Polytene Chromosomes (Chromatin/Indirect Immunofluorescence/Protein Blotting) PETER R

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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; fluorescein 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). In H4- some experiments, glands were fixed in 100 mM NaCl/2 mM KCl/10 mM MgCl2/10 mM sodium phosphate, pH 7.0/2% 2 NP-40/2% formaldehyde (30 min) as described (27). Subse- quent steps were performed at 37°C. Chromosome squashes were covered with 150 ,ul of diluted immune, preimmune, or d preadsorbed serum, incubated in a humid chamber (30 min), washed gently in three changes ofPBS (15 min), covered with 150 ,ul of fluorescein isothiocyanate-conjugated goat anti- rabbit IgG, incubated (30 min), washed three times in PBS, mounted in glycerol/i M Tris, pH 8.1 (9:1, vol/vol), and covered with a clean coverslip. Slides were examined on a Zeiss photomicroscope III, using a 40x Neofluar objective. UV-fluorescent images were photographed on Kodak Tri-X Pan film at ASA 800. For anti-H2A. 1, the exposure time was 8-20 sec; for anti-H2A.2, 12-25 sec. Unstained chromosomes 2 were photographed with Kodak Panatomic X film. RESULTS E9 H3 H2A2 H2B H2A.1 H4 19 D. melanogaster Nuclei Contain Two Distinct H2A Histones. FIG. 1. Electrophoretic analyses of reconstituted D. melano- The following experiments demonstrated that D2 could gaster histone octamers. In each analysis, part 1 shows histone substitute for histone H2A during the in vitro reconstitution complexes reconstituted with H2A.2; part 2, with H2A.1. (a) of Drosophila histone octamers. By this criterion, it is an Reconstitution complexes containing H2A.2 (or H2A.1), H2B, H3, H2A histone. Since D2 migrates less rapidly than the major and H4 were crosslinked and then electrophoresed in 5% acrylamide Drosophila H2A on NaDodSO4/PAGE (9), we have renamed gels. (b) Histone octamers, indicated by the arrow, formed with H2A.2 (or H2A.1), reversibly crosslinked with Lomant's reagent, it as H2A.2 and the major H2A histone as H2A.1. and purified electrophoretically. (c) The crosslinks of the octamer When equimolar amounts of histones H2A.1 (or H2A.2), bands in b were reversed, and the proteins were electrophoresed in H2B, H3, and H4 were reconstituted in vitro (see Experi- 18% acrylamide gels and stained. (d) Densitometric scans of the gels mental Procedures) and separated on 5% acrylamide gels, a in c, revealing the separation of H2B and H2A.2. Downloaded by guest on September 29, 2021 6880 .Cell Biology: Correction Proc. Natl. Acad. Sci. USA 83 (1986) gels; H2A.2 and H2B are partially resolved (9). Consequent- phase-dark bands at 49F, 50A, 50C, and SOF. Region ly, H2A.1 and H2B appear as two discrete bands (Fig. ic, 61A-63A includes the telomere of chromosome arm 3L. lane 1), whereas H2A.2 and H2B appear as a broad band (lane 2). However, H2A.2 and H2B were resolved by densitometric scanning ofthe stained gels (Fig. id), which also indicated that the H2A:H2B:H3:H4 stoichiometry of the reconstituted octamers was approximately 1:1:1:1. The H2A.2 preparation used for these experiments contained about 5% H2A.1 (data not shown). Consequently, a small H2A.1 peak is visible between the H2B and H4 peaks in the histones from octamers reconstituted with H2A.2 (Fig. id). We could not determine whether this peak reflected the formation of octamers containing H2A.1 and H2A.2. H2A.1 and H2A.2 Are Antigenically Distinct. Analyses of antibody binding to electrophoretically separated H2A revealed that anti-H2A. 1 bound H2A.1 but not H2A.2, whereas anti-H2A.2 bound H2A.2 but not H2A.1 (Fig. 2a). Analyses of antibody binding to electrophoretically separated chromosomal proteins established that affinity-purified anti-H2A.1 mom bound only H2A.1 and the protein identified as ubiquitinated H2A (28), whereas affinity-purified anti-H2A.2 bound only H2A.2 (Fig. 2b). Preimmune sera, preadsorbed anti-H2A.1, and preadsorbed anti-H2A.2 did not bind H2A. Anti-H2A.2 did not bind chromatographically purified H2B (data not shown). Anti-H2A.2 Binds Polytene Chromosome Interbands But Does Not Bind Bands. Indirect immunofluorescence analyses of antibody binding to polytene chromosome regions 49F-51F and 61A-63A demonstrated that anti-H2A.2 binding was interband-specific. Region 49F-51F (on chromosome arm 2R) contains "the goggles"-distinct phase-dark bands at 50A and SOC (29) and a puff at 50CD that is constitutively active after puff stage 1 (24). Anti-H2A.2 bound each interband between S1A and 51F and four distinct sites within the phase-light area between SOA and 50C. It did not bind the a b

