JOURNAL
OF
BACTERIOLOGY, Sept. 1991, p. 5918-5920
Vol. 173, No. 18
0021-9193/91/185918-03$02.00/0 Copyright C) 1991, American Society for Microbiology
Null Mutants of Saccharomyces cerevisiae Cu,Zn Superoxide Dismutase: Characterization and Spontaneous Mutation Rates EDITH BUTLER GRALLA* AND JOAN SELVERSTONE VALENTINE
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90024-1569 Received 29 April 1991/Accepted 3 July 1991
Deletion-replacement mutations of the Saccharomyces cerevisiae Cu,Zn superoxide dismutase gene were constructed. They were exquisitely sensitive to redox cycling drugs and showed slight sensitivity to other agents. The aerobic spontaneous mutation rate was three- to fourfold higher in sodlAl mutants, while the anaerobic rate was similar to that of the wild type.
The proposals that superoxide might be a major agent of dioxygen toxicity and that the primary function of superoxide dismutases (SODs) is to protect against superoxideinduced damage (7-9, 12) have been difficult to confirm, because the targets of such damage have not been characterized (6, 16). However, SOD-deficient mutants of both Escherichia coli and Saccharomyces cerevisiae are highly sensitive to dioxygen (2, 3), suggesting strongly that SODs play an important role in enabling the cells to grow aerobically. In Drosophila melanogaster, Cu,Zn SOD may protect against ionizing radiation (13) and increase longevity (14). To investigate the possibility that DNA is a primary target for superoxide-induced damage that is inhibitable by Cu,Zn SOD, we constructed and characterized Cu,Zn SOD-deficient mutations in the yeast S. cerevisiae. (There is some disagreement in the literature about the nomenclature of SOD genes in S. cerevisiae. We use SOD] for Cu,Zn SOD for consistency with the convention used for humans and other eucaryotes [18].) We report here our results with respect to mutation rates in sodi (Cu,Zn SOD-) null mutants and our conclusions concerning the role of SOD in preventing DNA damage. Deletion-replacement mutations. Deletion-replacement mutations in the Cu,Zn SOD gene were made by the onestep gene disruption procedure (15). The 3.7-kb EcoRI-PstI fragment of pUC-SOD4.3 (1) was recloned in the same vector to yield pUC-SOD3.7. A 0.7-kb AsuII fragment containing the C-terminal 75% of the Cu,Zn SOD-coding region was removed and replaced by a HindIll fragment containing the yeast URA3 gene, following end filling, to yield pUC-SODAA::URA3. Standard methods were used (11, 17). S. cerevisiae DBY746 (MAToa leu2-3,112 his3AJ trpl-289a ura3-52) was transformed (10) with HindIll-digested pUCSODAA::URA3 DNA, and URA+ colonies were isolated on plates containing synthetic complete medium (SC) minus uracil (17). To screen for sod] mutants, transformants were tested for oxygen and paraquat sensitivity. Of 18 URA+ isolates tested, 5 were paraquat and oxygen sensitive. Southern blot analyses (data not shown) revealed that the other colonies retained the wild-type SOD] gene. The mutation is called sodJAl:: URA3, sodJAl for short. Extracts from strains carrying this mutation showed no Cu,Zn SOD activity or immunoreactive material (data not shown). The sod] *
Corresponding author. 5918
point mutants described previously (2) were selected on the basis of paraquat sensitivity. Our deletion-replacement mutants showed the same greatly elevated sensitivity without previous selection, indicating that paraquat sensitivity is a direct consequence of the sodl mutation. EG1 (MATa leu23,112 his3AJ trpl-289 ura3-52 sodlAl::URA3) and EG3 (MATa leu2-3,112 his3AJ trpl-289 ura3-52 ade2::hisG gal2 sodlAl:: URA3) were isolated after two backcrosses to S1AA (MATa leu2-3,112 his3AJ trpl -289 ura3-52 ade2: :hisG), a related wild-type strain. As the work progressed, it became clear that drug resistance phenotypes varied between the wild-type parent strains. Therefore, other sodlAl strains, EG-AC1, EG-AE3, and EG-301, were produced subsequently in S1AA, DBY746, and DBY747 (MATa leu2-3,112 