. (1972) Biochim. Biophys. Acta 277, 384-402. gions 21 through 23) using different sera (b, c) after preparation by 5. Laemmli, U. K. (1970) Nature 227,680-685. each technique (b, d, e): phase contrast view of a formaldehyde- 6. Stumph, W. E., Elgin, S. C. R. & Hood, L. (1974) J. Immunol. fixed chromosome, stained with anti-NHCP-2 (a); UV dark field 113,1752-1756. optics view of the same chromosome (b); formaldehyde-fixed chro- 7. Williams, C. A. & Chase, M. W., eds. (1967) Methods in Im- mosome stained with anti-NHCP-1 (c); glutaraldehyde-fixed chro- munology and Immunochemistry I (Academic Press, New mosome stained with anti-NHCP-2 (d); traditionally fixed chro- York), pp. 318-324. mosome stained with anti-NHCP-2 (e). 8. McKenzie, S. L., Henikoff, S. & Meselson, M. (1975) Proc. Nat. Acad. Sci. USA 72, 1117-1121. Work in progress indicates that distinctive patterns of bands 9. Cohen, L. H. & Gotchel, B. V. (1971) J. Biol. Chem. 246, with more or less intense fluorescence are observed on stain- 1841-1848. ing polytene chromosomes with antisera raised against a 10. Holmquist, G. & Steffensen, D. M. (1973) J. Cell Biol. 59, particular molecular weight subfraction of the NHC pro- 147a. teins (to be published in detail elsewhere). 11. Judd, B. H., Shen, M. W. & Kaufman, T. C. (1972) Genetics All available evidence indicates that the technique dis- 71, 139-156. plays the distribution of the chromosomal proteins as they 12. Hochman, B. (1974) Cold Spring Harbor Symp. Quant. Biol. 38,581-589. occur in vivo. Unfortunately, no test supported by complete- 13. Lefevre, G., Jr. (1974) Cold Spring Harbor Symp. Quant. ly independent biochemical and genetic data (analogous to Biol. 38,591-599. the rRNA test of in situ RNA hybridization) is as yet possi- 14. Beerman, W. (1972) in Developmental Studies on Giant ble. Similarly, it has not yet been possible to assess accurately Chromosomes, ed. Beerman, W. (Springer-Verlag, New the ultimate sensitivity of the staining technique. Work by York), pp. 1-33. Goldman et al. (21) has demonstrated the uninterrupted flu- 15. Daneholt, B. (1975) Cell 4,1-9. orescent staining of microfilaments with antibodies against 16. Rudkin, G. T. (1972) in Developmental Studies on Giant actin. The sensitivity of immunofluorescence demonstrated Chromosomes, ed. Beerman, W. (Springer-Verlag, New by these results as well as theoretical calculations suggest the York), pp. 59-85. possibility of detecting NHC proteins that are present only 17. Dick, C. & Johns, E. W. (1968) Exp. Cell Res. 51, 626-632. once per haploid genome; a group of 1024 such proteins 18. Tata, J. R., Hamilton, M. J. & Cole, R. D. (1972) J. Mol. Biol. 67,231-246. would be present on a fully replicated polytene chromo- 19. Hancock, R. (1974) J. Mol. Biol. 86,649-663. some. 20. Peacock, W. J., Brutlag, D., Goldring, E., Appels, R., Hinton, The technique described for determination of the in situ C. W. & Lindsley, D. L. (1973) Cold Spring Harbor Symp. distribution of chromosomal proteins should be very useful Quant. Biol. 38,405-416. in studying the structure/function relationships of both the 21. Goldman, R. D., Lazarides, E., Pollack, R. & Weber, K. (1975) histones and the nonhistone chromosomal proteins, particu- Exp. Cell. Res. 90,333-344. Downloaded by guest on October 3, 2021