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1 2 3 FIG. 2. Specificities of anti-H2A.1 and anti-H2A.2. (a) Electrophoretically separated Drosophila histone H2A (lane 1) was transferred to a nitrocellulose membrane. After transfer, the membrane was split longitudinally through lane 3 and incubated with anti-H2A.1 (lanes 2 and 3) or anti-H2A.2 (lanes 3 and 4) serum. FIG. 3. Phase-contrast and UV fluorescence photographs of Antibody binding was visualized by peroxidase staining. Anti-H2A.2 chromosome regions 49F-51F (a) and 61A-63A (b) incubated with bound H2A.2 but not H2A.1; anti-H2A.1 bound H2A.1 but not anti-H2A.2 (panels 1-4) or anti-H2A.1 (panels 5-8). Panels 2 and 6 H2A.2. (b) Electrophoretically separated Drosophila chromosomal show UV fluorescence of chromosomes incubated with affinity- proteins (lane 3) were transferred to nitrocellulose. After transfer, the purified anti-H2A.2 and H2A.1, respectively; panels 3 and 7 show membrane was cut into strips, which were incubated with affinity- UV fluorescence of chromosomes incubated with anti-H2A.2 and purified anti-H2A.1 (lane 1) or anti-H2A.2 (lane 2). Antibody binding anti-H2A.1 sera, respectively. Panels 1, 4, 5, and 8 show corre- was visualized by peroxidase staining. Anti-H2A.1 bound H2A.1 and sponding phase-contrast photographs. Interband-specific binding the protein identified as ubiquitinated H2A (28); and anti-H2A.2 occurred with affinity-purified anti-H2A.2 (panel 2) and anti-H2A.2 bound H2A.2. uH2A, ubiquitinated H2A. sera (panel 3). (Bar = 5 ,um.) Downloaded by guest on September 29, 2021 Cell Biology: Correction Proc. Natl. Acad. Sci. USA 83 (1986) 6881 Anti-H2A.2 bound interbands but not phase-dark bands within this region. Comparable binding patterns were detected with affinity-purified anti-H2A.2 and anti-H2A.2 serum (Fig. 3 a and b, panels 2 and 3). Anti-H2A.1, in contrast, bound bands and interbands within 49F-51A and 61A-63A. It bound intensely to some bands, exemplified by 49F, 50D, and 51D. It did not bind to others, exemplified by 50A and 50C. The absence ofantibody binding to some highly compacted chromosomal bands has been noted previously (27). Anti-H2A.1 bound to the margins binding patterns were detected with of 50 CD. Comparable a I APZ_ afflnity-purified anti-H2A.1 and anti-H2A.1 sera (Fig. 3 a and vvs. b, panels 6 and 7). p~w The interband-specific binding of anti-H2A.2 was detected on each polytene chromosome arm (Fig. 4), with lightly fixed (26) and heavily fixed (27) chromosomes (data not shown). FIG. 5. Phase-contrast and UV photographs of chromosome arm Anti-H2A.2 bound preferentially to interbands, exemplified 3R incubated with anti-H2A.2 or anti-H2A.1 serum. Anti-H2A.2 by 88A and 89C, at all antiserum dilutions tested (1:4 to 1:32). dilutions were 1:32 (a and b) or 1:4 (c and d), anti-H2A.1 dilutions Anti-H2A.1 bound preferentially to bands, exemplified by were 1:65 (e and f) or 1:130 (g and h). Anti-H2A.2 bound preferen- 87F, 88C, and 88E, at an antiserum dilution of 1:130. tially to interbands at all dilutions tested. Anti-H2A.1 bound pref- However, at a 1:65 dilution, it bound bands, interbands, and erentially to bands at a 1:130 dilution, but bound bands, interbands, puffs (Fig. 5). Equivalent dilutions of preimmune sera, and puffs at a 1:65 dilution. (Bar = 5 A.m.) preadsorbed anti-H2A.1, and preadsorbed anti-H2A.2 did not bind to polytene chromosomes (data not shown; ref. 30). histone H2A (but not for histone H2B, H3, or H4) during the Analyses of chromosomes prepared from heat-shocked in vitro reconstitution of Drosophila histone octamers and larvae failed to detect anti-H2A.2 binding to heat shock- therefore is an H2A histone. On this basis, we have renamed induced puffs at 63C, 67B, 87A, 87C, 93D, and 95D. Parallel D2 as H2A.2 and the major Drosophila histone as H2A.1. analyses with anti-H2A.1 suggested that anti-H2A.1 binding H2A.1 and H2A.2 have different amino acid compositions varied with the puff. Puffs 63C, 93D, and 95D contained and tryptic peptide maps. At best, 30% of the tryptic peptides distinct fluorescent regions; 87A and 87C contained diffuse ofH2A.1 occur in H2A.2 (9). Given these differences, it is not fluorescence; 67B lacked fluorescence (Figs. 4 and 5). surprising that H2A.1 and H2A.2 are antigenically distinct. It is surprising, however, that antibodies against these two antigenically distinct Drosophila H2A histones have dramat- DISCUSSION ically different binding patterns along salivary gland polytene D. melanogaster Nuclei Contain Two Antigenically Distinct chromosomes. H2A Histones. The nucleosomal protein D2 can substitute for H2A.2 and Polytene Chromosome Interbands. Polytene

