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Interaction between X-ray- and restriction endonuclease-induced lesions in the formation of chromosomal aberrations
Mutation Research, 244 (1990) 197-200 Elsevier
197
MUTLET 0360
Interaction between X-ray- and restriction endonuclease-induced lesions in the formation of chromosomal aberrations C. Tanzarella, R. De Salvia, F. Degrassi, M. Fiore and F. Palitti 1 Centro di Genetica Evoluzionistica del CNR, c/o Dipartimento "Darwin', Universitfi "La Sapienza', 1-00185 Rome (Italy) and 1Dipartimento di Agrobiologk~ e Agrochimica, Universit,~ della Tuscia, I-Ol lO0 Viterbo (Italy) (Accepted I 1 January 1990)
Keyword~: Restriction endonucleases; X-rays; Interaction; Chromosomal aberrations
Summary Experiments were performed to analyze the possible interaction between lesions induced by X-rays and restriction endonucleases in the production of chromosome-type exchanges. A stronger interaction was found between X-rays and the AluI-induced 'blunt termini' lesions than between X-rays and the BamHIinduced 'cohesive termini' lesions.
With the development of techniques that allow the introduction of endonucleases into cells (Tanaka et al., 1975) and the subsequent work done by Natarajan et al. (1980) it became possible to gain direct evidence for the involvement of specific DNA lesions (i.e., double-strand breaks) in the induction of chromosomal damage. More recently Bryant (1984) and Natarajan and Obe (1984) were able to introduce restriction endonucleases (RE) into cells to mimic radiation damage. In the present paper we have investigated the interaction between X-ray-induced lesions (possible double-strand breaks) (dsb) with either 'blunt' or Correspondence: Prof. F. Palitti, Centro di Genetica Evoluzionistica del CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Universit~ 'La Sapienza', P.zz.le A. Moro, 1-00185 Rome (Italy).
'cohesive' termini induced by RE. To investigate this possible interaction we have analyzed the production of chromosome-type exchanges in Chinese hamster cells treated in the G1 phase of the cell cycle. Materials and methods Cell culture Chinese hamster ovary (CHO-K1) cells were grown in Ham's F10 medium supplemented with 10°70 fetal calf serum (FCS) (Flow Laboratories) in plastic flasks (25 cm). X-irradiation and restriction endonucleases Irradiation was carried out with a Gilardoni Xray apparatus operating at 180 kV and 8 mA with a 3-ram Al filter giving a dose rate of 1 cGy/min. Cells were exposed in suspension in 10-ml cen-
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
198 TABLE 1 Restriction endonuclease
Recognition sequence
Alul
5' 3' 5' 3'
BamHl
..AG*T..3' ..TC*A..5' ..G*GATC C..3' ..C CTAG*G..5'
trifuge tubes kept on ice. Restriction endonucleases used were AluI and BamHI (Boehringer) which induce 'blunt' or 'cohesive' ended dsbs respectively (Table 1).
Experimental procedure Exponentially growing cells were trypsinized and resuspended in complete medium. Aliquots of 16 x 106 cells were distributed in 10-ml centrifuge tubes; after centrifugation (10 min at 1000 rpm) the pellets were resuspended in 1 ml FCS, again centrifuged and as much as possible of the supernatant was discarded. The cell pellets were then treated with AluI (16 units/2 x 106 cells) or with BamHI (36 units/2 x 106 cells) for 10 min at 37°C. The concentrations of RE were adjusted with FCS to give an amount of fluid of 16 #1 (Obe et al., 1985; Obe and Winkel, 1985). Afterwards the cells were suspended in 10 ml of complete medium kept on ice, then 5-ml aliquots of these suspensions were exposed to 1 Gy of X-rays in centrifuge tubes kept on ice. Parallel aliquots of 16 x 106 cells were set up in the same way but were exposed to X-rays only or remained unirradiated. All the samples were then transferred into flasks (2 x 106 cells for each experimental point) and incubated in complete medium containing 5-bromo-
deoxyuridine (3/~g/ml) for 19 or 22 h at 37°C in 5°7o CO2 in order to score chromosomal aberrations only in first-mitosis cells. These 2 sampling times (19 and 22 h) were selected on the basis of previous experiments in order to analyze most of the G~-treated cells to avoid any selection effects.
