Mutation Research, 3 ° (1975) 83-88 'D Elsevier Scientific Publishing Cmnpany, Amsterdam---Printed in The Netherlands
83
C Y C L O H E X I M I D E R E S I S T A N C E IN C H I N E S E HAMSTER CELLS II. INDUCTION OF CHM R E S I S T A N C E IN C H I N E S E HAMSTER CELLS BY N-NITROSOMETHYLUREA
H U B E R T POCHE, NINA B. V A R S H A V E R * , MICHAEL T H E I L L E AND ERHARD GEISSLER Acatemy of Science of the GDR, Central Institute of Molecular Biology, Cell Genetics Department, Berlin-Buch (G.D.R.) and *Kurchatov Institute of Atomic Energy, Biological Department, Moscow (U.S.S.R.) (Received December iSth, 1974 ) (Revision received March 24th, I975) (Accepted April 7th, 1975)
SUMMARY
Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7"1o-7 and 8. lO.7 M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-1o -3 and 1.6-1.8-1o -s respectively after treatment with N-nitrosomethylurea (NMU) at ioo /~g/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.
INTRODUCTION
Among mutations investigated in chemical mutagenesis experiments involving somatic mammalian cells cultivated in vitro have been those causing resistance to 8-azaguanine, 6-mercaptopurine and other base analogues~,a,5,7,s,n,l~,lL No reports have so far been published on experiments performed on animal cell lines with the aim of inducing resistance to the antibiotic CHM. Such mutants should be important for an analysis of the mechanism of action of cycloheximide which represent a useful tool for molecular and cell-biological studies with animal systems. Furthermore, mutant cells that are resistant to CHM are believed to be essential for a further characterization of the translation mechanism in the mammalian cell including the structure and function of mammalian ribosomes. In a previous communication we reported that Chinese hamster cells mutate spontaneously to resistance to CHM, the mutation rate being o.5 to 1. 3 •IO-s (ref. 13). Interesting questions are--is it possible to induce resistance to cycloheximide experimentally and how high is the induced mutation rate ? In this paper we show how resistance to CHM in Chinese hamster cells may be induced the super-mutagen N-nitrosomethylurea. Abbreviations: CHM, cycloheximide, CHO, Chinese hamster ovarian; FBS, foetal bovine serum; MEM, minimal essential medium; NMU, N-nitrosomethylurea.
H. I'i~CH]£ el al.
84 MATERIAL AND METHOI)S
Cell cultures The cells used for the investigation were ovary cells of Chinese hatnster (CHO) obtained from Dr. Bateman (Paterson Laboratories, Christie Hospital and the Holt Radium Institute, Manchester, England) with a modal chroluosonae number of 21. The cells were cultivated as described by P6CHE et al. ~'~.
Qvtotoxicitv assay 6o-mm petri dishes were inoculated with 30o cells/dish. After incubation for 24 h the cells were treated with 50, 75, IOO, 125 and 15o/~g respectively of NMU (Chemapol, Prague) for 2.5 h. The mutagen was then removed by washing twice with serum-free Eagle-MEM, where upon E a g l e - M E M + I 5 % FBS (pH 7.1 7-3) was added. After incubation for 8 days in a 5% CO~-air mixture at 37 ° without a change of medium, the colonies were fixed and stained with methylene blue or orcein. The cytotoxicity was expressed by the number of colonies in the treated dishes as a percentage of the number of colonies in the untreated dishes.
Mutagenesis assay The scheme for induction and selection of CHM-resistant clones is shown in Fig. i. NMU (ioo/~g/ml) was applied for 2.5 h and subsequently removed by two washings with serum-free Eagle-MEM. Expression took place in E a g l e - M E M + I 5 % FBS (pH 7.I-7.3). The selective medium was E a g l e - M E M + I 5 % FBS (pH 7.1-7.3) with final concentrations of 7" 1°-7 and 8- lO-7 M CHM, and was changed every 3 or 4 days. The cultures were incubated at 37 ° in a 5% COs-air mixture for 12 days. The colonies were
2£ hours
(
~ 1
~
72 hours
~
.~
) o.95, in p a r t i c u l a r , in E x p t s . i 3 P > o.999).
