The impact of catalase expression on the replicative lifespan of Saccharomyces cerevisiae

Mechanisms of Ageing and Development 123 (2002) 365– 373 www.elsevier.com/locate/mechagedev

The impact of catalase expression on the replicative lifespan of Saccharomyces cere6isiae S.M. Van Zandycke, P.J. Sohier, K.A. Smart * School of Biological and Molecular Sciences, Oxford Brookes Uni6ersity, Headington, Oxford OX3 0BP, UK Received 10 November 2000; received in revised form 26 September 2001; accepted 26 September 2001

Abstract The role of catalase on Saccharomyces cere6isiae replicative lifespan was investigated using a wild-type haploid laboratory yeast W303a, a catalase A mutant, a catalase T mutant and an acatalasaemic mutant. Lifespan analysis was performed in two different environmental conditions. Under repressing conditions, on glucose media, catalase T activity, but not catalase A activity was necessary to assure longevity. However, under derepressing conditions, on ethanol media, both catalases were required for longevity assurance. Although catalase activity and carbon source influence yeast lifespan, the relationship between oxidative defence and replicative senescence is complex. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Saccharomyces cere6isiae; Replicative ageing; Catalase; Glutathione; Carbon source

1. Introduction Like mammalian cells the budding yeast Saccharomyces cere6isiae possesses a finite replicative potential: each cell is only capable of a limited number of divisions, before entering a physiological state termed senescence, finally leading to death (Mu¨ller et al., 1980). The number of daughters a mother cell produces is equivalent to the number of divisions undergone, and represents the maximum replicative lifespan, which is species and strain specific (Mortimer and Johnston, 1959). The ‘free radical’ theory of ageing (Har* Corresponding author. Tel.: + 44-1865-483-248; fax: + 44-1865-483-242. E-mail address: [email protected] (K.A. Smart).

man, 1956) postulates that ageing results from imperfect protection against oxidative damage to cellular macromolecules. The powerful oxidant H2O2 results from the enzymatic dismutation of the superoxide radical (O2’), and is converted to water and oxygen by catalases and glutathione (Grant et al., 1998). The catalase genes (CTA1 and CTT1 ) of the yeast S. cere6isiae encode a peroxisomal protein, catalase A (Cohen et al., 1988) and a cytosolic protein, catalase T (Hartig and Ruis, 1986), respectively. Catalase expression is controlled by oxygen, haeme and glucose (Hortner et al., 1982), however, both types of catalase are independently regulated (Izawa et al., 1996). The role of catalase A appears to detoxify H2O2 originated from the b-oxidation of fatty-acids (Jamieson, 1998), whereas catalase T has been

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involved in protection against various stresses such as heat-shock, osmotic-shock and H2O2 (Marchler et al., 1993; Godon et al., 1998). It has also been suggested that their activities are complementary (Izawa et al., 1996). Glutathione (GSH) is involved in many cellular processes and acts as a protectant against reactive oxygen species (Meister, 1988). It has been suggested that GSH, rather than catalase, represents the primary defence system to detoxify H2O2 in the cell, however, catalase activity is required in the absence of GSH (Grant et al., 1998). The impact of catalase on ageing has been studied in post-mitotic systems (Mackay and Bewley, 1989; Orr and Sohal, 1994; Seslija et al., 1999; Taub et al., 1999). These studies revealed the crucial role of catalase in assuring longevity, and it is believed that catalase provides one of the main enzymatic mechanisms for the removal of H2O2, because insects lack glutathione peroxidase activity (Orr et al., 1992; Orr and Sohal, 1992). The role of catalase in replicative ageing has not been established, although these enzymes have been reported to be dispensable for the maintenance of lifespan in yeast (Wawryn et al., 1999; Nestelbacher et al., 2000). Here, we demonstrate the role of catalases in replicative senescence in S. cere6isiae. The relationship between oxidative tolerance, oxidative defence and lifespan is examined on glucose and ethanol as sole carbon sources.

CTA1 CTT1–CTA1

cta1:: URA3 ctt1:: URA3; cta1:: URA3

Strains were maintained on YPD (2% glucose) or YPE (2% ethanol) (Barker et al., 1999).

