Location of the 18/28S Ribosomal RNA Genes in Two

Proc. NatL Acad. Sci. USA Vol. 78, No. 6, pp. 3751-3754, June 1981 Genetics

Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNADNA hybrids in situ (hybridoma/chromosomes/immunofluorescence/nucleolus) W. DORSEY STUART, JOHN G. BISHOP, HAMPTON L. CARSON, AND M. B. FRANK* Department of Genetics, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96822 Contributed by Hampton L. Carson, March 2, 1981

ABSTRACT Using both heterologous rabbit antisera and MATERIALS AND METHODS mouse monoclonal antibody to RNA-DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal Chromosomal Preparations. Salivary glands from larvae RNA fraction to a single large band on polytene autosome 3 in were excised in Grace's insect medium (GIBCO), fixed for 8-10 -Drosophila heteroneura and Drosophila silvestris. This portion of min in methanol/acetic acid, 3:1 (vol/vol), placed on acid- the chromosome is not physically connected with the nucleolus at washed slides with one drop of 45% acetic acid, and squashed the end of larval salivary gland development. In mature larvae, under siliconized coverslips. The preparations were held at little or no hybridization with the material in the nucleolus can be -20°C for at least 30 min and then immersed in liquid nitrogen. detected. In younger larvae, hybridization of the ribosomal RNA After 1 min, the slides were withdrawn and the coverslips were probe to the nucleolus itself can be observed. The chromosome removed by inserting a no. 10 surgical blade between the slide 3 locus is the only band in the polytene genome that shows variation and the coverslip. The slides were immediately immersed in in size and intensity of staining between populations and species. 95% ethanol. The slides remained in ethanol for at least 4 hr The interband chromosome regions that are immediately distal or prior to the in situ hybridization. Preparations may be stored proximal to the 18/28S tRNA locus have been involved in a dis- in ethanol for to 2 weeks proportionately large number of natural inversion breaks ob- up without deterioration of structure served in the euchromatic portion of the polytene chromosome. or hybridization results. In 104 species of Hawaiian Drosophila in which chromosome 3 RNA Probe. RNA was isolated from larvae quick-frozen in polytene sequences have been determined, 15 breaks occur in liquid nitrogen and stored at -70°C for up to 2 weeks. The these two regions. On a random basis, only one such break is ex- isolation method of Kirby (5) was used with the following mod- pected. We propose that this locus may be flanked by substantial ifications: sodium triisopropylnaphthalenesulfonate was substi- heterochromatic blocks which are not represented in the salivary tuted for sodium p-toluenesulfonate; the phenol/cresol extrac- gland chromosome. tion was repeated twice; and 3 M NaCl was substituted for 3 M sodium acetate to remove glycogen and tRNA. The high In the course of producing an antibody to RNA-DNA hybrid- molecular weight RNA salt precipitate was resuspended and duplexes, we have used in situ hybrids formed on polytene reprecipitated with 3 M NaCl a total of three times. The final chromosomes as an antigenic test system (1). The reports that precipitate was resuspended and made 0.12 M in NaCl, 0.01 fused immunoglobulin-producing cells would subsequently M in Tris-HC1 at pH 7.6, 1 mM in EDTA, and 0.02% in sodium produce both types ofparental immunoglobulins. have led to the dodecyl sulfate at an RNA concentration of2 mg/ml and applied development ofmonoclonal cell lines that produce large quan- to an oligo(dT)-cellulose column to remove polyadenylylated tities of specific antibody in culture (2, 3). Recently, we devel- RNA species (6). RNA not adsorbed to the column was collected oped a monoclonal cell line that produces antibody to RNA-DNA and precipitated with 2.5 vol of ethanol at -20°C overnight. hybrids (4) as part ofan experimental attempt to. identify in situ The precipitate was collected by centrifugation and resus- RNA-DNA hybrid-duplexes in human chromosomal prepara- pended at a concentration of 800 ,ug/ml in 0.01 M Tris-HCl, tions. As one assay for the presence of antibody, we used the pH 7.4/1 mM EDTA/0. 