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nuclei occur in numerous tissues of higher dipteran insects. predoctoral trainees of the National Institutes of Health. This They are considered interphase nuclei in which chromosomal research was supported by the National Institutes of Health, The DNA is endoreduplicated, while the chromosomal homologs Monsanto Company, and The University of Minnesota Graduate remain synapsed. In certain tissues, such as the salivary School. glands of late third instar Drosophila larvae, the polytene 1. McGhee, J. D. & Felsenfeld, G. (1980) Annu. Rev. Biochem. 49, chromosomes can be resolved microscopically into a series of 1115-1156. alternating dark bands and light interbands. Banding can be 2. Igo-Kemenes, T., Horz, W. & Zachau, H. G. (1982) Annu. Rev. detected in unfixed, unstained nuclei and reflects physical Biochem. 51, 89-121. differences along polytene chromosomes. DNA is more 3. Poccia, D., Salik, J. & Krystal, G. (1982) Dev. Biol. 82, 287-296. heavily compacted in bands (see ref. 31 for a review). Indirect 4. Newrock, K. M., Alfageme, C. R., Nardi, R. V. & Cohen, L. H. immunofluorescence analyses reveal that histones H1, H2B, (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 421-432. 5. Simpson, R. T. (1981) Proc. Natl. Acad. Sci. USA 78, 6803- H3, and H4 are distributed in the bands and interbands of 6807. polytene chromatin (reviewed by ref. 32). The interband- 6. Allis, C. D., Glover, C. V. C., Bowen, J. K. & Gorovsky, M. A. specific binding of anti-H2A.2, in contrast, strongly suggests (1980) Cell 20, 609-617. that the chromosomal distribution of H2A.2 matches the 7. Wenkert, D. & Allis, C. D. (1984) J. Cell Biol. 98, 2107-2117. distribution of interbands. It will be interesting to learn how 8. Alfageme, C. R., Zweidler, A., Mahowald, A. & Cohen, L. H. (1974) J. Biol. Chem. 249, 3729-3740. nucleosomes containing H2A.2 contribute to the less com- 9. Palmer, D., Snyder, L. A. & Blumenfeld, M. (1980) Proc. Natl. pacted structure of interbands. Acad. Sci. USA 77, 2671-2675. Genetic (33) and molecular (34) analyses suggest that each 10. Condie, J. M. (1984) Dissertation (The University of Minnesota). band-interband region contains one structural gene. The 11. Newrock, K. M., Friedman, N., Alfageme, C. R. & Cohen, L. H. interband-specific location of H2A.2, together with the evi- (1982) Dev. Biol. 89, 248-253. dence that the 12. Wu, R. S., Nishoka, D. & Bonner, W. M. (1982) J. Cell Biol. 93, band-interband region is the functional unit of 426-431. the polytene chromosome, suggest that the placement of 13. Urban, M. K., Franklin, S. G. & Zweidler, A. (1979) Biochemistry H2A.2 echoes the functional organization of the Drosophila 18, 3952-3960. genome. 14. Franklin, S. K. & Zweidler, A. (1977) Nature (London) 266, The Tetrahymena H2A variant hvl is correlated with 272-275. transcriptionally active macronuclei (6, 7). This correlation 15. Albright, S. C., Nelson, 0. P. & Garrard, W. T. (1979) J. Biol. Chem. 1065-1073. appears to between H2A.2 and 254, analogous the correlation 16. West, M. H. P. & Bonner, W. M. (1980) Biochemistry 19, interbands. Interbands are associated with ribonucleoprotein 3238-3245. particles (35), bound by antibodies against Drosophila RNA 17. Stein, A., Whitlock, J. P. & Bina, M. (1979) Proc. Natl. Acad. Sci. polymerase 11 (26) or RNADNA hybrids (36), and thus may USA 76, 5000-5004. contain some transcriptionally active DNA sequences. The 18. Lomant, A. J. & Fairbanks, G. (1976) J. Mol. Biol. 104, 243-261. correlations with transcriptional activity, plus the evidence 19. Weber, K., Pringle, J. & Osborn, M. J. (1972) Methods Enzymol. 26, 3-26. that similar H2A histones have a broad phylogenetic distri- 20. Bonner, W., West, M. & Stedman, J. (1980) Eur. J. Biochem. 109, bution (6, 11-16), suggest that the placement of "Drosophila 17-23. H2A.2-like" histones reflects the underlying functional or- 21. Stollar, B. D. & Ward, M. (1970) J. Biol. Chem. 245, 1261-1266. ganization of chromatin. 22. Switzer, R. C., Merrill, C. R. & Shifrin, S. (1979) Anal. Biochem. The interband-specific binding of anti-H2A.2 does not 98, 231-237. 23. Hawkes, R., Niday, E. & Gordon, J. (1982) Anal. Biochem. 