2.2. Micromanipulation YPD agar plates of no more than 5 mm thickness were inoculated with a single yeast colony and incubated for 24 h at 25 °C. The resultant microcolony was then examined using a Zeiss axioskop microscope with a long working distance 40× objective lens, by viewing through the petri dish and agar. Cells were manipulated using a micromanipulation needle. Virgin cells were isolated by manipulation of new buds away from mid-sized mother cell and incubated at 25 °C to allow division to commence. Careful monitoring of cell cycle progression and subsequent separation of newly generated daughter cells allowed the progression from virgin to aged mother cells to be investigated. Plates were incubated at 25 °C during the day and at 7 °C overnight to decrease growth rate and prevent excessive division. To prevent drying of agar during incubation, a filter paper was placed in the lid of each plate and regularly moistened with sterile deionised water. Lifespan data was analysed for statistical significance using the two-tailed Student’s t-test, assuming unequal variance between each data set.

2.3. Sterility test 2. Materials and methods

2.1. Strains and growth media S. cere6isiae strains were provided by Dr M. Breitenbach (University of Vienna, Austria). The ctt1, cta1, and ctt1 – cta1 mutants were isogenic with the wild type W303a (Nestelbacher et al., 2000). W303a

CTT1

MATa, ade 2 -1, can 1 -100, trp 1 -1, ura 3 -1, his 3 -11, 15, leu 2 -3, 112 ctt1:: URA3

Determination of a factor responsiveness was performed according to the method of Smeal et al. (1996). YPD agar plates were inoculated with a single yeast colony and incubated for 24 h at 25 °C. Newly budded cells (virgins) were isolated by micromanipulation (see Section 2.2); all subsequent daughters from these cells were removed to generate cells of different ages. A group of ten cells was moved to a distance of 50–100 mm from a filter moistened with a factor (2 mmg ml − 1). Cells that continued to bud were recorded as non-responders (sterile). If cells adopted a clear morphological shape termed ‘shmoos’ (Smeal et al., 1996), they were recorded as responders

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(non-sterile). The remainder of the lifespan of cells was determined.

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3. Results

3.1. Does catalase deletion influence lifespan on glucose?

2.4. Oxidati6e stress tolerance Tolerance to oxidative stress was determined according to a modified version of Izawa et al. (1996). Yeast cells were harvested by centrifugation at 3000 rpm for 5 min and washed twice in sterile deionised water. Cells were resuspended to a final concentration of 1×105 cells per ml in 20 ml of sterile deionised water (control) or hydrogen peroxide (100 mM) in a 100 ml flask. Cell suspensions were incubated in an orbital shaker (120 rpm) at 25 °C for a 2-h period. Viability was assessed using spread plates on YPD media, incubated at 25 °C and enumerated after 3 days.

2.5. Antioxidant le6els Catalase activity was determined using the method of Aebi (1984). Yeast cells were disrupted using glass beads (Greco et al., 1990). H2O2 solution (0.1 M), enzyme extract, and 50 mM phosphate buffer pH 7 were pipetted into a 3 ml cuvette. The reduction of H2O2 was followed at a wavelength of 240 nm for 1 min against a blank containing 50 mM phosphate buffer and enzyme extract. Catalase activity was expressed in catalase U per g of protein. Total Glutathione concentration (2GSH+ GSSG) was determined using the glutathione assay kit from Calbiochem and expressed as nmol per 1.5× 108 cells.

