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Mitotic spindle inhibitors and sister-chromatid exchange in human chromosomes
283
Mutation Research, 77 (1980) 283--286 © Elsevier/North-Holland Biomedical Press
Short Communication MITOTIC SPINDLE INHIBITORS AND SISTER-CHROMATID EXCHANGE IN HUMAN CHROMOSOMES
W I L L I A M F. M O R G A N
and P E T E R E. C R O S S E N
Cancer Society of N e w Zealand, Cytogenetics Unit, Christchurch Hospital, Christchurch
(New Zealand) (Received 31 July 1979) (Revision received 17 October 1979) (Accepted 22 October 1979)
The production of metaphase arrest in dividing cell populations by colchicine and its analogue colcemid is well documented [3] despite precise knowledge of the biochemical mechanisms involved. The antineoplastic alkaloids, vincristine and vinblastine, derived from the periwinkle plant (Vinca rosea Linn.), resemble colchicine in that they also causes metaphase arrest [4,5]. Like colchicine the Vinca alkaloids do n o t appear to inhibit the onset of mitosis b u t inhibit mitotic spindle formation to halt the dividing cell at metaphase. Since there are conflicting reports on the induction of sister-chromatid exchanges (SCEs) by these spindle poisons [2,13,16,18], we have studied the effect of vincristine ( a dimeric indole--indoline) and colcemid (demecolcine; desacetyl-methylcolchicine) on the rate of SCE in chromosomes from cultured human lymphocytes. Defibrinated gelatine sedimented blood from normal healthy persons was cultured for 72 h in Ham's F-10 medium. 5-Bromo-deoxyuridine (BrdU) was added after 24 h at a final concentration of 10 pg/ml and all cultures maintained in the dark to avoid photolysis of the BrdU substituted DNA [11]. Cultures were harvested as previously described [ 14]. Differential chromatid staining was produced by the method of Perry and Wolff [15]. Vincristine (vincristine sulfate, Lilly) was added for the final 24 h of culture at concentrations of 0.05, 0.5 and 1.0 pg/ml. When longer incubation times were investigated no differentially stained metaphases were found. Colcemid (Ciba Laboratories) was added at concentrations of 0.01 and 0.05 #g/ml 24 h after culture initiation and cultures harvested 48 h later. Higher doses of both colcemid and vincristine led to a marked reduction of the number of second division metaphases. There was no difference in the incidence of SCE between the control and treated cultures (p > 0.05) with either vincristine or colcemid (Table 1 and 2). Published reports of SCE induction by vincristine are confusing. A highly
284
TABLE 1 T H E I N C I D E N C E O F SCEs I N L Y M P H O C Y T E C H R O M O S O M E S E X P O S E D T O 0 . 0 5 , 0 . 5 0 a n d 1.00 /zg/ral V I N C R I S T I N E F O R T H E F I N A L 2 4 h O F C U L T U R E
Donor
C o n c e n t r a t i o n o f vincristine (;zg/ml) 0.00
1 2 3 4 5 6
0.05
0.50
1.00
SCE
±SD
SCE
±SD
SCE
±SD
SCE
±SD
12.05 9.40 11.80 9.50 9.30 9.55
4.75 2.23 2.95 2.88 2.83 2.84
11.50 10.85 11.75 9.20 10.60 9.05
2.26 2.35 3.06 2.61 2.75 2.87
14.00 tl.50 12.40 9.30 9.05 9.50
4.90 2.35 3.32 2.92 2.01 2.37
10.80 11.70 11.45 8.95 8.95 10.45
3.16 3.99 3.84 2.50 3.28 3.30
significant increase in SCE was reported in human lymphocytes treated for the final 19 h of culture with the same concentration (0.05 pg/ml) as used in this study [16] while treatment with 10 -7 mg/ml did not convincingly demonstrate an increase in SCE [13]. A small increase in SCE was also observed in the cloned hamster cell line A(TI)C 1--3 following exposure to vincristine at 0.05 or 0.10/~g/ml [2]. In contrast, Stoll et al. [18] found a significant decrease in the numbers of SCE in human lymphocyte chromosomes treated with vincristine concentrations of 1.25--5.0 /~g/ml. The range of SCE per cell was decreased and chromosomes with more than two exchanges were absent or very rare. In our experiments vincristine caused neither an increase nor decrease and these conflicting results are difficult to explain. There is evidence that vincristine can only enter a cell between prophase and metaphase [ 1,9] and once in a cell causes irreversible arrest of cell reproduction at metaphase [4,8,9]. Since SCE formation requires a cell to pass through a subsequent DNA synthesis (S) period following initial damage [20], it is unlikely that vincristine is capable of inducing a DNA lesion that would result in a SCE at mitosis.