1 M NaCl. At this point the RNA sam- salivary gland chromosomes of larvae of Drosophila silvestris ple was further purified by either (a) sucrose gradient fraction- and D. heteroneura, two members of the Hawaiian picture- ation or (b) preparative electrophoresis as follows. winged species group, hybridized in situ with homologous ri- (a) A 500-,ul sample ofRNA was heated to 38°C for 5 min and bosomal RNA. Antibodies reactive with RNA-DNA hybrids applied directly to a 17-ml linear sucrose gradient (5-20%) made were obtained from immunized rabbits and from clonal.cultures in the same buffer. Heating to 65°C was avoided because this ofimmunized mouse spleen cells fused with a mouse myeloma treatment results in the denaturation of the 28S rRNA species cell line. found in insects and the denatured RNA comigrates with the Using these immunological reagents in a secondary immu- 18S species during centrifugation (7). The gradient was centri- nofluorescent assay, we have identified the in situ hybridization fuged in a Beckman instrument using an SW 27.1 rotor at 25,000 site for 18/28S rRNA. The genes are located in a chromosome rpm for 21.5 hr at 4°C. Fractions were collected and those con- region that is involved in an unusually large proportion of in- taining 28S RNA were pooled, precipitated, and resuspended version breaks which have occurred in the polytene sequence in hybridization solution (1:1 mixture offormamide and 0;6 M during phylogeny of the Hawaiian picture-winged species. NaCV0.06 M Na citrate, pH 7) (1). (b) RNA (230 ,ug) was applied in 2.5 ml of sample buffer (8) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- * Present address: Department of Microbiology, College of Medicine, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. University of California at Irvine, Irvine, CA 92717. 3751 3752 Genetics: Stuart et al. Proc. Natl. Acad. Sci. USA 78 (1981)

to a 6 M urea/1% agarose gel (53 X 120 mm) formulated by the observed. (b) When the RNA normally present in the polytene method of Long and Dawid (8). The gel was run for 8 hr at 95 preparation was not predigested with RNase A, multiple flu- V (100 mA) and then stained for 15 min with ethidium bromide orescent bands were observed after hybridization. This indi- (8). The portion ofgel containing the large fragment ofthe dena- cated that the antibodies were not reactive with chromosomal tured 28S rRNA, 28S b (8), was placed in a dialysis bag with 3 DNA but did have the capacity to react with RNADNA du- ml ofgel buffer and electroeluted from the agarose for 3 hr. The plexes formed with more than one specific gene sequence. (c) eluted 28S fragment was precipitated with ethanol and resus- Absorption of antibody reagents with single-stranded pended in hybridization solution. poly(rA)-methylated albumin complexes or with RNA purified by either method gave similar hybridization poly(dT)-methylated albumin complexes did not reduce im- results. munofluorescent visualization ofin situ RNADNA hybrids. (d) In Situ Hybridization. Hybrids were prepared as reported Absorption of antibody reagents with poly(rA).poly(dT) du- (1). Estimates ofhybridization rates were obtained by incubat- plexes complexed to methylated albumin removed all activity ing preparations (sealed with rubber cement) at 70'C for 1 hr responsible for the secondary immunofluorescent bands. and then immediately shifting the preparations to 40'C by im- Therefore, the antibodies recognized RNA-DNA hybrids but mersion in a thermostatted water bath. Crt values were calcu- not other forms of RNA or DNA. lated as initial concentration of the RNA probe in mol per liter Localization ofthe 18/28S rRNA Locus. The in situ hybrid- multiplied by time (sec) ofincubation at40'C. The hybridization ization locus of the 18/28S rRNA is on chromosome 3 in both was terminated by immersion in an ice-water bath. D. silvestris (stock U28T2; Kilauea Forest Reserve, Hawaii) and Antibody Production. Rabbit antibody to poly(rA)-poly(dT) D. heteroneura (stock Q71G12; Olaa Tract, Hawaii). This chro- hybrids complexed to methylated bovine serum albumin was mosome is anautosomal element, designated B by Muller (11). prepared by the method ofStollar (9). Mouse monoclonal hybrid It is homologous to arm 2L ofD. melanogaster. The locus maps cell cultures to the same antigen were prepared by the method to a single band interstitially located in the basal third of the ofOi and Herzenberg (10). Immunized spleen cells were fused euchromatic arm. The relevant band isjustproximal to the distal with P3-NS-1 BALB/c myeloma cells (originally derived from break ofinversion 3b (figure 2 in ref. 12). The band is just distal the BALB/c MOPC-21 cell line) provided by Douglas Vann. to the three coincident breaks mapped for inversions 3i, 3c, and Positive fusion cultures were cloned by the limiting dilution 3a. We have examined several hundred preparations from both method (10). One positive clone (FS/B12-G10) was chosen for D. silvestris and D. heteroneura. After the fluorescent regions use in this study. were visualized, a phase condenser was inserted into the system Identification of Hybrids. Regions of in situ hybridization and phase-contrast photomicrographs were taken. The cover- were detected by secondary immunofluorescence as reported slip was then removed and the preparation was stained with (1). Goat anti-rabbit and goat anti-mouse Ig antisera conjugated aceto-orcein. A fresh coverslip was then added to the stained with fluorescein isothiocyanate were purchased from Antibod- preparation, producing a conventionally banded aspect. This ies Inc. Preparations were examined under a Zeiss dark-field procedure has allowed us to place the in situ hybrid band une- transmitted fluorescence system. [quartz/halogen source, KP quivocally at the chromosome 3 locus specified above. Fig. 1 500 excitation filter (Zeiss), and 530-nm barrier filter]. Photomi- illustrates fluorescent and phase-contrast micrographs of the crographs were taken with Tri-X Pan film (Kodak ASA 400). locus identified with rabbit antibody. In mature larval cells the nucleolus showed little or no flu- RESULTS orescence. In cells obtained from younger larvae, fluorescent staining of the nucleolus was very strong. This suggests that Specificity of the Antibodies. Both the rabbit, antisera and either the DNA sequences for 18/28S rRNA are present in the the mouse hybridoma antibodies were tested for specificity by nucleolus during early larval development but are not available the following control experiments. (a) When no RNA probe was for hybridization at the third-instar larval stage immediately present during the hybridization and the preparation was pre- prior to pupation or that our method is not capable ofvisualizing digested with RNase A (1), no secondary fluorescentbands were the sequences in the enlarged nucleolus. Fig. 2 illustrates hy-

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_ !R iJIIIL... '1r-1Ipi.AAfSI - 's't I.:A=M 4F; i Slin~~~~~hA! _ v i tS i~~~~~~w~tv *'li _ * 40 ' >11t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~MHs~~~~ band3-18 .x11350.);_1l'r D FIG. 1. Salivary gland chromosomes ofD. silvestris hybridized with homologous 28S rRNA and visualized with secondary immunofluorescence ofrabbit antibody to RNA-DNA hybrids. (Left) Fluorescence microscopy. (Right) Phase-contrast microscopy ofthe same field. N = nucleolus; arrow, band 3-18. ( x 1350.) Genetics: Stuart et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3753

may be characteristic of certain ancestral populations of which the Kauai grimshawi is an example. Natural Breaks Adjacent to the 18/28S rRNA Locus. The sequence ofpolytene banding orders ofchromosome 3 has been completely determined in 104 Hawaiian Drosophila, including 103 picture-winged species and the related D. mimica, a mem- ber of the "modified-mouthparts" group. Many different gene orders are known in this chromosome; most differences are due to fixed inversions between species (14). A survey ofthe break- points ofthese inversions reveals that a disproportionately large number ofthese natural breaks in chromosome 3 have occurred adjacent to the rRNA locus. A total of 39 inversions have been recorded, and distributions of the 78 breakpoints have been mapped. Of these, 10 have occurred in the basal heterochro- FIG. 2. Portion of the nucleus in salivary gland of an early third- matin near the centromere and cannot be localized accurately. instar larvalD. heteroneura; hybridization withD. silvestris 28S rRNA Of the 68 remaining breaks in the euchromatic chromosome and visualized with secondary immunofluorescence of mouse mono- arm, 15 have occurred adjacent to the rRNA locus, 4 on the clonal antibody. The preparation has been counterstained (1). The nu- cleolus and the indicated band are yellow-green, and the rest of the distal side and 11 on the proximal side. The