The suggestion that catalase activity may influence longevity comes from studies with Drosophila melanogaster (Mackay and Bewley, 1989; Sohal et al., 1995) and Caenorhabditis elegans (Taub et al., 1999). However, in both these systems, post mitotic senescence occurs. It has been previously reported that the lack of catalase does not influence replicative lifespan in S. cere6isiae (Wawryn et al., 1999; Nestelbacher et al., 2000). The lifespan of the S. cere6isiae haploid strain W303a, and three catalase mutant strains were examined on nutrient media with glucose as sole carbon source. The mean and maximum10% lifespans of the wild-type strain, W303a, was 21.429 7.72 and 33.739 1.22 (Table 1A), respectively, corroborating the observations of Sinclair and Guarente (1997), Nestelbacher et al. (2000). The mean and maximum10% lifespans of the mutant lacking both catalases was significantly shorter (20%) than that of the wild type (PB 0.05) (Fig. 1A, Table 1A). The catalase T mutant exhibited a similarly reduced mean and maximum10% lifespan (PB 0.05) (Fig. 1A, Table 1A). However, the proliferative capacity of the wild type was not affected by catalase A deletion (Fig. 1A, Table 1A). It has been suggested that sterility represents a biomarker for yeast replicative ageing (Smeal et al., 1996). Sterility tests were performed on catalase T mutant, double catalase knockout mutant

Table 1 Mean and maximum10% lifespans of W303a, ctt1, cta1, and acatalasaemic mutants on YP containing (A) 2% glucose and (B) 2% ethanol as sole carbon source Strains

W303a

ctt1

cta1

ctt1–cta1

(A) Number of cells Mean lifespan 9S.D. Maximum10% lifespan9S.D.

154 21.4297.72 33.73 91.22

72 18.11 9 6.58 26.57 9 1.40

72 21.85 96.75 33.29 9 2.56

68 17.47 9 6.72 26.43 9 1.90

(B) Number of cells Mean lifespan 9S.D. Maximum10% lifespan9S.D.

71 11.1893.28 16.7191.11

71 3.79 9 1.81 7.43 9 0.79

67 6.90 9 2.47 10.71 9 0.76

86 5.59 9 2.84 11.44 9 1.01

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Fig. 1. Mortality profiles showing the replicative lifespans of wild-type cells (W303a), catalase T (ctt1 ), catalase A (cta1 ), and acatalasaemic (ctt1 – cta1 ) mutant strains incubated on YP containing (A) 2% glucose and (B) 2% ethanol as sole carbon source.

and wild type strains. For both mutants and the wild type strain, the fraction of cells that did not respond to a factor began to increase once 60– 70% of the replicative lifespan had been achieved (Table 2). During the last 10% of the cells lifespan, sterility was observed for the majority of cells examined (Table 2).

3.2. Is yeast lifespan influenced by growth on ethanol? It has been reported that growth on ethanol extended mean (Mu¨ ller et al., 1980) and maximum (Mu¨ ller et al., 1980; Barker et al., 1999) lifespan, though this effect was observed to be strain dependent in polyploid (Rodgers et al., 1999) and haploid (Kirchman et al., 1999) strains. The influence of ethanol on the longevity of the haploid strain W303a and catalase deletion mutants was investigated. It was observed that

W303a exhibited a significant (PB 0.05) reduction in mean and maximum10% lifespan on YPethanol compared with YPD (Fig. 1B, Table 1B). Since catalase A appears to be dispensable for longevity assurance on glucose, it is postulated that derepressing growth conditions will necessitate CTA1 expression to maximise lifespan potential. To investigate this hypothesis, wild type and catalase deletion mutants were grown on YPethanol and divisional age at senescence was determined. It was observed that CTA1 expression is required to maintain lifespan potential on YPethanol (Fig. 1B, Table 1B). In addition, catalase T deletion results in a significant (PB 0.05) decrease (66%) in lifespan (Fig. 1B, Table 1B). The deletion of both catalases also resulted in a significant (PB0.05) reduction (50%) in lifespan (Fig. 1B, Table 1B) compared with the wild type though the impact on longevity was less than that observed with the single catalase T deletion.

