291
Mutation Research 63 (1979) 291--300
© Elsevier/North-Holland Biomedical Press
MITOCHONDRIAL MUTAGENESIS IN Saccbaromyces cerevisiae III. NITROUS ACID HANNA BARANOWSKA and ALEKSANDRA PUTRAMENT Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Rakowiecka 36, 02-532 Warszawa (Poland)
(Received 18 April 1979) (Revision received 20 July 1979) (Accepted 6 August 1979)
Summary Nitrous acid (NA) induced mutations efficiently in mitDNA, conferring resistance to erythromycin and weakly induces m i t - mutations. In some strains of yeast it also enhanced rho- mutations. The frequencies of nuclear and mitochondrial mutations induced with NA are compared.
We have worked o u t a mutational system in yeast that can be used to provide a comparison of the responses of nuclear and mitochondrial DNA towards mutagens. To begin with, we intended to see whether different types of wellknown mutagens can induce mutations in mitDNA [1]. It has been established so far that UV, MMS and DEB can induce mutations not only in nuclear DNA, but also in mitDNA, although with a lower efficiency [4,12]. In contrast with UV, neither MMS nor DEB induce rho- mutations. For our present purposes, NA is particularly useful, because it can act mutagenically in any of the following ways [for recent reviews see: 6,9,10,17]. (1) By direct action on DNA, deamination of bases, formation of interstrand cross-links and DNA--protein links. Mutations can be induced either (a) through changed pairing properties of the deaminated bases which remained in the DNA at the time of its replication, or (b) through mutagenic repair of the damage. (2) By indirect action. At low pH, NA can react with amines and amides. Abbreviatmns
cap r, cap s, alleles c o n f e r r i n g r e m s t a n c e o r s e n s l h w t y to c h l o r a m p h e n i c o l ; DEB, dlep o x y b u t a n e : e r y r, ery s, alleles conferring remstance o f sensitivity t o e r y t h r o m y c i n ; rnzt-, m i t o c h o n d n a l m u t a n t s , w h l c h d o n o t respire, but c a r r y o n f u n c t i o n a l m i t o c h o n d r i a l p r o t e i n s y n t h e s l s , m i t D N A , m i t o c h o n d r i a l D N A ; MMS, m e t h y l m e t h a n e s u l p h o n a t e ; N A , nitrous acld; rho O, m u t a n t s that h a v e lost all m i t D N A ; rho +, a p o p u l a t i o n o f s t a n d a r d m i t D N A m o l e c u l e s w i t h i n a cell; rho-, r e s p i r a t o r y - d e f i c i e n t m u t a n t s w i t h all m i t D N A m o l e c u l e s l e t h a l l y d a m a g e d w i t h i n t h e cell.
292 The nitrosamines thereby formed can act mutagenically either directly or after metabolic activation. There is abundant evidence showing that, in Saccharomyces cerevisiae, NA induces efficiently not only mutations, b u t also intra- and inter-genic recombination. It is also known that many rad mutants show an increased sensitivity to NA [15]. This indicates that, in yeast, NA-produced damage is repaired. This process can be mutagenic, but direct mutagenesis via unexcised deaminated bases also seems possible. So far there is no evidence for the indirect mutagenic action of NA on yeast, but such an action cannot be excluded. Thus, if NA proves to be mutagenic towards yeast mitDNA, one can further analyse the possible pathways of its mutagenicity, the possibility of formation of mutagenic nitrosamines being of particular interest. The results presented here show that NA induces mutations in mitDNA, but its mutagenic action is stronger on nuclear DNA. The induction of rho- mutations is observed only in some strains. Material and methods
Yeast strains. The strains of Saccharomyces cerevisiae used are described in Table 1. Strain AB1-4C is isogenic with 777-3A, and obtained from a revertant of o p l mutation. Media. YEPglu contained 1.0% yeast extract, 1% peptone and 2 or 10% glucose. The YEPglul0 medium was used to create glucose repression when induction of mit- mutants was studied. YEPglyc was like YEPglu, b u t with 2% glycerol instead of glucose. YEPdif medium was YEPglyc with 0.5% glucose. Minimal Difco Base Medium without amino acids, but with 2 or 5% glucose, was solidified with 2% agar.
