Experimental

MECHANISM

Cell Research 101 (1976) 55-58

OF DIFFERENTIAL

BUdR-SUBSTITUTED

VZCZA FABA

STAINING

OF

CHROMOSOMES

W. SCHEID Znstitut fiir Strahlenbiologie der Westfiilischen Wilhelms-Universitit,

44 Miinster, BRD

SUMMARY Chromosomes of the broad bean Vicia faba were isolated and air-dried on slides after incorporation of BUdR into DNA (BUdR substitution) for two rounds of reulication. Then the oreuarations were embedded in a buffer solution containing trypsin as weli as fluorescence dye (acridine orange or Hoechst 33258). We observed chromosomes with a fluorescence microscope at various times after embedding. After about 15 min one sister chromatid of some of the metaphase chromosomes showed enhanced darkening and disintegration within l-4 min (melting effect) during observation. We suppose that fragmentation of BUdR-substituted DNA by the acridine orange-visible light system in acridine orange staining and by irradiation with wavelengths around the transition from UV to visible light in Hoechst 33258 staining is responsible for this phenomenon. The disintegration of one sister chromatid in BUdR-substituted chromosomes can also be produced by UV irradiation during trypsin treatment when fluorescence dyes are not present.

Several methods have been worked out to demonstrate differences in staining with acridine orange, Hoechst 33258, Giemsa stain and other dyes between double (BB) and single (TB) or single (TB) and non(TT)-BUdR-substituted chromatids, especially to investigate sister chromatid exchanges (T, thymine-containing strand of the DNA duplex; B, bromouracil-containing strand). This differential staining may be induced by different mechanisms, e.g. “unequal spiralization delay” between BB and TB strands [12] or by “fluorescence quenching” [7]. The causes of differential staining produced by other methods (see [5] for literature), e.g. in Giemsa techniques, are not yet clear [6]. In the broad bean Viciafaba we observed that acridine orange and Hoechst 33258 stained BB chromatids rapidly dissolve dur-

ing observation with a fluorescence microscope while being treated with trypsin. This indicates that there exists another mechanism contributing to differential staining between TB and BB chromatids, namely destruction of BUdR-substituted DNA by single-strand breaks induced via the acridine orange-visible light (AOVL) system [ 1, 41 after acridine orange staining and possibly via a similar system when Hoechst 33258 is used. Single-strand breaks are also induced in BUdR-substituted DNA by irradiation with UV ranging from short to long wavelengths [3]. Therefore we investigated whether the disintegration of the BB chromatid during trypsin digestion of chromosomal proteins could be caused by UV irradiation when the influence of fluorescence dyes was excluded. Exp

Cell

Rcs

101 (1976)

56

W. Scheid

Table

1 Stained with Hoechst 33258

Orcein

Series

Pretreatment

A

B

C

A

B

c

1

Without

2 3

uv

6 5

0 0

6 5

5 6

Trypsin UV+trypsin

0 3”

5 3

5

0

5”

5

0

5

7

7

0

10

10

0

4

Differential staining between sister chromatids of Viciafuba after incorporation of BUdR for two rounds of DNA replication. A, Number of independent experiments in which on 3 slides some chromosomes were investigated; B, number of experiments in which differential staining was observed; C, number of experiments in which no differential staining was observed. ” The weak differential staining appeared between 2 and 6 h after addition of the orcein solution and disappeared after about 24 h. The cause for this phenomenon is not yet clear. b After prolonged observation with UV differential staining was sometimes visible.

MATERIALS

AND METHODS

Seeds of the broad bean Vicia faba (var. Exhibition Longpod) were cultured as described elsewhere [IO]. Ten day-old seedlings were put in aerated 100 pmol BUdR (Serva, Heidelberg) containing l/8-strength Hoagland solution and cultivated in a dark room at 19°C. After 27+0.5 h (a part of the chromosomes passed through two rounds of replication) colchicine up to a concentration of 0.03% was added for 21 h. Metaphase chromosomes of cells of primary root tips were fixed and isolated as described before [ll]. The solution containing the isolated chromosomes was dropped on clean slides and air-dried. One drop of a dye solution and then one drop of a trypsin solution were given on the preparations. The dye solution consisted of 30 pgjml acridine orange (Merck, Darmstadt) in neutral 0.066 mol phosphate buffer or 50 &ml Hoechst 33258 in deionized water. The trypsin solution consisted of 1 mg/mI trypsin (TPCK-treated, Serva, Heidelberg) dissolved in neutral 0.066 mol phosphate buffer. After covering with a coverslip the preparations were observed with the help of a Leitz Grtholux microscope equipped with incident illumination and a 200 W mercury light source. The filter combination chosen for observing acridine orange fluorescence was 1.5 mm BG 12 and TK 5 10(dichroic) excitor filters, K 515 and K 5 10 suppressor filters and for Hoechst 33258 fluorescence 2 mm UG 1 and TK 400 (dichroic) excitor filters, K 400 and K 430 suppressor filters. In order to studv the action of UV irradiation on BUdR-substituted *chromosomes not stained with fluorescence dyes, chromosomes were irradiated with UV during trypsin digestion and were afterwards stained with Hoechst 33258 or an orcein solution (besides control series). The above-mentioned suspension containing isolated chromosomes was dropped on clean slides without trypsin and was air-dried without UV irradiation (ser. 1) or while being irradiated with a Philips UV lamp (TUV 30 W) at a distance of 80 cm for 2 h (ser. 2). Exp Cd/ Res IO1 (1976)

The intensity of the UV irradiation was 126 pW/cm* (about 87 % at 254 nm, 8 % at 3 13 nm and 5 % at 366 ran). The same suspension containing 0.1 mg/ml trypsin was air-dried without UV irradiation (ser. 3) or while being irradiated with UV as described above (ser. 4). Then the preparations were embedded in a Hoechst 33258 solution (25 pg Hoechst 33258/m] deionized water) or an orcein solution prepared after Oster & Balaban [9].