119, disappear when antibody concentration is increased 8-fold 142-150. and thus does not reflect competition for limiting amounts of 24. Ashburner, M. (1972) in Results and Problems in Cell Differentia- antibody. In contrast, the preferential binding of anti-H2A.1 tion, ed. Beermann, W. (Springer, New York), pp. 101-151. to polytene chromosome bands disappears when antibody 25. Cohen, L. H. & Gotchel, B. V. (1971) J. Biol. Chem. 246, concentration is increased by a factor of 2 and thus reflects 1841-1848. competition for limiting amounts of antibody. This is con- 26. Jamrich, M., Greenleaf, A., Bautz, F. A. & Bautz, E. K. F. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 389-396. sistent with the fact that DNA (and presumably histone 27. Silver, L. E. & Elgin, S. C. R. (1976) Proc. Natl. Acad. Sci. USA H2A. 1) is more heavily concentrated in polytene chromo- 73, 423-427. some bands. 28. Levinger, L. & Varshavsky, A. (1982) Cell 28, 375-385. The interband-specific binding of anti-H2A.2 occurs with a 29. Lefevre, G., Jr. (1976) in The Genetics and Biology ofDrosophila, variety of fixation conditions and probably is not a cytolog- eds. Ashburner, M. & Novitski, E. (Academic, New York), pp. ical artifact. This concern is important because the interband- 31-66. 30. Donahue, P. R. (1986) Dissertation (The University of Minnesota). specific binding of anti-Z-DNA (37) is influenced by the 31. Laird, C. (1980) Cell 22, 865-874. conditions of chromosomal fixation (38, 39). Conceivably, 32. Cartwright, I., Abmayr, S., Fleischmann, G., Lowenhaupt, K., H2A.2 might be present in bands but inaccessible to anti- Elgin, S., Keene, M. & Howard, G. (1982) CRC Crit. Rev. bodies. This possibility is unlikely because H2A.1 in bands is Biochem. 13, 1-86. accessible to antibodies under the same conditions. 33. Judd, B. H. & Young, M. W. (1974) Cold Spring Harbor Symp. 573-579. It is not yet clear whether our to detect H2A.2 in Quant. Biol. 38, inability 34. Bossy, B., Hall, L. M. C. & Spierer, P. (1984) EMBO J. 3, heat shock puffs reflects dissociation ofH2A.2 from puff sites 2537-2541. during puffing or the dilution of H2A.2-specific fluorescence 35. Skaer, R. J. (1977) J. Cell Sci. 26, 251-266. over the large areas of puffs. It appears unlikely that H2A.2, 36. Vlassova, I. E., Umbetova, G. H., Zimmermann, V. H., Alonso, but not H2A.1, is extracted from puffs during cytological C., Belyaeva, E. S. & Zhimulev, I. F. (1985) Chromosoma 91, fixation. As a step in further investigating this problem, it will 251-258. 37. Nordheim, A., Pardue, M. L., Lafer, E. M., Moller, A., Stollar, be important to learn if puff formation and regression are R. D. & Rich, A. (1981) Nature (London) 294, 417-422. correlated with changes in the pattern of anti-H2A.2 binding. 38. Arndt-Jovin, D., Robert-Nicoud, M., Zarling, D. A., Greider, C., Weimer, E. & Jovin, T. M. (1983) Proc. Natl. Acad. Sci. USA 80, We thank Jose Bonner, Perry Hackett, Steve Henikoff, Vicki 4344-4348. Iwanij, Brooke Kirby, Lou Morejohn, Janet Schottel, and Walter 39. Lancillotti, F., Lopez, M. C., Alonso, C. & Stollar, B. D. (1985) J. Sauerbier for helpful suggestions. P.R.D. and J.M.C. were Cell Biol. 100, 1759-1766. Downloaded by guest on September 29, 2021 Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4744-4748, July 1986 Cell Biology 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 (0WC), 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). is an H2A histone. We therefore rename D2 as H2A.2 and the Preparation of Antibodies. H2A.2 was purified from total major H2A histone as H2A.1. H2A (9) by electrophoresis in discontinuous gels containing H2A.2 occurs with a frequency of approximately one 0.5% Triton X-100, 6% HOAc, and 8 M urea (20), excised molecule per five nucleosomes in 0- to 18-hr Drosophila from the gels, electrophoresed in 15% acrylamide/NaDod- cells It has been S04 gels (9), excised, electrophoresed into Tris/glycine tray embryos, adult heads, and SL2 (9, 10). 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. *To whom correspondence should be addressed. 