Preparation of slides and scoring procedure Colchicine (final concentration 5 x 10 -7 M) was added 3 h before harvesting. Metaphase chromosomes were then prepared by standard procedure using a hypotonic treatment (1°70 sodium citrate) of 10 min and stained according to the fluorescence plus Giemsa technique (Perry and Wolff, 1974). Only first post-treatment metaphases were analyzed with respect to chromosomal aberrations. For each treatment 100 metaphases, including heavily damaged cells, were analyzed and all classes of aberrations were classified according to Savage (1975).
Analysis o f the data In order to quantify the possible interaction between RE- and X-ray-induced lesions we have used a synergism factor (SF), defined as the ratio of the effect of the combined treatments to the sum of the effects of the 2 agents given separately. Results and discussion The results obtained at the two sampling times were pooled as they were not significantly different.
TABLE 2 C H R O M O S O M A L ABERRATIONS I N D U C E D BY X-RAYS (1 Gy), AIuI ENDONUCLEASE (16 units) OR X-RAYS AND A l u l ENDONUCLEASE COMBINED TREATMENTS IN THE Gl PHASE OF CHO CELLS Treatment
Control X-rays AluI A l u I + X-rays
Abnormal cells
Aberrations per 200 cells Gaps
Chromatid breaks + SE
Isochromatid breaks + SE
Chromatid exchanges + SE
Chromosome exchanges + SE
Total (excl. gaps)
9 50 56 93
4 6 8 8
1 12 13 9
4 14 24 131
0 3 + 1.7 11 + 3.3 10 + 3.1
0 20 + 4.4 26 + 5.0 110 + 10.4
5 49 74 260
+ + + +
1.0 3.46 3.6 3.0
+ 2.0 + 3.7 + 4.8 + 11.4
199 TABLE 3 CHROMOSOMAL ABERRATIONS INDUCED BY X-RAYS (1 Gy), BamHI ENDONUCLEASE (36 units) OR X-RAYS AND BamHI ENDONUCLEASE COMBINED TREATMENTS IN THE G~ PHASE OF CHO CELLS Treatment
Control X-rays BamHI BamHI + X-rays
Abnormal cells
Aberrations per 200 cells
7 47 41 61
1 12 7 6
Gaps
Chromatid breaks + SE 2 8 8 9
+ + + +
1.4 2.8 2.8 3.0
T a b l e 2 s h o w s t h a t A l u I + X - r a y t r e a t m e n t gave a frequency of chromosome-type aberrations higher t h a n the s u m o f t h e yields o b t a i n e d in the t r e a t m e n t s with the 2 agents w h e n given separately; in c o n t r a s t , B a m H I ÷ X - r a y t r e a t m e n t gave a freq u e n c y o f c h r o m o s o m e - t y p e a b e r r a t i o n s t h a t was m o r e o r less similar t o the s u m o f the yields o b t a i n ed in the t r e a t m e n t s with t h e 2 agents when given s e p a r a t e l y ( T a b l e 3). Since c h r o m o s o m e exchanges derive f r o m i n t e r a c t i o n b e t w e e n i n d u c e d D N A lesions we f o c u s e d o u r a t t e n t i o n o n this t y p e o f a b e r r a t i o n s . A s can be seen f r o m the S F for c h r o m o s o m e - t y p e exchanges, t h e r e is a very s t r o n g s y n e r g i s m b e t w e e n the lesions i n d u c e d b y A l u I a n d X - r a y s (SF = 2.39). N o s y n e r g i s m is o b s e r v e d between BamHIand X-ray-induced lesions (SF -- 1.12). O u r results i n d i c a t e t h a t the interact i o n b e t w e e n X - r a y - a n d R E - i n d u c e d lesions dep e n d s o n the t y p e o f dsb i n d u c e d b y the e n d o n u clease, n a m e l y ' b l u n t ' (AluI) vs. ' c o h e s i v e ' (BamHI) termini. A l s o in o u r e x p e r i m e n t s the d i s t r i b u t i o n o f c h r o m o s o m a l d a m a g e i n d u c e d b y R E is ' o v e r d i s p e r s e d ' as f o u n d b y o t h e r a u t h o r s (see B r y a n t , 1988). It is n o t e w o r t h y t h a t , while the f r e q u e n c y o f a b n o r m a l cells in b o t h c o m b i n e d t r e a t m e n t s is a d ditive o r less t h a n a d d i t i v e , the t o t a l n u m b e r o f c h r o m o s o m a l a b e r r a t i o n s is m u c h m o r e t h a n a d ditive o n l y in the c o m b i n e d t r e a t m e n t o f X - r a y s and AluI. T h e d i s t r i b u t i o n o f dicentrics after the v a r i o u s t r e a t m e n t s is s h o w n in Fig. 1. I n the case o f the c o m b i n e d t r e a t m e n t (AluI + X - r a y s ) t h e r e was a
lsochromatid breaks + SE 1 21 42 89
+ + + +
1.0 4.5 6.5 9.4
Chromatid exchanges + SE 0 1 + 1.0 1 + 1.0 2 + !.4
Chromosome exchanges + SE
Total (excl. gaps)
1 12 26 44
4 42 77 144
+ + + +
1.0 3.4 5.1 6.6
