Mapping of the pattern of DNA replication in polytene chromosomes from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

0 1985 Alan R. Liss, Inc. Cytometry 6584-590 (1985)

Mapping of the Pattern of DNA Replication in Polytene Chromosomes From Chironomus thummi Using Monoclonal Anti-Bromodeoxyuridine Antibodies

Linda Allison, Donna J. Arndt-Jovin, Howard Gratzner, Therese Ternynck, and Michel Robert-Nicoud Abteilung Molekulare Biologie, Max-Planck-Institut fiir Biophysikalische Chemie, D-3400 Giittingen, Federal Republic of Germany (L.A., D.J.A.-J., M.R.-N.), University of Miami School of Medicine, Miami, Florida 33101 (H.G.), and Institut

Pasteur, 75724 Paris CEDEX 15, France (T.T.)

Received for publication June 3, 1985; accepted June 18, 1985

We present results from a nonautoradi- ographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclo- nal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands. later in bands. Most chromosomal

bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subse- quent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.

Key terms: DNA replication, 5-bromodeoxyuridine, monoclonal anti-BrdUrd antibodies, polytene chromosomes

Successive endoreduplication cycles during the larval growth of dipteran insects give rise to the formation in certain tissues of giant polytenic chromosomes (3,23). In the salivary gland cells of Chironomus species, 12 to 13 rounds of DNA replication during embryonic and larval development (15) result in the formation of polytene chromosomes with up to Z13 chromatids. Due to their size, the constancy of their band-interband pattern, the equation of bands with genes, and the structural changes that accompany gene activation (puffing), these chromosomes present a unique opportunity for studying in situ the structural organization of active and inactive chromatin, the distribution of chromosomal proteins and of particular types of DNA sequences, the mechanisms of gene activation, and the dynamics of DNA replication (3,251.

DNA replication in polytene chromosomes of dipteran larvae has been the subject of numerous studies (1,2,4,5,8,17,20,21), which have made use of autoradiographic techniques to detect the incorporation of 3H-

thymidine. Various patterns of DNA synthesis have been described and tentatively ordered on a time scale. Pat- terns characteristic of early S-phase display more or less continuous labeling, while late S-phase patterns are of a discontinuous type. In Drosophila, a discontinuous pattern with label confined to puffs and interbands was observed by Hagele and Kalisch (10,19) and assigned to the very beginning of the replication phase. It was con- cluded from these experiments that the various patterns of DNA replication observed reflect the temporal stag- gering of both initiation and termination events in interbands, puffs, and in and among bands. However, a number of uncertainties remain regarding the assign- ment of the various patterns to specific time points within the S-phase, and the conclusions which can be drawn from these autoradiographic studies are re-

This work was supported by the Deutsche Forschungsgemeinschaft and the Max-Planck Gesellschaft.

DNA REPLICATION IN POLYTENE CHROMOSOMES 585

Fig. 2. Immunoflourescence detection of DNA synthesis in salivary gland polytene chromosomes of C. thumrni. In vivo BrdUrd incorporation: 4th instar larvae were kept for 90 min in the dark in culture water containing BrdUrd. Incorporation was detected using anti- BrdUrd antibody 76.7 (10 pgiml) and anti-mouse IgG fluorescein-conjugated goat F(ab’)z (B). H-33342 staining of the chromosomes is shown in A. Chromosome I11 shows bright immunofluorescence; nonincorporating chromosome I, from another nucleus of the same gland, shows fluorescence. C, centromeres; r, right end of chromosomes I and 111. Bar = 10 pm.

Fig. 1. Immunofluorescence detection of DNA synthesis in salivary gland duct cells of C. thummi. In vitro BrdUrd incorporation: explanted salivary glands were incubated for 15 min in the dark in BrdUrd-containing CR-medium. Incorporation was detected by using anti-BrdUrd antibody B44 (25 pglml) and anti-mouse IgG fluorescein- conjugated goat F(ab’)z fB). H-33342 staining of the nuclei is shown in A. Arrows designate replicating nuclei. Bar = 10 pm.

MATERIALS AND METHODS In Vivo and In Vitro Incorporation of BrdUrd

stricted by the inherent limitations of this technique, e.g., low resolving power and low sensitivity.

