289
Mutation Research, 51 (1978) 289--292 9 Elsevier/North-Holland Biomedical Press
Short Communication
N I T R O S O G U A N I D I N E MUTAGENESIS D U R I N G THE YEAST CELL CYCLE B.L.A. CARTER a and I.W. DAWES b
a Department of Genetics, University of Dublin, Trinity College, Dublin 2 (Ireland) and b Department of Microbiology, University of Edinburgh, Edinburgh (Scotland) (Received 28 July 1977) (Revision received 14 March 1978) (Accepted 17 March 1978)
There is evidence that treatment with the mutagen, N-methyl-N'-nitro-Nnitrosoguanidine (NG) results in enhanced mutagenesis of genes as they replicate in bacteria [3,6,8], and in three lower eukaryotes [11,10,1,4,9]. In the present paper it is reported that in Saccharomyces cerevisiae mutation to nuclear erythromycin resistance is enhanced during nuclear DNA replication and that this enhancement is dependent on DNA replication taking place at the time of mutagenesis. If replication is inhibited 15 min prior to the application of mutagen no peak of mutation is observed in that region of the cell cycle where nuclear DNA replication ordinarily occurs. It is also shown that in cells actively replicating DNA there is no detectable enhancement of spontaneous mutation during S phase. Exponential cultures of Saccharomyces cerevisiae strain ID1 genotype a adel grown in liquid YEP Glycerol media [4] were harvested by centrifugation and resuspended in 0.2 M potassium acetate buffer (pH 5.4) containing nitrosoguanidine (NG) (500 pg/ml) for 15 min at 30~ Since all stages of the cycle are represented in an exponential culture treating such a culture with NG for a fraction of the cycle is equivalent to treating cells at different stages of the cycle for a fraction of the cell cycle. The culture was then centrifuged and washed twice to remove NG before separation according to size and stage of the mitotic cell cycle by zonal centrifugation [2,4,12]. The distribution of mutants resistant to 0.4 mg m1-1 erythromycin is shown in Fig. 1. Erythromycin resistance can result from mutation in either nuclear or mitochondrial genes [15]. Nuclear mutations are found in the first region of enhanced mutation (A) which coincides with nuclear DNA replication [4] and is expected if NG treatment leads to enhanced mutation in genes as they replicate. The second region of mutation (B) contains mutants showing mitochondrial inheritance and this region m a y correspond to the time of mitochondrial gene replication as we have suggested earlier [4]. The percentage survivors was constant throughout the cell cycle with a mean of 36.7 and a standard deviation of 4.87.
290
We have examined also the effect of NG mutagenesis at different stages of the cycle after inhibition of D N A synthesis. Hydroxyurea (0.2 M) which inhibits D N A replication in less than 5 min [13,14] was added to an exponential culture of strain ID-1 15 min prior to NG mutagenesis. After treatment with mutagen for 15 min the culture was harvested and separated according to size and stage in the cycle. Fractions corresponding to different stages of the cycle were plated and examined for mutations to erythromycin-resistance (Fig. 1). Mutagenesis occurs at all stages of the cell cycle but the peak of mutation
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F i g . 1. N G - i n d u c e d m u t a g e n e s i s t o e r y t h r o m y c i n - r e s i s t a n c e as a f u n c t i o n o f s t a g e i n t h e cell c y c l e . V i a b l e cells in each fraction were determined by counting on YEPD plates. Erythromycin resistant (ER R) mutants in each fraction were scored after 4 days by plating between 106 and 107 survivors on to each of 6 YEPG plates containing 0.4 mg erythromycin m1-1. ER R mutants after treatment with NG at different s t a g e s o f t h e cell c y c l e (m). N G - i n d u c e d m u t a g e n e s i s t o E R 1~ a s a f u n c t i o n o f s t a g e i n t h e c y c l e i n cells i n which DNA replication has been inhibited (o). Fig. 2. The distribution of spontaneous cycle. An exponential culture of ID-1 trifugation and separated into fractions t i o n . V i a b l e cells i n e a c h f r a c t i o n w e r e mycin resistant (ER R) mutants in each taining 0.4 mg erythromyein m1-1 .