TABLE 2 T H E I N C I D E N C E O F SCEs I N L Y M P H O C Y T E C H R O M O S O M E S E X P O S E D T O 0 . 0 1 a n d 0 . 0 5 ~tg/ml COLCEMID F O R 48 h
Dono~
C o n c e n t r a t i o n o f c o l c e m i d 0~g/ml) 0.00
7 8 9 10
0.01
0.05
SCE
±SD
SCE
±SD
SCE
±SD
9.50 12.90 9.60 9.35
2.87 2.60 2.91 2.50
8.15 12.65 11.70 10.00
2.91 4.64 3.70 2.31
11.30 11.45 10.75 9.60
2.54 3.72 3.13 2.61
285 Colchicine and colcemid block cell division by affecting the mitotic spindle [12]. In addition, colcemid can prevent the onset of DNA synthesis, reduce the rate of synthesis o f cells already in S phase and inhibit the progress of cells through G2 [6,10]. Despite its ability to interfere with DNA synthesis, colcemid did not increase SCE in our lymphocyte cultures. The same results have been reported by Geard and Peadock [7] and Sparvoli and Gay [17] using Vicia faba and Haplopappus gracilis respectively. On the other hand there is evidence that colchicine can affect SCE formation. Taylor [19] treated Bellevalia rornana root tips with colchicine 2 h prior to the addition of H3-Tdr necessary for autoradiographic detection of SCEs. He observed that the frequency of exchanges occurring in the first interphase (twin exchanges) and second interphases (single exchanges) was the same in seconddivision metaphases. But when the colchicine was applied only during the second interphase, the number of twin exchanges was 5 times greater than the number of single exchanges. The significant of Taylors observation [19] is at present unclear. The primary effect of both vincristine and colcemid appears to be metaphase arrest by interference with, or inhibition of, mitotic spindle formation, with little interaction with DNA. Our data indicates that neither of these mitotic spindle inhibitors increase the number of SCE in human lymphocytes. Acknowledgements This work was supported by the Medical Research Council of New Zealand, the Canterbury and Westland Division of the Cancer Society of New Zealand and the Botany Department, University of Canterbury. We thank Mr. A. Stewart for his assistance with the statistical analysis and Dr. P.H. Fitzgerald for his comments and suggestions. References 1 Armstrong, J.G., Vincristine (NSC-67574) Symposium -- summary and prospectives, Cancer Chemother. Rep., 52 (1968) 527--535. 2 B a n e r j e e , A . , a n d W . F . B e n e d i c t , P r o d u c t i o n o f sister c h r o m a t i d e x c h a n g e s b y v a r i o u s c a n c e r c h e m o t h e r a p e u t i c a g e n t s , C a n c e r Res., 3 9 ( 1 9 7 9 ) 7 9 7 - - 7 9 9 . 3 Biesele, J . J . , M i t o t i c P o i s o n s a n d t h e C a n c e r P r o b l e m , Elsevier, A m s t e r d a m , 1 9 5 S . 4 C a r d i n a l i , G., G. C a r d i n a l i a n d M . A . E n e i n , S t u d i e s o n t h e a n t i m i t o t i c a c t i v i t y o f L e u r o c r i s t i n e (Vincristine), Blood, 21 (1963) 102--110. 5 C u t t s , J . H . , T h e e f f e c t o f v i n c a l e u k o b l a s t i n e o n d i v i d i n g cells in vlvo, C a n c e r R e s . , 2 1 ( 1 9 6 1 ) 1 6 8 - 172. 6 F i t z g e r a l d , P . H . , a n d L . A r B r e h a u t , D e p r e s s i o n o f D N A s y n t h e s i s a n d m i t o t i c i n d e x b y c o l c h i c i n e in c u l t u r e d h u m a n l y m p h o c y t e s , E x p . Cell R e s , , 5 9 ( 1 9 7 0 ) 2 7 - - 3 1 . 