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3.3. Is lifespan related to catalase acti6ity and glutathione le6els? In order to establish the relationship between catalase expression and lifespan potential, the activity of these enzymes was determined for the wild type and catalase mutant strains. No significant levels of activity could be measured in exponential phase, therefore, cells were grown to stationary phase on YPglucose and YPethanol. On glucose, both catalase A and T mutants exhibited a significantly (P B0.05) reduced catalase activity of 60% of that of the wild type (Fig. 2). The switch from fermentative to respiratory growth induced catalase T activity and reduced catalase A activity. The relative catalase activity observed for the cta1 and ctt1 mutants on ethanol was significantly (PB 0.05) reduced to 85 and 5% of that of the wild type, respectively (Fig. 2). No catalase activity was detected with the double catalase mutant on glucose or ethanol (Fig. 2). A direct relationship between the levels of catalase activity and lifespan could not be established on either carbon source. It is suggested that this may be due to an induction of GSH where catalase deficiencies occur. Since it is widely believed that GSH is the main means of detoxifying H2O2 in yeast (Bilinski et al., 1985; Izawa et al., 1996; Grant et al., 1998), it is possible that the levels of

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this tripeptide may influence longevity. Consequently, the levels of intracellular GSH were determined in stationary phase cells grown on YPglucose or YPethanol. No significant differences in the levels of GSH in wild type and catalase mutant cells grown on glucose was observed (Fig. 3). On ethanol, the intracellular concentration of GSH for the wild-type strain was observed to be half that on YPD (PB 0.05) (Fig. 3). Levels of GSH were higher in the three mutant strains compared with the wild type. However, this increase of 24 and 18% was only significant for the catalase T and acatalasaemic mutants, respectively (PB 0.05) (Fig. 3). A negative correlation between the glutathione levels and lifespan on ethanol was observed (r= − 0.9906) (Fig. 4).

3.4. Is lifespan related to oxidati6e stress tolerance? Since the relationship between lifespan and H2O2 antioxidant potential could not be clearly demonstrated on glucose, wild type and catalase mutants were exposed to exogeneous H2O2 stress in an attempt to establish the relationship between oxidative stress tolerance and longevity potential. It is suggested that lifespan may be a function of the cell’s capacity to tolerate oxidative damage rather than the level of defence per se. The four

Table 2 Sterility test: a factor responsiveness in old cells catalase T (ctt1 ) and acatalasaemic (ctt1–cta1 ) mutants, derived from wild type W303a

Cells from different replicative ages were examined for their ability to respond to a factor. R indicates that the cells responded by forming schmoos. N indicates that the cells did not respond to a factor through two complete passages through the cell cycle. Numbers in parentheses reflect the percentage of sterile cells (non responders).

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Fig. 2. Catalase activity of wild-type cells (W303a), catalase T (ctt1 ), catalase A (cta1 ), and acatalasaemic (ctt1 -cta1 ) mutant strains incubated on 2% glucose (YPD) or 2% ethanol (YPE) as sole carbon source. Data represents the mean of three replicates of three independent experiments. Catalase activity is expressed as units per g of protein 9S.D.

yeast strains were exposed to hydrogen peroxide to determine their level of resistance to this oxidant. Exponential phase populations of catalase mutants, grown on glucose media, have been observed to be insensitive to H2O2 (Izawa et al., 1996; Grant et al., 1998). These results have been confirmed in our study (data not shown). However, in stationary phase, a different pattern of response was observed. The catalase T and the double catalase mutants were adversely affected by this stress (Fig. 5), however, the wild type and catalase A mutant exhibited higher levels of resistance (Fig. 5) after 2 h exposure to H2O2. Growth on ethanol promoted a greater tolerance to H2O2 for the wild type, catalase T, and catalase A mutants (Fig. 5). All yeast strains examined on ethanol and glucose retained 100% viability after a 2-h period in water (data not shown).

4. Discussion It has been suggested that ageing is caused by deleterious changes generated by reactive oxygen species (Harman, 1956). By modifying the antioxidant capacity of the cells, the production of reactive oxygen species can be altered in order to study its influence on replicative capacity. To modify H2O2 production and detoxification, catalase deletion mutants were used, and the impact of catalase deletion on the replicative lifespan of S. cere6isiae was investigated in repressing and derepressing conditions.