We have investigated the possibility of using monoclonal antibodies directed against 5-bromodeoxyuridine (BrdUrd) to study by immunofluorescence DNA synthesis in polytene chromosomes and to map the successive patterns of DNA replication in salivary gland cells of Chi- ronomus thummi. The usefulness of monoclonal antibodies reactive with BrdUrd for studying DNA replication in cells was first recognized and demonstrated by Gratzner (7). A procedure was developed for simultaneous flow cy- tometric measurement of cellular DNA content and amount of BrdUrd incorporated using propidium iodide and anti-BrdUrd antibodies (6). Antibodies with the Same specificity have also been used to detect BrdUrd-labeled DNA immobilized on nitrocellulose filters (24). We show

Fourth instar larvae of Chironomus thummi (stages jL to mL) were taken from our laboratory stock. For details about the culture of C! thummi and a description of its developmental stages, see reference 16. For in vivo studies, BrdUrd was added to the culture water at a concentration of 1 mM; salivary glands were excised after various periods of time (30 min to 24 h) and immediately fixed. For in vitro experiments, salivary glands were explanted in insect saline (CR-medium: 87 mM NaC1, 3.2 mM KCl, 1 mM MgC12, 1 mM CaClz and 10 mM Tris-HCl, pH 7.3) or in one of various insect cell culture media: Grace’s medium, Schneider’s medium, Mitsu- hashi and Maramarosch’s medium (for composition of media see reference 11), and incubated in 20 P1 of the Same medium containing 40 pM BrdUrd7 PM 5- fluorodeoxyuridine (FdUrd), and 6 Pm uridine (Urd) for

in an accompanying manuscript (13) that immunofluorescence detection of BrdUrd incorporation is a conveni- ent method for studying and sequencing molecular events

cell differentiation. The results presented here show that anti-BrdUrd an-

tibodies can be used to study DNA replication in poly- Immunofluorescence tene chromosomes of insect larvae and that this new Before immunofl uorescence staining, the preparations technique presents numerous advantages over the au- were treated in 2 N HC1 at room temperature for 20-30 toradiographic procedure. DNA replication can be mea- min, neutralized by three 10 min washes in 100 mM sured in vivo or in vitro by adding BrdUrd to the culture Tris-HC1 pH 7.3, and washed three times in 100 ml CR- water of the larvae or to the incubation medium of medium for 10 min each. The monoclonal antibodies explanted salivary glands. Incorporation of BrdUrd is with specificity for BrdUrd used were B44 (Gratzner, 7) detectable by indirect immunofluorescence even after prepared from the supernatant culture medium of hy- short periods of exposure to this modified nucleoside. bridoma cells by precipitation with 50% (NH4)&04 and The procedure is quick, sensitive, and allows high reso- 76-7 (Ternynck, 241, obtained from ascites produced by lution mapping of the replication sites. injection of hybridoma cells and purified on a column of

various periods of time. In both types of experiments the salivary glands were fixed in ethano1:acetic acid 3:l(vol/ VOl) for 1 min and then in 45%(~01/~01) acetic acid for 7

on dry ice. related to the transition in DNA synthesis accompanying to lo min. After squashing, the Prepmations were frozen

Fig. 3. Successive patterns of DNA replication in polytene chromosomes of C. thurnrni salivary glands. Chromosomes shown in B-F are from explanted salivary glands incubated in BrdUrd for 1 h; chromosomes shown in A are from a gland exposed in vivo to BrdUrd for 1.5 h. Immunofluorescence staining with B44 antibody (B-F) or 76.7 anti-

body (A). In each panel, one complete set of chromosomes is shown. Panels A to F display successive DNA replication patterns during the S-phase. In A, puffs (arrows) and interbands are stained (pattern El); in B, most bands are fluorescent except those in centromeric and heterochromatic regions (dots) (pattern E2); in C, all the bands are

uniformly stained (pattern M1); in D, the staining is more differential (pattern M2); in E, only a certain fraction of the bands including bands in the four centromeres (arrows) display bright immunofluorescence (pattern of class Ll); in F, staining is restricted to a few heterochro-

matic bands (arrows) (pattern of class L2). In E and F, DNA staining with H-33342 is shown in insets for better orientation; arrows point to bands indicated in the corresponding immunofluorescence image. r, right end of chromosomes I, 11,111, and IV. Bars = 10 pm.

588 ALLISON ET AL

Protein A-Sepharose (Pharmacia, Uppsala). Antibodies were applied at a concentration of 25 pg/ml (B44 antibody) or 10 pg/ml(76-7 antibody) in CR-medium containing 1 mg bovine serum albumin per milliliter for 1 h at 37°C. The preparations were then given three 10 inin washes in 100 ml CR-medium and stained with anti- mouse IgG affinity purified goat F(ab’)z fractions labeled with fluorescein (TAGO, Inc., Burlingame, CA) for 1 h at 4°C or 30 min at 37°C. After washing, counterstain- ing with 3 pm H-33342 (Hoechst, Frankfurt) for 1 min, and rinsing in CR-medium, the preparations were mounted in Mowiol4-88 (Hoechst, Frankfurt). Immuno- fluorescence observations were made in a Zeiss epi-illu- mination photomicroscope using a high pressure mercury arc lamp (HBO 50 W/AC). Selective excitation- emission filter combinations (Zeiss 487702 and 487717) allowed the H-33342 and fluorescein images from the same chromosome to be viewed and photographed (Ko- dak Tri-X, 1600 ASA) without spectral overlap.