7 Gea~d, C . R . , a n d W.J. P e a c o c k , S i s t e r c h r o m a t i d e x c h a n g e s i n Vicia faba, M u t a t i o n Res., 7 ( 1 9 6 9 ) 215--223. 8 G e o r g e , P., L . J . J o u r n e y a n d M.N. G o l d s t e i n , In v i t r o s t u d i e s o n t h e m e c h a n i s m o f a c t i o n o f vineristine sulfate, Southern Med. J., 57 (1964) 1473. 9 G e o r g e , P., L~I. J o u r n e y a n d M.N. G o l d s t e i n , E f f e c t o f v i n c r i s t i n e o n t h e f i n e s t r u c t u r e o f H e L a cells during mitosis, J. Natl. Cancer Inst., 35 (1965) 355--375. 1 0 Hell, E., a n d D . G . C o x , E f f e c t s o f c o l c h i c i n e a n d c o l c e m i d o n s y n t h e s i s o f d e o x y r i b o n u c l e i c a c i d in the s k i n o f t h e g u i n e a p i g ' s e a r in v i t r o , N a t u r e ( L o n d o n ) , 1 9 7 ( 1 9 6 3 ) 2 8 7 . 11 I k u s h i m a , T., a n d S. Wolff, Sister c h r o m a t i d e x c h a n g e s i n d u c e d b y l i g h t f l a s h e s t o 5 - b r o m o d e o x y u r i d i n e a n d 5 - I o d o d e o x y u r i d i n c s u b s t i t u t e d C h i n e s e h a m s t e r c h r o m o s o m e s , E x p . Cell R c s . , 8 7 ( 1 9 7 4 ) 15--19.
286
12 Kihlman, B.A., Action of chemicals on dividing cells, Prentice-Hail, E n g l e w o o d Cliffs, NJ, 1966, pp. 105--113. 13 Kucerova, M., and Z. Polivkova, Mutagenic effects of different mutagens on h u m a n c h r o m o s o m e s in vitro as detected b y conventional and "harlequin" methods, in: D. Scott, B.A. Bridges and F.M. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier/North Holland, Amsterdam, 1977, pp. 319-325. 1 4 M o r g a n . W . F . , a n d P.E. C r o s s e n , T h e i n c i d e n c e o f sister c h r o m a t i d e x c h a n g e s in c u l t u r e d h u m a n l y m phocytes, Mutation Res., 42 (1977) 305--312. 1 5 P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h o m a t i d s , N a t u r e (London), 251 (1974) 156--158. 16 R a p o s a , T., Sister c h r o m a t i d e x c h a n g e s t u d i e s f o r m o n i t o r i n g D N A d a m a g e a n d r e p a i r c a p a c i t y a f t e r c y t o s t a t i c s i n v i t r o a n d in l y m p h o c y t e s o f l e u k a e m i c p a t i e n t s u n d e r c y t o s t a t i c t h e r a p y , M u t a t i o n R e s . , 57 ( 1 9 7 8 ) 2 4 1 - - 2 5 1 . 1 7 S P a r v o l i , E., a n d H . G a y , D i s t r i b u t i o n o f sister c h r o m a t i d e x c h a n g e s a l o n g t h e c h r o m o s o m e s o f Haplopappus gracilis, C a r y o l o g i a , 26 ( 1 9 7 3 ) 5 2 1 - - 5 3 0 . 1 8 StoU, C., D.S. B o r g a o n k a r a n d J . M . L e v y , E f f e c t o f v i n c r i s t i n e o n sister c h r o m a t i d e x c h a n g e s o f n o r mal human lymphocytes, Cancer Res., 36 (1976) 2710--2713. 1 9 T a y l o r , J . H . , T h e r e p l i c a t i o n a n d o r g a n i s a t i o n o f D N A in c h r o m o s o m e s , in: M o l e c u l a r G e n e t i c s , P a r t 1, A c a d e m i c Press, N e w Y o r k , 1 9 6 3 . 2 0 W o l f f , S., J. B o d y c o t e a n d R . B . P a i n t e r , S i s t e r - c h r o m a t i d e x c h a n g e s i n d u c e d in C h i n e s e h a m s t e r ceils b y U V i r r a d i a t i o n o n d i f f e r e n t s t a g e s o f t h e cell c y c l e , T h e n e c e s s i t y f o r cells t o p a s s t h r o u g h S, M u t a tion Res., 25 (1974) 73--81.
