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shown that Haemophilus influenzae and Myxococcus xanthus have a single system, KefX, which appears to be an intermediate between KefB and KefC21. The fact that electrophile-activated potassium loss has been identified in all Gram-negative bacteria tested to date provides further evidence that other bacteria possess KefB/KefC-like systems5. Although Gram-positive bacteria appear to lack KefB/KefClike systems, it is likely they possess other electrophile-protective mechanisms5. A Dps homologue has been identified in Bacillus subtilis, and alternative sigma factors have been found in Gram-positive bacteria22,23. In S. aureus, the alternative sigma factor sB is produced in response to growth phase, and a similar factor regulates the expression of stress genes in B. subtilis23. Even though all cells produce methylglyoxal there appear to be no mammalian homologues of these protective systems. In addition, the loss of one or more of these systems results in rapid killing of the bacterial cell. Electrophile-protective mechanisms therefore represent novel targets for future antibacterial drugs. Acknowledgements G.P.F. is a Wellcome Trust Toxicology Fellow. I thank Prof. Ian Booth for his helpful advice, other lab members (both past and present) for their contribution to our understanding of electrophile-protective mechanisms and Dr Conor O’Byrne for critical reading of this manuscript.
References 1 Ferguson, G.P. et al. (1998) J. Bacteriol. 180, 1030–1036 2 Stevens, K.L., Wilson, R.E. and Friedman, M. (1995) J. Agric. Food Chem. 43, 2424–2427 3 Zablotowicz, R.M. et al. (1995) Appl. Environ. Microbiol. 61, 1054–1060 4 Totemeyer, S. et al. (1998) Mol. Microbiol. 27, 553–562 5 Ferguson, G.P. et al. (1998) Arch. Microbiol. 170, 209–219 6 Ferguson, G.P. et al. (1996) J. Bacteriol. 178, 3957–3961 7 Ferguson, G.P. et al. (1993) Mol. Microbiol. 9, 1297–1303 8 Ferguson, G.P. et al. (1997) J. Bacteriol. 179, 1007–1012 9 Ness, L.S. et al. (1997) Appl. Environ. Microbiol. 63, 4083–4086 10 Ferguson, G.P., McLaggan, D. and Booth, I.R. (1995) Mol. Microbiol. 17, 1025–1033 11 Ferguson, G.P. and Booth, I.R. (1998) J. Bacteriol. 180, 4314–4318 12 MacLean, M. et al. (1998) Mol. Microbiol. 27, 563–571 13 Lo, T.W.C. et al. (1994) J. Biol. Chem. 269, 32299–32305 14 Papaulis, A., Al-Abed, Y. and Bucala, R. (1995) Biochemistry 34, 648–655 15 Misra, K.B. et al. (1995) Biochemistry 305, 999–1003 16 Misra, K.B. et al. (1996) Mol. Cell. Biochem. 156, 117–124 17 Booth, I.R. et al. (1996) in Handbook of Biological Physics (Vol. 2) (Konings, W.N., Kaback, H.R and Lolkema, J.S., eds), pp. 693–730, Elsevier Science 18 Oktyabrsky, O.N. et al. (1993) Mutat. Res. DNA Repair 293, 197–204 19 Henge-Aronis, R. (1996) Mol. Microbiol. 21, 887–893 20 Martinez, A. and Kolter, R. (1997) J. Bacteriol. 179, 5188–5194 21 Ness, L.S. and Booth, I.R. (1999) J. Biol. Chem. 274, 9524–9530 22 Chen, L. and Helmann, J.D. (1995) Mol. Microbiol. 18, 295–300 23 Kullik, I. and Gianchino, P. (1997) Arch. Microbiol. 167, 151–159
Molecular mechanisms of yeast longevity S. Michal Jazwinski
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t has frequently been ob- The genetic analysis of yeast longevity has Given that the best predictor of mortality is life span itself, the served that there are as illuminated the underlying molecular study of aging is on a firmer many theories of aging as mechanisms of aging that invoke the footing when longevity is used there are investigators studying importance of metabolic regulation, aging. This is because aging is genetic stability and stress resistance in as the assay, and this approach characterized by a multitude of determination of life span. The RAS genes is easily applied in these model changes that occur at many dif- have emerged as important modulators of systems. Manipulations that maintain or extend life span ferent levels. There is much to life-maintenance processes and of life have generally been applied, pick and choose from, and the span itself. because it is recognized that choice is often dictated by scienS.M. Jazwinski is in the Dept of Biochemistry and interference with any vital tific interest. The challenge has Molecular Biology, Louisiana State University function can curtail life span always been to establish a Medical Center, New Orleans, LA 70112, USA. and be mistaken for aging. The causal connection between the e-mail: [email protected] studies in genetic model sysfavored mechanism and aging. tems point to four broad With the heightened appreciation for genetic model systems in aging research, this physiological processes that are important for longevdifficulty has been surmounted (reviewed in Ref. 1). ity: metabolic regulation; control of stress responses; 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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Table 1. Phenotypic changes during yeast aging Characteristic5
Change
Cell size Cell shape Granular appearance Surface wrinkles Loss of turgor Cell fragility (prior to death) Cell lysis Bud scar number Cell wall chitin Vacuole size Generation (cell cycle) time Response to pheromones (haploids) Mating ability (haploids) Sporulation ability (diploids) Cell-cycle arrest at G1–S boundary (putative) Senescence factor Mutability of mtDNA UV resistance Telomere length Random budding4 Specific gene expression rRNA levels rDNA circles6 Cellular rRNA concentrationa Protein synthesis Ribosome activity, polysome recruitment Transcriptional silencing7,8
Increase Altered Develops Develop Develops None Occurs Increase Increase Increase Increase None9/decrease8 Decrease Increase Occurs Appears Decrease Increase/decrease None Increase Altered Increase Increase Decrease Decrease Decrease Decrease
the probability that an individual yeast cell will produce progeny, which is a function of its age in cell divisions or generations. This means that mortality rate increases exponentially with age, although a plateau in this increase is observed at later ages4. Yeasts undergo a variety of age changes, some of which clearly represent functional decline (Table 1). Thus, it is reasonable to speak of an aging process here. The phenomenology of yeast aging has recently been thoroughly reviewed5, so this review will focus on more recent developments. A dozen genes that determine yeast life span have been identified and cloned (Table 2). The first of these was LAG1 (Ref. 10). Notably, the recently identified human homolog can complement LAG1 to establish the longevity phenotype11. The conclusion that RAS1 and RAS2 function as longevity genes in yeast14 was already evident from studies involving an activated allele of mammalian ras (Ref. 21). The SIR4 gene was discovered to affect yeast longevity as a gain-offunction mutant, SIR4-42 (Ref. 15). The yeast longevity genes (Table 2) seem to perform a diverse array of functions, suggesting that several pathways are involved in determining yeast longevity. If more than one pathway proves to be involved, this points to more than one molecular mechanism determining yeast longevity.