4744 Cell Biology: Donahue et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4745 per pl 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 NaCl/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 pug 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 S 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 resolved on acrylamide/NaDodSO4 amide/NaDodSO4 gels. Replicate gel sections were stained (Coomassie blue R or silver stain; ref. 22) or transferred electrophoretically to nitrocellulose membranes (90 min; 1.3 a - b ez mA). The membranes were placed in 3% bovine serum . . albumin/0.25% carageenan (60 min), washed in TBS/0.05% 5- Tween 20 (TBST), incubated with 3-5 ml of diluted serum 8-- (1:100 in TBS/0.25% carageenan; 2 hr), washed twice in 6-_ uj 5 - % TBST (20 min), incubated with peroxidase-conjugated goat z anti-rabbit IgG (1:2000; Miles) in TBS (60 min), washed twice I-- 4- CIO in TBST, incubated in TBS/0.015% H202/0.05% 4-chloro- 0M 1-naphthol (5-10 min; 22-23°C), and washed twice with u. 3-- distilled H20 (23). 0 II Indirect Immunofluorescence. Antibody preparations were diluted with 0.5 M NaCl/10 mM sodium phosphate, pH 7.5. Anti-H2A.1 was diluted 1:60-120; anti-H2A.2, 1:3-32; fluorescein isothiocyanate-conjugated goat anti-rabbit IgG 1 1 .. (Miles), 1:100. D. melanogaster cultures were maintained at 17°C in yeasted cornmeal vials. Salivary glands were dissect- ed from normal or heat-shocked (20-30 min; 37°C; ref. 24) 1 2 1 2 late third instar larvae in G medium (25), transferred to G medium/0.05% Nonidet P-40 (NP-40) (2 min), fixed in C 0 PBS/3.7% formaldehyde (2 min), transferred to 45% H3- Ca2H2A.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). In H4 some experiments, glands were fixed in 100 mM NaCl/2 mM KCl/10 mM MgCl2/10 mM sodium phosphate, pH 7.0/2% 2 NP-40/2% formaldehyde (30 min) as described (27). Subse- quent steps were performed at 37°C. Chromosome squashes were covered with 150 A.l of diluted immune, preimmune, or d preadsorbed serum, incubated in a humid chamber (30 min), washed gently in three changes ofPBS (15 min), covered with 150 Al of fluorescein isothiocyanate-conjugated goat anti- rabbit IgG, incubated (30 min), washed three times in PBS, mounted in glycerol/1 M Tris, pH 8.1 (9:1, vol/vol), and -- covered with a clean coverslip. Slides were examined on a , ,--IV Zeiss photomicroscope III, using a 40x Neofluar objective. UV-fluorescent images were photographed on Kodak Tri-X Pan film at ASA 800. For anti-H2A.1, the exposure time was 8-20 sec; for anti-H2A.2, 12-25 sec. Unstained chromosomes 2 were photographed with Kodak Panatomic X film.

RESULTS E0 H3 H2A 2 H2B H2AJ H4 D. melanogaster Nuclei Contain Two Distinct H2A Histones. FIG. 1. Electrophoretic analyses of reconstituted D. melano- The following experiments demonstrated that D2 could gaster histone octamers. In each analysis, part 1 shows histone substitute for histone H2A during the in vitro reconstitution complexes reconstituted with H2A.2; part 2, with H2A.1. (a) of Drosophila histone octamers. By this criterion, it is an Reconstitution complexes containing H2A.2 (or H2A.1), H2B, H3, H2A histone. Since D2 migrates less rapidly than the major and H4 were crosslinked and then electrophoresed in 5% acrylamide gels. (b) Histone octamers, indicated by the arrow, formed with Drosophila H2A on NaDodSO4/PAGE (9), we have renamed H2A.2 (or H2A.1), reversibly crosslinked with Lomant's reagent, it as H2A.2 and the major H2A histone as H2A.1. and purified electrophoretically. (c) The crosslinks of the octamer When equimolar amounts of histones H2A.1 (or H2A.2), bands in b were reversed, and the proteins were electrophoresed in H2B, H3, and H4 were reconstituted in vitro (see Experi- 18% acrylamide gels and stained. (d) Densitometric scans of the gels mental Procedures) and separated on 5% acrylamide gels, a in c, revealing the separation of H2B and H2A.2. 4746 Cell Biology: Donahue et al. Proc. Natl. Acad. Sci. USA 83 (1986) gels; H2A.2 and H2B are partially resolved (9). Consequent- phase-dark bands at 49F, 50A, SOC, and SOF. Region ly, H2A.1 and H2B appear as two discrete bands (Fig. 1c, 61A-63A includes the telomere of chromosome arm 3L. lane 1), whereas H2A.2 and H2B appear as a broad band (lane 2). However, H2A.2 and H2B were resolved by densitometric scanning ofthe stained gels (Fig. id), which also indicated that the H2A:H2B:H3:H4 stoichiometry of the reconstituted octamers was approximately 1:1:1:1. The H2A.2 preparation used for these experiments contained about 5% H2A.1 (data not shown). Consequently, a small H2A.1 peak is visible between the H2B and H4 peaks in the histones from octamers reconstituted with H2A.2 (Fig. id). We could not determine whether this peak reflected the formation of octamers containing H2A.1 and H2A.2. H2A.1 and H2A.2 Are Antigenically Distinct. Analyses of antibody binding to electrophoretically separated H2A revealed that anti-H2A.1 bound H2A.1 but not H2A.2, whereas