(02) Characterization of surviving clones Nine cell clones from each of the series grown in selective m e d i m n (7" IO 7 a n d 8. lO -7 M CHM) were isolated from different p l a t e s a n d f u r t h e r c u l t i v a t e d according to different p r o g r a m m e s 1~. These clones were passed nine t i m e s between selective m e d i u m a n d n o r m a l m e d i u m a l t e r n a t e l y . No large differences in p l a t i n g efficiency were observed (the p l a t i n g efficiency r a n g e d from 25 to 4 0 % for different clones a n d in different passages into n o r m a l a n d selective medium). F r o z e n cells which were again passed t h r o u g h a selective m e d i u m (7" lO-7 a n d 8. lO -7 M CHM) of the same concent r a t i o n s as t h a t from which t h e y were isolated d i d not differ from the control cultures in selective m e d i u m with r e g a r d to their p l a t i n g efficiency. H o w e v e r , all clones isolated in a selective m e d i u m c o n c e n t r a t i o n of 8. lO -7 M CHM grew in a selective m e d i u m c o n c e n t r a t i o n of 7" IO-7 M CHM, whereas inversely, the clones isolated at the same t i m e in selective m e d i u m with a CHM c o n c e n t r a t i o n of 7" lO-7 3 I failed to grow in selective m e d i u m with a c o n c e n t r a t i o n of 8- IO 7 M CHM. DISCUSSION
The p u r p o s e of our e x p e r i m e n t s was to find out w h e t h e r C H M - r e s i s t a n t m u t a n t s of CHO cells could also arise after t r e a t m e n t w i t h m u t a g e n s a n d to d e t e r m i n e t h e i n d u c e d m u t a t i o n rate. The n u m b e r of C H M - r e s i s t a n t clones a c t u a l l y increased after t r e a t m e n t w i t h NMU. The i n d u c e d m u t a t i o n r a t e was a b o u t two orders of m a g n i t u d e higher t h a n the s p o n t a n e o u s m u t a t i o n r a t e la. The increase of clones n o t sensitive to CHM after m u t a g e n t r e a t m e n t is one more proof t h a t resistance to CHM is controlled by mutations. The s p o n t a n e o u s a n d i n d u c e d rates of m u t a t i o n to CHM resistance are of the same order of m a g n i t u d e as t h e p u b l i s h e d r a t e of m u t a t i o n of c u l t u r e d a n i m a l cells to resistance to 6 - m e r c a p t o p u r i n e 17. Different m u t a t i o n r a t e s were o b s e r v e d when different CHM c o n c e n t r a t i o n s were used in t h e selective m e d i u m : t h e r a t e of m u t a t i o n to resistance to 7" l°-7 M CHM (1.9-5.2. IO-a) was o b v i o u s l y higher c o m p a r e d with the value at 8. i o 7 M CHM (1.6-1.8. lO-3). This m i g h t p o s s i b l y be associated with t h e o b s e r v a t i o n t h a t none of the 9 t e s t e d clones isolated at a selective m e d i u m c o n c e n t r a t i o n of 7 ' lO-7 M CHM was able to live in a selective m e d i u m with a CHM c o n c e n t r a t i o n of 8. lO _7 M, whereas all 9 clones isolated at a CHM c o n c e n t r a t i o n of 8. lO .7 M r e t a i n e d their full v i a b i l i t y in 7" lO-7 M CHM. I t is u n l i k e l y t h a t , a m o n g clones t h a t can be isolated at t h e lower c o n c e n t r a t i o n of CHM, t h e r e will be no clones r e s i s t a n t to higher concentrations. However, in the set of m u t a n t s t e s t e d b y us no clone r e s p o n d e d in such a manner.