Genetic stability and gene dysregulation A molecular mechanism of yeast aging has recently a Owing to increase in cell size. been documented. Monomeric- and, especially, multimeric-circular species of extrachromosomal rDNA repeats, normally present in different amounts maintenance of the transcriptional silencing of in yeast strains, are found at extraordinarily high heterochromatic domains; and genetic stability1. levels in old cells6. Experimental generation of such The yeast Saccharomyces cerevisiae has played an circles markedly depresses life span. Furthermore, important role in the developments outlined above. partial inactivation of the Cdc6 protein, which would Individual yeasts are mortal, and their life span is cause a decline in their propagation, increases life measured by the number of times they divide or, in span. The possible pleiotropic effects of this treatother words, the number of daughter cells they pro- ment, however, are typical of the difficulties encounduce2,3. These daughter cells have, in principle, the tered in aging studies. Nevertheless, this study potential for a full life span. Thus, the yeast popu- demonstrates the significance of genetic instability in lation is immortal. There is an exponential decline in yeast aging. It also provides a plausible explanation for the appearance of nucleolar enlargement and fragmentation in old yeast cells16. Sinclair and Table 2. Yeast longevity genes Guarente6 suggest that the rDNA constitutes an ‘age’ locus to which Gene Function Refs the silencing proteins migrate from the telomeres and silent matingLAG1 Unknown 10 type loci in old cells. Currently, it LAC1 Unknown; homolog of LAG1 1,11 is unclear how this migration CDC7 Protein kinase; involved in cell-cycle control and transcriptional 12,13 silencing affects aging. The SIR4-42 muRAS1 GTP-binding (G-) protein; involved in signal transduction 14 tation, which extends life span15, RAS2 G-protein; involved in signal transduction 14 also causes migration of silencing SIR4 Transcriptional silencing 15 proteins to the nucleolus16. UTH4 Unknown 15,16 There is a loss of transcriptional YGL023 Unknown; homolog of UTH4 16 silencing at heterochromatic reSGS1 DNA helicase; involved in DNA recombination 17 gions of the yeast genome, includPHB1 Unknown; mitochondrial protein 18,19 ing subtelomeric chromatin7 and PHB2 Unknown; mitochondrial protein; homolog of PHB1 18,19 silent mating-type loci8, during RTG2 Unknown; effector of retrograde response 20 normal aging. The resultant gene
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dysregulation has been proposed as another molecular mechanism of yeast aging1. The SIR4 and CDC7 genes both affect silencing22 and are involved in determining yeast longevity12,15. Recently, it has been found that RAS2 affects the basal silencing level of subtelomeric heterochromatin4. These findings are suggestive; however, their interpretation is complicated. It appears that the enhanced resistance to stress found in SIR4-42 (Ref. 15) has a positive effect on yeast life span, but there is likely to be some overlap between the effects of gene dysregulation and migration of silencing complexes to the ‘age’ locus (and its consequences) on yeast longevity. SIR4-42 might be special in allowing a degree of silencing loss that facilitates the activation of stress response genes, while preventing wholesale gene dysregulation. Metabolic regulation A molecular mechanism determining yeast longevity has been identified in studies with petite yeasts20, whose mitochondria are not fully functional. This disruption in mitochondrial function activates an intracellular signaling pathway, termed retrograde regulation, that results in the induction of expression of nuclear genes23. The activation of this retrograde response by genetic or environmental manipulation is correlated with an extension of life span20. This life extension is completely suppressed by deletion of one of the downstream effectors of the retrograde response, RTG2. In addition to this gene, two other genes, RTG1 and RTG3, are required for retrograde regulation24,25. The Rtg2 protein might promote the formation of an active Rtg1–Rtg3 protein heterodimer, which is a transcription factor that induces gene expression from the retrograde response element26. Disruption of RAS2 completely suppresses the life-span extension observed in the petite yeast, indicating that the Ras2 protein affects events downstream of the mitochondrial signal20. In fact, the Ras2 protein modulates the expression of nuclear genes seen in the retrograde response20. Figure 1 summarizes our current understanding of the role of the retrograde response in determining yeast longevity. The nuclear genes regulated by the Rtg1–Rtg3 heterodimer encode a variety of metabolic enzymes that reside in the mitochondria, cytoplasm and peroxisomes27,28. The metabolic changes that the activation of this heterodimer portends are: a shift from glucose to acetate utilization, the activation of gluconeogenesis, and potentiation of the glyoxylate cycle to provide Krebs cycle intermediates. For yeast, this represents an effort to survive in the face of dwindling energy and carbon sources. It is also a means to signal changes in mitochondrial activity, although the nature of the physiological signal is not known. Some of the metabolic events that are triggered by retrograde regulation are also found during yeast sporulation, which itself is a response to severe caloric stress. The retrograde response constitutes an example of the importance of metabolic regulation in determining longevity. Indeed, any extension of life span in yeast must encompass such a molecular mechanism,
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Signal? Ras2
Rtg2 Rtg1
Rtg3
Rtg1
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Rtg3
Nuclear gene transcription
Life span Fig. 1. Interorganelle communication is a determinant of yeast longevity. A signal generated by the mitochondrion results in the activation of transcription of nuclear genes by the heterodimer Rtg1–Rtg3. These are basic helix-loop-helix, leucine zipper proteins25. The Rtg2 protein has been postulated to promote the formation of an active Rtg1–Rtg3 complex26. The nature of the physiological signal produced by the mitochondrion is not yet known. The nuclear genes controlled by the RTG genes encode proteins that are localized in the mitochondrion, cytoplasm and peroxisome27–29. They include: citrate synthase 1; citrate synthase 2 (glyoxylate cycle); isocitrate dehydrogenase; aconitase; acetyl-CoA synthetase; pyruvate carboxylase (gluconeogenic); acetyl-CoA oxidase; catalase A; and the major peroxisomal membrane protein, Pmp27. It is likely that there are many more genes controlled by the Rtg proteins. The activation of this retrograde response pathway results in the extension of yeast life span20. The Ras2 protein interacts with this pathway20, acting downstream of the mitochondrial signal and modulating the expression of the nuclear genes. It is not known whether it is the metabolic enzymes and/or other potential targets of the pathway that determine yeast life span. RAS2 is likely to determine life span by additional mechanisms.