Mutation Research, 77 (1980) 283--286 © Elsevier/North-Holland Biomedical Press
Short Communication MITOTIC SPINDLE INHIBITORS AND SISTER-CHROMATID EXCHANGE IN HUMAN CHROMOSOMES
W I L L I A M F. M O R G A N
and P E T E R E. C R O S S E N
Cancer Society of N e w Zealand, Cytogenetics Unit, Christchurch Hospital, Christchurch
(New Zealand) (Received 31 July 1979) (Revision received 17 October 1979) (Accepted 22 October 1979)
The production of metaphase arrest in dividing cell populations by colchicine and its analogue colcemid is well documented [3] despite precise knowledge of the biochemical mechanisms involved. The antineoplastic alkaloids, vincristine and vinblastine, derived from the periwinkle plant (Vinca rosea Linn.), resemble colchicine in that they also causes metaphase arrest [4,5]. Like colchicine the Vinca alkaloids do n o t appear to inhibit the onset of mitosis b u t inhibit mitotic spindle formation to halt the dividing cell at metaphase. Since there are conflicting reports on the induction of sister-chromatid exchanges (SCEs) by these spindle poisons [2,13,16,18], we have studied the effect of vincristine ( a dimeric indole--indoline) and colcemid (demecolcine; desacetyl-methylcolchicine) on the rate of SCE in chromosomes from cultured human lymphocytes. Defibrinated gelatine sedimented blood from normal healthy persons was cultured for 72 h in Ham's F-10 medium. 5-Bromo-deoxyuridine (BrdU) was added after 24 h at a final concentration of 10 pg/ml and all cultures maintained in the dark to avoid photolysis of the BrdU substituted DNA [11]. Cultures were harvested as previously described [ 14]. Differential chromatid staining was produced by the method of Perry and Wolff [15]. Vincristine (vincristine sulfate, Lilly) was added for the final 24 h of culture at concentrations of 0.05, 0.5 and 1.0 pg/ml. When longer incubation times were investigated no differentially stained metaphases were found. Colcemid (Ciba Laboratories) was added at concentrations of 0.01 and 0.05 #g/ml 24 h after culture initiation and cultures harvested 48 h later. Higher doses of both colcemid and vincristine led to a marked reduction of the number of second division metaphases. There was no difference in the incidence of SCE between the control and treated cultures (p > 0.05) with either vincristine or colcemid (Table 1 and 2). Published reports of SCE induction by vincristine are confusing. A highly
284
TABLE 1 T H E I N C I D E N C E O F SCEs I N L Y M P H O C Y T E C H R O M O S O M E S E X P O S E D T O 0 . 0 5 , 0 . 5 0 a n d 1.00 /zg/ral V I N C R I S T I N E F O R T H E F I N A L 2 4 h O F C U L T U R E
Donor
C o n c e n t r a t i o n o f vincristine (;zg/ml) 0.00
1 2 3 4 5 6
0.05
0.50
1.00
SCE
±SD
SCE
±SD
SCE
±SD
SCE
±SD
12.05 9.40 11.80 9.50 9.30 9.55
4.75 2.23 2.95 2.88 2.83 2.84
11.50 10.85 11.75 9.20 10.60 9.05
2.26 2.35 3.06 2.61 2.75 2.87
14.00 tl.50 12.40 9.30 9.05 9.50
4.90 2.35 3.32 2.92 2.01 2.37
10.80 11.70 11.45 8.95 8.95 10.45
3.16 3.99 3.84 2.50 3.28 3.30
significant increase in SCE was reported in human lymphocytes treated for the final 19 h of culture with the same concentration (0.05 pg/ml) as used in this study [16] while treatment with 10 -7 mg/ml did not convincingly demonstrate an increase in SCE [13]. A small increase in SCE was also observed in the cloned hamster cell line A(TI)C 1--3 following exposure to vincristine at 0.05 or 0.10/~g/ml [2]. In contrast, Stoll et al. [18] found a significant decrease in the numbers of SCE in human lymphocyte chromosomes treated with vincristine concentrations of 1.25--5.0 /~g/ml. The range of SCE per cell was decreased and chromosomes with more than two exchanges were absent or very rare. In our experiments vincristine caused neither an increase nor decrease and these conflicting results are difficult to explain. There is evidence that vincristine can only enter a cell between prophase and metaphase [ 1,9] and once in a cell causes irreversible arrest of cell reproduction at metaphase [4,8,9]. Since SCE formation requires a cell to pass through a subsequent DNA synthesis (S) period following initial damage [20], it is unlikely that vincristine is capable of inducing a DNA lesion that would result in a SCE at mitosis.