RESULTS Two types of experiments were carried out to test the

ability of monoclonal antibodies directed against BrdUrd to detect DNA synthesis in polytene chromosomes of dipteran larvae. For in vivo studies, 4th instar larvae of Chironomus (iL-mL) were maintained for 30 min to 24 h in water containing BrdUrd. For in vitro studies, salivary glands were explanted and incubated for 5 min to 4 h in media containing BrdUrd, FdUrd, and Urd at room temperature. After fixation, squashing, and dena- turation in acid, the incorporated BrdUrd was detected by indirect immunofluorescence using the monoclonal antibodies B44 (7) and 76-7 (24). Preparations of salivary gland duct cells and of brain cells were also made as a control for the incorporation of BrdUrd into diploid cells. In both diploid (Fig. 1) and polytenic cell (Fig. 2) populations, the nuclei and chromosomes that had incorporated BrdUrd displayed a bright fluorescence, while the background fluorescence over nonincorporating nuclei and over cytoplasmic structures was very low.

For detection of in vivo incorporation, a minimum exposure time of 45-60 min to BrdUrd was required. On the other hand, immunofluorescence staining could be observed in preparations of salivary glands exposed in vitro to BrdUrd for periods of time as short as 10 to 15 min. Various media were tested for optimal incorporation (see Materials and Methods). Immunofluorescence intensity was found to be highest after incubation of the glands in CR-medium or in the medium of Mitshuhashi and Maramarosch (11). The saline solution was used in short-term experiments; for periods of incubation ex- ceeding 90 min, the more complete insect cell culture medium was preferred.

Only a small percentage (510%) of the nuclei in explanted salivary glands incorporated BrdUrd during incubation periods of 1 h. This was also the case for nonpolytenic nuclei of the salivary gland duct cells (Fig. 1) and for brain cell nuclei (not shown). Clearly, as it has been found in earlier studies with 3H-thymidine

(1,5,17,22), DNA replication in these cell populations does not occur synchronously.

We have observed a number of different incorporation patterns that we have classified according to characteristic features and to order sequentially on the basis of evidence presented in earlier studies (14,18,19) and of synchronization experiments carried out in our laboratory. In order to describe the major classes of pattern (designated El , E2, M1, M2, L1, and LZ), we have chosen representative chromosome sets, which are displayed in Figure 3.

The pattern of incorporation (El) most likely to repre- sent the very beginning of the replication phase is shown in Figure 3A. Immunofluorescence staining is confined to chromosomal regions comprised of puffs and interbands. This pattern has been observed very rarely in both in vivo and in vitro experiments and must conse- quently correspond to a very short period at the beginning of the replication phase. The next pattern of incorporation (E2) is characterized by labeling of most chromosomal bands, including the telomeres of the three large chromosomes, with the exception of bands located in the centromeres and in regions of intercalary heterochromatin (Fig. 3B). In these regions DNA replication appears to be initiated at a later stage of the S-phase. A uniform immunofluorescence staining of the majority of the barids is characteristic for the next class (Ml) of incorporation patterns (Fig. 3C). This pattern is likely to correspond to the so-called continuous type described in autoradiographic studies (8,12,14). At this stage, and in contrast to autoradiographic labeling, the immunofluorescence staining allows localization of incorporation sites at the level of single bands.

The subsequent immunofluorescence patterns show an increasingly differential staining (Fig. 3D-F). In Fig- ure 3D, differences in fluorescence intensity among bands can clearly be seen (class M2). During the next period, replication comes to a stop in DNA-poor bands while it is continuing in DNA-rich bands (class Ll). The chromosomes shown in Figure 3E, e.g., display a total of about 60 strongly fluorescent regions. The latest patterns of replication (class L2) are characterized by fluorescent staining of only a few bands (Fig. 3F). In the insets to Figs. 3E and F, the H-33342 images of the chromosomes are shown to facilitate the localization of the sites of BrdUrd incorporation. Figure 4 shows one set of doubly stained chromosomes displaying a characteristic late pattern of replication (L2). Incorporation is confined to a few sites that are located in regions ID2, IC4c, IIC3, IIA3a-c, IIIBZ, IIIB3c, and IVEBde (map des- ignation according to Hagele (8)). In late immunofluorescence patterns, single bands often display a nonuniform spot-staining (Fig. 4) that may be the expression of an asynchronous termination of the replication among individual chromomeres of a band.