erythromycin-resistant m u t a n t s as a f u n c t i o n o f s t a g e i n t h e cell g r o w n t o 1 0 7 cells m 1 - 1 i n Y E P G a t 3 0 ~ was washed by cenof different size and thus stage in the cycle by zonal centrifugadetermined by counting on YEPD plates. Spontaneous erythrofraction were scored after 5 days by plating on YEPG plates con-
291
ordinarily seen a b o u t one-third of the way through the cycle is conspicuously absent. Neither is there a discrete peak of mutagenesis at the end of the cycle as found after NG treatment of cells undergoing DNA replication. The reasons for the somewhat greater number of mutants found at earlier stages of the cycle are n o t clear. The distribution of spontaneous erythromycin-resistant mutants as a function of cell-cycle stage has been examined by growing exponential cultures, separating them according to size and therefore stage by zonal centrifugation and then plating for erythromycin-resistance. The results (Fig. 2) indicate that the overall frequency of erythromycin-resistant mutants is, as expected, much lower than NG-induced mutagenesis. In addition, the level of mutagenesis is the same at all stages of the cycle: there is no evidence for elevated mutagenesis at certain stages of the cell cycle. It is, of course, likely that spontaneous mutation at a discrete stage of the cycle would be difficult to detect above the background already present. In conclusion, we have observed that nitrosoguanidine results in enhanced mutation during periods of active DNA synthesis. No region of enhanced mutation is observed for spontaneous mutation of cells actively replicating their DNA, nor for nitrosoguanidine mutagenesis of cells in which DNA synthesis is inhibited. Thus both the mutagen, nitrosoguanidine, and cells actively replicating their DNA are necessary for elevated mutagenesis. These observations are expected if nitrosoguanidine caused mutagenesis in genes as they replicate. Acknowledgements This research was supported by NATO Grant 1148. We thank I.D. Hardie and E. Gilmartin for technical assistance. References 1 B u r k e , W., a n d W.L. F a n g m a n , T e m p o r a l o r d e r in y e a s t c h r o m o s o m a l r e p l i c a t i o n , Cell, 5 ( 1 9 7 5 ) 2 6 3 - 269. 2 C a r t e r , B . L . A . , J. S e b a s t i a n a n d H . O . H a l v o r s o n , T h e r e g u l a t i o n o f t h e s y n t h e s i s o f a r g i n i n e c a t a b olising e n z y m e s d u r i n g t h e cell c y c l e o f Saecharomyces cerevisiae, A d v . in E n z y m e R e g u l a t i o n , 9 (1971) 253--263. 3 C e r d a - O l m e d o , E., P.C. H a n a w a l t a n d N. G u e r o l a , M u t a g e n e s i s o f t h e r e p l i c a t i o n p o i n t b y n i t r o s o g u a n i d i n e : m a p a n d p a t t e r n o f r e p l i c a t i o n o f t h e Escherichia coli c h r o m o s o m e , J. Mol. Biol., 3 3 (1968) 705--719. 4 D a w e s , I.W., a n d B . L . A . C a r t e r , N i t r o s o g u a n i d i n e m u t a g e n e s i s d u r i n g n u c l e a r a n d m i t o c h o n d r i a l g e n e replication, Nature (London), 250 (1974) 709--712. 5 Giles, K . W . , a n d A. M y e r s , A n i m p r o v e d d i p h e n y l a m i n e m e t h o d f o r t h e e s t i m a t i o n o f d e o x y r i b o nucleic acid, Nature (London), 206 (1965) 93. 6 G u e r o l a , N., J . L . I n g r a h a m a n d E. C e r d a - O l m e d a , I n d u c t i o n o f c l o s e l y - l i n k e d m u l t i p l e m u t a t i o n s b y n i t r o s o g u a n i d i n e , N a t u r e ( L o n d o n ) N e w Biol., 2 3 0 ( 1 9 7 1 ) 1 2 2 - - 1 2 5 . 7 H a x t w e l l , L . H . , Saccharomyces cerevisiae cell c y c l e , B a c t e r i o l . R e v . , 3 8 ( 1 9 7 4 ) 1 6 4 - - 1 9 8 . 8 H o h l f e l d , R . , a n d W. V i e l m e t t e r , B i d i r e c t i o n a l g r o w t h o f t h e E. coli c h r o m o s o m e , N a t u r e ( L o n d o n ) N e w Biol., 2 4 2 ( 1 9 7 3 ) 1 3 0 - - 1 3 2 . 9 K e e , S . G . , a n d J. H a b e r , Cell c y c l e - d e p e n d e n t i n d u c t i o n o f m u t a t i o n s a l o n g a y e a s t c h r o m o s o m e , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 1 1 7 9 - - 1 1 8 3 . 1 0 K i m b a l l , R . F . , S t u d i e s o n t h e m u t a g e n i c a c t i o n o f N-methyl-N-nitro-N-nitrosoguanidine in Param e c i u m aurelia w i t h e m p h a s i s o n r e p a i r p r o c e s s , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 2 6 1 - - 2 7 1 . 11 L e e , R . W . , a n d R . F . J o n e s , I n d u c t i o n o f M e n d e l i a n a n d n o n - M e n d e l i a n s t r e p t o m y c i n - r e s i s t a n t m u t a n t s d u r i n g t h e s y n c h r o n o u s cell c y c l e o f C h l a m y d o m o n a s reinhardtii. Mol. G e n . G e n t . , 1 2 1 ( 1 9 7 3 ) 9 9 - 108.