TABLE 2 T H E I N C I D E N C E O F SCEs I N L Y M P H O C Y T E C H R O M O S O M E S E X P O S E D T O 0 . 0 1 a n d 0 . 0 5 ~tg/ml COLCEMID F O R 48 h
Dono~
C o n c e n t r a t i o n o f c o l c e m i d 0~g/ml) 0.00
7 8 9 10
0.01
0.05
SCE
±SD
SCE
±SD
SCE
±SD
9.50 12.90 9.60 9.35
2.87 2.60 2.91 2.50
8.15 12.65 11.70 10.00
2.91 4.64 3.70 2.31
11.30 11.45 10.75 9.60
2.54 3.72 3.13 2.61
285 Colchicine and colcemid block cell division by affecting the mitotic spindle [12]. In addition, colcemid can prevent the onset of DNA synthesis, reduce the rate of synthesis o f cells already in S phase and inhibit the progress of cells through G2 [6,10]. Despite its ability to interfere with DNA synthesis, colcemid did not increase SCE in our lymphocyte cultures. The same results have been reported by Geard and Peadock [7] and Sparvoli and Gay [17] using Vicia faba and Haplopappus gracilis respectively. On the other hand there is evidence that colchicine can affect SCE formation. Taylor [19] treated Bellevalia rornana root tips with colchicine 2 h prior to the addition of H3-Tdr necessary for autoradiographic detection of SCEs. He observed that the frequency of exchanges occurring in the first interphase (twin exchanges) and second interphases (single exchanges) was the same in seconddivision metaphases. But when the colchicine was applied only during the second interphase, the number of twin exchanges was 5 times greater than the number of single exchanges. The significant of Taylors observation [19] is at present unclear. The primary effect of both vincristine and colcemid appears to be metaphase arrest by interference with, or inhibition of, mitotic spindle formation, with little interaction with DNA. Our data indicates that neither of these mitotic spindle inhibitors increase the number of SCE in human lymphocytes. Acknowledgements This work was supported by the Medical Research Council of New Zealand, the Canterbury and Westland Division of the Cancer Society of New Zealand and the Botany Department, University of Canterbury. We thank Mr. A. Stewart for his assistance with the statistical analysis and Dr. P.H. Fitzgerald for his comments and suggestions. References 1 Armstrong, J.G., Vincristine (NSC-67574) Symposium -- summary and prospectives, Cancer Chemother. Rep., 52 (1968) 527--535. 2 B a n e r j e e , A . , a n d W . F . B e n e d i c t , P r o d u c t i o n o f sister c h r o m a t i d e x c h a n g e s b y v a r i o u s c a n c e r c h e m o t h e r a p e u t i c a g e n t s , C a n c e r Res., 3 9 ( 1 9 7 9 ) 7 9 7 - - 7 9 9 . 3 Biesele, J . J . , M i t o t i c P o i s o n s a n d t h e C a n c e r P r o b l e m , Elsevier, A m s t e r d a m , 1 9 5 S . 4 C a r d i n a l i , G., G. C a r d i n a l i a n d M . A . E n e i n , S t u d i e s o n t h e a n t i m i t o t i c a c t i v i t y o f L e u r o c r i s t i n e (Vincristine), Blood, 21 (1963) 102--110. 5 C u t t s , J . H . , T h e e f f e c t o f v i n c a l e u k o b l a s t i n e o n d i v i d i n g cells in vlvo, C a n c e r R e s . , 2 1 ( 1 9 6 1 ) 1 6 8 - 172. 6 F i t z g e r a l d , P . H . , a n d L . A r B r e h a u t , D e p r e s s i o n o f D N A s y n t h e s i s a n d m i t o t i c i n d e x b y c o l c h i c i n e in c u l t u r e d h u m a n l y m p h o c y t e s , E x p . Cell R e s , , 5 9 ( 1 9 7 0 ) 2 7 - - 3 1 . 