389
Mutation Research, 62 (1979) 389--392 © Elsevier/North-Holland Biomedical Press
Short Communication
DETECTION OF SISTER CHROMATID EXCHANGES IN VIVO IN SOMATIC CELLS OF Drosophila melanogaster
HIDEO TSUJI and IZUO TOBARI
Division of Genetics, National Institute of Radiological Sciences, Anagawa, Chiba 260 (Japan) (Received 13 December 1978) (Revision received 10 April 1979) (Accepted 1 May 1979)
Recently established techniques for differential staining of sister chromatids [5,7,8,10,16], in which BrdU is incorporated into the chromosomal DNA and the chromosomes are stained by Fluorochrome or Giemsa, provide an accurate count of SCEs. These techniques have been widely used to investigate cell-cycle kinetics, the detection of environmental mutagens and the relationships of SCE formation with other genetical phenomena, such as DNA repair, DNA replication, mutation, recombination and chromosomal aberration. However, the biological significance of the SCE is not yet known. The experiments in vivo in which these techniques are used have been performed with various animals [1--3,6,13]. With Drosophila, however, no experiment in vivo has been done to date except one in which Wienberg [15] tried to apply the BrdU-Giemsa technique to the ganglion cells but failed. Were these techniques successful in D. melanogaster, more information about the mechanisms controlling the SCE formation would be obtained, because the various genetic backgrounds of D. melanogaster have been well analysed. The present study describes a newly developed technique that allows a clear demarcation of sister chromatids in ganglion cells of third.instar larvae of Oregon-R strain of Drosophila melanogaster after exposure to BrdU in vivo. The metaphase chromosomes of ganglia were prepared by a modified method of Tonomura and Tobari [14], and the sister chromatids were differentially stained by a modified technique of Perry and Wolff [10]. A large number of adult flies, kept for 3--5 days in a population cage with food cuPs having boiled yeast medium, were allowed to lay eggs for 1 h on the fresh yeast medium in food cups. At 80 h after egg-laying, 50 third-instar larvae, which emerged from these eggs, were transferred into a culture vial with 5 ml synthetic medium (Table 1) containing BrdU (300 pg/ml) and fed on this medium for 11.3--19.3 h at 25°C in the dark. The composition of the synthetic medium was the same as that of Sang's synthetic medium [11]. Ribonucleic acid was deleted because it may suppress the incorporation of BrdU into the chromosomal DNA. Agar was
390
TABLE 1 COMPOSITION OF SYNTHETIC MEDIUM Ingredient
Amount (mg/100 ml)
Ingredient
Amount (mg/100 ml)
Casein Glucose Cholesterol Lecithin (from soybean) Agar NaHCO 3 KH2PO 4 Na2HPO 4 • 12H20
5000 750 30 400 1400 140 100 287
Thiamine hydrochloride Riboflavin Nicotinic acid Calcium pantothenate Pyridoxine hydroehloride Biotin Folic acid
0.2 1.0 1.2 1.6 0.25 0.016 1.0
used at a lower concentration to make it easier for larvae to incorporate BrdU. The ganglia were dissected out from the third-instar larvae in Drosophila Ringer's solution and treated with colchicine (5 pg/ml) for 40 min in 0.7% NaC1 containing 20% fetal calf serum. The ganglia were transferred into the hypotonic solution (0.075 M KC1). After 10 min they were fixed in 45% acetic acid. After 5 min the ganglia were put on a wet slide with 50% methanol, and a
Fig. 1. Sister-chromatid d i f f e r e n t i a t i o n a n d S C E s i n g a n g l i o n cells o f D. melanog~ter. T h i r d - / n s t a r larvae w e r e f e d o n BrdU-containing m e d i u m for 1 5 . 3 h. Gan4Jlia dissected f r o m t h e s e larvae w e r e treated w i t h c o l c h i c i n e f o r 4 0 r a i n . T h e cells w e r e stained w i t h H o e e h s t 3 3 2 5 8 p l u s G i e m s a . (a) a n d ( b ) , m a l e cells. (c) and (d), f e m a l e cells, T h e arrows indicate the sites o f t h e S C E s .
391 TABLE 2 F R E Q U E N C I E S O F SCEs A T D I F F E R E N T T I M E S A F T E R I N I T I A T I O N O F T R E A T M E N T BY B t d U A T 3 0 0 D g / m l I N V I V O I N L A R V A L G A N G L I O N C E L L S O F D. rnelanogaster Sex
Number of ganglia scored
Number of cells s c o r e d
Number o f SCEs
M e a n SCEs p e r cell (± S.E.)