292
12 S e b a s t i a n , J., B . L . A . C a r t e r a n d H . O . H a l v o r s o n , T h e use o f y e a s t p o p u l a t i o n s f r a c t i o n a t e d b y z o n a l r o t o r c e n t r i f u g a t i o n t o s t u d y t h e cell c y c l e , J. B a c t e r i o l . , 1 0 8 ( 1 9 7 1 ) 1 0 4 5 - - 1 0 5 0 . 13 Slater, M.L., Recovery of yeast from transient inhibition of DNA synthesis, Nature (London), 247 (1974) 275--276. 1 4 Slater, M . L . , E f f e c t o f r e v e r s i b l e i n h i b i t i o n o f d e o x y r i b o n u c l i e c a c i d s y n t h e s i s o n t h e y e a s t cell c y c l e , J. B a c t e r i o l . , 1 1 3 ( 1 9 7 3 ) 2 6 3 - - 2 7 0 . 1 5 T h o m a s , D . Y . , a n d D. Wilkie, I n h i b i t i o n o f m i t o c h o n d r i a l s y n t h e s i s in y e a s t b y e r y t h r o m y c i n : C y t o p l a s m i c a n d n u c l e a r f a c t o r s c o n t r o l l i n g r e s i s t a n c e , G e n e t . Res., 11 ( 1 9 6 8 ) 3 3 - - 4 1 .
Mutation Research, 51 (1978) 289--292 9 Elsevier/North-Holland Biomedical Press
Short Communication
N I T R O S O G U A N I D I N E MUTAGENESIS D U R I N G THE YEAST CELL CYCLE B.L.A. CARTER a and I.W. DAWES b
a Department of Genetics, University of Dublin, Trinity College, Dublin 2 (Ireland) and b Department of Microbiology, University of Edinburgh, Edinburgh (Scotland) (Received 28 July 1977) (Revision received 14 March 1978) (Accepted 17 March 1978)
There is evidence that treatment with the mutagen, N-methyl-N'-nitro-Nnitrosoguanidine (NG) results in enhanced mutagenesis of genes as they replicate in bacteria [3,6,8], and in three lower eukaryotes [11,10,1,4,9]. In the present paper it is reported that in Saccharomyces cerevisiae mutation to nuclear erythromycin resistance is enhanced during nuclear DNA replication and that this enhancement is dependent on DNA replication taking place at the time of mutagenesis. If replication is inhibited 15 min prior to the application of mutagen no peak of mutation is observed in that region of the cell cycle where nuclear DNA replication ordinarily occurs. It is also shown that in cells actively replicating DNA there is no detectable enhancement of spontaneous mutation during S phase. Exponential cultures of Saccharomyces cerevisiae strain ID1 genotype a adel grown in liquid YEP Glycerol media [4] were harvested by centrifugation and resuspended in 0.2 M potassium acetate buffer (pH 5.4) containing nitrosoguanidine (NG) (500 pg/ml) for 15 min at 30~ Since all stages of the cycle are represented in an exponential culture treating such a culture with NG for a fraction of the cycle is equivalent to treating cells at different stages of the cycle for a fraction of the cell cycle. The culture was then centrifuged and washed twice to remove NG before separation according to size and stage of the mitotic cell cycle by zonal centrifugation [2,4,12]. The distribution of mutants resistant to 0.4 mg m1-1 erythromycin is shown in Fig. 1. Erythromycin resistance can result from mutation in either nuclear or mitochondrial genes [15]. Nuclear mutations are found in the first region of enhanced mutation (A) which coincides with nuclear DNA replication [4] and is expected if NG treatment leads to enhanced mutation in genes as they replicate. The second region of mutation (B) contains mutants showing mitochondrial inheritance and this region m a y correspond to the time of mitochondrial gene replication as we have suggested earlier [4]. The percentage survivors was constant throughout the cell cycle with a mean of 36.7 and a standard deviation of 4.87.