CYCLOHEXIMIDE RESISTANCE IN CHINESE HAMSTER CELLS. II
87
The different extent of resistance to CHM observed with clones isolated at two different concentrations of CHM might be due to the fact that resistance to different CHM concentrations involves the mutation of genes with unequal potencies as has already been proved for streptomycin resistance in bacteria 4. Resistance evolving on this pattern is called facultative multi-step resistance. Moreover, in consequence of mutations at different sites of one and the same gene, different mutant phenotypes could arise possessing a unique level of residual activity of an enzyme which may be responsible for sensitivity of cells to the antimetabolic agent. Such an effect seems to appear during the selection of mammalian cells for resistance to azaguanine 5. We are currently investigating the mechanism of CHM resistance in CHO cells and have found indications that the resistance in two of the mutant clones isolated at 7' lO-7 M and 8. lO-7 M CHM might be due to modification of the ribosomes 12. Such results have already been obtained by PONGRATZAND KLINGMi)LLER14with ribosomes from CHM-resistant Neurospora mutants. It will be interesting to find out whether this also applies to other clones isolated at 8-1o -7 M CHM and whether the clones isolated at 7" IO-' M CHM are significantely different. ACKNOWLEDGEMENTS
We express our gratitude to Professor N. I. SHAPIRO from the Kurchatov Institute of Atomic Energy, Moscow, and to Dr. HELGA KRAMER of the Research Institute for Pulmonary Diseases and Tuberculosis, Berlin-Buch, for valuable discussion. REFERENCES I CHATTOO, B. B., AND U. SINHA, M u t a g e n i c a c t i v i t y of N-methyl-N'-nitro-N-nitrosoguanidine a n d N - n i t r o s o m e t h y l u r e a in Aspergillus nidulans, Mutation Res., 23 (1974) 41-49. 2 CLIVE, D., W. G. FLAMM, M. R. MACHESKO AND N. J. BERNHEIM, A m u t a t i o n a l a s s a y s y s t e m u s i n g t h e t h y m i d i n e k i n a s e locus in m o u s e l y m p h o m a cells, Mutation Res., 16 (1972) 77-87. 3 CHU, E. H. Y., AND H. V. MALLING, M a m m a l i a n cell genetics, II. Chemical i n d u c t i o n of specific locus m u t a t i o n s in Chinese h a m s t e r cells in vitro, Proc. Natl. Acad. Sci. ( U . S . A . ) , 61 (1968) 13o6 I312. 4 DEMERC, M., Origin of bacterial e x i s t e n c e to antibiotics, J. Bacteriol., 56 (1948) 63 74. 5 GILLIN, F. D., D. J. ROUFA, A. L. BEAEDET AND C. T. CASKEY, 8 - A z a g u a n i n e resistance in m a m m a l i a n cells, I. H y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e , Genetics, 72 (1972) 239--252. 6 GUMANOV, L. L., A. E. BEDNYAK AND N. P. NORENKO, M u t a g e n i c a c t i o n of N - n i t r o s o a l k y l ureas, in Supermutagens, N a u k a , Moscow, 1966, pp. 34-42 ( R u s s i a n w i t h E n g l i s h s u m m a r y ). 7 HUBERMAN, E., L. ASPIRAS, CH. HEIDELBERGER, P. E. GROVER AND P. SIMS, M u t a g e n i c i t y to m a m m a l i a n cells of epoxides a n d o t h e r d e r i v a t i v e s of polycyclic h y d r o c a r b o n s , Proe. Natl. Acad. Sci. ( U . S . A . ) , 68 (1971 ) 3195-3199. 9 LOVELESS, A. AND C. L. HAMPTON, I n a c t i v a t i o n a n d m u t a t i o n of coliphage T 2 b y N - m e t h y l a n d N - e t h y l - N - n i t r o s o u r e a , Mutation Res., 7 (1969) i 12. io MINDLIN, S. Z. AND L. G. CHURKINA, T h e c o m p a r a t i v e m u t a g e n i c a c t i v i t y of N - a l k y l - N - n i t r o s o u r e a s a n d N-methyl-N-nitro-N-nitrosoguanidine in Escherichia coli K I 2 , Genetika, No. 9 (1968) 9 6 - 9 9 ( R u s s i a n w i t h E n g l i s h s u m m a r y ) . i I ORKINS, S. H. AND J. W. LITTLEFIELD, 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 in s y n c h r o n i z e d h a m s t e r cells, Exp. Cell Res., 66 (1971) 69-74. 12 POCHE, H., I. JUNGHAHN, E. GEISSLER AND H. BIELKA, C y c l o h e x i m i d e r e s i s t a n c e in Chinese h a m s t e r cells, I II. C h a r a c t e r i z a t i o n of cell-free p r o t e i n s y n t h e s i s b y p o l y s o m e s , Mol. Gen. Genet., 138 (1975) 173-177. 13 POCHE, H., N. B. VARSHAVER AND E. GEISSLER, C y c l o h e x i m i d e r e i s s t a n c e in Chinese h a m s t e r cells, I. S p o n t a n e o u s m u t a g e n e s i s , Mutation Res., 27 (1975) 399-406. 14 PONGRATZ, i . , AND W. KLINGMfJLLER, Role of r i b o s o m e s in c y c l o h e x i m i d e resistance of Neuros p o r a m u t a n t s , l]lol. Gen. Genet., 124 (1973) 359-363 .