because life span is measured in this organism by the number of daughter cells that are produced, which depends on metabolic capacity. Interestingly, activation of the retrograde response results in a dramatic increase in extrachromosomal rDNA circles30, indicating that this pathway can promote increased longevity, even when these circles are present. Control of stress responses Yeasts are exposed to a variety of stresses in their environment, including starvation, alluded to above, and UV radiation. Curiously, in yeast, the resistance to UV increases with age, reaching a peak at midlife31. However, UV resistance rapidly declines thereafter. The expression profile of RAS2 closely parallels this biphasic response. RAS2 is required for resistance to UV (Ref. 32). The link between stress resistance and longevity is also apparent in the ability to isolate yeast mutants with extended life span by selection for the secondary phenotype of resistance to starvation and cold stress15. The mutants obtained are also resistant to other stresses.
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Another stress that yeasts encounter is heat. Chronic exposure of yeast to regularly repeated episodes of sublethal heat stress results in a reduction in longevity33. This effect is exacerbated when RAS2 is deleted, but not when RAS1 is deleted. In the absence of the Ras2 protein, the cells display a marked delay in resumption of growth and division after the stress is lifted. There is no difference between RAS1 mutants, RAS2 mutants and wild-type cells in the activation of stress genes, nor in the downregulation of growthpromoting genes. However, there is a marked delay, in both the subsequent downregulation of the stress genes and upregulation of the growth-promoting genes in RAS2 mutant cells33, which simply do not recover well from the stress. The delay in downregu-
lation of the stress genes is due to a lack of proper control of transcription from the stress response regulatory element (STRE)34. This can be completely overcome by the overexpression of RAS2. Indeed, overexpression of this gene extends the life span well beyond that of the unstressed control cells33. This life extension is very similar to that observed on overexpression of RAS2 in the absence of any overt stress14. There is a difference, however: in the absence of stress, RAS2 extends life span by a cAMP-independent pathway14, while during chronic bouts of stress, the Ras2protein–cAMP pathway is necessary to achieve the rescue and life extension observed33. Unlike the effect of chronic stress, a transient, sublethal heat stress, delivered early in life, results in an increase in longevity35. This increase in life span is the result of a large decrease in the mortality Trends in Microbiology Acute stress rate, which persists for many generations but is not permanent. This constitutes a heritable Viability epigenetic effect. Both RAS1 and RAS2 are required to observe this effect. As in the chronic stress situation described above, cAMP Chronic stress RAS2 is necessary for timely recovery from the transient stress. cAMP HSP104, the gene encoding the (MAPK) Life span major yeast heat-shock protein that is involved in induced therRAS2 mal tolerance36, is essential for Rapid the life extension, as are funcLife span cAMP tioning mitochondria35. The synresponses (MAPK) PI thetic phenotype of shortened life span in petite yeasts subjected Transient stress to transient, sublethal heat stress RAS1 suggests an interaction between MAPK the life-extension mechanisms activated by this heat stress and Long-term PI Life span those controlled by the retroeffects (no stress) grade response35. The mechanisms might not operate concurrently. These studies on the role Fig. 2. Control of response to heat stress by the RAS genes. RAS2 is involved in the rapid of the RAS genes in the response response to changes in environmental conditions by facilitating recovery from heat stress33. This of yeast to heat stress have function of RAS2 is performed through modulation of the expression of stress genes34, among prompted the model shown in others, which prevents expression of these genes at levels sufficient to withstand an acute Fig. 2. Many of the features of (lethal) heat shock and reduces survival in the stationary phase37. This submaximal ability to this model have been confirmed resist lethal stress represents a trade-off for the ability of the yeast to withstand chronic, sublethal heat stress throughout their life span33. It is the cAMP-stimulatory effect of the Ras2 protein experimentally, while others that is involved in the response both to lethal and to chronic, sublethal heat shock33,34,37. RAS2 is await testing. In particular, it is also essential for the extension of life span provided by the conditioning effect of a transient, subproposed that RAS1 is involved lethal heat stress35. Under these conditions, it is also involved in recovery from heat stress. In the in the long-term response to absence of overt stress, RAS2 has a life span-extending effect, which entails a cAMP-independent pathway14. This is likely to be a mitogen-activated protein kinase (MAPK) pathway38. It is possible transient, sublethal heat stress. that the Ras2 protein operates through both the cAMP and MAP kinase pathways in the response This role is not likely to depend to both chronic and to transient heat stress. RAS1 is postulated to exert long-term effects on on the weak stimulation of longevity, because it is not required for the recovery that is part of the response to chronic adenylate cyclase, which does 33 stress but is essential for the persistent decrease in mortality rate following transient heat not allow RAS1 to substitute for stress35. RAS1 has a life span-shortening effect in the absence of stress14. It is hypothesized that the effects of the Ras1 protein are mediated through its stimulatory effect on inositol phosphoRAS2 in the response to chronic lipid (PI) turnover39,40, because it is not effective in replacing the Ras2 protein in the response to heat stress. Rather, it can be hychronic stress. It also stimulates adenylate cyclase much more weakly than the Ras2 protein pothesized that it is the stimu(Ref. 37). The shading highlights the area in which the actions of RAS1 and RAS2 overlap. The lation of inositol phospholipid blunt arrows denote negative effects. turnover, which the Ras1 protein
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promotes39,40, that is involved. Sphingolipids, which in yeast contain inositol, are required for resistance to a variety of stresses, including heat41. The Ras1 protein might effect a stable change in inositol lipid pools that persists for many generations in response to transient, sublethal heat stress.
represent a shift from a nutrient of high caloric content (glucose) to one of lower caloric content (acetate). Conclusions We do not know how many different molecular mechanisms of aging exist in yeast, however, two appear to be firmly established. These mechanisms coincide with two of the four broad physiological principles of longevity in yeast and in other organisms. The yeast longevity genes RAS1 and RAS2 modulate several processes that are important for longevity, responding to one known signal, nutritional status (Fig. 3). It is quite possible that a molecular mechanism of aging underlies each of these processes. For response to stress, a molecular mechanism is nearly in place in addition to the established role of the retrograde response in metabolic regulation. It is conceivable that genetic stability is also modulated by the RAS genes. Under constant conditions, there is an optimal level of Ras2 protein activity that results in maximal life span21, and the dependence on the Ras2 protein is nonlinear. A change in environmental conditions, with no manipulation of Ras2 protein levels results in either decreased or increased longevity, depending on the circumstances33,35. Thus, the RAS genes constitute a homeostatic device in yeast longevity.