anti-H2A.2 bound H2A.2 but not H2A.1 (Fig. 2a). Analyses of antibody binding to electrophoretically separated chromosomal proteins established that affinity-purified anti-H2A.1 bound only H2A.1 and the protein identified as ubiquitinated H2A (28), whereas afflinity-purified anti-H2A.2 bound only H2A.2 (Fig. 2b). Preimmune sera, preadsorbed anti-H2A.1, and preadsorbed anti-H2A.2 did not bind H2A. Anti-H2A.2 did not bind chromatographically purified H2B (data not shown). Anti-H2A.2 Binds Polytene Chromosome Interbands But Does Not Bind Bands. Indirect immunofluorescence analyses of antibody binding to polytene chromosome regions 49F-51F and 61A-63A demonstrated that anti-H2A.2 binding was interband-specific. Region 49F-51F (on chromosome arm 2R) contains "the goggles"-distinct phase-dark bands at 50A and 50C (29) and a puff at 5OCD that is constitutively active after puff stage 1 (24). Anti-H2A.2 bound each interband between S1A and S1F and four distinct sites within the phase-light area between 50A and SOC. It did not bind the a b

.2 i -Hl

...... ! 2 3

-uH2A

-H2A.2-H3 -H2A11H4

FIG. 2. Specificities of anti-H2A.1 and anti-H2A.2. (a) Electrophoretically separated Drosophila histone H2A (lane 1) was transferred to a nitrocellulose membrane. After transfer, the membrane was split longitudinally through lane 3 and incubated with anti-H2A.1 (lanes 2 and 3) or anti-H2A.2 (lanes 3 and 4) serum. FIG. 3. Phase-contrast and UV fluorescence photographs of Antibody binding was visualized by peroxidase staining. Anti-H2A.2 chromosome regions 49F-51F (a) and 61A-63A (b) incubated with bound H2A.2 but not H2A.1; anti-H2A.1 bound H2A.1 but not anti-H2A.2 (panels 1-4) or anti-H2A.1 (panels 5-8). Panels 2 and 6 H2A.2. (b) Electrophoretically separated Drosophila chromosomal show UV fluorescence of chromosomes incubated with affinity- proteins (lane 3) were transferred to nitrocellulose. After transfer, the purified anti-H2A.2 and H2A.1, respectively; panels 3 and 7 show membrane was cut into strips, which were incubated with affinity- UV fluorescence of chromosomes incubated with anti-H2A.2 and purified anti-H2A. 1 (lane 1) or anti-H2A.2 (lane 2). Antibody binding anti-H2A.1 sera, respectively. Panels 1, 4, 5, and 8 show corre- was visualized by peroxidase staining. Anti-H2A. 1 bound H2A. 1 and sponding phase-contrast photographs. Interband-specific binding the protein identified as ubiquitinated H2A (28); and anti-H2A.2 occurred with affinity-purified anti-H2A.2 (panel 2) and anti-H2A.2 bound H2A.2. uH2A, ubiquitinated H2A. sera (panel 3). (Bar = 5 ,um.) Cell Biology: Donahue et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4747 Anti-H2A.2 bound interbands but not phase-dark bands within this region. Comparable binding patterns were detected with affinity-purified anti-H2A.2 and anti-H2A.2 serum (Fig. 3 a and b, panels 2 and 3). Anti-H2A.1, in contrast, bound bands and interbands within 49F-51A and 61A-63A. It bound intensely to some bands, exemplified by 49F, SOD, and 51D. It did not bind to others, exemplified by 50A and 50C. The absence ofantibody binding to some highly compacted chromosomal bands has been noted previously (27). Anti-H2A.1 bound to the margins of 50 CD. Comparable binding patterns were detected with affinity-purified anti-H2A.1 and anti-H2A.1 sera (Fig. 3 a and b, panels 6 and 7). The interband-specific binding ofanti-H2A.2 was detected on each polytene chromosome arm (Fig. 4), with lightly fixed (26) and heavily fixed (27) chromosomes (data not shown). FIG. 5. Phase-contrast and UV photographs ofchromosome arm Anti-H2A.2 bound preferentially to interbands, exemplified 3R incubated with anti-H2A.2 or anti-H2A.1 serum. Anti-H2A.2 by 88A and 89C, at all antiserum dilutions tested (1:4 to 1:32). dilutions were 1:32 (a and b) or 1:4 (c and d); anti-H2A.1 dilutions Anti-H2A.1 bound preferentially to bands, exemplified by were 1:65 (e and f) or 1:130 (g and h). Anti-H2A.2 bound preferen- 87F, 88C, and 88E, at an antiserum dilution of 1:130. tially to interbands at all dilutions tested. Anti-H2A.1 bound pref- However, at a 1:65 dilution, it bound bands, interbands, and erentially to bands at a 1:130 dilution, but bound bands, interbands, puffs (Fig. 5). Equivalent dilutions of preimmune sera, and puffs at a 1:65 dilution. (Bar = 5 ,um.) preadsorbed anti-H2A.1, and preadsorbed anti-H2A.2 did not bind to polytene chromosomes (data not shown; ref. 30). histone H2A (but not for histone H2B, H3, or H4) during the Analyses of chromosomes prepared