Range
12--14
Female Male
10 12
135 137
20 16
0.148 + 0.033 0.117 ± 0.029
0--3 0--2
16
Female Male
7 10
106 155
14 24
0.132 ± 0.035 0.155 ± 0.032
0--2 0--2
20
Female Male
6 9
114 133
14 20
0.123 ± 0.033 0.150 ± 0.034
0--2 0--3
Time after BrdU treatment (h) a
a T o t a l t i m e f o r t h e B r d U and c o l c h i c i n e t r e a t m e n t s .
drop of a mixture of acetic acid, lactic acid and distilled water (2 : 1 : 1) was placed on them. Immediately after the cells o f the ganglia were dissociated from the tissue mass, the slide was soaked in a mixture of methanol and acetic acid (3 : 1, Carnoy's solution) for 20 rain to fix chromosomes and to remove the lactic acid. At 3--24 h after preparation of the chromosomes, the slide was stained with Hoechst 3 3 2 5 8 (1 #g/ml), in SSrensen's phosphate buffer (pH 6.8), for 5 min, then m o u n t e d in the same buffer with coverslip. The slide was exposed to a super-pressure mercury lamp (Osram HBO 200 W) for 6 min at a distance of 6 cm from the slide and stained with 2% Giemsa for 10 min. SCEs that occurred at the centromere as well as in the very small autosome IV's were omitted from the score. With our technique described here, we obtained clear-cut figures showing a distinct demarcation of sister chromatids in both the euchromatic and heterochromatic regions (Fig. 1). This is in contrast with Wienberg's experiment in which the differential staining of sister chromatids could only be seen in the euchromatic region, b u t n o t in the heterochromatic region. On the basis of the pattern of Giemsa differentiation of sister chromatids, metaphase cells could be classified as having undergone either one, t w o or three replication cycles. When the third-instar larvae were continuously exposed to BrdU at 300 ~ug/ml the metaphase cells with differentially staining sister chromatids first appeared 6 h after the initiation of BrdU treatment, and the proportion o f these cells had reached a peak at the 16th hour. At the 20th hour the cells with differentially staining sister chromatids were still observed. At this time those cells that had undergone one replication cycle in the presence of BrdU were also found in the same cell population. This suggests that the ganglion-cell population o f D. melanogaster is highly heterogeneous with respect to cell-cycle duration. Subsequently, we examined whether there was a difference in frequency of SCEs among the cells having different cell-cycle durations. As Table 2 shows, only little difference in the frequency of SCEs was found among the cells with different cell-cycle durations. Furthermore, there was no statistical difference between the sexes. The numbers of cells con-
392
taining different numbers of SCEs were distributed according to the Poisson expectation. In this study the mean frequency of SCEs after exposure in vivo to BrdU at 300 ~g/ml was estimated to be 0.14 per cell. The toxic effects of BrdU leading to the induction of SCEs [5,13] and chromosomal aberrations [4], the division delay [12] and the inhibition of DNA metabolism [9] are well known. Thus, the frequency of SCEs of 0.14 per cell, estimated after exposure to a high dose of BrdU, does n o t necessarily indicate the true spontaneous frequency of SCEs in Drosophila ganglion cells. We are n o w attempting to differentially stain the sister chromatids in ganglion cells with a lower dose of BrdU than 300 /~g/ml with the technique described here. We shall then be able to correlate the dose of BrdU with the frequency of SCEs. We are greatly i n d e b t e d to Dr. S. Nakai for his support and encouragement during the course o f this investigation.
References 1 Allen, J.W., and S.A, Latt, Analysis of sister chromatid exchange formation in vivo in mouse sperma~o8onia as a new system for environmental mutagens, Natuze (London), 260 (1976) 449--451. 2 Bayer, U., The in vivo induction of sister chromatid exchanges in the bone marrow of the Chinese hamster, II. N-Nitrosodiethyiamine (DEN) and N-isopropyl~-(2-methylhydmzino)~v-tolumide (NatulaB), two carcinogenic c o m p o u n d s with specific mutagenicity problems, Mutation Res., 56 (1978) 305--309. 3 Bloom, S.E., and T.C. Hsu, Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyurtdine, Chromosoma, 51 (1975) 261--267. 4 Hsu, T.C., and C.E. Somers, Effect of 5-bromodeoxyurldine on mammalian chromosomes, Proc. Natl. Acad. Scl. (U.S.A.), 47 (1961) 396--403. 5 Kato, H., SpontaneoUs sister ehromatid exchanges detected by a BUdR-iabeling method, Nature (London), 251 (1974) 70--72. 6 Kligerman, A.D., and S.E. Bloom, Sister chromatid differentiation and exchanges in adult midminBOWS (Umbra limi) after in vivo exposure to 5-bromodeoxyurldine, Chromosoma, 56 (1976) 101-109. 7 Korenberg, J,R., and E.F. Freedlender, Gicmsa technique for the detection of sister chromatid exchanges, Chromosoma, 48 (1974) 353--360. 8 Latt, S.A., Micro/!uorom6t~ic detection of deoxyribonucleic acid replication in h u m a n metaphase chromosomes, Pro¢. Natl. Acad. Sci. (U.S.A.), 70 (1973) 3395--3399. 9 Meuth, M., and H. Green, Induction of a deoxycytidinelcse state in cultured mammalian cells by hromodeoxyuridine, Cell, 2 (1974) 102--112. 10 Perry, P., and S. Wolff, New Giemsa method for the differential staining of sister chromatid, Nature (London), 251 (1974) 156--158. 11 Sang, J.H., Circumstances affecting the nutritional requirements of Drosophila melanogo.ster, Ann. N.Y. Acad. Sci., 77 (1959) 352--365. 12 Tice, R., E.L. Schneider and JaM. Rary, The utilization of bromodeoxyuridine incorporation into DNA for the analysis of cellular kinetics, Exp. Cell Res., 102 (1976) 232--236. 13 Tice, R,, J. Chaillet and E.L. Schneider, Demonstration of spontaneous sister chromatid exchanges in vivo, Exp. Cell Res., 102 (1976) 426--429, 14 Tonomura, Y., and Y.N. Tobari, Karyotype variations in Drosophil~ pseHdoananase nigrens from Kandy, Sri Lanks, Jpn. J. Genet., 53 (19'/9) 63--66. 15 Wlenberg, J., BrdU-Giamsa-technlque for the differentiation of sister chromatids in somatic cells of Drosophila melanogasfer, Mutation Res., 44 (1977) 283--286. 16 Wolff, S., and P. Perry, Differential Giemsa st~Inln~t of sister chromatids and the study of sister chromatid exchanges, Chromosoma, 48 (1974) 355--360.
Mutation Research, 62 (1979) 389--392 © Elsevier/North-Holland Biomedical Press
Short Communication
DETECTION OF SISTER CHROMATID EXCHANGES IN VIVO IN SOMATIC CELLS OF Drosophila melanogaster
HIDEO TSUJI and IZUO TOBARI
Division of Genetics, National Institute of Radiological Sciences, Anagawa, Chiba 260 (Japan) (Received 13 December 1978) (Revision received 10 April 1979) (Accepted 1 May 1979)
Recently established techniques for differential staining of sister chromatids [5,7,8,10,16], in which BrdU is incorporated into the chromosomal DNA and the chromosomes are stained by Fluorochrome or Giemsa, provide an accurate count of SCEs. These techniques have been widely used to investigate cell-cycle kinetics, the detection of environmental mutagens and the relationships of SCE formation with other genetical phenomena, such as DNA repair, DNA replication, mutation, recombination and chromosomal aberration. However, the biological significance of the SCE is not yet known. The experiments in vivo in which these techniques are used have been performed with various animals [1--3,6,13]. With Drosophila, however, no experiment in vivo has been done to date except one in which Wienberg [15] tried to apply the BrdU-Giemsa technique to the ganglion cells but failed. Were these techniques successful in D. melanogaster, more information about the mechanisms controlling the SCE formation would be obtained, because the various genetic backgrounds of D. melanogaster have been well analysed. The present study describes a newly developed technique that allows a clear demarcation of sister chromatids in ganglion cells of third.instar larvae of Oregon-R strain of Drosophila melanogaster after exposure to BrdU in vivo. The metaphase chromosomes of ganglia were prepared by a modified method of Tonomura and Tobari [14], and the sister chromatids were differentially stained by a modified technique of Perry and Wolff [10]. A large number of adult flies, kept for 3--5 days in a population cage with food cuPs having boiled yeast medium, were allowed to lay eggs for 1 h on the fresh yeast medium in food cups. At 80 h after egg-laying, 50 third-instar larvae, which emerged from these eggs, were transferred into a culture vial with 5 ml synthetic medium (Table 1) containing BrdU (300 pg/ml) and fed on this medium for 11.3--19.3 h at 25°C in the dark. The composition of the synthetic medium was the same as that of Sang's synthetic medium [11]. Ribonucleic acid was deleted because it may suppress the incorporation of BrdU into the chromosomal DNA. Agar was
390
TABLE 1 COMPOSITION OF SYNTHETIC MEDIUM Ingredient
Amount (mg/100 ml)
Ingredient
Amount (mg/100 ml)
Casein Glucose Cholesterol Lecithin (from soybean) Agar NaHCO 3 KH2PO 4 Na2HPO 4 • 12H20
5000 750 30 400 1400 140 100 287
Thiamine hydrochloride Riboflavin Nicotinic acid Calcium pantothenate Pyridoxine hydroehloride Biotin Folic acid
0.2 1.0 1.2 1.6 0.25 0.016 1.0
used at a lower concentration to make it easier for larvae to incorporate BrdU. The ganglia were dissected out from the third-instar larvae in Drosophila Ringer's solution and treated with colchicine (5 pg/ml) for 40 min in 0.7% NaC1 containing 20% fetal calf serum. The ganglia were transferred into the hypotonic solution (0.075 M KC1). After 10 min they were fixed in 45% acetic acid. After 5 min the ganglia were put on a wet slide with 50% methanol, and a
Fig. 1. Sister-chromatid d i f f e r e n t i a t i o n a n d S C E s i n g a n g l i o n cells o f D. melanog~ter. T h i r d - / n s t a r larvae w e r e f e d o n BrdU-containing m e d i u m for 1 5 . 3 h. Gan4Jlia dissected f r o m t h e s e larvae w e r e treated w i t h c o l c h i c i n e f o r 4 0 r a i n . T h e cells w e r e stained w i t h H o e e h s t 3 3 2 5 8 p l u s G i e m s a . (a) a n d ( b ) , m a l e cells. (c) and (d), f e m a l e cells, T h e arrows indicate the sites o f t h e S C E s .
391 TABLE 2 F R E Q U E N C I E S O F SCEs A T D I F F E R E N T T I M E S A F T E R I N I T I A T I O N O F T R E A T M E N T BY B t d U A T 3 0 0 D g / m l I N V I V O I N L A R V A L G A N G L I O N C E L L S O F D. rnelanogaster Sex
Number of ganglia scored
Number of cells s c o r e d
Number o f SCEs
M e a n SCEs p e r cell (± S.E.)
Range
12--14
Female Male
10 12
135 137
20 16
0.148 + 0.033 0.117 ± 0.029
0--3 0--2
16
Female Male
7 10
106 155
14 24
0.132 ± 0.035 0.155 ± 0.032
0--2 0--2
20
Female Male
6 9
114 133
14 20
0.123 ± 0.033 0.150 ± 0.034
0--2 0--3
Time after BrdU treatment (h) a
a T o t a l t i m e f o r t h e B r d U and c o l c h i c i n e t r e a t m e n t s .
drop of a mixture of acetic acid, lactic acid and distilled water (2 : 1 : 1) was placed on them. Immediately after the cells o f the ganglia were dissociated from the tissue mass, the slide was soaked in a mixture of methanol and acetic acid (3 : 1, Carnoy's solution) for 20 rain to fix chromosomes and to remove the lactic acid. At 3--24 h after preparation of the chromosomes, the slide was stained with Hoechst 3 3 2 5 8 (1 #g/ml), in SSrensen's phosphate buffer (pH 6.8), for 5 min, then m o u n t e d in the same buffer with coverslip. The slide was exposed to a super-pressure mercury lamp (Osram HBO 200 W) for 6 min at a distance of 6 cm from the slide and stained with 2% Giemsa for 10 min. SCEs that occurred at the centromere as well as in the very small autosome IV's were omitted from the score. With our technique described here, we obtained clear-cut figures showing a distinct demarcation of sister chromatids in both the euchromatic and heterochromatic regions (Fig. 1). This is in contrast with Wienberg's experiment in which the differential staining of sister chromatids could only be seen in the euchromatic region, b u t n o t in the heterochromatic region. On the basis of the pattern of Giemsa differentiation of sister chromatids, metaphase cells could be classified as having undergone either one, t w o or three replication cycles. When the third-instar larvae were continuously exposed to BrdU at 300 ~ug/ml the metaphase cells with differentially staining sister chromatids first appeared 6 h after the initiation of BrdU treatment, and the proportion o f these cells had reached a peak at the 16th hour. At the 20th hour the cells with differentially staining sister chromatids were still observed. At this time those cells that had undergone one replication cycle in the presence of BrdU were also found in the same cell population. This suggests that the ganglion-cell population o f D. melanogaster is highly heterogeneous with respect to cell-cycle duration. Subsequently, we examined whether there was a difference in frequency of SCEs among the cells having different cell-cycle durations. As Table 2 shows, only little difference in the frequency of SCEs was found among the cells with different cell-cycle durations. Furthermore, there was no statistical difference between the sexes. The numbers of cells con-
392
taining different numbers of SCEs were distributed according to the Poisson expectation. In this study the mean frequency of SCEs after exposure in vivo to BrdU at 300 ~g/ml was estimated to be 0.14 per cell. The toxic effects of BrdU leading to the induction of SCEs [5,13] and chromosomal aberrations [4], the division delay [12] and the inhibition of DNA metabolism [9] are well known. Thus, the frequency of SCEs of 0.14 per cell, estimated after exposure to a high dose of BrdU, does n o t necessarily indicate the true spontaneous frequency of SCEs in Drosophila ganglion cells. We are n o w attempting to differentially stain the sister chromatids in ganglion cells with a lower dose of BrdU than 300 /~g/ml with the technique described here. We shall then be able to correlate the dose of BrdU with the frequency of SCEs. We are greatly i n d e b t e d to Dr. S. Nakai for his support and encouragement during the course o f this investigation.