A reduction of lifespan was observed with catalase T and acatalasaemic mutants on glucose media. However, proliferative capacity was not affected by a catalase A deletion. Mutants and wild type strains displayed an increase in sterility with age, indicating that these strains were indeed senescing. These results are supported by the observations of Taub et al. (1999) with C. elegans and suggest a potential role for catalase T in longevity assurance. When cells are grown on glucose, both types of catalase are repressed (Hortner et al., 1982). Catalase A is a hundred times more repressed by glucose than catalase T (Hortner et al., 1982). Therefore, it is not surprising that catalase A deletion does not influence longevity on glucose. In derepressing conditions, such as growth on ethanol, CTA1 and CTT1 expression was necessary to assure lifespan of the wild type. It is suggested that the derepression of respiratory functions increases the production of reactive oxygen species, and under conditions of oxidative stress both types of catalase are required to assure replicative longevity. This was confirmed by Nestelbacher et al. (2000), who observed a reduction in lifespan potential with an acatalasaemic mutant under 55% oxygen, and therefore under oxidative stress, but not in ambient air. The wild-type strain also exhibited a reduced lifespan in ethanol compared with that on glucose, supporting the suggestion that some strains of S. cere6isiae are adversely affected by growth on ethanol resulting in a decreased longevity (Rodgers et al., 1999). The reasons for this strain

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Fig. 3. Intracellular levels of glutathione of wild-type cells (W303a), catalase T (ctt1 ), catalase A (cta1 ), and acatalasaemic (ctt1 –cta1 ) mutant strains incubated on 2% glucose (YPD) or 2% ethanol (YPE) as sole carbon source. Data represents the mean of three replicates of three independent experiments. Levels of GSH are expressed as nmol per 1.5 × 108 cells9 S.D.

dependent effect are not known. However, it is postulated that strains exhibiting a reduction in lifespan on ethanol are either sensitive to the increased levels of reactive oxygen species (ROS) as a result of the derepression of the respiratory chain, or lack tolerance to other physiological stress generated by this carbon source. Interestingly, recent studies suggested that catalases were dispensable for replicative lifespan on YPglucose (Wawryn et al., 1999) and SDglucose (Nestelbacher et al., 2000) and that overexpression of CTT1 as well as SOD1 did not have an impact on lifespan (Kirchman et al., 1999). However, our results suggest that the impact of catalase deletion on replicative lifespan is strain and media dependent. Catalase activity and GSH levels were measured to investigate a possible relationship between antioxidant defences and longevity

potential. Catalase activity was not observed to directly influence lifespan on either carbon sources. Catalase deletion did not result in an increase of GSH on glucose, supporting the observation that the absence of catalase does not influence glutathione levels in human erythrocytes (Gaetani et al., 1994). However, levels of GSH were higher on ethanol for catalase T and acatalasaemic mutant, suggesting that GSH compensates for impaired catalase T activity on this carbon source. One potential explanation for the lack of correlation between lifespan and GSH levels for glucose grown cells is that the concentration of GSH present is in excess of that required for H2O2 regulation on this carbon source. In yeast, sod1 mutants, grown on glucose, accumulate more oxidised GSH (Gralla, 1997) and exhibit reduced longevity (Barker et al., 1999; Wawryn et al.,

Fig. 4. Correlation between intracellular levels of glutathione and mean replicative lifespan of wild-type cells (W303a), catalase T (ctt1 ), catalase A (cta1 ), and acatalasaemic (ctt1 –cta1 ) mutant strains incubated on 2% ethanol (YPE) as sole carbon source. Data represents the mean of three replicates of three independent experiments. Levels of GSH are expressed as nmol per 1.5 × 108 cells9 S.D.

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Fig. 5. Viability of wild-type cells (W303a), catalase T (ctt1 ), catalase A (cta1 ), and acatalasaemic (ctt1 – cta1 ) mutant strains incubated to stationary phase on 2% glucose (YPD) or 2% ethanol (YPE) as sole carbon source, and resuspended in H2O2 (100 mM) for 2 h. Data represents the mean of three replicates and is expressed 9 the S.D.