from heat-shocked in vitro reconstitution of Drosophila histone octamers and larvae failed to detect anti-H2A.2 binding to heat shock- therefore is an H2A histone. On this basis, we have renamed induced puffs at 63C, 67B, 87A, 87C, 93D, and 95D. Parallel D2 as H2A.2 and the major Drosophila histone as H2A.1. analyses with anti-H2A.1 suggested that anti-H2A.1 binding H2A.1 and H2A.2 have different amino acid compositions varied with the puff. Puffs 63C, 93D, and 95D contained and tryptic peptide maps. At best, 30% ofthe tryptic peptides distinct fluorescent regions; 87A and 87C contained diffuse ofH2A.1 occur in H2A.2 (9). Given these differences, it is not fluorescence; 67B lacked fluorescence (Figs. 4 and 5). surprising that H2A.1 and H2A.2 are antigenically distinct. It is surprising, however, that antibodies against these two DISCUSSION antigenically distinct Drosophila H2A histones have dramat- ically different binding patterns along salivary gland polytene D. melanogaster Nuclei Contain Two Antigenically Distinct chromosomes. H2A Hitoacs. The nucleosomal protein D2 can substitute for H2A.2 and Polytene Chromosome Interbands. Polytene

FIG. 4. Binding of anti-H2A.2 and anti-H2A.1 sera to polytene chromosomes. (a and b) Phase- contrast and UV fluorescence of chromosomes incubated with anti- H2A.2. (c and d) Phase-contrast and UV fluorescence of chromosomes incubated with anti-H2A.1. Anti-H2A.2 binds interbands but does not bind bands or heat shock puffs; anti-H2A.1 binds interbands, bands, and heat shock puffs 63C, 87A, 93D, and 95D. Heat shock puffs are indicated. (Bar = 5 Aim.) 4748 Cell Biology: Donahue et al. Proc. Natl. Acad. Sci. USA 83 (1986) nuclei occur in numerous tissues of higher dipteran insects. predoctoral trainees of the National Institutes of Health. This They are considered interphase nuclei in which chromosomal research was supported by the National Institutes of Health, The DNA is endoreduplicated, while the chromosomal homologs Monsanto Company, and The University of Minnesota Graduate remain synapsed. In certain tissues, such as the salivary School. instar Drosophila larvae, the polytene glands of late third 1. McGhee, J. D. & Felsenfeld, G. (1980) Annu. Rev. Biochem. 49, chromosomes can be resolved microscopically into a series of 1115-1156. alternating dark bands and light interbands. Banding can be 2. Igo-Kemenes, T., Horz, W. & Zachau, H. G. (1982) Annu. Rev. detected in unfixed, unstained nuclei and reflects physical Biochem. 51, 89-121. differences along polytene chromosomes. DNA is more 3. Poccia, D., Salik, J. & Krystal, G. (1982) Dev. Biol. 82, 287-296. heavily compacted in bands (see ref. 31 for a review). Indirect 4. Newrock, K. M., Alfageme, C. R., Nardi, R. V. & Cohen, L. H. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 421-432. immunofluorescence analyses reveal that histones H1, H2B, 5. Simpson, R. T. (1981) Proc. Natl. Acad. Sci. USA 78, 6803- H3, and H4 are distributed in the bands and interbands of 6807. polytene chromatin (reviewed by ref. 32). The interband- 6. Allis, C. D., Glover, C. V. C., Bowen, J. K. & Gorovsky, M. A. specific binding of anti-H2A.2, in contrast, strongly suggests (1980) Cell 20, 609-617. that the chromosomal distribution of H2A.2 matches the 7. Wenkert, D. & Allis, C. D. (1984) J. Cell Biol. 98, 2107-2117. distribution of interbands. It will be interesting to learn how 8. Alfageme, C. R., Zweidler, A., Mahowald, A. & Cohen, L. H. (1974) J. Biol. Chem. 249, 3729-3740. nucleosomes containing H2A.2 contribute to the less com- 9. Palmer, D., Snyder, L. A. & Blumenfeld, M. (1980) Proc. Natl. pacted structure of interbands. Acad. Sci. USA 77, 2671-2675. Genetic (33) and molecular (34) analyses suggest that each 10. Condie, J. M. (1984) Dissertation (The University of Minnesota). band-interband region contains one structural gene. The 11. Newrock, K. M., Friedman, N., Alfageme, C. R. & Cohen, L. H. interband-specific location of H2A.2, together with the evi- (1982) Dev. Biol. 89, 248-253. 12. Wu, R. S., Nishoka, D. & Bonner, W. M. (1982) J. Cell Biol. 93, dence that the band-interband region is the functional unit of 426-431. the polytene chromosome, suggest that the placement of 13. Urban, M. K., Franklin, S. G. & Zweidler, A. (1979) Biochemistry H2A.2 echoes the functional organization of the Drosophila 18, 3952-3960. genome. 