290
We have examined also the effect of NG mutagenesis at different stages of the cycle after inhibition of D N A synthesis. Hydroxyurea (0.2 M) which inhibits D N A replication in less than 5 min [13,14] was added to an exponential culture of strain ID-1 15 min prior to NG mutagenesis. After treatment with mutagen for 15 min the culture was harvested and separated according to size and stage in the cycle. Fractions corresponding to different stages of the cycle were plated and examined for mutations to erythromycin-resistance (Fig. 1). Mutagenesis occurs at all stages of the cell cycle but the peak of mutation
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F i g . 1. N G - i n d u c e d m u t a g e n e s i s t o e r y t h r o m y c i n - r e s i s t a n c e as a f u n c t i o n o f s t a g e i n t h e cell c y c l e . V i a b l e cells in each fraction were determined by counting on YEPD plates. Erythromycin resistant (ER R) mutants in each fraction were scored after 4 days by plating between 106 and 107 survivors on to each of 6 YEPG plates containing 0.4 mg erythromycin m1-1. ER R mutants after treatment with NG at different s t a g e s o f t h e cell c y c l e (m). N G - i n d u c e d m u t a g e n e s i s t o E R 1~ a s a f u n c t i o n o f s t a g e i n t h e c y c l e i n cells i n which DNA replication has been inhibited (o). Fig. 2. The distribution of spontaneous cycle. An exponential culture of ID-1 trifugation and separated into fractions t i o n . V i a b l e cells i n e a c h f r a c t i o n w e r e mycin resistant (ER R) mutants in each taining 0.4 mg erythromyein m1-1 .
erythromycin-resistant m u t a n t s as a f u n c t i o n o f s t a g e i n t h e cell g r o w n t o 1 0 7 cells m 1 - 1 i n Y E P G a t 3 0 ~ was washed by cenof different size and thus stage in the cycle by zonal centrifugadetermined by counting on YEPD plates. Spontaneous erythrofraction were scored after 5 days by plating on YEPG plates con-
291
ordinarily seen a b o u t one-third of the way through the cycle is conspicuously absent. Neither is there a discrete peak of mutagenesis at the end of the cycle as found after NG treatment of cells undergoing DNA replication. The reasons for the somewhat greater number of mutants found at earlier stages of the cycle are n o t clear. The distribution of spontaneous erythromycin-resistant mutants as a function of cell-cycle stage has been examined by growing exponential cultures, separating them according to size and therefore stage by zonal centrifugation and then plating for erythromycin-resistance. The results (Fig. 2) indicate that the overall frequency of erythromycin-resistant mutants is, as expected, much lower than NG-induced mutagenesis. In addition, the level of mutagenesis is the same at all stages of the cycle: there is no evidence for elevated mutagenesis at certain stages of the cell cycle. It is, of course, likely that spontaneous mutation at a discrete stage of the cycle would be difficult to detect above the background already present. In conclusion, we have observed that nitrosoguanidine results in enhanced mutation during periods of active DNA synthesis. No region of enhanced mutation is observed for spontaneous mutation of cells actively replicating their DNA, nor for nitrosoguanidine mutagenesis of cells in which DNA synthesis is inhibited. Thus both the mutagen, nitrosoguanidine, and cells actively replicating their DNA are necessary for elevated mutagenesis. These observations are expected if nitrosoguanidine caused mutagenesis in genes as they replicate. Acknowledgements This research was supported by NATO Grant 1148. We thank I.D. Hardie and E. Gilmartin for technical assistance. References 1 B u r k e , W., a n d W.L. F a n g m a n , T e m p o r a l o r d e r in y e a s t c h r o m o s o m a l r e p l i c a t i o n , Cell, 5 ( 1 9 7 5 ) 2 6 3 - 269. 