88
H. Pi)CHE el al.
15 RAPOPORT, i. A., Peculiarities and m e c h a n i s m of action of supernlutagens, in Supermutaget~s, Nauka, Moscow, I966 pp. 9 az (Russian with English s u m m a r y ) . I6 SHAPIRO, N. L, O. N. PETROVA AND A. E. KHALIZEV, I n d u c t i o n of gene m u t a t i o n s in mammalian cells in vitro, Preprint No. z 78.% K u r c h a t o v I n s t i t u t e of Atomic Energy, Moscow, Ict68 (Russian with English s u m m a r y ) . 17 SHAPIRO, N. I., A. E. KHALIZEV, E. g. LUss, E. S. MANUILOVA, (). N. PETROVA AND N. lg. VARSHAVER, Mutagenesis in cultured m a m m a l i a n cells, II. Induction of gene m u t a t i o n s in Chinese h a m s t e r cells, J~lutation Res., I6 (r97 z) 8 9 - I o I .
83
C Y C L O H E X I M I D E R E S I S T A N C E IN C H I N E S E HAMSTER CELLS II. INDUCTION OF CHM R E S I S T A N C E IN C H I N E S E HAMSTER CELLS BY N-NITROSOMETHYLUREA
H U B E R T POCHE, NINA B. V A R S H A V E R * , MICHAEL T H E I L L E AND ERHARD GEISSLER Acatemy of Science of the GDR, Central Institute of Molecular Biology, Cell Genetics Department, Berlin-Buch (G.D.R.) and *Kurchatov Institute of Atomic Energy, Biological Department, Moscow (U.S.S.R.) (Received December iSth, 1974 ) (Revision received March 24th, I975) (Accepted April 7th, 1975)
SUMMARY
Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7"1o-7 and 8. lO.7 M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-1o -3 and 1.6-1.8-1o -s respectively after treatment with N-nitrosomethylurea (NMU) at ioo /~g/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.
INTRODUCTION
Among mutations investigated in chemical mutagenesis experiments involving somatic mammalian cells cultivated in vitro have been those causing resistance to 8-azaguanine, 6-mercaptopurine and other base analogues~,a,5,7,s,n,l~,lL No reports have so far been published on experiments performed on animal cell lines with the aim of inducing resistance to the antibiotic CHM. Such mutants should be important for an analysis of the mechanism of action of cycloheximide which represent a useful tool for molecular and cell-biological studies with animal systems. Furthermore, mutant cells that are resistant to CHM are believed to be essential for a further characterization of the translation mechanism in the mammalian cell including the structure and function of mammalian ribosomes. In a previous communication we reported that Chinese hamster cells mutate spontaneously to resistance to CHM, the mutation rate being o.5 to 1. 3 •IO-s (ref. 13). Interesting questions are--is it possible to induce resistance to cycloheximide experimentally and how high is the induced mutation rate ? In this paper we show how resistance to CHM in Chinese hamster cells may be induced the super-mutagen N-nitrosomethylurea. Abbreviations: CHM, cycloheximide, CHO, Chinese hamster ovarian; FBS, foetal bovine serum; MEM, minimal essential medium; NMU, N-nitrosomethylurea.