14. Franklin, S. K. & Zweidler, A. (1977) Nature (London) 266, The Tetrahymena H2A variant hvl is correlated with 272-275. transcriptionally active macronuclei (6, 7). This correlation 15. Albright, S. C., Nelson, 0. P. & Garrard, W. T. (1979) J. Biol. Chem. 254, 1065-1073. appears analogous to the correlation between H2A.2 and 16. West, M. H. P. & Bonner, W. M. (1980) Biochemistry 19, interbands. Interbands are associated with ribonucleoprotein 3238-3245. particles (35), bound by antibodies against Drosophila RNA 17. Stein, A., Whitlock, J. P. & Bina, M. (1979) Proc. Natl. Acad. Sci. polymerase II (26) or RNADNA hybrids (36), and thus may USA 76, 5000-5004. contain some transcriptionally active DNA sequences. The 18. Lomant, A. J. & Fairbanks, G. (1976) J. Mol. Biol. 104, 243-261. the evidence 19. Weber, K., Pringle, J. & Osborn, M. J. (1972) Methods Enzymol. correlations with transcriptional activity, plus 26, 3-26. that similar H2A histones have a broad phylogenetic distri- 20. Bonner, W., West, M. & Stedman, J. (1980) Eur. J. Biochem. 109, bution (6, 11-16), suggest that the placement of "Drosophila 17-23. H2A.2-like" histones reflects the underlying functional or- 21. Stollar, B. D. & Ward, M. (1970) J. Biol. Chem. 245, 1261-1266. ganization of chromatin. 22. Switzer, R. C., Merrill, C. R. & Shifrin, S. (1979) Anal. Biochem. The interband-specific binding of anti-H2A.2 does not 98, 231-237. 23. Hawkes, R., Niday, E. & Gordon, J. (1982) Anal. Biochem. 119, disappear when antibody concentration is increased 8-fold 142-150. and thus does not reflect competition for limiting amounts of 24. Ashburner, M. (1972) in Results and Problems in Cell Differentia- antibody. In contrast, the preferential binding of anti-H2A.1 tion, ed. Beermann, W. (Springer, New York), pp. 101-151. to polytene chromosome bands disappears when antibody 25. Cohen, L. H. & Gotchel, B. V. (1971) J. Biol. Chem. 246, concentration is increased by a factor of 2 and thus reflects 1841-1848. for amounts of This is con- 26. Jamrich, M., Greenleaf, A., Bautz, F. A. & Bautz, E. K. F. (1978) competition limiting antibody. Cold Spring Harbor Symp. Quant. Biol. 42, 389-396. sistent with the fact that DNA (and presumably histone 27. Silver, L. E. & Elgin, S. C. R. (1976) Proc. Natl. Acad. Sci. USA H2A.1) is more heavily concentrated in polytene chromo- 73, 423-427. some bands. 28. Levinger, L. & Varshavsky, A. (1982) Cell 28, 375-385. The interband-specific binding of anti-H2A.2 occurs with a 29. Lefevre, G., Jr. (1976) in The Genetics and Biology ofDrosophila, variety of fixation conditions and probably is not a cytolog- eds. Ashburner, M. & Novitski, E. (Academic, New York), pp. because the interband- 31-66. ical artifact. This concern is important 30. Donahue, P. R. (1986) Dissertation (The University of Minnesota). specific binding of anti-Z-DNA (37) is influenced by the 31. Laird, C. (1980) Cell 22, 865-874. conditions of chromosomal fixation (38, 39). Conceivably, 32. Cartwright, I., Abmayr, S., Fleischmann, G., Lowenhaupt, K., H2A.2 might be present in bands but inaccessible to anti- Elgin, S., Keene, M. & Howard, G. (1982) CRC Crit. Rev. bodies. This possibility is unlikely because H2A.1 in bands is Biochem. 13, 1-86. accessible to antibodies under the same conditions. 33. Judd, B. H. & Young, M. W. (1974) Cold Spring Harbor Symp. Quant. Biol. 38, 573-579. It is not yet clear whether our inability to detect H2A.2 in 34. Bossy, B., Hall, L. M. C. & Spierer, P. (1984) EMBO J. 3, heat shock puffs reflects dissociation ofH2A.2 from puffsites 2537-2541. during puffing or the dilution of H2A.2-specific fluorescence 35. Skaer, R. J. (1977) J. Cell Sci. 26, 251-266. over the large areas of puffs. It appears unlikely that H2A.2, 36. Vlassova, I. E., Umbetova, G. H., Zimmermann, V. H., Alonso, but not H2A.1, is extracted from puffs during cytological C., Belyaeva, E. S. & Zhimulev, I. F. (1985) Chromosoma 91, in further this it will 251-258. fixation. As a step investigating problem, 37. Nordheim, A., Pardue, M. L., Lafer, E. M., Moller, A., Stollar, be important to learn if puff formation and regression are R. D. & Rich, A. (1981) Nature (London) 294, 417-422. correlated with changes in the pattern ofanti-H2A.2 binding. 38. Arndt-Jovin, D., Robert-Nicoud, M., Zarling, D. A., Greider, C., Weimer, E. & Jovin, T. M. (1983) Proc. Natl. Acad. Sci. USA 80, We thank Jose Bonner, Perry Hackett, Steve Henikoff, Vicki 4344-4348. Iwanij, Brooke Kirby, Lou Morejohn, Janet Schottel, and Walter 39. Lancillotti, F., Lopez, M. C., Alonso, C. & Stollar, B. D. (1985) J. Sauerbier for helpful suggestions. P.R.D. and J.M.C. were Cell Biol. 100, 1759-1766.