DISCUSSION We have provided evidence in this manuscript that the

immunofluorescence technique for the detection of

Fig. 4. Mapping of late DNA replication pattern (class L2) in C. thummi polytene chromosomes by immunofluorescence and H-33342 staining. Explanted salivary glands were incubated for 1 h in BrdUrd- containing medium. Immunofluorescence staining with B44 antibody

(B); DNA staining with H-33342 is shown in A. At this late stage of the replication phase, incorporation is confined to a few sites located in regions ID2, IC4c, IIC3, IIASac, IIIB2, IIIB3c and NE2de (arrows). Note spot-staining of replicating bands. Bar = 10 pm.

BrdUrd incorporation into DNA presents numerous advantages over autoradiography for refined analysis of the temporal course as well as the specific localization of DNA replication sites within the genome. To obtain information about replication of eucaryotic DNA at the molecular level (e.g., replicon size, number of replication forks per gene), we require high spatial resolution. The alignment of chromomeres and high endoreduplication of the DNA in polytene chromosomes provide a resolution with the light microscope and immunofluorescence of a single band or gene that could be extended using immunoelectronmicroscopic techniques to even higher resolution.

Monoclonal antibodies reactive with BrdUrd can be detected after very short exposure (less than 2% of the total S-phase) of explanted salivary glands to this modified nucleoside. Thus, we have been able to follow the dynamics of DNA replication in salivary gland cells of 4th instar larvae of Chironomus thummi from both in vivo and in vitro labeling. A large number of unique replication patterns can be distinguished and analyzed.

These patterns have been classified and tentatively ordered on a time scale. Due to the lack of recognizable cell cycle events to which the temporal order of replication can be related, it is difficult to establish this order unequivocably. The sequence described here is based on the synchronization of DNA synthesis in Chironomus larvae (Allison et al., in preparation).

During a first short period at the beginning of the S- phase, DNA synthesis appears to be confined to puffs and interbands (pattern class El). Replication is detectable in bands at a later stage. This may reflect the fact that initiation sites are located in interbands or may be due to a delayed initiation in the condensed chromosomal regions andlor to large differences in the rate of elongation between open and condensed chromatin. In the next stage, replication is occurring in most of the bands with the exception of those located in centromeres and in regions of intercalary heterochromatin (E2). DNA synthesis in these regions appears to be initiated at a still later stage. The uniform pattern of immunofluores-

- cence staining seen at mid S-phase demonstrates that

590 ALLISON ET AL.

DNA synthesis is now proceeding in all bands (MI). In 4. Cave MD: Chromosome replication and synthesis of non-histone contrast to autoradiography, the immunofluorescence proteins in giant polytene chromosomes. Chromosoma 25:392-401,

1968. technique allows even at this stage a clear resolution of 5. Darnow m, Clever u: Chromosome activity and cell function in the staining of individual bands. polytene cells. 111. Growth and replication. Dev Biol 21:331-348,

(Ml,Ll,LZ) observed in later stages ofthe S-phase reflect 6. Dolbeare F, Gratzner H, Pallavicini MG, Gray Jw: Flow cytomet- the asynchronous termination of DNA synthesis in in- ric measurement of total DNA content and incorporated bromode-

oxyuridine. Proc Natl Acad Ski USA 80:5573-5577, 1983. dividual bands- This asynchrony can be due to differ- 7, Gratzner H G Monoclonal antibody to 5.bromo- and 5-iododeoxyu.

Thc more differential patterns of replication 1970.

ences in the length of the replicons andor to differences , in the rate of elongation among bands. The end of the replication phase is characterized by immunofluorescence staining of only a few bands in centromeric and heterochromatic regions (L2). The nonuniform spot-labeling within bands observed in late replication patterns (see Fig. 4) may indicate that there is also an asynchronous termination of DNA synthesis among individual chromomeres of a band.

The majority of the late replicating bands are located in DNA-rich regions that show intense fluorescence staining with H-33342 (see Fig. 4). However, the DNA- poor bands in region IIAa-c, which can be seen to replicate during the final stage of the S-phase (see Fig. 4), are a striking exception to this observation. These bands have also been shown by autoradiography to synthesize DNA for a longer period of time than bands containing up to 3.8 times more DNA (8.9). Since apparently no extra DNA is accumulated in this region, these bands may replicate at a slower rate than the majority of bands or might release newly synthesized DNA from the chromosome. These two possibilities can be distinguished by quantification of pulse-labeled chromosomes using a fluorescent image acquisition and analysis system (Allison et al., in preparation).

