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

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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 distribu- tion 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.
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