his3AJ trpl-289 ura3-52 Canr gal2), respectively, and were not backcrossed. To help prevent second-site pseudoreversion events, these strains were maintained microaerobically (CampyPak; BBL). Sensitivity to oxidative conditions. The sodlAl mutants were viable in aerobic incubation but grew more slowly than wild-type cells with glucose as the carbon source (Table 1). The difference was not as pronounced with 3% glycerol as the carbon source, perhaps because the sodlAl strains retain the functional gene for mitochondrial Mn SOD, which is expressed during growth on glycerol but not during growth on glucose (20). The sodlAl strains were extremely sensitive to paraquat and menadione, as measured by disk assays or killing by challenge in liquid culture (data not shown). Gradient plates (Table 2) were formed in square petri dishes by pouring two layers of YPD (17) agar-the first a slanted one containing the agent at the indicated concentration and the second without the agent filling in the slope. Yeast cultures were diluted 1:100 in 0.5% agar, and even lines of cells were inoculated along the gradient by using a bent glass rod. The length of the growth area was measured after 3 days. The lowest level of paraquat tested (10 puM in the bottom layer) was sufficient to prevent all growth of sodJAl cells, while SOD] (wild-type) strains were resistant to nearly 1,000 ,uM (>90% growth on a 1,000 ,uM plate). This represents a more-than-100-fold difference in sensitivity. The extreme sensitivity to redox cyclers was observed only in the presence of dioxygen (Table 2). sodlAl strains also exhibited dioxygen-dependent lysine auxotrophy and inability to survive in 100% dioxygen (Table 2) that was observed with the previously isolated point mutants (2, 4). Sensitivity to other agents. Aerobic growth slightly but
VOL. 173, 1991
NOTES
TABLE 3. Spontaneous mutation ratesa
TABLE 1. Aerobic doubling timea Generation time (h) with the following genotype at the SOD) locus:
Carbon source (concn)
Wild type
sodlAJ
2.0 1.6 3.5
2.8 2.7 3.8
Glucose (2%) Glucose (10%o) Glycerol (3%)
a Cells were grown in SC medium (17). Doubling times are averages of at least two experiments with each of at least two cell lines.
consistently increased the sensitivity of the already stressed sodlAl strains to certain other DNA-damaging agents, such as methyl methanesulfonate and tert-butyl hydroperoxide (Table 2). However, the seemingly unrelated agent canavanine (an arginine analog) gave a similar result (data not shown), leading us to believe that this is a "straw that broke the camel's back" effect rather than a specific sensitivity. These differences were not evident in anaerobic conditions. The increase in anaerobic tert-butyl hydroperoxide resistance in the sodlAl strain was not reproducibly observed in all genetic backgrounds. Spontaneous mutation rates. Spontaneous mutation rates were measured in sod] and parent strains by using the fluctuation test method described by Von Borstel (19). The strains used in this work contained the trpl-289 allele, a revertible amber mutation of the TRPI gene. Individual cultures were grown in 24-well tissue culture dishes in SC medium (17) limited in tryptophan (1.25 ,uM) and increased fourfold in other required nutrients (Ade, Leu, His, Ura, Met, and Lys) to prevent growth limitation by their depletion. Growth proceeded until the Trp supply was exhausted, after which only cells that had reverted to TRP+ grew and formed large colonies over a thin background. The concentration of Trp, and thus the final nonrevertant cell density, was adjusted so that revertants arose in about half of the wells. The reversion rate was calculated from the zeroth term of the Poisson distribution. For experiments in which TABLE 2. Phenotypic characteristics' Relative growth (%) Aerobic Anaerobic
Agent (concn) or condition
Paraquat (10 ,uM) Paraquat (1,000 ,uM) Menadione (20 ,uM) tert-Butyl hydroperoxide (2.5 mM) 5. Methyl methanesulfonate (0.04%)
1. 2. 3. 4.