Correlates in other organisms Certain mutants (daf) in the developmental pathway for dauer larva formation in the nematode Caenorhabditis elegans cause an increase in the life span of the adult (reviewed in Ref. 1). The dauer is a dispersal form that allows the nematode to survive periods of starvation, crowding and heat stress. The daf pathway encompasses genes that encode an insulin-receptor-like protein42, a phosphatidylinositol-3-hydroxide kinase43 and a transcription factor belonging to the forkhead family44,45. The identity of these proteins suggests that metabolic regulation plays an important role in nematode aging. Indeed, the metabolic changes that are associated with enhanced life span in the nematode encompass lipid and glycogen metabolism44. Such metabolic consequences are also associated with extended longevity in the fruit fly (reviewed in Ref. 1). The activation of the daf pathway results in alterations in metabolic enzyme activity46 similar to those found on activation of the retrograde response in yeast. A mutation in another nematode gene, clk-1, which does not reside in the daf pathway, Nutritional Changing external also extends life span, and this status and internal gene is a homolog of the yeast Cellular resources environment gene COQ7, a nuclear-encoded regulator of mitochondrial funcNutrients Energy tion47. The long-lived daf and clk-1 mutants have an increased resistance to UV, heat and oxidative stress48. Fruit flies that are selected for extended longevity are also more resistant to a variety of stresses (reviewed in Ref. 1). A Growth RAS Epigenetic gene long-lived Drosophila mutant, regulation methuselah, is more resistant to starvation, heat and oxidative stress than the wild type49. Cell division Genetic stability Caloric restriction of mammals results in extension of life span (reviewed in Ref. 50). These Cell organization Response to stress animals are more resistant to heat stress51. They also display a Trends in Microbiology Competing demands variety of metabolic changes, such as reduced blood glucose Fig. 3. Coordination of cellular activities by the RAS genes. This heuristic model shows that RAS genes ‘channel’ cellular resources (nutrients and energy) to the competing demands of various and insulin levels (reviewed in cellular activities, which are essential for maintenance of homeostasis and determination of Ref. 50), and have higher insulin longevity. The Ras1 and Ras2 proteins do not literally channel resources to these cellular activreceptor levels and increased acities, because they are not part of the pathways that define them but instead modulate them. tivity of the gluconeogenic enThese cellular activities include growth or metabolism 20, cell division 14, cell organization 4, zyme, phosphoenolpyruvate carresponse to stress31,33,35, epigenetic control of gene expression4 and, possibly, genetic stability6. 52,53 This channeling of resources occurs under conditions of changing external and internal environboxykinase . Caloric restriction ment, which alter the balance between the demands on the resources. The RAS genes respond involves a reduction in the to one known signal, nutritional status37. The dependencies are not shown as pathways; instead, caloric content of the diet. The the Ras proteins are depicted driving a cycle (not unlike the Krebs cycle), into which various enzymatic changes associated pathways enter or exit. with the retrograde response
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Questions for discussion • How can there be more than one mechanism of aging in a genetically homogenous population that is maintained under constant conditions? • Why is the production of extrachromosomal rDNA circles not deleterious for longevity under certain conditions? • What is the relationship between metabolic control and stress resistance? • What criteria need to be satisfied in order to classify a gene or a process as a determinant of longevity and/or a cause of aging? • What are the similarities and differences between life extension by caloric restriction, the retrograde response and the daf pathway? Acknowledgements Research in my laboratory is supported by grants from the National Institute on Aging of the National Institutes of Health (U.S.P.H.S.). References 1 Jazwinski, S.M. (1996) Science 273, 54–59 2 Mortimer, R.K. and Johnston, J.R. (1959) Nature 183, 1751–1752 3 Muller, I. et al. (1980) Mech. Ageing Dev. 12, 47–52 4 Jazwinski, S.M. et al. (1998) Exp. Gerontol. 33, 571–580 5 Jazwinski, S.M. (1996) in Handbook of the Biology of Aging (4th edn) (Schneider, E.L. and Rowe, J.W., eds), pp. 39–54, Academic Press 6 Sinclair, D.A. and Guarente, L. (1997) Cell 91, 1033–1042 7 Kim, S., Villeponteau, B. and Jazwinski, S.M. (1996) Biochem. Biophys. Res. Commun. 219, 370–376 8 Smeal, T. et al. (1996) Cell 84, 633–642 9 Muller, I. (1985) Antonie von Leeuwenhoek J. Microbiol. Serol. 51, 1–10 10 D’mello, N.P. et al. (1994) J. Biol. Chem. 269, 15451–15459 11 Jiang, J.C. et al. (1998) Genome Res. 8, 1259–1272 12 Egilmez, N.K. and Jazwinski, S.M. (1989) J. Bacteriol. 171, 37–42 13 Jazwinski, S.M., Egilmez, N.K. and Chen, J.B. (1989) Exp. Gerontol. 24, 423–436 14 Sun, J. et al. (1994) J. Biol. Chem. 269, 18638–18645 15 Kennedy, B.K. et al. (1995) Cell 80, 485–496 16 Kennedy, B.K. et al. (1997) Cell 89, 381–391 17 Sinclair, D.A., Mills, K. and Guarente, L. (1997) Science 277, 1313–1316 18 Coates, P.J. et al. (1997) Curr. Biol. 7, 607–610 19 Berger, K.H. and Yaffe, M.P. (1998) Mol. Cell. Biol. 18, 4043–4052
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Coming soon in Trends in Microbiology •Endogenous retroviruses: are they the cause of Multiple Sclerosis?, by R.S. Fujinami and J.E. Libbey •Variation and evolution of the citric-acid cycle: a genomic perspective, by M.A. Huynen, T. Dandekar and P. Bork •Evolution of Wolbachia pipientis transmission dynamics in insects, by E.A. McGraw and S.L. O’Neill Don’t miss these and many more articles of interest; subscribe to Trends in Microbiology using the form bound in this issue.
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shown that Haemophilus influenzae and Myxococcus xanthus have a single system, KefX, which appears to be an intermediate between KefB and KefC21. The fact that electrophile-activated potassium loss has been identified in all Gram-negative bacteria tested to date provides further evidence that other bacteria possess KefB/KefC-like systems5. Although Gram-positive bacteria appear to lack KefB/KefClike systems, it is likely they possess other electrophile-protective mechanisms5. A Dps homologue has been identified in Bacillus subtilis, and alternative sigma factors have been found in Gram-positive bacteria22,23. In S. aureus, the alternative sigma factor sB is produced in response to growth phase, and a similar factor regulates the expression of stress genes in B. subtilis23. Even though all cells produce methylglyoxal there appear to be no mammalian homologues of these protective systems. In addition, the loss of one or more of these systems results in rapid killing of the bacterial cell. Electrophile-protective mechanisms therefore represent novel targets for future antibacterial drugs. Acknowledgements G.P.F. is a Wellcome Trust Toxicology Fellow. I thank Prof. Ian Booth for his helpful advice, other lab members (both past and present) for their contribution to our understanding of electrophile-protective mechanisms and Dr Conor O’Byrne for critical reading of this manuscript.
References 1 Ferguson, G.P. et al. (1998) J. Bacteriol. 180, 1030–1036 2 Stevens, K.L., Wilson, R.E. and Friedman, M. (1995) J. Agric. Food Chem. 43, 2424–2427 3 Zablotowicz, R.M. et al. (1995) Appl. Environ. Microbiol. 61, 1054–1060 4 Totemeyer, S. et al. (1998) Mol. Microbiol. 27, 553–562 5 Ferguson, G.P. et al. (1998) Arch. Microbiol. 170, 209–219 6 Ferguson, G.P. et al. (1996) J. Bacteriol. 178, 3957–3961 7 Ferguson, G.P. et al. (1993) Mol. Microbiol. 9, 1297–1303 8 Ferguson, G.P. et al. (1997) J. Bacteriol. 179, 1007–1012 9 Ness, L.S. et al. (1997) Appl. Environ. Microbiol. 63, 4083–4086 10 Ferguson, G.P., McLaggan, D. and Booth, I.R. (1995) Mol. Microbiol. 17, 1025–1033 11 Ferguson, G.P. and Booth, I.R. (1998) J. Bacteriol. 180, 4314–4318 12 MacLean, M. et al. (1998) Mol. Microbiol. 27, 563–571 13 Lo, T.W.C. et al. (1994) J. Biol. Chem. 269, 32299–32305 14 Papaulis, A., Al-Abed, Y. and Bucala, R. (1995) Biochemistry 34, 648–655 15 Misra, K.B. et al. (1995) Biochemistry 305, 999–1003 16 Misra, K.B. et al. (1996) Mol. Cell. Biochem. 156, 117–124 17 Booth, I.R. et al. (1996) in Handbook of Biological Physics (Vol. 2) (Konings, W.N., Kaback, H.R and Lolkema, J.S., eds), pp. 693–730, Elsevier Science 18 Oktyabrsky, O.N. et al. (1993) Mutat. Res. DNA Repair 293, 197–204 19 Henge-Aronis, R. (1996) Mol. Microbiol. 21, 887–893 20 Martinez, A. and Kolter, R. (1997) J. Bacteriol. 179, 5188–5194 21 Ness, L.S. and Booth, I.R. (1999) J. Biol. Chem. 274, 9524–9530 22 Chen, L. and Helmann, J.D. (1995) Mol. Microbiol. 18, 295–300 23 Kullik, I. and Gianchino, P. (1997) Arch. Microbiol. 167, 151–159
Molecular mechanisms of yeast longevity S. Michal Jazwinski
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t has frequently been ob- The genetic analysis of yeast longevity has Given that the best predictor of mortality is life span itself, the served that there are as illuminated the underlying molecular study of aging is on a firmer many theories of aging as mechanisms of aging that invoke the footing when longevity is used there are investigators studying importance of metabolic regulation, aging. This is because aging is genetic stability and stress resistance in as the assay, and this approach characterized by a multitude of determination of life span. The RAS genes is easily applied in these model changes that occur at many dif- have emerged as important modulators of systems. Manipulations that maintain or extend life span ferent levels. There is much to life-maintenance processes and of life have generally been applied, pick and choose from, and the span itself. because it is recognized that choice is often dictated by scienS.M. Jazwinski is in the Dept of Biochemistry and interference with any vital tific interest. The challenge has Molecular Biology, Louisiana State University function can curtail life span always been to establish a Medical Center, New Orleans, LA 70112, USA. and be mistaken for aging. The causal connection between the e-mail: [email protected] studies in genetic model sysfavored mechanism and aging. tems point to four broad With the heightened appreciation for genetic model systems in aging research, this physiological processes that are important for longevdifficulty has been surmounted (reviewed in Ref. 1). ity: metabolic regulation; control of stress responses; 0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. TRENDS
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Table 1. Phenotypic changes during yeast aging Characteristic5
Change
Cell size Cell shape Granular appearance Surface wrinkles Loss of turgor Cell fragility (prior to death) Cell lysis Bud scar number Cell wall chitin Vacuole size Generation (cell cycle) time Response to pheromones (haploids) Mating ability (haploids) Sporulation ability (diploids) Cell-cycle arrest at G1–S boundary (putative) Senescence factor Mutability of mtDNA UV resistance Telomere length Random budding4 Specific gene expression rRNA levels rDNA circles6 Cellular rRNA concentrationa Protein synthesis Ribosome activity, polysome recruitment Transcriptional silencing7,8
Increase Altered Develops Develop Develops None Occurs Increase Increase Increase Increase None9/decrease8 Decrease Increase Occurs Appears Decrease Increase/decrease None Increase Altered Increase Increase Decrease Decrease Decrease Decrease
the probability that an individual yeast cell will produce progeny, which is a function of its age in cell divisions or generations. This means that mortality rate increases exponentially with age, although a plateau in this increase is observed at later ages4. Yeasts undergo a variety of age changes, some of which clearly represent functional decline (Table 1). Thus, it is reasonable to speak of an aging process here. The phenomenology of yeast aging has recently been thoroughly reviewed5, so this review will focus on more recent developments. A dozen genes that determine yeast life span have been identified and cloned (Table 2). The first of these was LAG1 (Ref. 10). Notably, the recently identified human homolog can complement LAG1 to establish the longevity phenotype11. The conclusion that RAS1 and RAS2 function as longevity genes in yeast14 was already evident from studies involving an activated allele of mammalian ras (Ref. 21). The SIR4 gene was discovered to affect yeast longevity as a gain-offunction mutant, SIR4-42 (Ref. 15). The yeast longevity genes (Table 2) seem to perform a diverse array of functions, suggesting that several pathways are involved in determining yeast longevity. If more than one pathway proves to be involved, this points to more than one molecular mechanism determining yeast longevity.
Genetic stability and gene dysregulation A molecular mechanism of yeast aging has recently a Owing to increase in cell size. been documented. Monomeric- and, especially, multimeric-circular species of extrachromosomal rDNA repeats, normally present in different amounts maintenance of the transcriptional silencing of in yeast strains, are found at extraordinarily high heterochromatic domains; and genetic stability1. levels in old cells6. Experimental generation of such The yeast Saccharomyces cerevisiae has played an circles markedly depresses life span. Furthermore, important role in the developments outlined above. partial inactivation of the Cdc6 protein, which would Individual yeasts are mortal, and their life span is cause a decline in their propagation, increases life measured by the number of times they divide or, in span. The possible pleiotropic effects of this treatother words, the number of daughter cells they pro- ment, however, are typical of the difficulties encounduce2,3. These daughter cells have, in principle, the tered in aging studies. Nevertheless, this study potential for a full life span. Thus, the yeast popu- demonstrates the significance of genetic instability in lation is immortal. There is an exponential decline in yeast aging. It also provides a plausible explanation for the appearance of nucleolar enlargement and fragmentation in old yeast cells16. Sinclair and Table 2. Yeast longevity genes Guarente6 suggest that the rDNA constitutes an ‘age’ locus to which Gene Function Refs the silencing proteins migrate from the telomeres and silent matingLAG1 Unknown 10 type loci in old cells. Currently, it LAC1 Unknown; homolog of LAG1 1,11 is unclear how this migration CDC7 Protein kinase; involved in cell-cycle control and transcriptional 12,13 silencing affects aging. The SIR4-42 muRAS1 GTP-binding (G-) protein; involved in signal transduction 14 tation, which extends life span15, RAS2 G-protein; involved in signal transduction 14 also causes migration of silencing SIR4 Transcriptional silencing 15 proteins to the nucleolus16. UTH4 Unknown 15,16 There is a loss of transcriptional YGL023 Unknown; homolog of UTH4 16 silencing at heterochromatic reSGS1 DNA helicase; involved in DNA recombination 17 gions of the yeast genome, includPHB1 Unknown; mitochondrial protein 18,19 ing subtelomeric chromatin7 and PHB2 Unknown; mitochondrial protein; homolog of PHB1 18,19 silent mating-type loci8, during RTG2 Unknown; effector of retrograde response 20 normal aging. The resultant gene
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dysregulation has been proposed as another molecular mechanism of yeast aging1. The SIR4 and CDC7 genes both affect silencing22 and are involved in determining yeast longevity12,15. Recently, it has been found that RAS2 affects the basal silencing level of subtelomeric heterochromatin4. These findings are suggestive; however, their interpretation is complicated. It appears that the enhanced resistance to stress found in SIR4-42 (Ref. 15) has a positive effect on yeast life span, but there is likely to be some overlap between the effects of gene dysregulation and migration of silencing complexes to the ‘age’ locus (and its consequences) on yeast longevity. SIR4-42 might be special in allowing a degree of silencing loss that facilitates the activation of stress response genes, while preventing wholesale gene dysregulation. Metabolic regulation A molecular mechanism determining yeast longevity has been identified in studies with petite yeasts20, whose mitochondria are not fully functional. This disruption in mitochondrial function activates an intracellular signaling pathway, termed retrograde regulation, that results in the induction of expression of nuclear genes23. The activation of this retrograde response by genetic or environmental manipulation is correlated with an extension of life span20. This life extension is completely suppressed by deletion of one of the downstream effectors of the retrograde response, RTG2. In addition to this gene, two other genes, RTG1 and RTG3, are required for retrograde regulation24,25. The Rtg2 protein might promote the formation of an active Rtg1–Rtg3 protein heterodimer, which is a transcription factor that induces gene expression from the retrograde response element26. Disruption of RAS2 completely suppresses the life-span extension observed in the petite yeast, indicating that the Ras2 protein affects events downstream of the mitochondrial signal20. In fact, the Ras2 protein modulates the expression of nuclear genes seen in the retrograde response20. Figure 1 summarizes our current understanding of the role of the retrograde response in determining yeast longevity. The nuclear genes regulated by the Rtg1–Rtg3 heterodimer encode a variety of metabolic enzymes that reside in the mitochondria, cytoplasm and peroxisomes27,28. The metabolic changes that the activation of this heterodimer portends are: a shift from glucose to acetate utilization, the activation of gluconeogenesis, and potentiation of the glyoxylate cycle to provide Krebs cycle intermediates. For yeast, this represents an effort to survive in the face of dwindling energy and carbon sources. It is also a means to signal changes in mitochondrial activity, although the nature of the physiological signal is not known. Some of the metabolic events that are triggered by retrograde regulation are also found during yeast sporulation, which itself is a response to severe caloric stress. The retrograde response constitutes an example of the importance of metabolic regulation in determining longevity. Indeed, any extension of life span in yeast must encompass such a molecular mechanism,
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Signal? Ras2
Rtg2 Rtg1
Rtg3
Rtg1
and/or
Rtg3
Nuclear gene transcription
Life span Fig. 1. Interorganelle communication is a determinant of yeast longevity. A signal generated by the mitochondrion results in the activation of transcription of nuclear genes by the heterodimer Rtg1–Rtg3. These are basic helix-loop-helix, leucine zipper proteins25. The Rtg2 protein has been postulated to promote the formation of an active Rtg1–Rtg3 complex26. The nature of the physiological signal produced by the mitochondrion is not yet known. The nuclear genes controlled by the RTG genes encode proteins that are localized in the mitochondrion, cytoplasm and peroxisome27–29. They include: citrate synthase 1; citrate synthase 2 (glyoxylate cycle); isocitrate dehydrogenase; aconitase; acetyl-CoA synthetase; pyruvate carboxylase (gluconeogenic); acetyl-CoA oxidase; catalase A; and the major peroxisomal membrane protein, Pmp27. It is likely that there are many more genes controlled by the Rtg proteins. The activation of this retrograde response pathway results in the extension of yeast life span20. The Ras2 protein interacts with this pathway20, acting downstream of the mitochondrial signal and modulating the expression of the nuclear genes. It is not known whether it is the metabolic enzymes and/or other potential targets of the pathway that determine yeast life span. RAS2 is likely to determine life span by additional mechanisms.
because life span is measured in this organism by the number of daughter cells that are produced, which depends on metabolic capacity. Interestingly, activation of the retrograde response results in a dramatic increase in extrachromosomal rDNA circles30, indicating that this pathway can promote increased longevity, even when these circles are present. Control of stress responses Yeasts are exposed to a variety of stresses in their environment, including starvation, alluded to above, and UV radiation. Curiously, in yeast, the resistance to UV increases with age, reaching a peak at midlife31. However, UV resistance rapidly declines thereafter. The expression profile of RAS2 closely parallels this biphasic response. RAS2 is required for resistance to UV (Ref. 32). The link between stress resistance and longevity is also apparent in the ability to isolate yeast mutants with extended life span by selection for the secondary phenotype of resistance to starvation and cold stress15. The mutants obtained are also resistant to other stresses.
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Another stress that yeasts encounter is heat. Chronic exposure of yeast to regularly repeated episodes of sublethal heat stress results in a reduction in longevity33. This effect is exacerbated when RAS2 is deleted, but not when RAS1 is deleted. In the absence of the Ras2 protein, the cells display a marked delay in resumption of growth and division after the stress is lifted. There is no difference between RAS1 mutants, RAS2 mutants and wild-type cells in the activation of stress genes, nor in the downregulation of growthpromoting genes. However, there is a marked delay, in both the subsequent downregulation of the stress genes and upregulation of the growth-promoting genes in RAS2 mutant cells33, which simply do not recover well from the stress. The delay in downregu-
lation of the stress genes is due to a lack of proper control of transcription from the stress response regulatory element (STRE)34. This can be completely overcome by the overexpression of RAS2. Indeed, overexpression of this gene extends the life span well beyond that of the unstressed control cells33. This life extension is very similar to that observed on overexpression of RAS2 in the absence of any overt stress14. There is a difference, however: in the absence of stress, RAS2 extends life span by a cAMP-independent pathway14, while during chronic bouts of stress, the Ras2protein–cAMP pathway is necessary to achieve the rescue and life extension observed33. Unlike the effect of chronic stress, a transient, sublethal heat stress, delivered early in life, results in an increase in longevity35. This increase in life span is the result of a large decrease in the mortality Trends in Microbiology Acute stress rate, which persists for many generations but is not permanent. This constitutes a heritable Viability epigenetic effect. Both RAS1 and RAS2 are required to observe this effect. As in the chronic stress situation described above, cAMP Chronic stress RAS2 is necessary for timely recovery from the transient stress. cAMP HSP104, the gene encoding the (MAPK) Life span major yeast heat-shock protein that is involved in induced therRAS2 mal tolerance36, is essential for Rapid the life extension, as are funcLife span cAMP tioning mitochondria35. The synresponses (MAPK) PI thetic phenotype of shortened life span in petite yeasts subjected Transient stress to transient, sublethal heat stress RAS1 suggests an interaction between MAPK the life-extension mechanisms activated by this heat stress and Long-term PI Life span those controlled by the retroeffects (no stress) grade response35. The mechanisms might not operate concurrently. These studies on the role Fig. 2. Control of response to heat stress by the RAS genes. RAS2 is involved in the rapid of the RAS genes in the response response to changes in environmental conditions by facilitating recovery from heat stress33. This of yeast to heat stress have function of RAS2 is performed through modulation of the expression of stress genes34, among prompted the model shown in others, which prevents expression of these genes at levels sufficient to withstand an acute Fig. 2. Many of the features of (lethal) heat shock and reduces survival in the stationary phase37. This submaximal ability to this model have been confirmed resist lethal stress represents a trade-off for the ability of the yeast to withstand chronic, sublethal heat stress throughout their life span33. It is the cAMP-stimulatory effect of the Ras2 protein experimentally, while others that is involved in the response both to lethal and to chronic, sublethal heat shock33,34,37. RAS2 is await testing. In particular, it is also essential for the extension of life span provided by the conditioning effect of a transient, subproposed that RAS1 is involved lethal heat stress35. Under these conditions, it is also involved in recovery from heat stress. In the in the long-term response to absence of overt stress, RAS2 has a life span-extending effect, which entails a cAMP-independent pathway14. This is likely to be a mitogen-activated protein kinase (MAPK) pathway38. It is possible transient, sublethal heat stress. that the Ras2 protein operates through both the cAMP and MAP kinase pathways in the response This role is not likely to depend to both chronic and to transient heat stress. RAS1 is postulated to exert long-term effects on on the weak stimulation of longevity, because it is not required for the recovery that is part of the response to chronic adenylate cyclase, which does 33 stress but is essential for the persistent decrease in mortality rate following transient heat not allow RAS1 to substitute for stress35. RAS1 has a life span-shortening effect in the absence of stress14. It is hypothesized that the effects of the Ras1 protein are mediated through its stimulatory effect on inositol phosphoRAS2 in the response to chronic lipid (PI) turnover39,40, because it is not effective in replacing the Ras2 protein in the response to heat stress. Rather, it can be hychronic stress. It also stimulates adenylate cyclase much more weakly than the Ras2 protein pothesized that it is the stimu(Ref. 37). The shading highlights the area in which the actions of RAS1 and RAS2 overlap. The lation of inositol phospholipid blunt arrows denote negative effects. turnover, which the Ras1 protein
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promotes39,40, that is involved. Sphingolipids, which in yeast contain inositol, are required for resistance to a variety of stresses, including heat41. The Ras1 protein might effect a stable change in inositol lipid pools that persists for many generations in response to transient, sublethal heat stress.
represent a shift from a nutrient of high caloric content (glucose) to one of lower caloric content (acetate). Conclusions We do not know how many different molecular mechanisms of aging exist in yeast, however, two appear to be firmly established. These mechanisms coincide with two of the four broad physiological principles of longevity in yeast and in other organisms. The yeast longevity genes RAS1 and RAS2 modulate several processes that are important for longevity, responding to one known signal, nutritional status (Fig. 3). It is quite possible that a molecular mechanism of aging underlies each of these processes. For response to stress, a molecular mechanism is nearly in place in addition to the established role of the retrograde response in metabolic regulation. It is conceivable that genetic stability is also modulated by the RAS genes. Under constant conditions, there is an optimal level of Ras2 protein activity that results in maximal life span21, and the dependence on the Ras2 protein is nonlinear. A change in environmental conditions, with no manipulation of Ras2 protein levels results in either decreased or increased longevity, depending on the circumstances33,35. Thus, the RAS genes constitute a homeostatic device in yeast longevity.