References 1 Allen, J.W., and S.A, Latt, Analysis of sister chromatid exchange formation in vivo in mouse sperma~o8onia as a new system for environmental mutagens, Natuze (London), 260 (1976) 449--451. 2 Bayer, U., The in vivo induction of sister chromatid exchanges in the bone marrow of the Chinese hamster, II. N-Nitrosodiethyiamine (DEN) and N-isopropyl~-(2-methylhydmzino)~v-tolumide (NatulaB), two carcinogenic c o m p o u n d s with specific mutagenicity problems, Mutation Res., 56 (1978) 305--309. 3 Bloom, S.E., and T.C. Hsu, Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyurtdine, Chromosoma, 51 (1975) 261--267. 4 Hsu, T.C., and C.E. Somers, Effect of 5-bromodeoxyurldine on mammalian chromosomes, Proc. Natl. Acad. Scl. (U.S.A.), 47 (1961) 396--403. 5 Kato, H., SpontaneoUs sister ehromatid exchanges detected by a BUdR-iabeling method, Nature (London), 251 (1974) 70--72. 6 Kligerman, A.D., and S.E. Bloom, Sister chromatid differentiation and exchanges in adult midminBOWS (Umbra limi) after in vivo exposure to 5-bromodeoxyurldine, Chromosoma, 56 (1976) 101-109. 7 Korenberg, J,R., and E.F. Freedlender, Gicmsa technique for the detection of sister chromatid exchanges, Chromosoma, 48 (1974) 353--360. 8 Latt, S.A., Micro/!uorom6t~ic detection of deoxyribonucleic acid replication in h u m a n metaphase chromosomes, Pro¢. Natl. Acad. Sci. (U.S.A.), 70 (1973) 3395--3399. 9 Meuth, M., and H. Green, Induction of a deoxycytidinelcse state in cultured mammalian cells by hromodeoxyuridine, Cell, 2 (1974) 102--112. 10 Perry, P., and S. Wolff, New Giemsa method for the differential staining of sister chromatid, Nature (London), 251 (1974) 156--158. 11 Sang, J.H., Circumstances affecting the nutritional requirements of Drosophila melanogo.ster, Ann. N.Y. Acad. Sci., 77 (1959) 352--365. 12 Tice, R., E.L. Schneider and JaM. Rary, The utilization of bromodeoxyuridine incorporation into DNA for the analysis of cellular kinetics, Exp. Cell Res., 102 (1976) 232--236. 13 Tice, R,, J. Chaillet and E.L. Schneider, Demonstration of spontaneous sister chromatid exchanges in vivo, Exp. Cell Res., 102 (1976) 426--429, 14 Tonomura, Y., and Y.N. Tobari, Karyotype variations in Drosophil~ pseHdoananase nigrens from Kandy, Sri Lanks, Jpn. J. Genet., 53 (19'/9) 63--66. 15 Wlenberg, J., BrdU-Giamsa-technlque for the differentiation of sister chromatids in somatic cells of Drosophila melanogasfer, Mutation Res., 44 (1977) 283--286. 16 Wolff, S., and P. Perry, Differential Giemsa st~Inln~t of sister chromatids and the study of sister chromatid exchanges, Chromosoma, 48 (1974) 355--360.