high f r e q u e n c y o f h e a v i l y d a m a g e d cells ( m o r e t h a n 3 dicentrics); this m i g h t be e x p e c t e d if R E m o l e c u l e p e n e t r a t i o n is n o t the s a m e in all cells o f the population. Considering that after treatment with R E the cells were k e p t at 0 ° C in o r d e r to prevent r e p a i r , one m i g h t a r g u e t h a t the X - r a y i n d u c e d lesions can i n t e r a c t better with A l u I i n d u c e d lesions ( ' b l u n t ' ) t h a n with B a m H I - i n d u c e d lesions ( ' c o h e s i v e ' ) . O f course it c a n n o t be excluded t h a t , as suggested b y B r y a n t (1984), the d s b with ' c o h e s i v e ' t e r m i n i i n d u c e d b y B a m H I w o u l d be
A ~
•
30-
x-rays
~,u,
~ 20-
O-
- 1
2
3
4
5
S Z
20
dJI 1
2
3
4
~
x-rays
~
Barn HI
~
x-rays ÷BarnHI
5
Number of dicentrics per cell
Fig. 1. Frequencies of cells with different number of dicentrics. (A) X-ray-, AluI- or X-ray and Alul-treated cells. (B) X-ray-, BamHI- or X-ray and BamHI-treated cells.
200
rapidly repaired with no chance of interaction with other lesions induced by the enzyme itself or with those induced by X-rays. Usually RE which induce dsb with 'cohesive' termini are less efficient in inducing chromosomal aberrations (Bryant, 1984; Natarajan and Obe, 1984). Therefore the synergism between RE-induced lesions with 'blunt' termini and X-ray-induced lesions could suggest that, as the repair of this type of dsb could be slower, there is enough time for these types of lesions to interact among themselves or with X-rayinduced lesions. Our data also indirectly confirm the suggestion advanced by Bryant et al. (1988) that if the frequency of dsbs is maintained at a high level the opportunity for misjoining is larger producing a higher frequency of chromosomal exchanges (Fig. 1). Acknowledgements This work was partially supported by Progetto Finalizzato 'Oncologia' CNR, Ministero della Ricerca and by a grant of CNR Progetto Bilaterale Italia-Germania. References Bryant, P.E. (1984) Enzymatic restriction of mammalian DNA using Pvull and BamHI: evidence for the double-strand
break origin of chromosomal aberrations, Int. J. Radiat. Biol., 46, 57-65. Bryant, P.E. (1988) Use of restriction endonucleases to study relationship between double-strand breaks, chromosomal aberrations and other end-points in mammalian cells, Int. J. Radiat. Biol., 54, 869-890. Natarajan, A.T., and G. Obe (1984) Molecular mechanisms involved in the production of chromosomal aberrations, Chromosoma, 90, 120-127. Natarajan, A.T., G. Obe, A.A. van Zeeland, F. Palitti, M. Meijers and E.A.M. Verdegaal-lmmerzeel (1980) Molecular mechanisms involved in the production of chromosomal aberrations. II. Utilization of Neurospora endonuclesae for the study of aberration production by X-ray in G1 and G2 stages of the cell cycle, Mutation Res., 69, 293-305. Obe, G., and E.U. Winkel (1985) The chromosome breaking activity of the restriction endonuclease Alul in CHO cells is independent of the S-phase of the cell cycle, Mutation Res., 152, 25-29. Obe, G., F. Palitti, C. Tanzarella, F. Degrassi and R. De Salvia (1985) Chromosomal aberrations induced by restriction endonucleases, Mutation Res., 150, 359-368. Perry, P., and S. Wolff (1974) New Giemsa method for detection of sister chromatids, Nature (London), 251, 156-158. Savage, J.R. (1975) Classification and relationship of induced chromosomal structural changes, J. Med. Genet., 13, 103-122. Tanaka, K., M. Sekiguchi and Y. Okada (1975) Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus), Proc. Natl. Acad. Sci. (U.S.A.), 72, 4071-4075. Communicated by F.H. Sobels
197
MUTLET 0360
Interaction between X-ray- and restriction endonuclease-induced lesions in the formation of chromosomal aberrations C. Tanzarella, R. De Salvia, F. Degrassi, M. Fiore and F. Palitti 1 Centro di Genetica Evoluzionistica del CNR, c/o Dipartimento "Darwin', Universitfi "La Sapienza', 1-00185 Rome (Italy) and 1Dipartimento di Agrobiologk~ e Agrochimica, Universit,~ della Tuscia, I-Ol lO0 Viterbo (Italy) (Accepted I 1 January 1990)
Keyword~: Restriction endonucleases; X-rays; Interaction; Chromosomal aberrations
Summary Experiments were performed to analyze the possible interaction between lesions induced by X-rays and restriction endonucleases in the production of chromosome-type exchanges. A stronger interaction was found between X-rays and the AluI-induced 'blunt termini' lesions than between X-rays and the BamHIinduced 'cohesive termini' lesions.