6. Growth without lysine 7. Growth in 100TO 02
5919
Condition
(temp [°C]) Aerobic (30) Anaerobic (30) Aerobic (24)
No. of mutations/cell/generation, 10-8 (SD) Fold increase Wild type sodlAl
3.4 (1.3) 1.8 (0.4) 1.6 (0.5)
13.2 (4.6)
3.9
2.1 (0.7) 5.3 (1.4)
1.3 3.2
a The reversion rate was calculated from the zeroth term of the Poisson distribution according to the formula M = (-ln NJN)/2C, where M is the mutation rate, No is the number of wells with no colonies, N is the total number of wells, and C is the number of cells in wells without revertants (determined by direct counts with a hemacytometer). Points are averages of four to six separate experiments and include at least three different wild-typesodlAl pairs.
anaerobic conditions were used, 0.1% Tween 80 and 30 mg of ergosterol per liter were added to the medium. Supplementation did not affect aerobic mutation rates. The results are shown in Table 3. The sodlAl strains had mutation rates during aerobic growth about threefold greater than that determined for wild-type cells. Under anaerobic conditions, the rates were similar for sodlAl and wild-type strains. Essentially the same results were obtained with sodlIAl and SODJ + strains in the various backgrounds tested. Aerobic mutation rates differed with different growth temperatures, but the ratio between sodlAl and SOD]+ mutation rates
remained similar. E. coli strains lacking both Mn SOD and Fe SOD have dramatic increases in aerobic mutation rates. Those lacking only Mn SOD have more modest increases, while those lacking only Fe SOD show no change (5). Work with D. melanogaster has implied a possible connection between SOD and ionizing radiation damage (13). However, ours is the first demonstration of the role of Cu,Zn SOD in a eucaryote as a protector against mutational change. The increase measured here is modest but nonetheless significant. While recognizing that any increase in the accumulation of mutations in a species is significant for the long-term survival of the species, we believe that the effect of sodlAl on mutation rates is unlikely to account for the other phenotypes observed in these mutants. In particular, the acute toxicity of redox cycling drugs and the reduced growth rates are probably best accounted for by other mechanisms. The effect might be more dramatic in strains lacking mitochondrial Mn SOD as well as Cu,Zn SOD. Work is under way in our laboratory to isolate and characterize such mutants. Nevertheless, the strong phenotype of the sodlAl mutant, even in the presence of Mn SOD, argues for separate roles for these two proteins, and in particular for the importance of cytoplasmic Cu,Zn SOD.
Wild type
sod],AJ
Wild type
sodlAJ
100 86 100 54
0 0 0 46
100 82 100 39
100 79 100 46
97
69
86
86
Our thanks to Bruce Demple, Harvard University School of Public Health, for laboratory space where part of this work was performed and for many helpful discussions. Thanks to Sonya Popoff for helpful discussions and for introducing gradient plates to
+ +
-
+
+
us.
aResults are reported for the DBY747 wild-type strain and its isogenic
This work was supported by USPHS grant GM28222 to J.S.V. E.B.G. received partial support from the California Institute for Cancer Research.
similar. Lines 1 to 5 summarize gradient plate results, reported as percentages of the inoculated line showing growth. The concentrations are those that were initially present in the bottom, slanted layer; the maximum concentration was slightly under this because of diffusion through the upper layer. For line 6, cells were streaked on plates containing SC and SC minus lysine, supplemented with Tween 80 and ergosterol and grown aerobically or anaerobically. For line 7, cells were streaked on YPD plates and grown in desiccators filled with air or oxygen.
REFERENCES 1. Bermingham-McDonogh, O., E. B. GralHa, and J. S. Valentine. 1988. The copper, zinc superoxide dismutase gene of S. cerevisiae: cloning, sequencing, and biological activity. Proc. Natl. Acad. Sci. USA 85:4789-4793. 2. BiliUski, T., Z. Krawiec, A. Liczmanski, and J. Litwinska. 1985. Is hydroxyl radical generated by the Fenton reaction in vivo?