1999). It is postulated that the levels of oxidised GSH, and therefore oxidative stress tolerance, rather than total GSH, which represents the antioxidant potential of the cell, may be more closely related to longevity in yeast. Given the lack of relationship between GSH levels and catalase mutant strains on glucose, it was suggested that replicative lifespan may be related to the level of tolerance to hydrogen peroxide. Oxidative stress tolerance and longevity appear to be related to glucose. Acatalasaemic and ctt1 mutants were highly sensitive to H2O2 compared with the wild type and cta1 mutant strains, potentially as a result of CTT1 induction by H2O2 (Godon et al., 1998). These results suggest that catalase T may compensate for the lack of catalase A, however, the very low levels of Cta1p make it difficult for the ctt1 mutant to compensate for the lack of Ctt1p. The wild type, ctt1, and cta1 mutant strains were more resistant to H2O2 when grown on ethanol. This improvement in oxidative stress tolerance has been previously reported by Jamieson (1992), and is likely to be due to a HAP1 -mediated increase in catalase expression. However, a correlation between lifespan potential and resistance to oxidative stress was not observed on ethanol. Replicative lifespan appeared to be related to tolerance to hydrogen peroxide on glucose media, and to GSH levels on ethanol media. However, given the marked strain and media dependency observed with catalase mutant strains, there is still a need for a universal marker of oxidative stress

that would be linked to replicative lifespan. It is postulated that the relationship between oxidative stress and longevity may be due to a tolerance to oxidative damage rather than to the extent of oxidative defence exhibited by the cell. This remains the subject of further investigation.

Acknowledgements The authors would like to thank Dr M. Breitenbach for providing the strains used in this study. S.M. Van Zandycke is supported by an Oxford Brookes University studentship and P.J. Sohier is supported by the Socrates undergraduate exchange programme. The authors acknowledge the technical assistance of Elizabeth Taylor who was supported by a Wellcome Trust vacation scholarship.

References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121 – 126. Barker, M.G., Brimage, L.J.E., Smart, K.A., 1999. Effect of Cu,Zn superoxide disputes disruption mutation on the replicative capacity of Saccharomyces cere6isiae. FEMS Microbiol. Lett. 177, 199 – 204. Bilinski, T, Krewiec, Z., Liczmanski, A., Litwinska, J., 1985. Is hydroxyl radical generated by the Fenton reaction in vivo? Biochem. Biophys. Res. Commun. 130, 533 – 539. Cohen, G., Rapatz, W., Ruis, H., 1988. Sequence of the Saccharomyces cere6isiae CTA1 gene and amino acid sequence of catalase A derived from it. Eur. J. Biochem. 176, 159 – 163.

S.M. Van Zandycke et al. / Mechanisms of Ageing and De6elopment 123 (2002) 365–373 Gaetani, G.F., Kirkman, H.N., Mangerini, R., Ferraris, A.M., 1994. Importance of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood 84, 325 –330. Godon, C., Lagniel, G., Lee, J., Buhler, J.M., Kieffer, S., Perrot, M., Boucherie, H., Toledano, M.B., Labarre, J., 1998. The H2O2 stimulation in Saccharomyces cere6isiae. J. Biol. Chem. 273, 22480 –22489. Gralla, E.B., 1997. Superoxide dismutases: studies in the yeast Saccharomyces cere6isiae. In: Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 495– 525. Grant, C.M., Perrone, G., Dawes, I.W., 1998. Glutathione and catalase provide overlapping defenses for protection against hydrogen peroxide in the yeast Saccharomyces cere6isiae. Biochem. Biophys. Res. Commun. 253, 893 – 898. Greco, M.A., Hrab, D.I., Magner, W., Kosman, D., 1990. Cu,Zn superoxide dismutase and copper deprivation and toxicity in Saccharomyces cere6isiae. J. Bacteriol. 172, 317– 325. Harman, D., 1956. Aging: a theory based on free radicals and radiation chemistry. J. Gerontol. 11, 298 –300. Hartig, A., Ruis, H., 1986. Nucleotide sequence of the Saccharomyces cere6isiae CTT1 gene and deduced amino-acid sequence of yeast catalase T. Eur. J. Biochem. 160, 487 – 490. Hortner, H., Ammerer, G., Hartter, E., Hamilton, B., Rytka, J., Bilinski, T., Ruis, H., 1982. Regulation of synthesis of catalases and iso-1-cytochrome c in Saccharomyces cere6isiae by glucose, oxygen and heme. Eur. J. Biochem. 128, 179 – 184. Izawa, S., Inoue, Y., Kimura, A., 1996. Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cere6isiae. Biochem. J. 320, 61 – 67. Jamieson, D.J., 1992. Saccharomyces cere6isiae has distinct responses to both hydrogen peroxide and menadione. J. Bacteriol. 174, 6678 –6681. Jamieson, D.J., 1998. Oxidative stress responses of the yeast Saccharomyces cere6isiae. Yeast 14, 1511 –1527. Kirchman, P.A., Kim, S., Lai, C., Jazwinski, S.M., 1999. Interorganelle signalling is a determinant of longevity in Saccharomyces cere6isiae. Genetics 152, 179 –190. Mackay, W.J., Bewley, G.C., 1989. The genetics of catalase in Drosophila melanogaster, isolation and characterisation of acatalasemic mutants. Genetics 122, 643 –652. Marchler, G., Schuller, C., Adam, G., Ruis, H.A., 1993. A Saccharomyces cere6isiae UAS element controlled by