TABLE 1 LIST OF STRAINS USED Strain
Nuclear genotype
Mitochondnal genotype
O n g m of strata
197/2d 777-3A AB1-4C
a l p h a , ade2-1 a, ade l , op l a l p h a , ade l, m e t
rho+capSery s rho+capServ s rho+capSery s
B.S. Cox P.P. S l o m m s k l A. K r u s z e w s k a
IL126-2A S/M-13D DT15
alpha, ural a l p h a , leu l , ade l a, leu4, hzs
rho+caprery r rho+capSery s rho 0
P.P. S l o m m s l o J. L l t w m s k a G. F m k
BZ34
arg4-4/arg4-17, t r p S - 4 8 / t r p 5 - 4 8 , hisS-2~ h~sS-2, l y s l - 1 / l y s l - 1 , a d e 2 - 1 / a d e 2 - 1 , + / p l , thr4/+, + / l e u l - 1 2 , +~met1, ura3/+
rho+capSery s
S. F o g e l
SBTD
hom2-1/hom2-2, tyr4/4, leul/+, ade5/+, a d e l / + , +lade2
rho+capSeryS
H Baranowska
IL166-6C IL166-6C ILlS7-4C
a, ural a, ural a, his1
rho+capSeryS rho 0 rho+capreryS
P.P. S l o n u n s l u P.P. S l o m m s k l P.P. S l o n i m s l u
293 Mutagenesis. The cells to be treated with NA were pre-grown in liquid YEPglyc medium or in YEPglul0 medium (strain o p l ) . When still in the exponential phase (1--5 X 107 cells/ml), they were collected by centrifugation and suspended in 0.1 M acetate buffer (pH 4). To this suspension a freshly prepared solution of NaNO2 was added to give a final concentration of 0.02 M. The treatment was terminated by adding to the cell suspension an equal volume of 0.1 M phosphate buffer (pH 7.5). Immediately afterwards the cells were harvested by centrifugation, resuspended in liquid YEPdif medium and incubated for 1 h at 30°C. Then the YEPdif medium was removed, the cells were suspended in Ringer solution and plated. For selection of drug-resistant mutants the cells were plated on YEPglyc medium (pH 6.2) containing 5 mg erythromycin or 2 mg chloramphenicol per ml. For selection of ade ÷ and met" revertants, suitably supplemented minimal medium was used. The cells of strain 777-3A were plated on YEPglul0 medium. Suitably diluted samples were plated on YEPdif medium to estimate cell survival and the percentage of respiratory
Mutation Research 63 (1979) 291--300
© Elsevier/North-Holland Biomedical Press
MITOCHONDRIAL MUTAGENESIS IN Saccbaromyces cerevisiae III. NITROUS ACID HANNA BARANOWSKA and ALEKSANDRA PUTRAMENT Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Rakowiecka 36, 02-532 Warszawa (Poland)
(Received 18 April 1979) (Revision received 20 July 1979) (Accepted 6 August 1979)
Summary Nitrous acid (NA) induced mutations efficiently in mitDNA, conferring resistance to erythromycin and weakly induces m i t - mutations. In some strains of yeast it also enhanced rho- mutations. The frequencies of nuclear and mitochondrial mutations induced with NA are compared.
We have worked o u t a mutational system in yeast that can be used to provide a comparison of the responses of nuclear and mitochondrial DNA towards mutagens. To begin with, we intended to see whether different types of wellknown mutagens can induce mutations in mitDNA [1]. It has been established so far that UV, MMS and DEB can induce mutations not only in nuclear DNA, but also in mitDNA, although with a lower efficiency [4,12]. In contrast with UV, neither MMS nor DEB induce rho- mutations. For our present purposes, NA is particularly useful, because it can act mutagenically in any of the following ways [for recent reviews see: 6,9,10,17]. (1) By direct action on DNA, deamination of bases, formation of interstrand cross-links and DNA--protein links. Mutations can be induced either (a) through changed pairing properties of the deaminated bases which remained in the DNA at the time of its replication, or (b) through mutagenic repair of the damage. (2) By indirect action. At low pH, NA can react with amines and amides. Abbreviatmns
cap r, cap s, alleles c o n f e r r i n g r e m s t a n c e o r s e n s l h w t y to c h l o r a m p h e n i c o l ; DEB, dlep o x y b u t a n e : e r y r, ery s, alleles conferring remstance o f sensitivity t o e r y t h r o m y c i n ; rnzt-, m i t o c h o n d n a l m u t a n t s , w h l c h d o n o t respire, but c a r r y o n f u n c t i o n a l m i t o c h o n d r i a l p r o t e i n s y n t h e s l s , m i t D N A , m i t o c h o n d r i a l D N A ; MMS, m e t h y l m e t h a n e s u l p h o n a t e ; N A , nitrous acld; rho O, m u t a n t s that h a v e lost all m i t D N A ; rho +, a p o p u l a t i o n o f s t a n d a r d m i t D N A m o l e c u l e s w i t h i n a cell; rho-, r e s p i r a t o r y - d e f i c i e n t m u t a n t s w i t h all m i t D N A m o l e c u l e s l e t h a l l y d a m a g e d w i t h i n t h e cell.