sodJAl derivative EG301. Results for the other wild-type-sodlAl pairs were
5920
NOTES
Biochem. Biophys. Res. Commun. 130:533-539. 3. Carlioz, A., and D. Touati. 1986. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 5:623-630. 4. Chang, E. C., and D. J. Kosman. 1990. 02-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae. J. Bacteriol. 172:1840-1845. 5. Farr, S. B., R. D'Ari, and D. Touati. 1986. Oxygen-dependent mutagenesis in E. coli lacking superoxide dismutase. Proc. Natl. Acad. Sci. USA 83:8268-8272. 6. Fee, J. A. 1982. Is superoxide important in oxygen poisoning? Trends Biochem. Sci. 7:84-86. 7. Fridovich, I. 1978. The biology of oxygen radicals. Science 201:875-880. 8. Fridovich, I. 1986. Superoxide dismutases. Adv. Enzymol. 58:61-97. 9. Halliwell, B., and J. M. C. Gutteridge. 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219:1-14. 10. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. The transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. 11. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12. McCord, J. M., B. B. Keele, Jr., and I. Fridovich. 1971. An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Natl. Acad. Sci. USA
J. BACTERIOL.
68:1024-1027. 13. Peng, T. X., A. Moya, and F. J. Ayala. 1986. Irradiation resistance conferred by superoxide dismutase: possible adaptive role of a natural polymorphism in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 83:684-687. 14. Phillips, J. P., S. D. Campbell, D. Michaud, M. Charbonneau, and A. J. Hilliker. 1989. Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. Proc. Natl. Acad. Sci. USA 86: 2761-2765. 15. Rothstein, R. 1983. One step gene disruption. Methods Enzymol. 101:202-211. 16. Sawyer, D. T., and J. S. Valentine. 1981. How super is superoxide? Accounts Chem. Res. 14:393-400. 17. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast genetics-laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Sinet, P. M., J. Couturier, B. Dutrillaux, M. Poissonnier, 0. Raoul, M.-O. Rethore, D. Allard, J. Lejeune, and H. Jerome. 1976. Trisomie 21 et superoxyde dismutase-1 (IPO-A), tentative de localisation sur la sous bande 21q22.1. Exp. Cell. Res. 97:47-55. 19. Von Borstel, R. C. 1978. Measuring spontaneous mutation rates in yeast. Methods Cell Biol. 20:1-24. 20. Westerbeek-Marres, C. A. M., M. M. Moore, and A. P. Autor. 1988. Regulation of manganese superoxide dismutase in Saccharomyces cerevisiae: the role of respiratory chain activity. Eur. J. Biochem. 174:611-620.
OF
BACTERIOLOGY, Sept. 1991, p. 5918-5920
Vol. 173, No. 18
0021-9193/91/185918-03$02.00/0 Copyright C) 1991, American Society for Microbiology
Null Mutants of Saccharomyces cerevisiae Cu,Zn Superoxide Dismutase: Characterization and Spontaneous Mutation Rates EDITH BUTLER GRALLA* AND JOAN SELVERSTONE VALENTINE
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90024-1569 Received 29 April 1991/Accepted 3 July 1991
Deletion-replacement mutations of the Saccharomyces cerevisiae Cu,Zn superoxide dismutase gene were constructed. They were exquisitely sensitive to redox cycling drugs and showed slight sensitivity to other agents. The aerobic spontaneous mutation rate was three- to fourfold higher in sodlAl mutants, while the anaerobic rate was similar to that of the wild type.