RESULTS AND DISCUSSION After a digestion time of about 15 min BUdR-substituted chromosomes embedded in a solution containing trypsin and a fluorescence dye (acridine orange or Hoechst 33258) showed enhanced darkening of one chromatid when observed with a fluorescence microscope. This chromatid dissolved within 14 min (melting effect) while being observed (Leitz N Pl 4010.65 objective). Particles of the chromatid were carried off by the dye solution. Sister chromatid exchanges were often visible. Without trypsin treatment differential staining was not observed. Kihlman & Kronborg [5) incorporated BUdR in chromosomal DNA in the presence of FUdR and uridine to get differential staining in Vicia fuba chromosomes in their fluorescence plus Giemsa (FPG) technique. This

Differential staining of BUdR-substituted chromosomes

57

mosome can be caused without the cooperation of a fluorescence dye, chromosomes were irradiated with UV (the spectrum contained bands at 254, 313 and 366 nm) while being treated with trypsin and were afterwards stained. For results of the microscopical analysis see table 1 and fig. 1. The disintegration of one of the sister chromatids, probably the BB chromatid (chromosomes have passed through two rounds of DNA replication in the presence of BUdR) is visible after UV plus trypsin treatment. As irradiation with UV both of short and of long wavelengths induces singlestrand breaks in BUdR-substituted DNA more frequently than in non-substituted DNA [3], BB strands may dissolve during trypsin treatment while TB strands are held together by the T strand which is less sensitive to fragmentation. (Chromosomes were irradiated with UV during air-drying; a dislocation of the broken particles was therefore possible.) In the above-mentioned acridine orange series (fluorescence excitation above 400 nm) the participation of the AO-VL system [l, 41 must be assumed to explain the enhanced destruction of the BB chromatid during observation with the fluorescence microscope. Also in the Hoechst 33258 Fig. 1. Orcein-stained S chromosome of Vi& faba after BUdR incorporation, isolation and subsequent series a similar effect cannot be excluded UV irradiation while being treated with trypsin. The extensive dissolving of one chromatid can be seen because the direct action of the UV irradiation used (fluorescence excitation at about clearly. 35uOO nm) is not very strong, as indicated in experiments by Mennigmann [8]. Menprocedure failed to show positive results nigmann showed that the transforming acin our experiments (unpublished results) tivity of bifiliarly substituted (BB) DNA of when trypsin was not present. Perhaps one Bacillus subtilis is much less reduced by or the other of the various additional treat- UV of 350 nm than by UV of 320 nm. ments used by Kihlman & Kronborg in their Very recently a publication of Goto et al. experiments brought about the same effect [2] came to our attention. These authors, working with BUdR-substituted rat chroas trypsin did. To decide whether the destruction of one mosomes, found that irradiation with visible chromatid in the BUdR-substituted chro- or UV light is an essential step in the E.rp Cc/I Rrs 101 (1976)

58

W. Scheid

Hoechst 33258 Giemsa method. They suggest that photolysis of DNA is the principal cause of differential chromatid staining. Such a photolysis has indeed been observed in our acridine orange and Hoechst 33258 series. We thank Dr H. Loewe, Farbwerke Hoechst, for a gift of the dye Hoechst 33258.

REFERENCES 1. Freifelder, D, Davison, P F & Geiduschek, E P, Biophys j I (I%I) 389. 2. Goto, K, Akematsu, T, Shimazu, H & Sugiyama, T, Chromosoma 53 (1975) 223.

E.rp Cdl

Rrs

101 (197ti)

3. Hutchinson, F, Quart rev biophy\ 6 ( lY73) 201 4. Kihlman. B A. Actions of che&cals on dividing cells (ed W D McElroy & C P Swanson). Prentice-Hall, Englewood Cliffs, N.J. (1966). Kihlman, B A & Kronborp, D. Chromosoma 51 (1975) I. Korenberg, J R & Freedlender. E F, Chromosoma 48 (1974) 355. Latt, S A & Wohlleb, J C, Chromosoma 52 ( 1975) 297. Mennigmann, H-D, Mol gen genet 99 (1967) 76. Oster, I I & Balaban, G, Drosoph inform serv 37 (1963) 142. 10. Scheid, W & Traut. H, Mutation res 6 (1968) 481. 11. Traut, H & Scheid, W, Mutation res 17(1973) 335. 12. Zakharov, A F & Egolina, N A, Chromosoma 38 (1972) 341. Received March 12, 1976 Accepted March 16, 1976