292 The nitrosamines thereby formed can act mutagenically either directly or after metabolic activation. There is abundant evidence showing that, in Saccharomyces cerevisiae, NA induces efficiently not only mutations, b u t also intra- and inter-genic recombination. It is also known that many rad mutants show an increased sensitivity to NA [15]. This indicates that, in yeast, NA-produced damage is repaired. This process can be mutagenic, but direct mutagenesis via unexcised deaminated bases also seems possible. So far there is no evidence for the indirect mutagenic action of NA on yeast, but such an action cannot be excluded. Thus, if NA proves to be mutagenic towards yeast mitDNA, one can further analyse the possible pathways of its mutagenicity, the possibility of formation of mutagenic nitrosamines being of particular interest. The results presented here show that NA induces mutations in mitDNA, but its mutagenic action is stronger on nuclear DNA. The induction of rho- mutations is observed only in some strains. Material and methods
Yeast strains. The strains of Saccharomyces cerevisiae used are described in Table 1. Strain AB1-4C is isogenic with 777-3A, and obtained from a revertant of o p l mutation. Media. YEPglu contained 1.0% yeast extract, 1% peptone and 2 or 10% glucose. The YEPglul0 medium was used to create glucose repression when induction of mit- mutants was studied. YEPglyc was like YEPglu, b u t with 2% glycerol instead of glucose. YEPdif medium was YEPglyc with 0.5% glucose. Minimal Difco Base Medium without amino acids, but with 2 or 5% glucose, was solidified with 2% agar.
TABLE 1 LIST OF STRAINS USED Strain
Nuclear genotype
Mitochondnal genotype
O n g m of strata
197/2d 777-3A AB1-4C
a l p h a , ade2-1 a, ade l , op l a l p h a , ade l, m e t
rho+capSery s rho+capServ s rho+capSery s
B.S. Cox P.P. S l o m m s k l A. K r u s z e w s k a
IL126-2A S/M-13D DT15
alpha, ural a l p h a , leu l , ade l a, leu4, hzs
rho+caprery r rho+capSery s rho 0
P.P. S l o m m s l o J. L l t w m s k a G. F m k
BZ34
arg4-4/arg4-17, t r p S - 4 8 / t r p 5 - 4 8 , hisS-2~ h~sS-2, l y s l - 1 / l y s l - 1 , a d e 2 - 1 / a d e 2 - 1 , + / p l , thr4/+, + / l e u l - 1 2 , +~met1, ura3/+
rho+capSery s
S. F o g e l
SBTD
hom2-1/hom2-2, tyr4/4, leul/+, ade5/+, a d e l / + , +lade2
rho+capSeryS
H Baranowska
IL166-6C IL166-6C ILlS7-4C
a, ural a, ural a, his1
rho+capSeryS rho 0 rho+capreryS
P.P. S l o n u n s l u P.P. S l o m m s k l P.P. S l o n i m s l u
293 Mutagenesis. The cells to be treated with NA were pre-grown in liquid YEPglyc medium or in YEPglul0 medium (strain o p l ) . When still in the exponential phase (1--5 X 107 cells/ml), they were collected by centrifugation and suspended in 0.1 M acetate buffer (pH 4). To this suspension a freshly prepared solution of NaNO2 was added to give a final concentration of 0.02 M. The treatment was terminated by adding to the cell suspension an equal volume of 0.1 M phosphate buffer (pH 7.5). Immediately afterwards the cells were harvested by centrifugation, resuspended in liquid YEPdif medium and incubated for 1 h at 30°C. Then the YEPdif medium was removed, the cells were suspended in Ringer solution and plated. For selection of drug-resistant mutants the cells were plated on YEPglyc medium (pH 6.2) containing 5 mg erythromycin or 2 mg chloramphenicol per ml. For selection of ade ÷ and met" revertants, suitably supplemented minimal medium was used. The cells of strain 777-3A were plated on YEPglul0 medium. Suitably diluted samples were plated on YEPdif medium to estimate cell survival and the percentage of respiratory