euchromatic po- nucleus is red. (Fluorescence microscopy; X 1500.) lytene chromosome 3 shows 138 interband regions into which breaks can easily be mapped. On a random basis, the probability bridization of 28S rRNA to the nucleolus in young larval cells of a given break falling into one of these interband regions is identified with mouse monoclonal antibodies. Hybridization to 1/138. The number of breaks expected in each region is 0.49 a single band in the genome is visible. This can be contrasted or about one-half of one break. Therefore, only 1 of the 68 ob- to Fig. 1 where the 18/28S locus is clearly visible on chromo- served breaks would be expected in the two interband regions some 3 but no fluorescence is visible in the adjacent nucleolus. adjacent to band 3-18 whereas 15 have been observed. Hybridization was performed at Crt values ranging from 1 X 10-4 to 4 x 101 to determine if the rate of hybridization DISCUSSION was consistent with that expected for 18/28S rRNA. Szabo et al. (13) have reported Crt curves and rate constants for both 5S The antibodies produced in rabbits and those obtained by hy- and 18/28S rRNA ofD. melanogaster hybridized in situ under bridoma cell culture are specific for RNADNA hybrids as de- conditions similar to those used here. We observed faint fluo- termined by the series ofcontrol experiments. Antibodies ofthis rescence of both the nucleolus and the chromosome 3 band at type have already proven useful for identification of in situ hy- a Crt value of1 x 10-2. D. melanogaster 18/28S rRNA has been brids (1, 15). In the present study they were used to identify hybridized in situ to 15% ofsaturation when the Crt value equals the 18/28S rRNA locus in D. heteroneura and D. silvestris. The 1 x 102 (13). Crt value at which both the nucleolus and band are first visu- Size Differences in the Band Containing the 18/28S rRNA alized is consistent with the visualized band and the nucleolus Locus. Prior to its identification as the locus of the large rRNA being in situ hybrids of 18/28S RNADNA. The location ofthe genes, standard cytological observations had been made on this large rRNA genes is a single band, 3-18, lying interstitially in band, designated 3-18, in 104 species of Hawaiian Drosophila. the euchromatic section of chromosome 3. The data indicate This is the only band in the polytene genome known to display that the DNA sequences are also present in the nucleolus in the variation in physical size. This variation is observed between early third-instar larvae. Hybridization to these sequences is not certain island populations of the species D. grimshawi and the strongly visualized in the later third-instar larval cells. This sug- very closely related species D. pullipes ofthe island of Hawaii. gests that, in the nucleolus, these DNA sequences are dispersed In D. grimshawi specimens from the islands ofKauai and Oahu or degraded prior to pupation. and in D. pullipes, band 3-18 is apparently double in size com- In D. melanogaster, the 18/28S rRNA locus appears to be pared to the equivalent band in D. grimshawi from the Maui identical to the nucleolus organizer (NO) region, being marked complex. F, hybrids between the types produce a striking het- by the well-studied mutant bobbed (16). This region maps erozygosity (Fig. 3). The heavy, presumably redundant, band within a large heterochromatic segment at the base of the ac- rocentric X chromosome and is covered by a homologous region on the Y. A similar situation obtains in D. virilis (17). In D. hydei, the X chromosome is metacentric. One arm (XL) is wholy heterochromatic and the nucleolus organizer is located close to the distal end (18). Both virilis and hydei belong to the subgenus Drosophila and as such are much more closely related to the Hawaiian drosophilids than is melanogaster. Accordingly, the .A location of the 18/28S rRNA locus in an apparently interstitial position on an autosome requires explanation. The accumulation of breaks near the 18/28S locus suggests that this locus may be flanked by relatively long heterochro- matic segments which, like centromeric heterochromatin, are not represented in the polytene chromosome. Breaks in the FIG. 3. Base (at left) ofpolytene autosome 3 ofD.grimshawi, show- centromeric heterochromatin are numerous not only at the base ing heteromorphism of band 3-18. The chromosome shown is from an of chromosome 3 as previously mentioned but also in the het- interisland hybrid from a female (C134.7D) from Keanae, Maui, by a male (L37B4) from Pouli Stream, Kauai. The arrowhead indicates the erochromatic portions of the bases of the other four major ac- heavier band on the homologue derived from the Kauai strain. rocentric chromosomes in the Hawaiian drosophilids, including (x 1200.) the X chromosome. 3754 Genetics: Stuart et al. Proc. Natl. Acad. Sci. USA 78 (1981)