2 C a r t e r , B . L . A . , J. S e b a s t i a n a n d H . O . H a l v o r s o n , T h e r e g u l a t i o n o f t h e s y n t h e s i s o f a r g i n i n e c a t a b olising e n z y m e s d u r i n g t h e cell c y c l e o f Saecharomyces cerevisiae, A d v . in E n z y m e R e g u l a t i o n , 9 (1971) 253--263. 3 C e r d a - O l m e d o , E., P.C. H a n a w a l t a n d N. G u e r o l a , M u t a g e n e s i s o f t h e r e p l i c a t i o n p o i n t b y n i t r o s o g u a n i d i n e : m a p a n d p a t t e r n o f r e p l i c a t i o n o f t h e Escherichia coli c h r o m o s o m e , J. Mol. Biol., 3 3 (1968) 705--719. 4 D a w e s , I.W., a n d B . L . A . C a r t e r , N i t r o s o g u a n i d i n e m u t a g e n e s i s d u r i n g n u c l e a r a n d m i t o c h o n d r i a l g e n e replication, Nature (London), 250 (1974) 709--712. 5 Giles, K . W . , a n d A. M y e r s , A n i m p r o v e d d i p h e n y l a m i n e m e t h o d f o r t h e e s t i m a t i o n o f d e o x y r i b o nucleic acid, Nature (London), 206 (1965) 93. 6 G u e r o l a , N., J . L . I n g r a h a m a n d E. C e r d a - O l m e d a , I n d u c t i o n o f c l o s e l y - l i n k e d m u l t i p l e m u t a t i o n s b y n i t r o s o g u a n i d i n e , N a t u r e ( L o n d o n ) N e w Biol., 2 3 0 ( 1 9 7 1 ) 1 2 2 - - 1 2 5 . 7 H a x t w e l l , L . H . , Saccharomyces cerevisiae cell c y c l e , B a c t e r i o l . R e v . , 3 8 ( 1 9 7 4 ) 1 6 4 - - 1 9 8 . 8 H o h l f e l d , R . , a n d W. V i e l m e t t e r , B i d i r e c t i o n a l g r o w t h o f t h e E. coli c h r o m o s o m e , N a t u r e ( L o n d o n ) N e w Biol., 2 4 2 ( 1 9 7 3 ) 1 3 0 - - 1 3 2 . 9 K e e , S . G . , a n d J. H a b e r , Cell c y c l e - d e p e n d e n t i n d u c t i o n o f m u t a t i o n s a l o n g a y e a s t c h r o m o s o m e , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 1 1 7 9 - - 1 1 8 3 . 1 0 K i m b a l l , R . F . , S t u d i e s o n t h e m u t a g e n i c a c t i o n o f N-methyl-N-nitro-N-nitrosoguanidine in Param e c i u m aurelia w i t h e m p h a s i s o n r e p a i r p r o c e s s , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 2 6 1 - - 2 7 1 . 11 L e e , R . W . , a n d R . F . J o n e s , I n d u c t i o n o f M e n d e l i a n a n d n o n - M e n d e l i a n s t r e p t o m y c i n - r e s i s t a n t m u t a n t s d u r i n g t h e s y n c h r o n o u s cell c y c l e o f C h l a m y d o m o n a s reinhardtii. Mol. G e n . G e n t . , 1 2 1 ( 1 9 7 3 ) 9 9 - 108.
292
12 S e b a s t i a n , J., B . L . A . C a r t e r a n d H . O . H a l v o r s o n , T h e use o f y e a s t p o p u l a t i o n s f r a c t i o n a t e d b y z o n a l r o t o r c e n t r i f u g a t i o n t o s t u d y t h e cell c y c l e , J. B a c t e r i o l . , 1 0 8 ( 1 9 7 1 ) 1 0 4 5 - - 1 0 5 0 . 13 Slater, M.L., Recovery of yeast from transient inhibition of DNA synthesis, Nature (London), 247 (1974) 275--276. 1 4 Slater, M . L . , E f f e c t o f r e v e r s i b l e i n h i b i t i o n o f d e o x y r i b o n u c l i e c a c i d s y n t h e s i s o n t h e y e a s t cell c y c l e , J. B a c t e r i o l . , 1 1 3 ( 1 9 7 3 ) 2 6 3 - - 2 7 0 . 1 5 T h o m a s , D . Y . , a n d D. Wilkie, I n h i b i t i o n o f m i t o c h o n d r i a l s y n t h e s i s in y e a s t b y e r y t h r o m y c i n : C y t o p l a s m i c a n d n u c l e a r f a c t o r s c o n t r o l l i n g r e s i s t a n c e , G e n e t . Res., 11 ( 1 9 6 8 ) 3 3 - - 4 1 .