With the development of techniques that allow the introduction of endonucleases into cells (Tanaka et al., 1975) and the subsequent work done by Natarajan et al. (1980) it became possible to gain direct evidence for the involvement of specific DNA lesions (i.e., double-strand breaks) in the induction of chromosomal damage. More recently Bryant (1984) and Natarajan and Obe (1984) were able to introduce restriction endonucleases (RE) into cells to mimic radiation damage. In the present paper we have investigated the interaction between X-ray-induced lesions (possible double-strand breaks) (dsb) with either 'blunt' or Correspondence: Prof. F. Palitti, Centro di Genetica Evoluzionistica del CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Universit~ 'La Sapienza', P.zz.le A. Moro, 1-00185 Rome (Italy).
'cohesive' termini induced by RE. To investigate this possible interaction we have analyzed the production of chromosome-type exchanges in Chinese hamster cells treated in the G1 phase of the cell cycle. Materials and methods Cell culture Chinese hamster ovary (CHO-K1) cells were grown in Ham's F10 medium supplemented with 10°70 fetal calf serum (FCS) (Flow Laboratories) in plastic flasks (25 cm). X-irradiation and restriction endonucleases Irradiation was carried out with a Gilardoni Xray apparatus operating at 180 kV and 8 mA with a 3-ram Al filter giving a dose rate of 1 cGy/min. Cells were exposed in suspension in 10-ml cen-
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
198 TABLE 1 Restriction endonuclease
Recognition sequence
Alul
5' 3' 5' 3'
BamHl
..AG*T..3' ..TC*A..5' ..G*GATC C..3' ..C CTAG*G..5'
trifuge tubes kept on ice. Restriction endonucleases used were AluI and BamHI (Boehringer) which induce 'blunt' or 'cohesive' ended dsbs respectively (Table 1).
Experimental procedure Exponentially growing cells were trypsinized and resuspended in complete medium. Aliquots of 16 x 106 cells were distributed in 10-ml centrifuge tubes; after centrifugation (10 min at 1000 rpm) the pellets were resuspended in 1 ml FCS, again centrifuged and as much as possible of the supernatant was discarded. The cell pellets were then treated with AluI (16 units/2 x 106 cells) or with BamHI (36 units/2 x 106 cells) for 10 min at 37°C. The concentrations of RE were adjusted with FCS to give an amount of fluid of 16 #1 (Obe et al., 1985; Obe and Winkel, 1985). Afterwards the cells were suspended in 10 ml of complete medium kept on ice, then 5-ml aliquots of these suspensions were exposed to 1 Gy of X-rays in centrifuge tubes kept on ice. Parallel aliquots of 16 x 106 cells were set up in the same way but were exposed to X-rays only or remained unirradiated. All the samples were then transferred into flasks (2 x 106 cells for each experimental point) and incubated in complete medium containing 5-bromo-
deoxyuridine (3/~g/ml) for 19 or 22 h at 37°C in 5°7o CO2 in order to score chromosomal aberrations only in first-mitosis cells. These 2 sampling times (19 and 22 h) were selected on the basis of previous experiments in order to analyze most of the G~-treated cells to avoid any selection effects.
Preparation of slides and scoring procedure Colchicine (final concentration 5 x 10 -7 M) was added 3 h before harvesting. Metaphase chromosomes were then prepared by standard procedure using a hypotonic treatment (1°70 sodium citrate) of 10 min and stained according to the fluorescence plus Giemsa technique (Perry and Wolff, 1974). Only first post-treatment metaphases were analyzed with respect to chromosomal aberrations. For each treatment 100 metaphases, including heavily damaged cells, were analyzed and all classes of aberrations were classified according to Savage (1975).