H. I'i~CH]£ el al.
84 MATERIAL AND METHOI)S
Cell cultures The cells used for the investigation were ovary cells of Chinese hatnster (CHO) obtained from Dr. Bateman (Paterson Laboratories, Christie Hospital and the Holt Radium Institute, Manchester, England) with a modal chroluosonae number of 21. The cells were cultivated as described by P6CHE et al. ~'~.
Qvtotoxicitv assay 6o-mm petri dishes were inoculated with 30o cells/dish. After incubation for 24 h the cells were treated with 50, 75, IOO, 125 and 15o/~g respectively of NMU (Chemapol, Prague) for 2.5 h. The mutagen was then removed by washing twice with serum-free Eagle-MEM, where upon E a g l e - M E M + I 5 % FBS (pH 7.1 7-3) was added. After incubation for 8 days in a 5% CO~-air mixture at 37 ° without a change of medium, the colonies were fixed and stained with methylene blue or orcein. The cytotoxicity was expressed by the number of colonies in the treated dishes as a percentage of the number of colonies in the untreated dishes.
Mutagenesis assay The scheme for induction and selection of CHM-resistant clones is shown in Fig. i. NMU (ioo/~g/ml) was applied for 2.5 h and subsequently removed by two washings with serum-free Eagle-MEM. Expression took place in E a g l e - M E M + I 5 % FBS (pH 7.I-7.3). The selective medium was E a g l e - M E M + I 5 % FBS (pH 7.1-7.3) with final concentrations of 7" 1°-7 and 8- lO-7 M CHM, and was changed every 3 or 4 days. The cultures were incubated at 37 ° in a 5% COs-air mixture for 12 days. The colonies were
2£ hours
(
~ 1
~
72 hours
~
.~
) o.95, in p a r t i c u l a r , in E x p t s . i 3 P > o.999).
(02) Characterization of surviving clones Nine cell clones from each of the series grown in selective m e d i m n (7" IO 7 a n d 8. lO -7 M CHM) were isolated from different p l a t e s a n d f u r t h e r c u l t i v a t e d according to different p r o g r a m m e s 1~. These clones were passed nine t i m e s between selective m e d i u m a n d n o r m a l m e d i u m a l t e r n a t e l y . No large differences in p l a t i n g efficiency were observed (the p l a t i n g efficiency r a n g e d from 25 to 4 0 % for different clones a n d in different passages into n o r m a l a n d selective medium). F r o z e n cells which were again passed t h r o u g h a selective m e d i u m (7" lO-7 a n d 8. lO -7 M CHM) of the same concent r a t i o n s as t h a t from which t h e y were isolated d i d not differ from the control cultures in selective m e d i u m with r e g a r d to their p l a t i n g efficiency. H o w e v e r , all clones isolated in a selective m e d i u m c o n c e n t r a t i o n of 8. lO -7 M CHM grew in a selective m e d i u m c o n c e n t r a t i o n of 7" IO-7 M CHM, whereas inversely, the clones isolated at the same t i m e in selective m e d i u m with a CHM c o n c e n t r a t i o n of 7" lO-7 3 I failed to grow in selective m e d i u m with a c o n c e n t r a t i o n of 8- IO 7 M CHM. DISCUSSION
The p u r p o s e of our e x p e r i m e n t s was to find out w h e t h e r C H M - r e s i s t a n t m u t a n t s of CHO cells could also arise after t r e a t m e n t w i t h m u t a g e n s a n d to d e t e r m i n e t h e i n d u c e d m u t a t i o n rate. The n u m b e r of C H M - r e s i s t a n t clones a c t u a l l y increased after t r e a t m e n t w i t h NMU. The i n d u c e d m u t a t i o n r a t e was a b o u t two orders of m a g n i t u d e higher t h a n the s p o n t a n e o u s m u t a t i o n r a t e la. The increase of clones n o t sensitive to CHM after m u t a g e n t r e a t m e n t is one more proof t h a t resistance to CHM is controlled by mutations. The s p o n t a n e o u s a n d i n d u c e d rates of m u t a t i o n to CHM resistance are of the same order of m a g n i t u d e as t h e p u b l i s h e d r a t e of m u t a t i o n of c u l t u r e d a n i m a l cells to resistance to 6 - m e r c a p t o p u r i n e 17. Different m u t a t i o n r a t e s were o b s e r v e d when different CHM c o n c e n t r a t i o n s were used in t h e selective m e d i u m : t h e r a t e of m u t a t i o n to resistance to 7" l°-7 M CHM (1.9-5.2. IO-a) was o b v i o u s l y higher c o m p a r e d with the value at 8. i o 7 M CHM (1.6-1.8. lO-3). This m i g h t p o s s i b l y be associated with t h e o b s e r v a t i o n t h a t none of the 9 t e s t e d clones isolated at a selective m e d i u m c o n c e n t r a t i o n of 7 ' lO-7 M CHM was able to live in a selective m e d i u m with a CHM c o n c e n t r a t i o n of 8. lO _7 M, whereas all 9 clones isolated at a CHM c o n c e n t r a t i o n of 8. lO .7 M r e t a i n e d their full v i a b i l i t y in 7" lO-7 M CHM. I t is u n l i k e l y t h a t , a m o n g clones t h a t can be isolated at t h e lower c o n c e n t r a t i o n of CHM, t h e r e will be no clones r e s i s t a n t to higher concentrations. However, in the set of m u t a n t s t e s t e d b y us no clone r e s p o n d e d in such a manner.
CYCLOHEXIMIDE RESISTANCE IN CHINESE HAMSTER CELLS. II
87
The different extent of resistance to CHM observed with clones isolated at two different concentrations of CHM might be due to the fact that resistance to different CHM concentrations involves the mutation of genes with unequal potencies as has already been proved for streptomycin resistance in bacteria 4. Resistance evolving on this pattern is called facultative multi-step resistance. Moreover, in consequence of mutations at different sites of one and the same gene, different mutant phenotypes could arise possessing a unique level of residual activity of an enzyme which may be responsible for sensitivity of cells to the antimetabolic agent. Such an effect seems to appear during the selection of mammalian cells for resistance to azaguanine 5. We are currently investigating the mechanism of CHM resistance in CHO cells and have found indications that the resistance in two of the mutant clones isolated at 7' lO-7 M and 8. lO-7 M CHM might be due to modification of the ribosomes 12. Such results have already been obtained by PONGRATZAND KLINGMi)LLER14with ribosomes from CHM-resistant Neurospora mutants. It will be interesting to find out whether this also applies to other clones isolated at 8-1o -7 M CHM and whether the clones isolated at 7" IO-' M CHM are significantely different. ACKNOWLEDGEMENTS
We express our gratitude to Professor N. I. SHAPIRO from the Kurchatov Institute of Atomic Energy, Moscow, and to Dr. HELGA KRAMER of the Research Institute for Pulmonary Diseases and Tuberculosis, Berlin-Buch, for valuable discussion. REFERENCES I CHATTOO, B. B., AND U. SINHA, M u t a g e n i c a c t i v i t y of N-methyl-N'-nitro-N-nitrosoguanidine a n d N - n i t r o s o m e t h y l u r e a in Aspergillus nidulans, Mutation Res., 23 (1974) 41-49. 2 CLIVE, D., W. G. FLAMM, M. R. MACHESKO AND N. J. BERNHEIM, A m u t a t i o n a l a s s a y s y s t e m u s i n g t h e t h y m i d i n e k i n a s e locus in m o u s e l y m p h o m a cells, Mutation Res., 16 (1972) 77-87. 3 CHU, E. H. Y., AND H. V. MALLING, M a m m a l i a n cell genetics, II. Chemical i n d u c t i o n of specific locus m u t a t i o n s in Chinese h a m s t e r cells in vitro, Proc. Natl. Acad. Sci. ( U . S . A . ) , 61 (1968) 13o6 I312. 4 DEMERC, M., Origin of bacterial e x i s t e n c e to antibiotics, J. Bacteriol., 56 (1948) 63 74. 5 GILLIN, F. D., D. J. ROUFA, A. L. BEAEDET AND C. T. CASKEY, 8 - A z a g u a n i n e resistance in m a m m a l i a n cells, I. H y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e , Genetics, 72 (1972) 239--252. 6 GUMANOV, L. L., A. E. BEDNYAK AND N. P. NORENKO, M u t a g e n i c a c t i o n of N - n i t r o s o a l k y l ureas, in Supermutagens, N a u k a , Moscow, 1966, pp. 34-42 ( R u s s i a n w i t h E n g l i s h s u m m a r y ). 7 HUBERMAN, E., L. ASPIRAS, CH. HEIDELBERGER, P. E. GROVER AND P. SIMS, M u t a g e n i c i t y to m a m m a l i a n cells of epoxides a n d o t h e r d e r i v a t i v e s of polycyclic h y d r o c a r b o n s , Proe. Natl. Acad. Sci. ( U . S . A . ) , 68 (1971 ) 3195-3199. 9 LOVELESS, A. AND C. L. HAMPTON, I n a c t i v a t i o n a n d m u t a t i o n of coliphage T 2 b y N - m e t h y l a n d N - e t h y l - N - n i t r o s o u r e a , Mutation Res., 7 (1969) i 12. io MINDLIN, S. Z. AND L. G. CHURKINA, T h e c o m p a r a t i v e m u t a g e n i c a c t i v i t y of N - a l k y l - N - n i t r o s o u r e a s a n d N-methyl-N-nitro-N-nitrosoguanidine in Escherichia coli K I 2 , Genetika, No. 9 (1968) 9 6 - 9 9 ( R u s s i a n w i t h E n g l i s h s u m m a r y ) . i I ORKINS, S. H. AND J. W. LITTLEFIELD, 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 in s y n c h r o n i z e d h a m s t e r cells, Exp. Cell Res., 66 (1971) 69-74. 12 POCHE, H., I. JUNGHAHN, E. GEISSLER AND H. BIELKA, C y c l o h e x i m i d e r e s i s t a n c e in Chinese h a m s t e r cells, I II. C h a r a c t e r i z a t i o n of cell-free p r o t e i n s y n t h e s i s b y p o l y s o m e s , Mol. Gen. Genet., 138 (1975) 173-177. 13 POCHE, H., N. B. VARSHAVER AND E. GEISSLER, C y c l o h e x i m i d e r e i s s t a n c e in Chinese h a m s t e r cells, I. S p o n t a n e o u s m u t a g e n e s i s , Mutation Res., 27 (1975) 399-406. 14 PONGRATZ, i . , AND W. KLINGMfJLLER, Role of r i b o s o m e s in c y c l o h e x i m i d e resistance of Neuros p o r a m u t a n t s , l]lol. Gen. Genet., 124 (1973) 359-363 .
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15 RAPOPORT, i. A., Peculiarities and m e c h a n i s m of action of supernlutagens, in Supermutaget~s, Nauka, Moscow, I966 pp. 9 az (Russian with English s u m m a r y ) . I6 SHAPIRO, N. L, O. N. PETROVA AND A. E. KHALIZEV, I n d u c t i o n of gene m u t a t i o n s in mammalian cells in vitro, Preprint No. z 78.% K u r c h a t o v I n s t i t u t e of Atomic Energy, Moscow, Ict68 (Russian with English s u m m a r y ) . 17 SHAPIRO, N. I., A. E. KHALIZEV, E. g. LUss, E. S. MANUILOVA, (). N. PETROVA AND N. lg. VARSHAVER, Mutagenesis in cultured m a m m a l i a n cells, II. Induction of gene m u t a t i o n s in Chinese h a m s t e r cells, J~lutation Res., I6 (r97 z) 8 9 - I o I .