.

373

protein kinase A activates transcription in response to a variety of stress conditions. EMBO J. 249, 1997 – 2003. Meister, A., 1988. Glutathione metabolism and its selective modification. J. Biol. Chem. 263, 17205 – 17208. Mortimer, R.K., Johnston, J.R., 1959. Life span of individual yeast cells. Nature 183, 1751 – 1752. Mu¨ ller, I., Zimmermann, M., Becker, D., Flomer, M., 1980. Calendar lifespan versus budding lifespan of Saccharomyces cere6isiae. Mech. Ageing Dev. 12, 47 – 52. Nestelbacher, R., Laun, P., Vondrakonva, D., Pichova, D., Schuller, C., Breitenbach, M., 2000. The influence of oxygen toxicity on yeast mother cell-specific aging. Exp. Gerontol. 35, 63 – 70. Orr, W.C., Sohal, R.S., 1992. The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster. Arch. Biochem. Biophys. 297, 35 – 41. Orr, W.C., Sohal, R.S., 1994. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263, 1128 – 1130. Orr, W.C., Arnold, L.A., Sohal, R.S., 1992. Relationship between catalase activity, lifespan, and some parameters associated with antioxidant defenses in Drosophila melanogaster. Mech. Ageing Dev. 63, 287 – 296. Rodgers, D.L., Kennedy, A., Hodgson, J.A., Smart, K.A., 1999. Lager brewing yeast ageing and stress tolerance. Proc. EBC Congr. 27, 671 – 679. Seslija, D., Blagojevic, D., Spasic, M., Tucie, N., 1999. Activity of superoxide dismutase and catalase in the bean weevil (Acanthoscelides obtectus) selected for postponed senescence. Exp. Gerontol. 34, 185 – 195. Sinclair, D.A., Guarente, L., 1997. Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91, 1033 – 1042. Smeal, T., Claus, J., Kennedy, B., Cole, F., Guarente, L., 1996. loss of transcriptional silencing causes sterility in old mother cells of S. cere6isiae. Cell 84, 633 – 642. Sohal, R.S., Argawal, A., Argawal, S., Orr, W.C., 1995. Simultaneous overexpression of copper-containing and zinc-containing superoxide dismutase and catalase retards age-related oxidative damage and increases metabolic potential in Drosophila melanogaster. J. Biol. Chem. 270, 15671 – 15674. Taub, J., Lau, J.F., Ma, C., Hahn, J.H., Hoque, R., Rothblatt, J., Chalfie, M., 1999. A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-C and clk-1 mutants. Nature 399, 162 – 166. Wawryn, J., Krzepilko, A., Mytszka, A., Bilinski, T., 1999. Deficiency in superoxide dismutases shortens life span of yeast cells. Acta Biochim. Pol. 46, 249 – 253.