In conclusion, the results presented here demonstrate the advantages of the immunofluorescence technique over autoradiography for mapping the patterns of DNA synthesis in chromosomes at the level of resolution of one band in the light microscope. Use of immunoelec- tronmicroscopy could extend the resolution to the mapping of replicons. In addition, this technique also permits in situ quantification of incorporation by fluorescence microscopy and computer-assisted image analysis.

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ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of

B. Staehr.

LITERATURE CITED 1. Achary PMR, Majumdar K, Duttagupta A, Mukherjee A S Repli-

cation of DNA in larval salivary glands of Drosophila after in vivo synchronization. Chromosoma 82:505-514,1981.

2. Arcos-Teran L: DNS-Replikation und die Natur der spat replizier- enden Orte im X-Chromosom von Drosophila melanogaster. Chro- mosoma 37:233-296, 1972.

3. Beermann W (ed): Developmental Studies on Giant Chromosomes. Springer-Verlag, Berlin, 1972.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20

21

22.

23

24

25

ridine: A new reagent for detection of DNA replication. Science 218:474-475, 1982. Hagele K: DNS-Replikationsmuster der Speicheldriisen-chromosomen von Chironomiden. Chromosoma 31:91-138, 1970. Hagele K: Querscheibenspezifische Unterschiede in der 3H-Thym- idin-Markierung wahrend der Larvenentwicklung von Chirone mus thummi piger. Chromosoma 39:63-82, 1972. Hagele K, Kalisch WE: Initial phases of DNA synthesis in Droso- phila melanogaster. I. Differential participation in replication of the Xchromosome in males and females. Chromosoma 47:403- 413,1974. Hink WF: A compilation of invertebrate cell lines and culture media. In: Invertebrate Tissue Culture. Research Applications, Maramorosch K (ed). Academic Press, New York, 1976, pp 319- 369. Kalisch WE, Hagele K: Correspondence of banding patterns to 3H- thymidine labeling patterns in polytene chromosomes. Chromo- soma 57:19-23, 1976. Kaufman SJ, Robert-Nicoud M: DNA replication and differentiation in rat myoblasts studied with monoclonal antibodies against 5-bromodeoxyuridine, actin and cr2-macroglobulin. Cytometry 6:570-577, 1985. Key1 HG, Pelling C: Differentielle DNS-Replikation in den Spei-

-

- cheldriisen-Chromosomen von Chironomus thummi. Chromosoma 14:347-359, 1963. Kiknadze 11, Vlasova IE, Sherudilo AI: Quantitative analysis of DNA content in the salivary gland chromosomes of Chironornus thummi at larval and prepupal stages. Cell Differ 3:323-334, 1975. Kroeger H: Zur Normalentwicklung von Chironomus thurnrni Kieffer. 2 Morphol Tiere 74:65-88, 1973. Kroeger H, Gettmann W, Kalter CH: Zur Kontrolle der DNA- Replikation in Riesenchromosomen explantierter Speicheldriisen. Cytohiologie 7:117-126, 1973. Mulder MP, van Diujn P, Gloor JH: The replicative organization of DNA in polytene chromosomes of Drosophila hydei. Genetica 39:385-428, 1968. Nash D, Bell J: Larval age and the pattern of DNA synthesis in polytene chromosomes. Can J Genet Cytol 10:82-90, 1968. Pelling C: Differentielle DNS-Replikation in den Speicheldriisen- Chromosomen von Chironomus thumrni. Chromosoma 14:347-359, 1963. Plaut W, Nash D, Fanning T Ordered replication of DNA in polytene chromosomes of Drosophila rnelanogaster. J Mol Biol 16:85-93, 1966. Rudkin G Results and Problems in Cell Differentiation. Vol 4, Beermann W, Reinert J, Ursprung H (eds). Springer-Verlag, New York, 1972, pp 59-85. Rudkin GT: Cyclic synthesis of DNA in polytene chromosomes of diptera. In: The Cell Cycle in Development and Differentiation, Robert M (ed). Cambridge University Press, Cambridge, 1973, pp 279-292. Traincard F, Ternynck T, Danchin A, Avrameas S: Une technique immunoenzymatique pour la mise en evidence de l’hybridation moleculaire entre acides nucleiques. Ann Immunol (Paris) 134:399- 405,1983. Zegarelli-Schmidt EC, Goodman R: The Diptera as a model system in cell and molecular biology. Int Rev Cytol 71:245-362, 1981.

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Mapping of the pattern of DNA replication in polytene chromosomes from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies