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Polytene Chromosomes (Chromatin/Indirect Immunofluorescence/Protein Blotting) PETER R Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6878-6882, September 1986 Cell Biology: Correction Because of the poor quality of reproduction of Figs. 3-5 in the original publication (pp. 4744-4748), this paper is reprinted here in its entirety. Drosophila histone H2A.2 is associated with the interbands of polytene chromosomes (chromatin/indirect immunofluorescence/protein blotting) PETER R. DONAHUE, DOUGLAS K. PALMER*, JOHN M. CONDIE, LINDA M. SABATINIt, AND MARTIN BLUMENFELDt Department of Genetics and Cell Biology, The University of Minnesota, St. Paul, MN 55108 Communicated by Howard A. Schneiderman, February 24, 1986 ABSTRACT Drosophila chromatin contains two antigeni- specific polyclonal antibodies to polytene chromosomes. cally distinct H2A histones, H2A.1 and H2A.2. Indirect im- These analyses revealed intriguing contrasts between the munofluorescence analyses revealed that anti-H2A.1 binding chromosomal distributions of H2A.1 and H2A.2. was distributed throughout polytene chromosomes, whereas anti-H2A.2 binding was interband-specific. Thus, H2A.2 prob- EXPERIMENTAL PROCEDURES ably contributes to the less compacted structure of interbands. Since each band-interband region is thought to contain a single Chromosomal Protein Preparation. Chromosomal proteins gene, our results suggest that the distribution of H2A.2 echoes were prepared by mixing chromatin (9), 1:1, with NaDodSO4 the functional organization of the Drosophila genome. Similar gel sample buffer, placing the mixture in a boiling water bath H2A histones occur in eukaryotes ranging from protozoa to (5 min), and centrifuging it in an Eppendorf centrifuge (1 mammals. Their placement might be an important determi- min). nant of chromatin structure. Histone Octamer Reconstitution. Reconstitution mixtures contained 25 ,uM (each) D. melanogaster histone (9), 2 mg of Eukaryotic DNA is compacted into nucleosomes by octa- poly(glutamic acid) (Sigma, type III-B) per ml, 10 mM mers containing two molecules of each of the four histone triethanolamine-HCl (pH 8.0), 0.1 M NaCl, and 10 mM types: H2A, H2B, H3, and H4 (reviewed in refs. 1 and 2). 2-mercaptoethanol (17). They were shaken gently (12-14 hr; Primary sequence histone variants can generate nucleosome 20-22°C), diluted 1:10 with 55 mM Na2B407 (pH 9.5), diversity. In the sea urchin, sperm-specific histones are combined with 1/50th vol of dimethyl suberimidate (Sigma; replaced by maternal cleavage-stage histones during male 50 mg/ml in dimethyl sulfoxide) at four 15-min intervals, pronuclear decondensation (3). In the developing embryos, incubated (15 min), precipitated with 20% trichloroacetic acid stage-specific histones (4) affect nucleosomal structure and (0°C), washed (acetone/0.2% HCl and acetone), and air stability (5). In Tetrahymena, an H2A-like histone, hvl, dried. In some experiments, histone octamers were cross- occurs in the transcriptionally active macronucleus but not in linked reversibly with dithiobis(succinimidylpropionate) the transcriptionally inactive micronucleus (6). The appear- (Lomant's reagent; ref. 18). In these cases, 2-mercapto- ance of hvl in the developing macronucleus coincides with ethanol was omitted from the reconstitution mixture. the onset of RNA synthesis (7), suggesting a relationship Crosslinked histone complexes were separated electropho- between gene activity and the distribution of specific retically on 10 x 0.1 cm 5% acrylamide slab gels containing histones. 0.1% NaDodSO4 and 0.1 M sodium phosphate (pH 7.1) (19), In Drosophila melanogaster, only one histone sequence stained with Coomassie brilliant blue R, and destained. variant has been reported. It was discovered by Alfageme et Bands containing reversibly crosslinked histone octamers, al. (8) and designated D2, or "Drosophila 2." D2 is nucleo- produced with Lomant's reagent, were excised, incubated in somal and H2A-like in its amino acid composition (9). In this 62.5 mM Tris, pH 6.8/5% glycerol/10 mM 2-mercap- article, we establish that it functions as an H2A during the in toethanol (60 min; 22-23°C), and electrophoresed in 18% vitro reconstitution ofDrosophila histone octamers and thus acrylamide/NaDodSO4 gels (9). We therefore rename D2 as H2A.2 and the Preparation of Antibodies. H2A.2 was purified from total is an H2A histone. H2A (9) by electrophoresis in discontinuous gels containing major H2A histone as H2A.1. 