The proposals that superoxide might be a major agent of dioxygen toxicity and that the primary function of superoxide dismutases (SODs) is to protect against superoxideinduced damage (7-9, 12) have been difficult to confirm, because the targets of such damage have not been characterized (6, 16). However, SOD-deficient mutants of both Escherichia coli and Saccharomyces cerevisiae are highly sensitive to dioxygen (2, 3), suggesting strongly that SODs play an important role in enabling the cells to grow aerobically. In Drosophila melanogaster, Cu,Zn SOD may protect against ionizing radiation (13) and increase longevity (14). To investigate the possibility that DNA is a primary target for superoxide-induced damage that is inhibitable by Cu,Zn SOD, we constructed and characterized Cu,Zn SOD-deficient mutations in the yeast S. cerevisiae. (There is some disagreement in the literature about the nomenclature of SOD genes in S. cerevisiae. We use SOD] for Cu,Zn SOD for consistency with the convention used for humans and other eucaryotes [18].) We report here our results with respect to mutation rates in sodi (Cu,Zn SOD-) null mutants and our conclusions concerning the role of SOD in preventing DNA damage. Deletion-replacement mutations. Deletion-replacement mutations in the Cu,Zn SOD gene were made by the onestep gene disruption procedure (15). The 3.7-kb EcoRI-PstI fragment of pUC-SOD4.3 (1) was recloned in the same vector to yield pUC-SOD3.7. A 0.7-kb AsuII fragment containing the C-terminal 75% of the Cu,Zn SOD-coding region was removed and replaced by a HindIll fragment containing the yeast URA3 gene, following end filling, to yield pUC-SODAA::URA3. Standard methods were used (11, 17). S. cerevisiae DBY746 (MAToa leu2-3,112 his3AJ trpl-289a ura3-52) was transformed (10) with HindIll-digested pUCSODAA::URA3 DNA, and URA+ colonies were isolated on plates containing synthetic complete medium (SC) minus uracil (17). To screen for sod] mutants, transformants were tested for oxygen and paraquat sensitivity. Of 18 URA+ isolates tested, 5 were paraquat and oxygen sensitive. Southern blot analyses (data not shown) revealed that the other colonies retained the wild-type SOD] gene. The mutation is called sodJAl:: URA3, sodJAl for short. Extracts from strains carrying this mutation showed no Cu,Zn SOD activity or immunoreactive material (data not shown). The sod] *
Corresponding author. 5918
point mutants described previously (2) were selected on the basis of paraquat sensitivity. Our deletion-replacement mutants showed the same greatly elevated sensitivity without previous selection, indicating that paraquat sensitivity is a direct consequence of the sodl mutation. EG1 (MATa leu23,112 his3AJ trpl-289 ura3-52 sodlAl::URA3) and EG3 (MATa leu2-3,112 his3AJ trpl-289 ura3-52 ade2::hisG gal2 sodlAl:: URA3) were isolated after two backcrosses to S1AA (MATa leu2-3,112 his3AJ trpl -289 ura3-52 ade2: :hisG), a related wild-type strain. As the work progressed, it became clear that drug resistance phenotypes varied between the wild-type parent strains. Therefore, other sodlAl strains, EG-AC1, EG-AE3, and EG-301, were produced subsequently in S1AA, DBY746, and DBY747 (MATa leu2-3,112 his3AJ trpl-289 ura3-52 Canr gal2), respectively, and were not backcrossed. To help prevent second-site pseudoreversion events, these strains were maintained microaerobically (CampyPak; BBL). Sensitivity to oxidative conditions. The sodlAl mutants were viable in aerobic incubation but grew more slowly than wild-type cells with glucose as the carbon source (Table 1). The difference was not as pronounced with 3% glycerol as the carbon source, perhaps because the sodlAl strains retain the functional gene for mitochondrial Mn SOD, which is expressed during growth on glycerol but not during growth on glucose (20). The sodlAl strains were extremely sensitive to paraquat and menadione, as measured by disk assays or killing by challenge in liquid culture (data not shown). Gradient plates (Table 2) were formed in square petri dishes by pouring two layers of YPD (17) agar-the first a slanted one containing the agent at the indicated concentration and the second without the agent filling in the slope. Yeast cultures were diluted 1:100 in 0.5% agar, and even lines of cells were inoculated along the gradient by using a bent glass rod. The length of the growth area was measured after 3 days. The lowest level of paraquat tested (10 puM in the bottom layer) was sufficient to prevent all growth of sodJAl cells, while SOD] (wild-type) strains were resistant to nearly 1,000 ,uM (>90% growth on a 1,000 ,uM plate). This represents a more-than-100-fold difference in sensitivity. The extreme sensitivity to redox cyclers was observed only in the presence of dioxygen (Table 2). sodlAl strains also exhibited dioxygen-dependent lysine auxotrophy and inability to survive in 100% dioxygen (Table 2) that was observed with the previously isolated point mutants (2, 4). Sensitivity to other agents. Aerobic growth slightly but
VOL. 173, 1991
NOTES
TABLE 3. Spontaneous mutation ratesa
TABLE 1. Aerobic doubling timea Generation time (h) with the following genotype at the SOD) locus:
Carbon source (concn)
Wild type
sodlAJ
2.0 1.6 3.5
2.8 2.7 3.8
Glucose (2%) Glucose (10%o) Glycerol (3%)
a Cells were grown in SC medium (17). Doubling times are averages of at least two experiments with each of at least two cell lines.
consistently increased the sensitivity of the already stressed sodlAl strains to certain other DNA-damaging agents, such as methyl methanesulfonate and tert-butyl hydroperoxide (Table 2). However, the seemingly unrelated agent canavanine (an arginine analog) gave a similar result (data not shown), leading us to believe that this is a "straw that broke the camel's back" effect rather than a specific sensitivity. These differences were not evident in anaerobic conditions. The increase in anaerobic tert-butyl hydroperoxide resistance in the sodlAl strain was not reproducibly observed in all genetic backgrounds. Spontaneous mutation rates. Spontaneous mutation rates were measured in sod] and parent strains by using the fluctuation test method described by Von Borstel (19). The strains used in this work contained the trpl-289 allele, a revertible amber mutation of the TRPI gene. Individual cultures were grown in 24-well tissue culture dishes in SC medium (17) limited in tryptophan (1.25 ,uM) and increased fourfold in other required nutrients (Ade, Leu, His, Ura, Met, and Lys) to prevent growth limitation by their depletion. Growth proceeded until the Trp supply was exhausted, after which only cells that had reverted to TRP+ grew and formed large colonies over a thin background. The concentration of Trp, and thus the final nonrevertant cell density, was adjusted so that revertants arose in about half of the wells. The reversion rate was calculated from the zeroth term of the Poisson distribution. For experiments in which TABLE 2. Phenotypic characteristics' Relative growth (%) Aerobic Anaerobic
Agent (concn) or condition
Paraquat (10 ,uM) Paraquat (1,000 ,uM) Menadione (20 ,uM) tert-Butyl hydroperoxide (2.5 mM) 5. Methyl methanesulfonate (0.04%)
1. 2. 3. 4.
6. Growth without lysine 7. Growth in 100TO 02
5919
Condition
(temp [°C]) Aerobic (30) Anaerobic (30) Aerobic (24)
No. of mutations/cell/generation, 10-8 (SD) Fold increase Wild type sodlAl
3.4 (1.3) 1.8 (0.4) 1.6 (0.5)
13.2 (4.6)
3.9
2.1 (0.7) 5.3 (1.4)
1.3 3.2
a The reversion rate was calculated from the zeroth term of the Poisson distribution according to the formula M = (-ln NJN)/2C, where M is the mutation rate, No is the number of wells with no colonies, N is the total number of wells, and C is the number of cells in wells without revertants (determined by direct counts with a hemacytometer). Points are averages of four to six separate experiments and include at least three different wild-typesodlAl pairs.
anaerobic conditions were used, 0.1% Tween 80 and 30 mg of ergosterol per liter were added to the medium. Supplementation did not affect aerobic mutation rates. The results are shown in Table 3. The sodlAl strains had mutation rates during aerobic growth about threefold greater than that determined for wild-type cells. Under anaerobic conditions, the rates were similar for sodlAl and wild-type strains. Essentially the same results were obtained with sodlIAl and SODJ + strains in the various backgrounds tested. Aerobic mutation rates differed with different growth temperatures, but the ratio between sodlAl and SOD]+ mutation rates
remained similar. E. coli strains lacking both Mn SOD and Fe SOD have dramatic increases in aerobic mutation rates. Those lacking only Mn SOD have more modest increases, while those lacking only Fe SOD show no change (5). Work with D. melanogaster has implied a possible connection between SOD and ionizing radiation damage (13). However, ours is the first demonstration of the role of Cu,Zn SOD in a eucaryote as a protector against mutational change. The increase measured here is modest but nonetheless significant. While recognizing that any increase in the accumulation of mutations in a species is significant for the long-term survival of the species, we believe that the effect of sodlAl on mutation rates is unlikely to account for the other phenotypes observed in these mutants. In particular, the acute toxicity of redox cycling drugs and the reduced growth rates are probably best accounted for by other mechanisms. The effect might be more dramatic in strains lacking mitochondrial Mn SOD as well as Cu,Zn SOD. Work is under way in our laboratory to isolate and characterize such mutants. Nevertheless, the strong phenotype of the sodlAl mutant, even in the presence of Mn SOD, argues for separate roles for these two proteins, and in particular for the importance of cytoplasmic Cu,Zn SOD.