7 Gea~d, C . R . , a n d W.J. P e a c o c k , S i s t e r c h r o m a t i d e x c h a n g e s i n Vicia faba, M u t a t i o n Res., 7 ( 1 9 6 9 ) 215--223. 8 G e o r g e , P., L . J . J o u r n e y a n d M.N. G o l d s t e i n , In v i t r o s t u d i e s o n t h e m e c h a n i s m o f a c t i o n o f vineristine sulfate, Southern Med. J., 57 (1964) 1473. 9 G e o r g e , P., L~I. J o u r n e y a n d M.N. G o l d s t e i n , E f f e c t o f v i n c r i s t i n e o n t h e f i n e s t r u c t u r e o f H e L a cells during mitosis, J. Natl. Cancer Inst., 35 (1965) 355--375. 1 0 Hell, E., a n d D . G . C o x , E f f e c t s o f c o l c h i c i n e a n d c o l c e m i d o n s y n t h e s i s o f d e o x y r i b o n u c l e i c a c i d in the s k i n o f t h e g u i n e a p i g ' s e a r in v i t r o , N a t u r e ( L o n d o n ) , 1 9 7 ( 1 9 6 3 ) 2 8 7 . 11 I k u s h i m a , T., a n d S. Wolff, Sister c h r o m a t i d e x c h a n g e s i n d u c e d b y l i g h t f l a s h e s t o 5 - b r o m o d e o x y u r i d i n e a n d 5 - I o d o d e o x y u r i d i n c s u b s t i t u t e d C h i n e s e h a m s t e r c h r o m o s o m e s , E x p . Cell R c s . , 8 7 ( 1 9 7 4 ) 15--19.
286
12 Kihlman, B.A., Action of chemicals on dividing cells, Prentice-Hail, E n g l e w o o d Cliffs, NJ, 1966, pp. 105--113. 13 Kucerova, M., and Z. Polivkova, Mutagenic effects of different mutagens on h u m a n c h r o m o s o m e s in vitro as detected b y conventional and "harlequin" methods, in: D. Scott, B.A. Bridges and F.M. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier/North Holland, Amsterdam, 1977, pp. 319-325. 1 4 M o r g a n . W . F . , a n d P.E. C r o s s e n , T h e i n c i d e n c e o f sister c h r o m a t i d e x c h a n g e s in c u l t u r e d h u m a n l y m phocytes, Mutation Res., 42 (1977) 305--312. 1 5 P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h o m a t i d s , N a t u r e (London), 251 (1974) 156--158. 16 R a p o s a , T., Sister c h r o m a t i d e x c h a n g e s t u d i e s f o r m o n i t o r i n g D N A d a m a g e a n d r e p a i r c a p a c i t y a f t e r c y t o s t a t i c s i n v i t r o a n d in l y m p h o c y t e s o f l e u k a e m i c p a t i e n t s u n d e r c y t o s t a t i c t h e r a p y , M u t a t i o n R e s . , 57 ( 1 9 7 8 ) 2 4 1 - - 2 5 1 . 1 7 S P a r v o l i , E., a n d H . G a y , D i s t r i b u t i o n o f sister c h r o m a t i d e x c h a n g e s a l o n g t h e c h r o m o s o m e s o f Haplopappus gracilis, C a r y o l o g i a , 26 ( 1 9 7 3 ) 5 2 1 - - 5 3 0 . 1 8 StoU, C., D.S. B o r g a o n k a r a n d J . M . L e v y , E f f e c t o f v i n c r i s t i n e o n sister c h r o m a t i d e x c h a n g e s o f n o r mal human lymphocytes, Cancer Res., 36 (1976) 2710--2713. 1 9 T a y l o r , J . H . , T h e r e p l i c a t i o n a n d o r g a n i s a t i o n o f D N A in c h r o m o s o m e s , in: M o l e c u l a r G e n e t i c s , P a r t 1, A c a d e m i c Press, N e w Y o r k , 1 9 6 3 . 2 0 W o l f f , S., J. B o d y c o t e a n d R . B . P a i n t e r , S i s t e r - c h r o m a t i d e x c h a n g e s i n d u c e d in C h i n e s e h a m s t e r ceils b y U V i r r a d i a t i o n o n d i f f e r e n t s t a g e s o f t h e cell c y c l e , T h e n e c e s s i t y f o r cells t o p a s s t h r o u g h S, M u t a tion Res., 25 (1974) 73--81.