0.5% Triton X-100, 6% HOAc, and 8 M urea (20), excised H2A.2 occurs with a frequency of approximately one from the gels, electrophoresed in 15% acrylamide/NaDod- molecule per five nucleosomes in 0- to 18-hr Drosophila S04 gels (9), excised, electrophoresed into Tris/glycine tray embryos, adult heads, and SL2 cells (9, 10). It has been buffer in a sealed dialysis membrane, precipitated with 6 vol conserved during the evolution of Drosophila. Its electro- of acetone/0.2% HCl (12-16 hr; -20°C), centrifuged, dis- phoretic mobility in Triton X-100/acetic acid/urea gels is solved in H20, precipitated with 20% trichloroacetic acid, sensitive to the [Triton X-100]/[urea] ratio. Since H2A collected by centrifugation, washed with acetone/0.2% HCl, histones with similar electrophoretic properties in Triton washed twice with acetone, and air dried. Preimmune sera X-100/acetic acid/urea gels and amino acid compositions were collected, antibodies were induced, and immune sera have been found in Tetrahymena (6), sea urchins (11, 12), were prepared as described (21). Rabbits were injected with birds (13), and mammals (14-16), H2A.2 may belong to an 200 ,g of electrophoretically pure H2A.1 or H2A.2, given evolutionarily conserved histone H2A family. booster injections 10 and 20 days after the initial immuniza- Even though histone subtypes have been analyzed exten- tion, and bled 1 wk later. In some experiments, antibodies sively, their functions are still unclear. To approach this were preadsorbed from immune sera with 1-2 ug of antigen problem we studied the binding of H2A.1- and H2A.2- *Present address: Hutchinson Cancer Center, Seattle, WA 98104. The publication costs of this article were defrayed in part by page charge tPresent address: Laboratory of Genetics, The University of Wis- payment. This article must therefore be hereby marked "advertisement" consin, Madison, WI 53706. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom correspondence should be addressed. 6878 Downloaded by guest on September 29, 2021 Cell Biology: Correction Proc. Natl. Acad. Sci. USA 83 (1986) 6879 per dul of serum and removed by centrifugation in a micro- characteristic ladder of nine prominent bands was produced. centrifuge (5 min). The two fastest migrating bands contained monomeric To affinity-purify antibodies, immune sera were dialyzed histones. The seven slower migrating bands were multimers, into phosphate-buffered saline (PBS) (150 mM NaCi/10 mM ranging from dimers to octamers (Fig. la). When any histone sodium phosphate, pH 7.3), adjusted to 0.5% Tween 20/10 class was omitted from the reconstitution mixtures, only units of aprotinin per ml/0.02% NaN3, and incubated with traces of multimers larger than tetramers were detected (data nitrocellulose (Schleicher & Schuell) containing 4-8 ,.g of not shown). Therefore, H2A.2 supports the in vitro recon- H2A histone per cm2 (12-16 hr; 370C). Bound antibodies were stitution of Drosophila histone octamers. eluted from the nitrocellulose with 5 M Nal in Tris-buffered To confirm that H2A.2 was incorporated into the recon- saline (TBS) (0.5 M NaCl/20 mM Tris, pH 7.5) and 100 pug of stituted octamers, we purified octamers that were reversibly bovine serum albumin per ml (5 min; 22-240C), concentrated crosslinked with Lomant's reagent (indicated by the arrow in 3-fold with Sephadex G-10, desalted with Sephadex G-25, Fig. lb), reversed the crosslinks with 2-mercaptoethanol, and and concentrated 10- to 15-fold with an Amicon A-25 analyzed the released proteins electrophoretically. These microconcentrator. analyses revealed that reconstituted octamers contain H2A. 1 To assay antibody specificity, Drosophila histones or total (or H2A.2), H2B, H3, and H4 (Fig. 1 c and d). H2A.1 and chromosomal proteins were electrophoresed in acryl- H2B are completely amide/NaDodSO4 gels. Replicate gel sections were stained resolved on acrylamide/NaDodSO4 (Coomassie blue R or silver stain; ref. 22) or transferred a - b Xir electrophoretically to nitrocellulose membranes (90 min; 1.3 R mA). The membranes were placed in 3% bovine serum t albumin/0.25% carageenan (60 min), washed in TBS/0.05% 8- Tween 20 (TBST), incubated with 3-5 ml of diluted serum (1:100 in TBS/0.25% carageenan; 2 hr), washed twice in 7-: -t # ti TBST (20 min), incubated with peroxidase-conjugated goat anti-rabbit IgG (1:2000; Miles) in TBS (60 min), washed twice 4- in TBST, incubated in TBS/0.015% H202/0.05% 4-chloro- 1, 1-naphthol (5-10 min; 22-23°C), and washed twice with U- 3- 0 distilled H20 (23). 1i5-if .. Indirect Immunofluorescence. Antibody preparations were diluted with 0.5 M NaCl/10 mM sodium phosphate, pH 7.5. z 14 2 I Anti-H2A.1 was diluted 1:60-120; anti-H2A.2, 1:3-32; fluo- rescein isothiocyanate-conjugated goat anti-rabbit IgG (Miles), 1:100. D. melanogaster cultures were maintained at I 17°C in yeasted cornmeal vials. Salivary glands were dissect- 1 2 ed from normal or heat-shocked (20-30 min; 37°C; ref. 24) late third instar larvae in G medium (25), transferred to G 0 medium/0.05% Nonidet P-40 (NP-40) (2 min), fixed in C PBS/3.7% formaldehyde (2 min), transferred to 45% H3- H2A.2- HOAc/3.7% formaldehyde (1.5 min), transferred to 45% H2B- acetic acid/10 mM MgCl2, dehydrated in 95% ethanol (30 H2A.1- min; -70°C; ref. 26), and processed as described (27).
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