Analysis o f the data In order to quantify the possible interaction between RE- and X-ray-induced lesions we have used a synergism factor (SF), defined as the ratio of the effect of the combined treatments to the sum of the effects of the 2 agents given separately. Results and discussion The results obtained at the two sampling times were pooled as they were not significantly different.
TABLE 2 C H R O M O S O M A L ABERRATIONS I N D U C E D BY X-RAYS (1 Gy), AIuI ENDONUCLEASE (16 units) OR X-RAYS AND A l u l ENDONUCLEASE COMBINED TREATMENTS IN THE Gl PHASE OF CHO CELLS Treatment
Control X-rays AluI A l u I + X-rays
Abnormal cells
Aberrations per 200 cells Gaps
Chromatid breaks + SE
Isochromatid breaks + SE
Chromatid exchanges + SE
Chromosome exchanges + SE
Total (excl. gaps)
9 50 56 93
4 6 8 8
1 12 13 9
4 14 24 131
0 3 + 1.7 11 + 3.3 10 + 3.1
0 20 + 4.4 26 + 5.0 110 + 10.4
5 49 74 260
+ + + +
1.0 3.46 3.6 3.0
+ 2.0 + 3.7 + 4.8 + 11.4
199 TABLE 3 CHROMOSOMAL ABERRATIONS INDUCED BY X-RAYS (1 Gy), BamHI ENDONUCLEASE (36 units) OR X-RAYS AND BamHI ENDONUCLEASE COMBINED TREATMENTS IN THE G~ PHASE OF CHO CELLS Treatment
Control X-rays BamHI BamHI + X-rays
Abnormal cells
Aberrations per 200 cells
7 47 41 61
1 12 7 6
Gaps
Chromatid breaks + SE 2 8 8 9
+ + + +
1.4 2.8 2.8 3.0
T a b l e 2 s h o w s t h a t A l u I + X - r a y t r e a t m e n t gave a frequency of chromosome-type aberrations higher t h a n the s u m o f t h e yields o b t a i n e d in the t r e a t m e n t s with the 2 agents w h e n given separately; in c o n t r a s t , B a m H I ÷ X - r a y t r e a t m e n t gave a freq u e n c y o f c h r o m o s o m e - t y p e a b e r r a t i o n s t h a t was m o r e o r less similar t o the s u m o f the yields o b t a i n ed in the t r e a t m e n t s with t h e 2 agents when given s e p a r a t e l y ( T a b l e 3). Since c h r o m o s o m e exchanges derive f r o m i n t e r a c t i o n b e t w e e n i n d u c e d D N A lesions we f o c u s e d o u r a t t e n t i o n o n this t y p e o f a b e r r a t i o n s . A s can be seen f r o m the S F for c h r o m o s o m e - t y p e exchanges, t h e r e is a very s t r o n g s y n e r g i s m b e t w e e n the lesions i n d u c e d b y A l u I a n d X - r a y s (SF = 2.39). N o s y n e r g i s m is o b s e r v e d between BamHIand X-ray-induced lesions (SF -- 1.12). O u r results i n d i c a t e t h a t the interact i o n b e t w e e n X - r a y - a n d R E - i n d u c e d lesions dep e n d s o n the t y p e o f dsb i n d u c e d b y the e n d o n u clease, n a m e l y ' b l u n t ' (AluI) vs. ' c o h e s i v e ' (BamHI) termini. A l s o in o u r e x p e r i m e n t s the d i s t r i b u t i o n o f c h r o m o s o m a l d a m a g e i n d u c e d b y R E is ' o v e r d i s p e r s e d ' as f o u n d b y o t h e r a u t h o r s (see B r y a n t , 1988). It is n o t e w o r t h y t h a t , while the f r e q u e n c y o f a b n o r m a l cells in b o t h c o m b i n e d t r e a t m e n t s is a d ditive o r less t h a n a d d i t i v e , the t o t a l n u m b e r o f c h r o m o s o m a l a b e r r a t i o n s is m u c h m o r e t h a n a d ditive o n l y in the c o m b i n e d t r e a t m e n t o f X - r a y s and AluI. T h e d i s t r i b u t i o n o f dicentrics after the v a r i o u s t r e a t m e n t s is s h o w n in Fig. 1. I n the case o f the c o m b i n e d t r e a t m e n t (AluI + X - r a y s ) t h e r e was a
lsochromatid breaks + SE 1 21 42 89
+ + + +
1.0 4.5 6.5 9.4
Chromatid exchanges + SE 0 1 + 1.0 1 + 1.0 2 + !.4
Chromosome exchanges + SE
Total (excl. gaps)
1 12 26 44
4 42 77 144
+ + + +
1.0 3.4 5.1 6.6