Assuming a hydei-like condition to be ancestral to the Ha- 3-18 could have been moved finally to its present interstitial waiian one, an arbitrary choice, we propose the following position after an inversion having one break in the euchromatin scheme for the evolution of the interstitial autosomal position and the other in the heterochromatin proximal to band 3-18 ofthe 18/28S rRNA locus in heteroneura and silvestris (Fig. 4). (step 3). The proposed scheme requires only a single break in First, the locus, with its flanking heterochromatin could have a euchromatic section; all the rest are confined to heterochro- been translocated to the short arm of chromosome 3 (step 1). matin and, as such, would be expected to become easily estab- Following this, a pericentric inversion (step 2) could then trans- lished and to have only minimal effects on viability. This scheme fer this segment to the base of the euchromatic arm. Indeed, also provides for a widely observed phenomenon, the conser- this is the observed position of band 3-18 in D. mimica which, vation of the integrity of the major chromosomal elements of on the other grounds, has been judged to carry primitive se- Muller (11) during chromosomal evolution. quences of Hawaiian Drosophila (19). From this position, band We thank Roberta Brashear, Nevis Fregien, Greg Dolecki, John ; NO Hunt, Tom Humphreys, Barbara Johnson, Terrence Lyttle, Mary Lou -C Pardue, Linden Teramoto, and Douglas Vann for helpful assistance and discussion. This work was supported in part by National Science Foun- dation Grants PCM 79-16350, DEB 79-26692, and DEB 78-22820. TRANSLOCATION W t 1. Stuart, W. D. & Porter, D. L. (1978) Exp. Cell Res. 113, 219-222. 2. Cotton, R. G. H. & Milstein, C. (1973) Nature (London) 244, 4243. 3. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495-497. 4. Frank, M. B., Bishop, J. G. & Stuart, W. D. (1980) Genetics 94, _ s33-s34 (abstr.). INVERSION W 5. Kirby, K. S. (1968) Methods Enzymol. 12, 94-95. 6. Mezl, V. A. & Hunt, J. A. (1978) Biochem.J. 175, 159-169. 7. Shine, J., Hunt, J. A. & Dalgarno, L. (1974) Biochem. 1. 141, 617-625. 8. Long, E. 0. & Dawid, I. (1980) Cell 18, 1185-1196. 9. Stollar, B. D. (1970) Science 169, 609-611. T t 10. Oi, V. T. & Herzenberg, L. A. (1980) in Selected Methods in Cel- M. INVERSION W lular Immunology, eds. Mishell, B. B. & Shiigi, S. (Freeman, San Francisco), pp. 351-372. 11. Muller, H. J. (1940) in The New Systematics, ed. Huxley, J. (Ox- ford, London), pp. 185-268. 12. Carson, H. L. & Stalker, H. D. (1968) Univ. Texas Publ. 6818, --cm 335-354. NO 13. Szabo, P., Elder, R., Steffensen, D. M. & Uhlenbeck, 0. C. (1977)J. Mol. Biol. 115, 539-563. FIG. 4. Hypothetical model for the origin of an acrocentric auto- 14. Carson, H. L. & Kaneshiro, K. Y. (1976) Annu. Rev. Ecol. Syst. some (as in D. silvestris) carrying the nucleolar organizer (NO) in an 7, 311-345. interstitial position (bottom of figure). X, metacentric X chromosome 15. Rudkin, G. T. & Stollar, B. D. (1977) Nature (London) 265, carrying NO region near end of heterochromatic arm. 3, Acrocentric 473-473. autosome 3. Step 1 (large arrow), production of a translocation chro- 16. Ritossa, F. (1976) in The Genetics and Biology of Drosophila, mosome (3x) resulting from rejoining at breakpoints shown by small eds. Ashburner, M. & Novitski, E. (Academic, London), Vol. lb, arrows on X and 3. Step 2 (large arrow), production of a chromosome pp. 801-846. resulting from a pericentric inversion at breakpoints shown. Step 3 17. Gall, J. G., Cohen, E. H. & Polan, M. L. (1971) Chromosoma 33, (large arrow), production of final chromosome resulting from a para- 319-344. centric inversion at breakpoints shown. Circle, centromere; heavy solid 18. Hennig, W., Link, B. & Leoncini, P. (1975) Chromosoma 51, areas, heterochromatin; narrow line, euchromatin. For details, see 57-63. text. 19. Stalker, H. D. (1972) Genetics 70, 457-490.