Correlates in other organisms Certain mutants (daf) in the developmental pathway for dauer larva formation in the nematode Caenorhabditis elegans cause an increase in the life span of the adult (reviewed in Ref. 1). The dauer is a dispersal form that allows the nematode to survive periods of starvation, crowding and heat stress. The daf pathway encompasses genes that encode an insulin-receptor-like protein42, a phosphatidylinositol-3-hydroxide kinase43 and a transcription factor belonging to the forkhead family44,45. The identity of these proteins suggests that metabolic regulation plays an important role in nematode aging. Indeed, the metabolic changes that are associated with enhanced life span in the nematode encompass lipid and glycogen metabolism44. Such metabolic consequences are also associated with extended longevity in the fruit fly (reviewed in Ref. 1). The activation of the daf pathway results in alterations in metabolic enzyme activity46 similar to those found on activation of the retrograde response in yeast. A mutation in another nematode gene, clk-1, which does not reside in the daf pathway, Nutritional Changing external also extends life span, and this status and internal gene is a homolog of the yeast Cellular resources environment gene COQ7, a nuclear-encoded regulator of mitochondrial funcNutrients Energy tion47. The long-lived daf and clk-1 mutants have an increased resistance to UV, heat and oxidative stress48. Fruit flies that are selected for extended longevity are also more resistant to a variety of stresses (reviewed in Ref. 1). A Growth RAS Epigenetic gene long-lived Drosophila mutant, regulation methuselah, is more resistant to starvation, heat and oxidative stress than the wild type49. Cell division Genetic stability Caloric restriction of mammals results in extension of life span (reviewed in Ref. 50). These Cell organization Response to stress animals are more resistant to heat stress51. They also display a Trends in Microbiology Competing demands variety of metabolic changes, such as reduced blood glucose Fig. 3. Coordination of cellular activities by the RAS genes. This heuristic model shows that RAS genes ‘channel’ cellular resources (nutrients and energy) to the competing demands of various and insulin levels (reviewed in cellular activities, which are essential for maintenance of homeostasis and determination of Ref. 50), and have higher insulin longevity. The Ras1 and Ras2 proteins do not literally channel resources to these cellular activreceptor levels and increased acities, because they are not part of the pathways that define them but instead modulate them. tivity of the gluconeogenic enThese cellular activities include growth or metabolism 20, cell division 14, cell organization 4, zyme, phosphoenolpyruvate carresponse to stress31,33,35, epigenetic control of gene expression4 and, possibly, genetic stability6. 52,53 This channeling of resources occurs under conditions of changing external and internal environboxykinase . Caloric restriction ment, which alter the balance between the demands on the resources. The RAS genes respond involves a reduction in the to one known signal, nutritional status37. The dependencies are not shown as pathways; instead, caloric content of the diet. The the Ras proteins are depicted driving a cycle (not unlike the Krebs cycle), into which various enzymatic changes associated pathways enter or exit. with the retrograde response
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Questions for discussion • How can there be more than one mechanism of aging in a genetically homogenous population that is maintained under constant conditions? • Why is the production of extrachromosomal rDNA circles not deleterious for longevity under certain conditions? • What is the relationship between metabolic control and stress resistance? • What criteria need to be satisfied in order to classify a gene or a process as a determinant of longevity and/or a cause of aging? • What are the similarities and differences between life extension by caloric restriction, the retrograde response and the daf pathway? Acknowledgements Research in my laboratory is supported by grants from the National Institute on Aging of the National Institutes of Health (U.S.P.H.S.). References 1 Jazwinski, S.M. (1996) Science 273, 54–59 2 Mortimer, R.K. and Johnston, J.R. (1959) Nature 183, 1751–1752 3 Muller, I. et al. (1980) Mech. Ageing Dev. 12, 47–52 4 Jazwinski, S.M. et al. (1998) Exp. Gerontol. 33, 571–580 5 Jazwinski, S.M. (1996) in Handbook of the Biology of Aging (4th edn) (Schneider, E.L. and Rowe, J.W., eds), pp. 39–54, Academic Press 6 Sinclair, D.A. and Guarente, L. (1997) Cell 91, 1033–1042 7 Kim, S., Villeponteau, B. and Jazwinski, S.M. (1996) Biochem. Biophys. Res. Commun. 219, 370–376 8 Smeal, T. et al. (1996) Cell 84, 633–642 9 Muller, I. (1985) Antonie von Leeuwenhoek J. Microbiol. Serol. 51, 1–10 10 D’mello, N.P. et al. (1994) J. Biol. Chem. 269, 15451–15459 11 Jiang, J.C. et al. (1998) Genome Res. 8, 1259–1272 12 Egilmez, N.K. and Jazwinski, S.M. (1989) J. Bacteriol. 171, 37–42 13 Jazwinski, S.M., Egilmez, N.K. and Chen, J.B. (1989) Exp. Gerontol. 24, 423–436 14 Sun, J. et al. (1994) J. Biol. Chem. 269, 18638–18645 15 Kennedy, B.K. et al. (1995) Cell 80, 485–496 16 Kennedy, B.K. et al. (1997) Cell 89, 381–391 17 Sinclair, D.A., Mills, K. and Guarente, L. (1997) Science 277, 1313–1316 18 Coates, P.J. et al. (1997) Curr. Biol. 7, 607–610 19 Berger, K.H. and Yaffe, M.P. (1998) Mol. Cell. Biol. 18, 4043–4052
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Coming soon in Trends in Microbiology •Endogenous retroviruses: are they the cause of Multiple Sclerosis?, by R.S. Fujinami and J.E. Libbey •Variation and evolution of the citric-acid cycle: a genomic perspective, by M.A. Huynen, T. Dandekar and P. Bork •Evolution of Wolbachia pipientis transmission dynamics in insects, by E.A. McGraw and S.L. O’Neill Don’t miss these and many more articles of interest; subscribe to Trends in Microbiology using the form bound in this issue.
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