high f r e q u e n c y o f h e a v i l y d a m a g e d cells ( m o r e t h a n 3 dicentrics); this m i g h t be e x p e c t e d if R E m o l e c u l e p e n e t r a t i o n is n o t the s a m e in all cells o f the population. Considering that after treatment with R E the cells were k e p t at 0 ° C in o r d e r to prevent r e p a i r , one m i g h t a r g u e t h a t the X - r a y i n d u c e d lesions can i n t e r a c t better with A l u I i n d u c e d lesions ( ' b l u n t ' ) t h a n with B a m H I - i n d u c e d lesions ( ' c o h e s i v e ' ) . O f course it c a n n o t be excluded t h a t , as suggested b y B r y a n t (1984), the d s b with ' c o h e s i v e ' t e r m i n i i n d u c e d b y B a m H I w o u l d be
A ~
•
30-
x-rays
~,u,
~ 20-
O-
- 1
2
3
4
5
S Z
20
dJI 1
2
3
4
~
x-rays
~
Barn HI
~
x-rays ÷BarnHI
5
Number of dicentrics per cell
Fig. 1. Frequencies of cells with different number of dicentrics. (A) X-ray-, AluI- or X-ray and Alul-treated cells. (B) X-ray-, BamHI- or X-ray and BamHI-treated cells.
200
rapidly repaired with no chance of interaction with other lesions induced by the enzyme itself or with those induced by X-rays. Usually RE which induce dsb with 'cohesive' termini are less efficient in inducing chromosomal aberrations (Bryant, 1984; Natarajan and Obe, 1984). Therefore the synergism between RE-induced lesions with 'blunt' termini and X-ray-induced lesions could suggest that, as the repair of this type of dsb could be slower, there is enough time for these types of lesions to interact among themselves or with X-rayinduced lesions. Our data also indirectly confirm the suggestion advanced by Bryant et al. (1988) that if the frequency of dsbs is maintained at a high level the opportunity for misjoining is larger producing a higher frequency of chromosomal exchanges (Fig. 1). Acknowledgements This work was partially supported by Progetto Finalizzato 'Oncologia' CNR, Ministero della Ricerca and by a grant of CNR Progetto Bilaterale Italia-Germania. References Bryant, P.E. (1984) Enzymatic restriction of mammalian DNA using Pvull and BamHI: evidence for the double-strand
break origin of chromosomal aberrations, Int. J. Radiat. Biol., 46, 57-65. Bryant, P.E. (1988) Use of restriction endonucleases to study relationship between double-strand breaks, chromosomal aberrations and other end-points in mammalian cells, Int. J. Radiat. Biol., 54, 869-890. Natarajan, A.T., and G. Obe (1984) Molecular mechanisms involved in the production of chromosomal aberrations, Chromosoma, 90, 120-127. Natarajan, A.T., G. Obe, A.A. van Zeeland, F. Palitti, M. Meijers and E.A.M. Verdegaal-lmmerzeel (1980) Molecular mechanisms involved in the production of chromosomal aberrations. II. Utilization of Neurospora endonuclesae for the study of aberration production by X-ray in G1 and G2 stages of the cell cycle, Mutation Res., 69, 293-305. Obe, G., and E.U. Winkel (1985) The chromosome breaking activity of the restriction endonuclease Alul in CHO cells is independent of the S-phase of the cell cycle, Mutation Res., 152, 25-29. Obe, G., F. Palitti, C. Tanzarella, F. Degrassi and R. De Salvia (1985) Chromosomal aberrations induced by restriction endonucleases, Mutation Res., 150, 359-368. Perry, P., and S. Wolff (1974) New Giemsa method for detection of sister chromatids, Nature (London), 251, 156-158. Savage, J.R. (1975) Classification and relationship of induced chromosomal structural changes, J. Med. Genet., 13, 103-122. Tanaka, K., M. Sekiguchi and Y. Okada (1975) Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus), Proc. Natl. Acad. Sci. (U.S.A.), 72, 4071-4075. Communicated by F.H. Sobels