Wild type
sod],AJ
Wild type
sodlAJ
100 86 100 54
0 0 0 46
100 82 100 39
100 79 100 46
97
69
86
86
Our thanks to Bruce Demple, Harvard University School of Public Health, for laboratory space where part of this work was performed and for many helpful discussions. Thanks to Sonya Popoff for helpful discussions and for introducing gradient plates to
+ +
-
+
+
us.
aResults are reported for the DBY747 wild-type strain and its isogenic
This work was supported by USPHS grant GM28222 to J.S.V. E.B.G. received partial support from the California Institute for Cancer Research.
similar. Lines 1 to 5 summarize gradient plate results, reported as percentages of the inoculated line showing growth. The concentrations are those that were initially present in the bottom, slanted layer; the maximum concentration was slightly under this because of diffusion through the upper layer. For line 6, cells were streaked on plates containing SC and SC minus lysine, supplemented with Tween 80 and ergosterol and grown aerobically or anaerobically. For line 7, cells were streaked on YPD plates and grown in desiccators filled with air or oxygen.
REFERENCES 1. Bermingham-McDonogh, O., E. B. GralHa, and J. S. Valentine. 1988. The copper, zinc superoxide dismutase gene of S. cerevisiae: cloning, sequencing, and biological activity. Proc. Natl. Acad. Sci. USA 85:4789-4793. 2. BiliUski, T., Z. Krawiec, A. Liczmanski, and J. Litwinska. 1985. Is hydroxyl radical generated by the Fenton reaction in vivo?
sodJAl derivative EG301. Results for the other wild-type-sodlAl pairs were
5920
NOTES
Biochem. Biophys. Res. Commun. 130:533-539. 3. Carlioz, A., and D. Touati. 1986. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 5:623-630. 4. Chang, E. C., and D. J. Kosman. 1990. 02-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae. J. Bacteriol. 172:1840-1845. 5. Farr, S. B., R. D'Ari, and D. Touati. 1986. Oxygen-dependent mutagenesis in E. coli lacking superoxide dismutase. Proc. Natl. Acad. Sci. USA 83:8268-8272. 6. Fee, J. A. 1982. Is superoxide important in oxygen poisoning? Trends Biochem. Sci. 7:84-86. 7. Fridovich, I. 1978. The biology of oxygen radicals. Science 201:875-880. 8. Fridovich, I. 1986. Superoxide dismutases. Adv. Enzymol. 58:61-97. 9. Halliwell, B., and J. M. C. Gutteridge. 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219:1-14. 10. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. The transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. 11. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12. McCord, J. M., B. B. Keele, Jr., and I. Fridovich. 1971. An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Natl. Acad. Sci. USA
J. BACTERIOL.
68:1024-1027. 13. Peng, T. X., A. Moya, and F. J. Ayala. 1986. Irradiation resistance conferred by superoxide dismutase: possible adaptive role of a natural polymorphism in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 83:684-687. 14. Phillips, J. P., S. D. Campbell, D. Michaud, M. Charbonneau, and A. J. Hilliker. 1989. Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. Proc. Natl. Acad. Sci. USA 86: 2761-2765. 15. Rothstein, R. 1983. One step gene disruption. Methods Enzymol. 101:202-211. 16. Sawyer, D. T., and J. S. Valentine. 1981. How super is superoxide? Accounts Chem. Res. 14:393-400. 17. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast genetics-laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Sinet, P. M., J. Couturier, B. Dutrillaux, M. Poissonnier, 0. Raoul, M.-O. Rethore, D. Allard, J. Lejeune, and H. Jerome. 1976. Trisomie 21 et superoxyde dismutase-1 (IPO-A), tentative de localisation sur la sous bande 21q22.1. Exp. Cell. Res. 97:47-55. 19. Von Borstel, R. C. 1978. Measuring spontaneous mutation rates in yeast. Methods Cell Biol. 20:1-24. 20. Westerbeek-Marres, C. A. M., M. M. Moore, and A. P. Autor. 1988. Regulation of manganese superoxide dismutase in Saccharomyces cerevisiae: the role of respiratory chain activity. Eur. J. Biochem. 174:611-620.
